Genetic control of virulence in erwinia chrysanthemi pv zeae

GENETIC CONTROL OF VIRULENCE IN ERWINIA CHRYSANTHEMI PV. ZEAE MUMTAZ BEGUM BINTE MOHAMED HUSSAIN INSTITUTE OF MOLECULAR AND CELL BIOLOGY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2007 GENETIC CONTROL OF VIRULENCE IN ERWINIA CHRYSANTHEMI PV. ZEAE MUMTAZ BEGUM BINTE MOHAMED HUSSAIN (B.Sc. Hons.) (UNIVERSITY OF LEEDS) A THESIS SUBMITTED FOR THE DEGREE OF MASTERS OF SCIENCE INSTITUTE OF MOLECULAR AND CELL BIOLOGY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEGEMENT I would like to express my heartfelt gratitude to my supervisor, A/P Zhang Lian Hui for his invaluable guidance, insight and encouragement throughout the duration of this project. I sincerely thank the members of my postgraduate Supervisory Committee, A/P Leung Ka Yuen and A/P Wang Yue for their constructive suggestions and guidance. I am extremely grateful to Ms. Xu Jin Ling for her excellent help in generation of mutants as well as in many other aspects of this project; to Dr. Dong Yi Hu, Dr. Zhang Hai Bao and Ms. Zhang Xi Fen for assistance in gene cloning of my mutants. I would also like to thank Dr. Wang Lian Hui and Mr. Yan Fang for advice and assistance in the chemistry part of my project and to other past and present members of the microbial quorum sensing laboratory for their advice, discussion and friendship. Many thanks are also due to the DNA sequencing facility and the histology unit of the IMCB for their excellent service. Last but not least, I would like to thank my sister for her love, encouragement and understanding. ii TABLE OF CONTENTS Contents Page Acknowledgement ii Table of Contents iii Summary ix List of Tables xii List of Figures xiii List of Symbols xvi Chapter 1 General introduction 1 1.1 Taxonomy of E. chrysanthemi 3 1.2 The host range of E. chrysanthemi and related pathovars 5 1.3 Disease symptoms and progression 6 1.4 Virulence genes 9 1.5 Regulation of virulence genes 13 1.6 General mechanisms of quorum sensing 17 1.7 Erwinia chrysanthemi pv. zeae 20 1.8 Aims and scope of the thesis 21 Chapter 2 Materials and methods 22 2.1 Bacterial strains and culture media 22 2.2 Generation of mutants of EC1 defective in AHL signalling 22 2.3 Generation of avirulent mutants of EC1 26 2.4 Southern blotting and hybridization 26 iii 2.5 Sequence analysis of tox- mutants of EC1 27 2.6 Identification of autoinducer mutants of EC1 27 2.7 Complementation of EC1 autoinducer mutants 30 2.8 Complementation of the horEC1 mutant EM53 31 2.9 Biochemical analysis of EC1 and nucleotide sequence 32 analysis of the 16S rDNA 2.10 AHL bioassay 32 2.11 Toxin bioassay 33 2.12 Motility assays 34 2.13 Biofilm formation assay 34 2.14 Transmission electron microscopy analysis 35 2.15 LPS analysis 35 2.16 Alcian blue assay for EPS quantification 36 2.17 Enzyme assay 36 2.18 CAS assay 37 2.19 Pathogenicity assay against potato tubers and Chinese cabbage 38 2.20 Bacterial pathogenicity assay against rice seeds germination 38 2.21 Extraction of EC1 toxin(s) 39 2.22 Physio-chemical treatment of EC1 toxin(s) 39 Chapter 3 Identification and characterization of the AHL-type 40 quorum sensing system in E. chrysanthemi pv. zeae 3.1 Introduction 40 3.2 Results 41 iv 3.2.1 Phenotype and genetic differences between EC1 and 41 E. chrysanthemi strains 3.2.2 Screening and cloning of the genes involved in AHL 43 biosynthesis in EC1 3.2.3 Mutation of echIEC1 did not significantly affect toxin 45 production by E. chrysanthemi pv. zeae 3.2.4 Mutation of echIEC1 resulted in enhanced swimming motility 48 3.2.5 Autoinducer mutants showed no significant difference in flagella 49 and LPS production but displayed increased EPS production 3.2.6 AHL-deficient mutants showed decreased virulence against 54 potato tubers and Chinese cabbage 3.2.7 AHL-deficient mutants still possessed the ability to inhibit rice 57 seed germination 3.2.8 AHL-deficient mutants showed no significant difference with the 57 wild-type parental strain in pectate lyase and protease production 3.3 Summary Chapter 4 Screening of the genes involved in 60 62 E. chrysanthemi pv. zeae toxin production and regulation 4.1 Introduction 62 4.2 Results 63 4.2.1 Screening of the Tox- mutants of strain EC1 63 4.2.2 Single Tn5 insertion in the genome of EC1 mutants was 65 responsible for the Tox- phenotype v 4.2.3 Sequence analysis of Tox- mutants revealed the genes 67 implicated in polyketide antibiotics biosynthesis 4.3 Summary Chapter 5 Sequence analysis and characterization of the 71 73 gene of E. chrysanthemi pv. zeae encoding a novel polyketide synthase 5.1 Introduction 73 5.2 Results 74 5.2.1 Cloning and sequencing of EC1 chromosomal fragment 74 containing the polyketide synthase gene 5.2.2 Domain structure analysis of the polyketide synthase 75 5.2.3 The polyketide synthase mutants were attenuated in their 77 virulence against potato tubers and Chinese cabbage 5.2.4 Polyketide mutants are defective in inhibition of rice 79 seed germination 5.2.5 Pigment and siderophore production in the polyketide 80 mutants were affected 5.3 Summary Chapter 6 81 A key transcriptional regulator that modulates the toxin production and virulence of E. chrysanthemi pv. zeae 6.1 Introduction 82 82 vi 6.2 Results 83 6.2.1 Cloning and sequencing of the DNA fragment containing 83 the hor homologue 6.2.2 Expression of the wild-type horEC1 gene in EM53 restored 86 the toxin production 6.2.3 HorEC1 played an essential role for infection of 87 E. chrysanthemi pv. zeae on both dicot and monocot plants 6.2.4 HorEC1 did not appear to play a significant role in regulation of 92 pectate lyase and protease production 6.2.5 Mutation of horEC1 had no effect on production of AHL quorum 92 sensing signals 6.2.6 Mutation of horEC1 affected swimming ability, biofilm 94 formation and pigment production 6.3 Summary Chapter 7 E. chrysanthemi pv. zeae produces a toxin(s) 98 99 that inhibits rice seeds germination 7.1 Introduction 99 7.2 Results 100 7.2.1 Maximal toxin production occurred at stationary phase in 100 minimal medium 7.2.2 Virulence factor can be extracted using Amberlite XAD7 beads 102 7.2.3 Toxin was stable under various conditions 103 7.2.4 The toxin extract inhibited the root germination of rice seeds 105 vii 7.3 Summary Chapter 8 General discussion and conclusion 107 108 8.1 Summary of major findings 108 8.2 Quorum sensing in Erwinia strains and its role in regulation 109 of bacterial virulence 8.3 EC1 is likely to produce polyketide toxins 111 8.4 The role of the HorEC1 transcriptional regulator 113 8.5 The general characteristics of toxin(s) 114 8.6 Conclusion 115 Bibliography 117 Appendix 1 138 Appendix 2 142 Appendix 3 148 Appendix 4 151 Appendix 5 155 Appendix 6 156 viii SUMMARY The bacterial pathogens belonging to Erwinia chrysanthemi infect many dicot plants but hardly damage any monocot crops. This study focused on a bacterial strain EC1 isolated from the rice plants showing typical foot rot symptoms. The 16S rDNA analysis showed that EC1 shared a high homology with, but was distinct from several E. chrysanthemi strains. In addition, strain EC1 showed phenotypical differences with the well characterized E. chrysanthemi strains EC3937 and EC16, including the abilities to inhibit the growth of bacteria and fungi, and rice seeds germination. These results plus the fact that EC1 is able to infect monocot rice, established that strain EC1 belongs to E. chrysanthemi pv. zeae, which is a rarely characterized subspecies of E. chrysanthemi. Disruption using Tn5 mutagenesis, of one of the regulatory systems known to affect virulence ability, the ExpI-ExpR quorum sensing system in EC1, did not affect the inhibition ability of the pathogen on rice seeds germination, suggesting the possibility of other regulatory mechanisms. The ExpI-ExpR quorum sensing system of EC1 that shows about 90 % homology to the similar system in E. chrysanthemi EC3937, did however affect other phenotypes such as swimming ability, EPS production and pathogenesis against potato tubers and Chinese cabbage. Further Tn5 transposon mutangenesis to identify genes involved in toxin production and regulation, revealed the genes encoding regulation and biosynthesis of polyketide antibiotics/toxin(s). Cloning and sequencing analysis identified a gene encoding a peptide sharing about 60 % homology to the polyketide synthase P3-A6- ix PKS from Chromobacterium violaceum. We obtained three independent E. chrysanthemi with Tn5 inserted in various regions of this gene and in all cases, the manifestation of phenotypes due to the Tn5 insertion was similar, including the diminished inhibitory effect on bacterial, fungal growth and rice seeds germination, and the decreased virulence on dicot plants. In addition, we identified a transcriptional regulator HorEC1, which is a member of the MarR/SlyA transcriptional regulator family. It shows a high homology to other members of this family such as the Rap of Serratia sp. (92 % identity) and the Hor of E. carotovora subsp. carotovora (84 % identity). The horEC1 mutant showed enhanced swimming ability but decreased biofilm formation, pigment production and virulence against potato tubers and Chinese cabbage. Besides, the mutant lost the ability to inhibit microbial growth and rice seeds germination. These phenotype changes were restored by overexpression of the intact horEC1 gene in the mutant, demonstrating the global regulatory role of HorEC1 on diverse bacterial activities. Furthermore, we found that the strain EC1 produced significantly more toxin in minimum medium than rich medium. We then established a chromatographic protocol for partial purification of the toxin(s) from strain EC1. The toxin(s) appeared to be a heat stable molecule and could tolerate both acid and alkaline treatment. These findings would help further purification and characterization of this intersect molecule. Importantly, we found that the toxin specifically inhibited the root germination of rice seeds, though had less effect on rice shoot germination and elongation. The genetic and biochemical results from this study demonstrate for the x first time that the bacterial pathogen E. chrysanthemi pv. zeae produces a toxin(s) which appears to play a key role in bacterial infection of monocot plants. xi LIST OF TABLES Table No. Title Page 1.1 Erwinia chrysanthemi EC16 enzymes inducible by pectate and involved in the depolymerization or de-esterification of pectic polymers and oligomers 8 2.1 Bacterial strains and plasmids used in this study 23-25 2.2 Oligonucleotides used in this study 28 -30 4.1 Classification of the Tox- mutants based on the size of hybridization bands 67 4.2 Sequence analysis of Tox- mutants of the strain EC1 69-70 6.1 List of bacterial strains 88 xii LIST OF FIGURES Figure No. Title Page 1.1 Pectin catabolism in E. chrysanthemi 10 1.2 The Type II secretion system of E. chrysanthemi 12 1.3 E. chrysanthemi 3937 siderophore and pigment gene clusters 13 1.4 A simplified model for the regulatory network controlling pectinase, indigoidine, achromobactin and chrysobactin synthesis in E. chrysanthemi 14 1.5 A quorum-sensing model 19 3.1 16S rDNA based phylogenetic position of EC1 42-43 3.2 Virulence bioassay 43 3.3 AHL assay of representative AHL-deficient mutants and complementary strains 46 3.4 Physical map and sequence analysis of the DNA fragment containing the genes involved in AHL quorum sensing signal biosynthesis and regulation 46-48 3.5 Mutation of the gene encoding AHL biosynthesis enhanced EC1 swimming motility 50-51 3.6 OHHL modulates the swimming motility of E. chrysanthemi pv. zeae 51 3.7 Electron micrographs of EC1 and AHL- mutants 52 3.8 LPS assay using SDS-PAGE and stained with silver solution 53 3.9 Alcian blue assay 54 xiii 3.10 Pathogenesis assay using potato tubers and Chinese cabbage 55-56 3.11 E. chrysanthemi pv. zeae inhibited rice seed germination 58 3.12 Analysis of exoenzymes produced by E. chrysanthemi pv. zeae 59 4.1 Examples of growth inhibition assay by strain EC1 and its mutants 64-65 4.2 Southern blot analysis of the Tox- mutants of strain EC1 66 5.1 Generic map of the regions flanking Tn5 insertion sites in mutants EM9, EM11 and EM107 74-75 5.2 Protein domain analysis 76-77 5.3 Pathogenesis assay using potato tubers and Chinese cabbage 78 5.4 Pathogenesis assay on rice seed germination 79 5.5 Pigment and siderophore production assays 80 6.1 The genome structure of Tn5 inserted chromosomal region of the Tox- mutant EM53 83 6.2 Domain analysis of the transcriptional regulator HorEC1 85 of E. chrysanthemi pv. zeae strain EC1, SlyA of Serratia spp. and Hor of E. carotovora subsp. carotovora 6.3 Sequence alignment of HorEC1 and homologues 86 6.4 Toxin bioassay against C. albicans and E. coli DH5α. 89 6.5 Pathogenesis assay using potato tubers and Chinese cabbage 90 6.6 Pathogenesis assay on rice seeds germination 91-92 6.7 Analysis of exoenzymes produced by E. chrysanthemi pv. zeae 93 6.8 AHL signal bioassay 94 xiv 6.9 Mutation of horEC1 resulted in enhanced bacterial swimming motility 95 6.10 Mutation of horEC1 decreased biofilm formation 96-97 6.11 Pigmentation assay 97 7.1 Bacterial growth and toxin production in LB medium and minimal medium 101 7.2 Bioassay of chromatography fractions and extracted toxin 102-103 7.3 Bioassay against E. coli DH5α and C. albicans 104 7.4 Treatment of toxin in acid and alkaline solution 105 7.5 Rice seeds germination 106-107 xv LIST OF SYMBOLS AND ABBREVIATIONS Symbol Explanation 5’ five prime µg microgram µl microlitre µg/mg microgram per milligram µg/ml microgram per millilitre µm micrometre µM micromolar 1X one time ∆A/∆T rate of increase in absorbance at 235 nm 3’ three prime 2X two times A235 absorbance at 235 nm A440 absorbance at 440 nm aa amino acid AHL N-acylhomoserine lactones AHL- autoinducer defective AI autoinducer AMP adenosine monophosphate Ampr ampicillin resistant ATCC American Type Culture Collection xvi atm atmospheric BLAST Basic Local Alignment Search Tool bp base pair CAS chrome azural S c.f.u. colony forming units CoA coenzyme A CRP cyclic AMP receptor protein cm centimetre ddH2O double distilled water DHL N-decanoyl-homoserine lactone DIG digoxigenin DMSO dimethyl sulphoxide EPS exopolysaccharide EtOH ethyl alcohol g gram Genr gentamycin resistant h hour Hgr hygromycin resistant HHL N-(hexanoyl)-homoserine lactone IM Inner Membrane KCl potassium chloride KDG 2-keto-3-deoxygluconate Kanr kanamycin resistant xvii kb kilobase L litre LB Luria-Bertani LPS lipopolysaccharide Mg2SO4 Magnesium sulphate Mg2SO4.7H2O Magnesium sulphate 7 water min minutes mJ millijoule M molar concentration MM minimal medium mg milligram mg/ml milligram per millilitre ml millilitre mM millimolar mm millimetre mol/min mole per minute nm nanometer N normal NCBI National Center for Biotechnology Information NGM nematode growth media OD optical density OHHL N-(3-oxohexanoyl)-homoserine lactone OM Outer Membrane xviii ORF open reading frame PBS Phosphate Buffered Saline PCR polymerase chain reaction PL pectate lyase PK proteinase K PKS polyketide synthase pH potential of hydrogen pv. pathovar RB ribosome binding rDNA ribosomal deoxyribonucleic acid RNA ribonucleic acid rpm revolutions per minute r.t. room temperature SDS sodium dodecyl sulphate SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis Smr streptomycin resistant spp. species subsp. subspecies SSC standard sodium citrate T2S type II secretion pathway TAIL-PCR thermal asymmetric interlaced PCR TEM transmission electron microscopy TM trademark xix Tetr tetracyclin resistant tox- toxin defective U unit UDG digalacturonate UTP uridyl triphosphate UV ultra violet vol volume wt weight wt/vol weight per volume X-Gal 5-bromo-4-chloro-3-indolyl-β-galactopyranoside YEB yeast extract broth xx CHAPTER 1 General introduction Bacterial diseases are one of the major threats to human health and animal life worldwide. Bacteria also cause plant diseases which could lead to severe reduction in global food production (Strange, R.N. et al., 2005; Fauci, A.S., 2001). The pathogens hence become serious economical, political, social and ecological problems which are further exacerbated with the emergence of antibiotic resistant bacteria (Yoneyama, H. et al., 2006; Monaghan, R.L., et al., 2006; Cohen, M.L., 2000). Due to the rapid increase of antibiotic resistant bacteria in recent years, efforts have been made on curtailing the use of antibiotics as this has been identified to be one of the main contributory factors to the emergence of bacterial antibiotic resistance. This concern extends to the agricultural practice where antibiotics are sprayed on growing crops as a preventive measure against plant diseases (McManus, P.S. et al., 2002). It is feared that not only will such practice result in an increase in antibiotic resistant phytopathogens, like streptomycin-resistant Erwinia amylovora that causes fire blight disease of apple and pear (McManus, P.S. et al., 2002), but may also contribute to antibiotic resistance in human pathogens by increasing the resistance gene pool and the chance of horizontal gene transfer. While there has been no conclusive evidence that this may be the case, USA for example, has taken a 1 conservative view by banning the use of gentamycin sulphate in agriculture since the antibiotic is also used in human medicine (McManus, P.S. et al., 2002). In addition to reducing the inappropriate use of antibiotics, which act by either killing or stopping bacterial growth, several other strategies have been explored for the control of plant bacterial diseases. One traditional strategy is to exploit plant defence resistance mechanisms. Due to their long-term association with pathogens, plants have evolved sophisticated mechanisms to perceive pathogen invasions and to translate the perception into defence responses (Dang, J.L. et al., 2001). The plant varieties that show strong disease resistance have been commonly used as parental lines in crop breeding programmes. The plant defence responses usually involve inducible production of bactericidal substances such as phytoalexins and free radicals, and generation of physical barriers to prevent bacterial invasions. A more recent strategy known as “quorum quenching”, on the other hand, aims to stop or reduce expression of virulence genes by bacterial pathogens and hence confiscate the biochemical weapons with which the pathogens invade and infect their host plants (Dong, Y.H. et al., 2001; Zhang L.H., 2003). This strategy is based on the understanding that single-celled bacterial pathogens rely on a population density dependent cell-cell communication mechanism, termed quorum sensing (Fuqua, W.C. et al., 1994), to coordinate many important biological activities including expression of virulence genes. This promising development illustrates the importance of identification of key bacterial virulence factors and the mechanisms of genetic regulation. 2 Bacterial stalk rot is one of the important rice bacterial diseases. It occurs in many rice planting countries and regions including China, India, Indonesia, Philippines and Korea. The disease is caused by the bacterial pathogen Erwinia chrysanthemi. The pathovar, E. chrysanthemi pv. zeae, also causes severe infections in maize (Sinha, S.K. et al., 1977). However, in contrast to its closely related pathovar, E. chrysanthemi, which infects many crops and plants worldwide, E. chrysanthemi pv. zeae is much less characterized, in particular, at the molecular and genetic levels. It is not clear what the key virulence factors are and how much this pathovar is similar to its related pathovar E. chrysanthemi. For effective control of this important bacterial pathogen, it is essential to determine its key virulence genes and their regulatory mechanisms. 1.1 Taxonomy of E. chrysanthemi The genus Erwinia was introduced in 1917 by the Society of American Bacteriologists to accommodate grouping of phytopathogenic bacteriae with the species being named in accordance with the host plant from which they had been isolated. While it was proposed as early as 1945 that non-pectolytic bacteria that cause dry necrosis be classified as true Erwinia while pectolytic bacteria that cause soft rot be grouped in a new genus, Pectobacterium, it has not been officially taken up or strictly adhered to (Starr, M.P. et al., 1972; Waldee, E.L. et al., 1945). As a result, certain bacteria have been grouped under both genuses by different research groups so that the terms have been used interchangeably. For example, bacteria isolated from the plant Chrysanthemum morifolium have been identified as E. 3 chrysanthemi in some cases and P. chrysanthemi in others, although the 16S rDNA sequence analysis supports the relatedness of these two groups of bacterial isolates (Hauben, L. et al., 1998). For better understanding of pathogen-host interaction and developing appropriate treatments, there is a need to identify and characterize the pathogens. Toward this end, there have been considerable research efforts on developing physiological and biochemical identification methods and characterization of the various species classified under the genus Erwinia (Avrova, A.O. et al., 2002; Lee, Y.A. et al., 2006). In 2004, an attempt was made to classify E. chrysanthemi based on a range of characteristics from biochemical properties to phenotypic variations and it was proposed that P. chrysanthemi be assigned to a new genus, Dickeya chrysanthemi (D. chrysanthemi) (Samson, R. et al., 2004). In this classification, Dickeya contains 6 species, namely D. chrysanthemi, D. dadantii, D. dianthicola, D. dieffenbachiae, D. paradisiacal and D. zeae. The last species is a novel biovar that infects both Zea mays and Chrysanthemum morifolium. However, this proposal has yet to be accepted by the Bergey’s Manual of Determinative Bacteriology even though it has been unofficially accepted and used in certain cases (Palacio-Bielsa, A. et al., 2006). One reason for this delayed acceptance is that the Bergey’s Manual is not updated on a yearly basis, the last one being published in 1994 (Bergey’s Manual, 1994). Using the latest taxonomy list obtained from The International Society for Plant Pathology (http://www.isppweb.org/names_bacterial_pant2005.asp), the genus 4 E. chrysanthemi consists of 6 pathovars, namely, E. chrysanthemi pv. chrysanthemi, E. chrysanthemi pv. dianthicola, E. chrysanthemi pv. dieffenbachiae, E. chrysanthemi pv. paradisiaca, E. chrysanthemi pv. parthenii and E. chrysanthemi pv. zeae. It is worth noting that for all the pathovars listed, the genus Erwinia is used interchangeably with Pectobacterium except for E. chrysanthemi pv. paradisiaca which is used interchangeably with Brenneria paradisiacal. This official taxonomical classification will be used hereafter in this study. For convenience and being consistent with numerous previous literature, the pathovar E. chrysanthemi pv. chrysanthemi will be in general, referred to as E. chrysanthemi. 1.2 The host range of E. chrysanthemi and related pathovars E. chrysanthemi is a gram-negative, rod-shaped, motile bacteria with a broad hostrange, and is responsible for soft rot disease in a variety of commercially important plants such as Chrysanthemum, potato tubers (Solanaceae tuberosum) and African violet (Saintpaulia ionantha) (CABI/EPPO; Bergey’s Manual 1994; Collmer, A. et al., 1994; Whitehead, N.A. et al., 2002). The findings show that E. chrysanthemi only affects dicotylenous plants. E. chrysanthemi has been isolated across a wide range of geographical areas such as Asia (Malaysia), Africa (Senegal), North America (USA), South America (Peru, Cuba), Antarctica, Europe (Finland, Scotland, France, Spain) and Australia (Avrova, A.O. et al., 2002). While constant regrouping adds difficulties in finding the host ranges of other E. chrysanthemi pathovars, it is clear that E. chrysanthemi pv. zeae is the key 5 pathovar to infect monocotyledonous plants, i.e., zeae mays and oryza sativ (Gray, J.S.S. et al., 1993; Liu, Q.G. et al., 2004). Nevertheless, at least under artificial inoculation conditions as shown in the following chapters, the E. chrysanthemi pv. zeae isolates used in this study can also cause infections in dicotyledonous plants such as potato and Chinese cabbage. 1.3 Disease symptoms and progression E. chrysanthemi is known to cause soft rot disease which is characterized by foulsmelling rot and the eventual collapse of plant tissues. The way in which this occurs is through a number of stages whereby E. chrysanthemi adapts itself to the varying microenvironments of the infected plant during the course of infection by the production of an arsenal of virulence factors. The first stage of maceration by E. chrysanthemi involves the entry of the bacteria to the parenchymatous tissues of plants that have been physiologically compromised, such as by bruising, excess water or high temperature (Collmer, A. et al., 1994). The next stage involves local maceration as a result of depolymerization of plant cell walls, followed by necrosis of the entire plant (Barras, F. et al., 1994). Due to the complexity of plant cell walls, which consists of polysaccharides, the main ones being cellulose, hemicellulose and pectin, a variety of enzymes are accordingly produced by E. chrysanthemi for the efficient breakdown of cell walls (Robert-Baudouy, J. et al., 2000). Most work on the enzymes involved in maceration have been done using the E. chrysanthemi strains 3937 (EC3937) and EC16 (EC16), which will be used in this study as reference strains. 6 The major enzymes have been found to be pectinases (Table 1.1), which degrade various components of pectin using different reaction mechanisms. Other hydrolytic enzymes are also produced, such as cellulase isozymes, protease isozymes, xylanases and phospolipases (Robert-Baudouy, J., 2000; Hugouvieux-Cotte-Pattat, N., 1996; Collmer, A. et al., 1994). It has also been reported that E. chrysanthemi is capable of causing systemic disease by spreading through the vascular system of a plant. The physiological symptoms of such infection are yellowing of new leaves, wilting and a mushy, foulsmelling stem rot (Slade, M.B. et al., 1984). Genetic and physiological studies show that systemic infection of E. chrysanthemi is dependent on two abilities, namely iron acquisition and production of the pigment, indigoidine (Expert, D. et al., 1985; Enard, C. et al., 1988; Enard, C. et al., 2000; Reverchon, S. et al., 2002). Due to iron scarcity in the environment and its role as an essential element, most organisms have derived the ability to sequester iron by production of low-molecular-weight highaffinity iron-chelating agents called siderophores. These are produced in response to iron limitation in order to capture Fe3+ ions. In a plant-bacteria interaction, the successful competition for iron between the two organisms could determine the outcome of an invasion (Enard, C. et al., 1988). In E. chrysanthemi 3937, two siderophores are produced, namely chrysobactin and achromobactin. The structures of both these iron chelators as well as the pigment, indigoidine have been elucidated and characterized (Persmark, M. et al., 1989; Franza, T. et al., 2005; Munzinger, M et al., 2000; Expert, D. et al., 1996; Kuhn, R. et al., 1965). It has also been observed that mutants affected in 7 chrysobactin, achromobactin or indigoidine production were impaired in its ability to cause systemic invasion in Saintpaulia ionantha (Franza, T. et al., 2005; Enard, C. et al., 1988; Reverchon, S. et al., 2002). Table 1.1 Erwinia chrysanthemi EC16 enzymes inducible by pectate and involved in the depolymerization or de-esterification of pectic polymers and oligomers (Collmer, A. et al., 1994). Enzymes Genesa Reaction mechanism Substrate and products Direct role in maceration Pectate lyase (isozymes PelA-E) pelABCE βelimination of internal glycosidic bonds Pectate/various Yes, oligomers depending on isozyme Exo-poly-α-Dgalacturonosidase pehX Hydrolysis of penultimate glycosidic bond Pectate/dimers No Oligogalacturonide ogl lyase βelimination Oligomers/ monomers No Pectin methylesterase Hydrolysis of methylester Pectin No (polymethoxygalacturonide)/ pectate pem a The gene designations were based on the enzyme reaction mechanism: pel (pectate enzyme lyase); peh (pectic enzyme hydrolase); pem (pectic enzyme methylesterase) and ogl (oligogalacturonide lyase). 8 1.4 Virulence genes Most of the genes that have been identified to play a role in pathogenesis of E. chrysanthemi are those that encode for the major virulence factors of the bacteria. These are listed in Table 1.1 and summarized in Figure 1.1. Among the several pectate lyases, PelE appears to be the most important isozyme in potato tuber maceration as a null mutation of pelE reduces half of the maceration capacity of the wild-type strain (Payne, J.H. et al., 1987), which is followed by PelB, PelC and PelA. However, the importance of each isozyme in pathogenesis may vary depending on the host plants. For example, while the pelBC mutation does not significantly affect the virulence of pathogen on Saintpaulia, it does so on chicory (Beaulieu, C. et al., 1993). This also suggests that the redundancy provided by the arsenal of isozymes may be important for effecting pathogenesis in different hosts. As these pectin degrading enzymes need to reach their target, the plant tissues, the genes encoding their secretion are also essential for virulence. In E. chrysanthemi, the type II secretion pathway, which is also known as the Out system, is the main secretion system involved in secreting the pectate lyases. Protein transportation by the type II secretion pathway (T2S), also known as the general secretory pathway (Gsp), is a two step process, common among gram negative bacteria. The first step involves translocation of proteins across the inner bacterial membrane by the Sec system followed by transportation of the proteins from the periplasm to the exterior by an outer membrane secretin (Cianciotto, N.P., 2005). 9 Figure 1.1 Pectin catabolism in E. chrysanthemi. Pectin is degraded sequentially by a variety of pectinases either those secreted (eg. PelA, PelB, PelC, PelD, PelE, PelL and PelX) to the external medium by the Out system or those located in the outer membrane (PemB), or in the periplasm. The catabolism of oligogalacturonides takes place in the cytoplasm. (Compiled with modifications based on the following references: Ito, Y. et al., 1999 and Chatterjee, A.K. et al., 1985). _____________________________________________________________________ 10 Biochemical and yeast two-hybrid analyses show that the type II secretion system of E. chrysanthemi consists of 12 core components making up the outer membrane secretins (GspDSC), the cytoplasmic ATPase (GspE), the inner transmembrane proteins (GspFLM), and the major (GspG) and minor (GspHIJK) pseudophilins as depicted in Figure 1.2 (Py B. et al., 2001). Beyond these, there may be other non-conserved proteins involved based on the latest protein-protein analysis using yeast two-hybrid system (Douet, V. et al., 2004). Disruption of these genes reduces the virulence and maceration capacities of E. chrysanthemi (de Kievit, T. R. et al., 2000; Lindeberg, M. et al., 1992). It is also interesting to note that while the Out proteins of E. chrysanthemi shows high homology to those of E. carotovora, the system is unable to secrete the pectinases of E. carotovora and vice-versa, indicating strong species specificity of the Out proteins (Lindeberg, M. et al., 1998). Other genes that are involved in virulence are those associated with systemic infection of E. chrysanthemi such as siderophores and pigment. The siderophore, chrysobactin is encoded by an 8-kb fct-cbsCEBA operon, with cbsCEBA being the biosynthesis genes of the catechol moiety of chrysobactin and fct being the receptor gene (Figure 1.3a). The fct-cbsCEBA operon is regulated by a bidirectional promoter which also controls operon cbsHF (Figure 1.3a). The product of cbsH is involved in intracellular iron homeostasis (Franza, T. et al., 1991; Rauscher, L. et al., 2002). 11 Figure 1.2 The Type II secretion system of E. chrysanthemi. The secretion machinery can be depicted as comprising three buildings blocks: the inner membrane (IM) platform (GspE, GspF, GspL, GspM; white box); the pseudopilus (GspG, GspH, GspI, GspJ, GspK; grey box); and the gated outer membrane (OM) pore (GspC, GspD, GspS, dotted box). Cel5 is shown as an example of a secreted protein ‘en route’ to the cell exterior (modified based on Py, B. et al., 2001). The other siderophore, achromobactin is encoded by a 13-kb long operon comprising eight genes of which six are biosynthesis genes (acs), one is necessary for extracellular release of achromobactin (yhcA) and one encodes the outer membrane receptor for its ferric complex (acr). The promoter of the operon lies immediately upstream of the acsF gene (Figure 1.3b) (Franza, T. et al., 2005). The gene coding the pigment indigoidine, is located in a 6.3-kb cluster close to the regulatory gene pecS-pecM locus, immediately downstream of pecM. It comprises the indA gene encoding a protein of unknown function and the biosynthetic genes, indB and indC (Reverchon, S. et al., 2002). 12 In all these cases, disruption of the genes encoding for siderophore or pigment production affects the pathogenesis ability of E. chrysanthemi. For example, mutation of indA abolishes the systemic infection of the pathogen in Saintpaulia ionantha (Reverchon, S. et al., 2002). (a) chrysobactin gene cluster (8-kb long) (b) achromobactin gene cluster (13-kb long) (c) indigoidine gene cluster (6.3-kb long) Figure 1.3 E. chrysanthemi 3937 siderophore and pigment gene clusters. (a) chrysobactin and (b) achromobactin coding regions (modified based on Rauscher, L. et al., 2002; Franza, T. et al., 2005) and (c) the indigoidine gene cluster (modified based on Reverchon, S. et al., 2002). Oval and square represent promoter regions. _____________________________________________________________________ 1.5 Regulation of virulence genes As the major virulence determinants of E. chrysanthemi are pectate lyases, the attempts to decipher the virulence regulatory mechanisms not surprisingly, focused mainly on pectate lyases. Several regulatory models were proposed. A representative model incorporating the siderophore and pigment regulation, in addition to the pectate lyase regulation is depicted in Figure 1.4. 13 Figure 1.4 A simplified model for the regulatory network controlling pectinase, indigoidine, achromobactin and chrysobactin synthesis in E. chrysanthemi. The functional conformation of each regulator is indicated by a square (inactive form) or a circle (active form). Promoter activation and repression are indicated by arrows and bars respectively. This model includes the potential relationships occurring between the different regulatory circuits (ExpI-ExpR, PecS, PecT, CRP, Fur, KdgR and RsmA-rsmB RNA). The signals recognized by PecS and PecT are not yet identified but appear to be linked to plant sensing (modified based on Robert-Baudouy, J. et al 2000). _____________________________________________________________________ 14 Briefly, Figure 1.4 shows that in the absence of pectin degradation products such as KDG (2-keto-3-deoxygluconate), the transcriptional repressor, KdgR, represses the pel genes which encode for petate lyases. It also represses the aepH expression, which is part of the RsmA-aepH post-transcriptional regulatory system. In the absence of pectin, RsmA is expressed and binds to and facilitates the degradation of pel RNA and the RNA of the quorum sensing gene involved in AHL production, expI. aepH that is an untranslated RNA molecule, positively activates pectate lyases production by antagonizing the effects of RsmA. It does this by sequestering RsmA, thereby preventing RsmA from degrading the pel and expI RNAs (Liu, Y. et al., 1998; Reverchon, S. et al., 1998; Chatterjee, A. et al., 2002). Based on this, it is worth mentioning that the regulation of the pel genes consists of a network of various interacting factors which fine tune the entire virulence factor production system. Another regulator, the cyclic AMP receptor protein (CRP) has been demonstrated to be an activator for petate lyases production as a crp mutant showed reduced pectate lyases activity and maceration ability (Reverchon, S. et al., 1997). It acts by binding directly to the promoter regions of the pel genes. A second level of regulation that CRP confers is by competing with KdgR for their overlapping binding sites on the promoter, thereby displacing the KdgR repressor level (Reverchon, S. et al., 1997; Nasser, W. et al., 1997). Research on iron deficiency and pectate lyase production reveals that the iron regulator, Fur, represses not only the siderophores, achromobactin and chrysobactin production (Rauscher, L. et al 2002; Franza, T. et al., 2005), but also likely the pectate lyases, in high iron environment. This may be achieved by direct binding 15 because sequence analysis of pelD and pelE reveals the presence of Fur boxes near their promoter regions. These Fur boxes are the consensus sequences to which Fur has the ability to bind (Robert-Baudouy, J. et al., 2000; Venkatesh, B. et al., 2006). Although the cognate signal ligand remains unknown, the transcriptional regulator, PecT appears to regulate the pectate lyase genes by binding directly to its promoter based on band shift assay (Castillo, A. et al., 1998). In addition, PecT is also implicated in other biological activities associated with virulence such as motility and EPS production (Condemine G. et al., 1999). Similarly, the inducer of PecS, which belongs to the MarR family of transcriptional repressors, has not been identified and characterized. PecS has been shown to negatively regulate the expression of pectate lyases, indigoidine and out genes but positively regulate the production of polygalacturonases. In all instances, it does so by direct binding to the promoter regions of the genes (Reverchon, S. et al., 1994; Robert-Baudouy, J. et al., 2000). The dual roles of the PecS regulator therefore illustrate the complexity of virulence regulation in E. chrysanthemi. The discovery that a regulator, Hor, which seems to affect similar phenotypes as PecS, unveils another layer of complexity in virulence regulatory networks (Thomson, N.R. et al., 1997). The remaining regulatory elements depicted in Figure 1.4 are related to the quorum sensing system, which will be discussed in the following section. From this proposed regulatory network, some of the regulators like KdgR, RsmA and CRP were discovered to have homologues in other Enterobacteria, 16 suggesting the likely general and conserved roles. This implies that what is discovered in Erwinia spp. may be extrapolated to other bacterial pathogens. 1.6 General mechanisms of quorum sensing Quorum sensing is a term used to describe a type of cell-cell communication that involves small signal molecules called autoinducers (AIs). Bacteria produce and accumulate AIs in a population-dependent manner which upon reaching a critical threshold concentration alters the expression level of target genes (Zhang, L.H., 2003; Reading, N.C. et al., 2006). This widely conserved mechanism is exemplified by the regulation of bioluminescence in Vibrio fischeri and Vibrio harveyi by AIs (Nealson K.H. et al., 1970; Nealson K.H. et al., 1979). In this system, AIs such as N-acylhomoserine lactones (AHLs) are synthesized by the LuxI protein and binds to and activates an AHL-dependent transcription factor called LuxR which in turn, binds to the promoters of target genes to initiate quorum-sensing-dependent gene expression as illustrated in Figure 1.5 (Zhang, L.H., 2003). It is worthy to note that in the past two decades, several classes of autoinducers have been identified. The best-characterized autoinducers are the AHLs which are produced by more than 70 bacterial species, the majority of which are Gram-negative bacterial pathogens (Dong, Y.H. et al., 2000; Zhang, L.H., 2003). Most of these signalling molecules are involved in the regulation of bacterial virulence (Whitehead, N.A. et al., 2001; Zhang, L.H. et al., 2004). 17 In E. chrysanthemi, there is a 5- to 60- fold increase in pectate lyase production during exponential growth phase of the bacteria which corresponds to the appearance of the physiological symptom, soft rot on Saintpaulia ionantha after extensive bacterial multiplication (Hugouvieux-Cotte-Pattat, N. et al., 1996). Since the accumulation of bacteria is linked to quorum sensing, research on possible correlation between the production of the main virulence factors, pectate lyases and quorum sensing was undertaken. The homologues of the quorum sensing system, LuxI-LuxR in E. chrysanthemi was found to be the ExpI-ExpR system which mediates the production of two out of the three AIs present, namely, N-(3-oxohexanoyl)-homoserine lactone (OHHL) and N-(hexanoyl)-homoserine lactone (HHL). However, the system implicated with the third AI, N-(decanoyl)-homoserine lactone (DHL) has yet to be discovered (Nasser, W. et al., 1998; Whitehead, N.A. et al., 2002; Whitehead, N.A., 2001). It was discovered that the null mutation of expI abolished OHHL and HHL production but did not significantly affect the overall pectate lyase activity. Transcriptional analysis showed that mutation of expI decreased the expression of the genes encoding PelA and PelB, which are minor contributors to virulence, but did not have significant effect on the expression of PelE which is the main contributor to virulence (Nasser, W. et al., 1998; Boccara, M. et al., 1988; Payne, J.H. et al., 1987). It was further shown that null mutation of ExpR did not show any phenotype changes. These findings suggest the presence of other factors involved in pectate lyase regulation and that the ExpI-ExpR system constitutes only part of a complex 18 regulatory system controlling the pectate lyase production in E. chrysanthemi (Nasser, W. et al., 1998). Figure 1.5 A quorum-sensing model. signalling systems (Zhang, L.H., 2003). It is based on acyl homoserine lactone 19 1.7 Erwinia chrysanthemi pv. zeae Erwinia chrysanthemi pv. zeae was first documented in 1954 to cause soft rot in wheat (Sabet, A.K., 1954). Since then, it has been isolated from a wide variety of hosts and geographical regions (Kalia, V. et al., 2006; Hiroyuki, U. et al., 2006). In maize, the typical symptoms are premature withering and the drying up of the uppermost leaves, followed by the lower leaves. The rot either extends from the base upwards or from the top downwards (Sinha, S.K. et al., 1977). The host range studies showed that the maize pathogen also caused soft rot in many dicot plants including potato, carrot, cabbage, tobacco and tomato (Sinha, S.K. et al., 1977). In rice, the disease caused by E. chrysanthemi pv. zeae is known as rice foot rot (Goto, M., 1979; Liu, Q.G. et al., 2004). The typical symptoms are rice stem rot or foot rot, leave withering and white ear. The pathogen can also infect rice seeds during seed germination and causes rotten seeds or seedlings (Liu, Q.G. et al., 2004). Frequent occurrences of rice foot rot disease have been reported and the disease could cause severe economical losses (Liu, Q.G. et al., 2004). However, apart from some efforts made in characterizing the EPS (Gray, J.S.S. et al., 1993) and in identification of the bacteria (Samson, R. et al., 2004; Avrova, A.O. et al., 2002; Lee, Y.A. et al., 2006; Palacio-Bielsa, A. et al., 2006), little has been done on molecular identification and biochemical characterization of its virulence determinants and their corresponding regulatory systems. 20 1.8 Aims and scope of the thesis The aims of this project are to identify the virulence genes of E. chrysanthemi pv. zeae strain EC1, and to characterize the corresponding genetic and regulatory mechanisms. Moreover, it is interesting to compare the differences in virulence between the closely related E. chrysanthemi pv. zeae and E. chrysanthemi; the latter has been characterized extensively at the molecular level. The thesis has been divided into 8 chapters. Chapters 1 and 2 provide the general introduction and experimental methods, respectively. Chapter 3 is on the characterization of the gene encoding for production of AHL-type quorum sensing signals and the role of the signal in regulation of bacterial biological functions. Chapter 4 describes the screening of toxin-defective mutants following transposon mutagenesis and preliminary characterizations. Chapter 5 presents sequence analysis and characterization of the gene encoding a beta-ketoacyl synthase essential for toxin production. Chapter 6 focuses on cloning and characterization of a transcriptional regulator involved in regulation of bacterial virulence and toxin production. Chapter 7 reports the preliminary work on purification of the toxin and characterization of its physio-chemical properties. Chapter 8 is the general discussion of the thesis. 21 CHAPTER 2 Materials and methods 2.1 Bacterial strains and culture media The bacterial strains and plasmids used in this study are listed in Table 2.1. E. coli and C. albicans were routinely grown at 37°C in LB medium (per litre contains 10 g Bacto tryptone, 5 g yeast extract and 10 g NaCl, pH 7.0). E. chrysanthemi strains were grown at 28°C in YEB (per litre contains 10 g Bacto tryptone, 5 g yeast extract, 5 g sucrose, 5 g NaCl and 1 mM MgSO4.7H2O) or minimal medium as indicated (Zhang, L.H. et al., 1991). Antibiotics were added at the following concentrations when required: gentamycin 50 µg/ml; kanamycin 50 µg/ml; ampicillin 100 µg/ml. 2.2 Generation of mutants defective in AHL signalling in EC1 Transposon, Tn5, carried by the suicide plasmid pRL27 was introduced from the host strain E. coli DH5α into the genome of EC1 by triparental mating using the conjugation helper strain E. coli RK 2013 (Ditta, G. et al., 1980; Garfinkel, D. et al., 1980). Triparental mating was performed by mixing overnight cultures of corresponding bacterial strains onto an LB agar plate and incubated at 28°C for 6 h. Transconjugants were then selected on MM agar plates containing 100 µg/ml kanamycin. 22 Table 2.1 Bacterial strains and plasmids used in this study* Strain or Plasmid Remarks Reference C. albicans strain CAI4 ATCC MYA-682 C. violaceum (CV533) CV026 (Sm mini-Tn5 Hg cviI:: Tn5xy/E r Kan ) r r McClean, K.H, et al., 1997 Escherichia coli DH5α supE44 ∆lacU169 ( Φ80lacZ ∆M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 HB101 (pRK2013) Thr leu thi recA hsdR hsdM pro, Kan Laboratory Collection EC1 An isolate from rice plant belonging to E. chrysanthemi pv. zeae This study EC16 A wild type strain of E. chrysanthemi pv. chrysanthemi from Chrysanthemum morifolium Chatterjee, A.K. et al., 1983 EC3937 A wild type strain of E. chrysanthemi pv. chrysanthemi from Saintpaulia Lemattre, M. et al., 1972 WM3 Tn5 (delivered by pRL27) insertion mutant of EC1, AHL This study WM6 Tn5 (delivered by pRL27) insertion mutant of EC1, AHL This study WM8 Tn5 (delivered by pRL27) insertion mutant of EC1, AHL This study WM3-echI Mutant WM3 containing expression r construct pDSK-Gen :echI This study WM6-echI Mutant WM6 containing expression r construct pDSK-Gen :echI This study WM8-echI Mutant WM8 containing expression r construct pDSK-Gen :echI This study r Laboratory Collection E. chrysanthemi 23 Table 2.1 (Cont’d) Bacterial strains and plasmids used in this study* Strain or Plasmid Remarks Reference EC1-echI EC1 containing expression r construct pDSK-Gen :echI This study EM9 Tn5 (delivered by pTGN) insertion mutant of EC1, tox This study EM11 Tn5 (delivered by pTGN) insertion mutant of EC1, tox This study EM107 Tn5 (delivered by pTGN) insertion mutant of EC1, tox This study EM40 Tn5 (delivered by pTGN) insertion mutant of EC1, tox This study EM13 Tn5 (delivered by pTGN) insertion mutant of EC1, tox This study EM20 Tn5 (delivered by pTGN) insertion mutant of EC1, tox This study EM104 Tn5 (delivered by pTGN) insertion mutant of EC1, tox This study EM53 Tn5 (delivered by pTGN) insertion mutant of EC1, tox This study EM53H1-4 Mutant EM53 containing expression construct pUC19:horEC1 This study EM53H2-1a Mutant EM53 containing expression construct pUC19:horEC1 This study EC1H1-2 EC1 containing expression construct pUC19:horEC1 This study EC1H2-8 EC1 containing expression construct pUC19:horEC1 This study traR tra::lacZ, AHL indicator Piper, K.R. et al 1993 A. tumefaciens NTI (CF11) 24 Table 2.1 (Cont’d) Bacterial strains and plasmids used in this study Strain or Plasmid Remarks Reference BW020767(pRL27) Harboring Tn5-RL27 (kan -oriR6 K) for mutagenesis Larsen, R.A. et al., 2002; Zhang, H.B. et al., 2004 pDSK-Gen Broad-host-range IncQ cloning vector, r Gen Laboratory Collection pGEM7+ Amp , cloning vector Promega pDSK Broad-host-range IncQ cloning vector, r Kan Keen, N.T. et al., 1998 pUC19 Amp Yanisch-Perron, C. et al., 1985 pDST Broad-host-range IncQ cloning vector, r Tet Laboratory Collection r r Cosmid C285 pTGN r r r Amp Laboratory Collection pBSL202 carrying a 2-kb XbaI-SmaI fragment containg the gfp-nptII fusion operon Tang, X. et al., 1999 * Ampr denotes ampicillin resistance, Genr denotes gentamycin resistance, Hgr denotes hygromycin resistance, Kanr denotes kanamycin resistance, Smr denotes streptomycin resistance, Tetr denotes tetracyclin resistance, AHL- denotes autoinducer defective, and tox- denotes toxin defective. _____________________________________________________________________ Transconjugants were then transferred onto fresh LB agar plates containing the relevant antibiotics and screened for its inability to produce OHHL using the AHL bioassay method listed in 2.10. Mutants that were unable to activate the indicator strain were selected for further analysis. 25 2.3 Generation of avirulent mutants of EC1 Transposon mutagenesis of EC1 was performed as described in section 2.2, except that another mini transposon Tn5 carried by pTGN (Tang, X. et al., 1999) was used in this experiment. Transconjugants were transferred onto fresh LB agar plates containing the relevant antibiotics and screened for its inability to inhibit C. albicans and E. coli DH5α growth using the spotting technique of the virulence factor bioassay assay method listed in 2.11. Mutants that were defective in the production of an inhibition zone were selected for further characterization. 2.4 Southern blotting and hybridization Bacterial genomic DNA was isolated using the MasterPureTM DNA Purification Kit (EPICENTRE® Biotechnologies). An aliquot of 20 µg of genomic DNA was digested with ClaI or EcoRI and separated by electrophoresis in a 0.8 % agarose gel and transferred onto a Hybond-N+ membrane (Amersham Pharmacia Biotech). It was then covalently linked to the membrane by exposure to UV light (312 nm) using the Stratalinker® UV cross linker set at 1200 mJ (Stratagene). A 0.4-kb fragment containing the gentamycin gene of the Tn5 transposon which was amplified using PCR primers Gt-f and Gt-R (Table 2.2), was labelled with digoxigenin-11-dUTP of the DIG DNA labelling kit (Roche). The labelled fragment was then used as the probe for hybridization at 42°C overnight. Pre-hybridization and hybridization were performed using the DIG easy Hyb kit (Roche) according to the manufacturer’s recommendations. 26 Following hybridization, the membrane was washed twice with 2X SSC-0.1 % SDS at room temperature for 5 min (1X SSC contains 0.15 M NaCl, 0.015 M sodium citrate). This was followed by two washes in 0.1X SSC-1 % SDS at 65 °C for 15 min. Detection was performed using the DIG Detection Kit (Roche). 2.5 Sequence analysis of tox- mutants of EC1 In order to identify the sequences flanking the Tn5 insert, thermal asymmetric interlaced PCR (TAIL-PCR) was performed as previously described (Liu, Y.G. et al., 1995), except that the following nested primers were used: Gt-447, Gt-464 and Gt487 (Table 2.2). The 13 arbitrary primers designated AD1-AD13 used in this study are listed in Table 2.2. For sequencing the TAIL-PCR products, the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) was used and the reaction was carried out on a MJ Research PTC 100 Thermal Cycler. Sequence determination was carried out by the DNA Sequencing Facility at the IMCB using an automated DNA sequencer 3700 (ABI), according to the manufacturer’s protocol. Sequences were analysed using NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). 2.6 Identification of autoinducer mutants of EC1 The genomic DNA of the mutants were extracted using MasterPureTM DNA Purification Kit (EPICENTRE® Biotechnologies), digested with BamHI and ligated using T4 DNA ligase. The ligated plasmid was transformed into E. coli DH5α 27 competent cells and the transformants were selected on LB agar supplemented with 100 µg/ml kanamycin. Table 2.2 Oligonucleotides used in this study Primer Sequence Gt-f 5’-GCAGTCGCCCTAAAACAAA-3’ Gt-R 5’-AAGTTGGGCATACGGGAAGAAGTG-3’ Gt-447 5’-GTGCAAGCAGATTACGGTGACGAT-3’ Gt-464 5’-TGACGATCCCGCAGTGGCTCTC-3’ Gt-487 5’- ATACAAAGTTGGGCATACG-3’ AD1 5’- AGWGNAGWANCAWAGG-3’ AD2 5’- CANGCTWSGTNTSCAA-3’ AD3 5’- GTCGASWGANAWGNA-3’ AD4 5’- GTNCGASWCANAWGTT-3’ AD5 5’- NCAGCTWSCTNTSCTT-3’ AD6 5’- NGTASASWGTNAWCAA-3’ AD7 5’- NGTCGASWGANAWGAA-3’ AD8 5’- NTCGASTWTSGWGTT-3’ AD9 5’- SCACNTCSTNGTNTCT-3’ AD10 5’- STTGNTASTNCTNTGC-3’ Purpose For creation of the probe used in Southern blot Nested primers for TAIL-PCR amplification of the inserted Tn5 (carried by pTGN) flanking sequences Arbitrary primers for TAIL-PCR amplification of the inserted Tn5 flanking sequences 28 Table 2.2 (Cont’d) Oligonucleotides used in this study Primer Sequence Purpose AD11 5’- TGWGNAGWANCASAGA-3’ AD12 5’- WAGTGNAGWANCANAGA-3’ AD13 5’- WGTGNAGWANCANAGA-3’ Arbitrary primers for TAIL-PCR amplification of the inserted Tn5 flanking sequences tpnR1_172 5’-AACAAGCCAGGGATGTAACG-3’ tpnR1_132 5’-CAGCAACACCTTCTTCACGA-3’ EchR-1 20 5’-CCCATACTTGCCCAGTAGAG-3 EchI-B-1 28 5’-CGGGATCCTCACCAGGTGAGCTATTGCG-3’ EchI-B-2 28 5’-CGGAATTCGCTTGGGGTTGAAATGAACC-3’ 16sf95 5’-TGACGAGTGGCGGACGGGTG-3’ 16sr394 5’-CCATGGTGTGACGGGCGGTGTG-3’ 16sf342 5’-TACGGGAGGCAGCAGTGGGGAATA-3’ For PCR amplification of the inserted Tn5 (carried by pRL27) flanking sequences For sequencing echR For PCR amplification of the echI coding region For 16S rDNA fragment amplification and sequencing 29 Table 2.2 (Cont’d) Oligonucleotides used in this study Primer Sequence Purpose Hor600Hf 5’-TGACAAGCTTGCTGGGGGGAGTCCAAAC-3’ For PCR amplification of the horEC1 promoter and coding region horHf 5’-TGACAAGCTTATGGAATTGCCGTTAGGTTCTG-3’ horBr 5’-CACGGATCCAGCAACTCGAATCATTGAG-3’ For PCR amplification of the horEC1 coding region _____________________________________________________________________ Following PCR amplification with the primers tpnR1_17-2 and tpnR1_13-2 (Table 2.2), the PCR product was sequenced and analysed using NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). To identify downstream genes of echI, further sequencing was performed using EchR-1 20 primer (Table 2.2) designed based on the sequence already identified above. 2.7 Complementation of EC1 autoinducer mutants Primers EchI-B-1 28 and EchI-B-2 28 (Table 2.2) were designed based on sequence data of echI and used to amplify its coding region. The PCR product was then subjected to BamHI and EcoRI digestion as was the vector pDSK-Genr. The digested PCR product and vector were then purified and ligated in such a way that the coding region of echI was placed under the control of the lac promoter carried by the vector. 30 The ligation mixture was transferred to E. coli and transformations were selected on LB agar supplemented with 25 µg/ml gentamycin and confirmed by DNA sequencing. The corresponding complementary strains of WM3, WM6 and WM8 were generated by conjugal triparental mating as described in 2.2 except that transformants were selected on MM agar plates supplemented with 100 µg/ml kanamycin and 25 µg/ml gentamycin. The resultant strains WM3-echI, WM6-echI and WM8-echI were confirmed by PCR as described in section 2.6. The same expression construct was also introduced into the wild-type strain EC1 as a control. 2.8 Complementation of the horEC1 mutant EM53 Two sets of primers as listed in Table 2.2 were designed with one for amplification of the horEC1 coding region (primers horHf and horBr) and the other for horEC1 and its promoter (Hor600-Hf and horBr). The horEC1 gene was amplified using PCR and subjected to BamHI and HindIII digestion, purified and ligated to plasmid pUC19 which was also digested with BamHI and HindIII. The ligated plasmid was then transformed into E. coli DH5α competent cells and transformants were selected on LB agar supplemented with 100 µg/ml ampicillin. The plasmid purified from E. coli DH5α was then digested using BamHI and HindIII to confirm the presence of the horEC1 gene. Following confirmation, the plasmid containing the horEC1 gene was transformed into EM53 and EC1 competent cells, respectively. The EM53 complementary strains were selected on LB agar supplemented with 100 µg/ml 31 ampicillin and 50 µg/ml kanamycin, and the EC1 overexpressing horEC1 was selected on LB containing 100 µg/ml ampicillin. 2.9 Biochemical analysis and nucleotide sequence analysis of the 16S rDNA of EC1 EC1 was analysed using the standardized identification kit (Api 20E, BIOMERIEUX INDUSTRY) for Enterobacteriaceae and other non-fastidious Gram negative rods according to the manufacturer’s recommendation. The kit consists of 21 miniaturized biochemical tests. The results obtained were compared to the database provided with the kit. Nucleotide analysis was performed by first amplifying a 1.4-kb fragment of 16S rDNA using primers 16sf95 and 16sr394 (Table 2.2) from genomic DNA of the bacterial strains. This fragment was then sequenced using both the primers, in addition to primer 16sf342. The sequence was then analysed using nucleotide- nucleotide blast of NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). A phylogenetic tree was constructed using the ClustalW program from DNASTAR based on the 16S DNA sequence of EC1 and its best homologues from NCBI database. 2.10 AHL bioassay AHL bioassay was performed as described previously (Zhang, L.H. et al., 1993). Agrobacterium tumefaciens NTI, containing a lacZ fusion with the tra gene, was used as an indicator strain for AHL activity (Piper, K.R. et al., 1993). Briefly, plates 32 containing 20 ml of minimal agar medium supplemented with 5-bromo-4-chloro-3indolyl -D-galactopyranoside (X-Gal, 40 µg/ml) were used for the bioassay. The solidified medium in the plates was cut into separate slices (1 cm in width). Bacteria were streaked or bacterial culture (5 µl) was added to one end of an agar slice, and then the cultures of the AHL indicator strain were spotted (0.6 µl of OD600 0.4) at progressively further distances from the loaded samples. The plates were incubated at 28°C for 24 h. The distance of the indicator spots that turned blue was measured to determine the relative amount of AHL produced (Dong, Y.H. et al., 2000; Zhang, L.H. et al., 1993). 2.11 Toxin bioassay The bioassay plate was prepared by pouring about 20 ml of LB agar medium, which after solidification was overlaid with 5 ml 1 % agarose containing 1.0 x 108 cells of fresh C. albicans, E. coli DH5α or Chromobacterium violaceum (CV533). Wild-type EC1 and its derivatives were grown overnight on YEB agar plates. The bacterial cells were then spotted onto the plates using toothpicks. Plates containing C. albicans and E. coli DH5α were incubated overnight at 37°C. CV533 was incubated at 28°C for 3 days. For liquid assay, wells of 4 mm in diameter were punched in the bioassay plates. The supernatants of overnight bacterial cultures grown in minimal medium to OD600 = 1.5 were filter-sterilized. For each sample, duplicates of 15 µl were taken and added to bioassay wells. The plates were then incubated as mentioned above. 33 A clear zone of inhibition of EC1 or derivatives on plates containing C. albicans, E. coli DH5α or CV533 is indicative of bacterial toxin. 2.12 Motility assays For determination of swimming and swarming motility, semisolid Bacto Tryptone agar plates were either spotted with bacteria using a toothpick or inoculated with 1 µl of an overnight bacteria culture as previously described (Rashid, M.H. et al., 2000) except that incubation at 28°C was 6 h for the swimming assay plates and 16 h for the swarming assay plates. For complementation assay with OHHL, 200 µl solution of 20 µM OHHL was spread on each swimming agar plate and allowed to air-dry for 1 h before spotting bacterial cells. Each experiment was repeated three times in triplicates. 2.13 Biofilm formation assay Biofilm formation assay was performed in Fisherbrand® borosilicate glass culture tubes using SOBG medium (per litre, 20 g tryptone, 5 g yeast extract, 0.5 g NaCl, 2.4 g MgSO4, 0.186 g KCl and 50 ml 40 % glycerol). Glass slides were then immersed in the tubes containing SOBG medium. This was done to provide a removable surface for observing biofilm formation under a microscope. The culture tubes containing the glass slides and medium were inoculated with 1:1000 dilution of overnight bacteria culture. The tubes were then incubated at room temperature (approximately 25°C) and observed daily for biofilm formation (Yap, M.N. et al., 2005). 34 The glass slides were then removed from the culture tubes to be stained for visualization of biofilm. Non-adherent cells on the slides were removed by rinsing with distilled water. Biofilms were stained with a 0.1 % (wt/vol) crystal violet solution for 15 min followed by rinsing twice with 75 % EtOH and once with distilled water and photographed. For microscopy analysis of bacterial biofilm, the glass slides were removed from the culture tubes and viewed under a Leica MZFLIII Fluorescence stereomicroscope or an Olympus Microscope BX50 under magnification of x 10 and x 40, respectively (Choy, W.K. et al., 2004). 2.14 Transmission electron microscopy analysis Bacterial cells were collected from LB agar plates and suspended in distilled water, 10 µl of the suspension were used for negative staining and subsequent examination. Transmission electron microscopy (TEM) analysis at x 20 magnification was carried out by the Histology Laboratory at the IMCB. 2.15 LPS analysis LPS was extracted using the LPS Extraction Kit (iNtRON BIOTECHNOLOGY) according to the manufacturer’s recommendation. The LPS extracted was run on a 12 % sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained using a silver staining kit (BIO-RAD) to determine the quantity and quality of LPS samples. 35 2.16 Alcian blue assay for EPS quantification Bacterial culture (3 ml) at OD600 = 1.2 was centrifuged at 12,000 rpm for 2 min. The bacterial pellet was resuspended with 1 ml distilled water and stained according to a modified method of Reddy, K.J. et al., 1996. Alcian blue solution, pH 2.5 (1 mg/ml was dissolved in 3 % acetic acid) (10 µl) was added to the bacterial suspension and incubated at r.t. for 30 min. The sample was then centrifuged at 12,000 rpm for 2 min. For washing, the pellet was resuspended with 1 ml distilled water and recentrifuged as described above. The washing step was repeated twice. Following the last centrifuged step, the pellet was resuspended in 1 ml distilled water and its optical density was read at 450 nm and 620 nm. The relative EPS content was calculated by subtracting the optical density read at 620 nm from the read at 450 nm. 2.17 Enzyme assay Quantitative pectate lyase assay was conducted as describe previously (Chatterjee, A.K. et al., 1985) with some modification. Briefly, bacteria was grown to OD600 = 1.2 and the supernatant filter-sterilized. The pectate lyase (PL) activity was determined by measuring the change in absorbance at A235 after addition of 0.5 ml of the filter-sterilized bacterial supernatant to 2 ml of substrate [0.5 mg/ml polygalacturonic acid (Sigma) was used as substrate in 50 mM CAPS buffer (pH 10.8) containing 1 mM CaCl2]. The mixture was then incubated for 30 min at 30°C and the OD235 then measured. An increase in absorbance is indicative of pectate lyase activity. One unit of PL activity is defined as the amount of enzyme that produced 1 36 µmol of unsaturated digalacturonate (UDG) per minute at 30°C. The activity in units/ml is calculated as follows: Activity = ∆A/∆T x 1/4.6 x 2.5/0.5 x Dilution, where ∆A/∆T = rate of increase in absorbance at 235 nm 4.6 = absorption coefficient of the unsaturated bond at the 4th -5th position of the uronic acid residue (ie ε235 = 4.6 mmol-1 x cm-1). 2.5 = the total volume of the reaction mixture. 0.5 = the volume of the sample. Dilution = dilution of the original enzyme preparation. Proteolytic activity was determined according to the method of Caldas, C. et al., 2002. In this method, the activity is measured by adding 50 µl aliquots of bacterial supernatants to an equal volume of Tris-HCl (pH8) buffer containing 2 % azocasein and 0.2 M NaCl. The reaction mixture was then incubated at 37°C for 1 h. Undigested azocasein was precipitated by adding 130 µl of 10 % trichloroacetic acid to the incubations and centrifuged at 10,000 x g for 10 min. The supernatants (100 µl) were transferred to a 96-well microtitration plate containing 200 µl of 1 M NaOH and the absorbance at A440 was read. One azocasein unit is defined as the amount of enzyme producing an increase of 0.01 OD units per hour. 2.18 CAS assay Siderophore production was measured using the chrome azurol S (CAS) liquid assay as described by Schwyn, B. et al., 1987. 37 2.19 Pathogenicity assay against potato tubers and Chinese cabbage Potato (Solanum tuberosum) tubes were obtained from local stores. After being washed with tap water and dried on a paper towel, the potato tubers were surface sterilized with 70 % ethanol and then sliced evenly to about 5 mm in thickness. Each slice was then placed in a Petri dish lined with sterilized Whatman no. 3 filter paper moistened with distilled water. Bacterial cells (2 µl at OD600 =1.2) were added to the sliced potato tuber after piercing it with a needle. The potato tubers were then incubated at 28ºC. The potato tubers were observed daily for maceration ability. Each treatment was repeated 3 times with triplicates. The treatment for Chinese cabbage (Brassica oleracea L.) was similar except that it was cut to 2 cm2 pieces. LB media is used as a negative control. 2.20 Bacterial pathogenicity assay against rice seeds germination Thirty rice seeds were randomly picked and placed in 100 ml bottles. The rice seeds were inoculated with 20 ml of bacterial culture of OD600=1.2 for 6 h at r.t. The rice seeds were then washed three times with distilled water and transferred onto two moistened Whatman Paper no. 3 filter paper in a Petri dish. The seeds were then incubated at 28°C under 16 h light and 8 h dark conditions and observed daily. The rice seeds and germinated seedlings were watered with distilled water when necessary. LB media is used as a negative control. 38 2.21 Extraction of EC1 toxin(s) An overnight culture of EC1 grown in YEB media was diluted 1/50-fold into 750 ml of fresh MM in a 2 L flask. EC1 was then anaerobically incubated at 28°C, 100 rpm for 24 h. The cells were then removed by centrifugation at 6000 rpm twice and the supernatant filter-sterilized using a 0.22 µm pore filter. The cell-free supernatants were passed slowly through a 10 x 20 cm column containing about 114 g of XAD7 (Sigma) at a flow-rate of 1 ml/min. The column was first washed with 3 L ddH2O, followed by 500 ml of methanol. The EC1 toxin(s) was then eluted using 2 L of acetone. The acetone was evaporated from the elute solutions by rotary evaporator, and the residues were dissolved in 3 ml DMSO. Samples at each stage were collected and assayed for toxicity. 2.22 Physio-chemical treatment of EC1 toxin(s) For testing heat stability, EC1 toxin(s) (10 µl) was subjected to autoclave treatment at 121°C for 15 min. The toxin was then assayed against C. albicans and E. coli DH5α. For enzyme stability test, the toxin (10 µl) was treated with proteinase K at 37°C for 4 h. Then, proteinase K was inactivated by heating at 100°C for 7 min. For acid and alkaline stability analysis, the toxin (10 µl) was added to 1 ml PBS buffer of various pH (pH 1, pH 3, pH 6, pH 7, pH 9, pH 11 and pH 13) for 1 h before performing any bioassay. The respective buffers were used as controls. 39 CHAPTER 3 Identification and characterization of the AHL-type quorum sensing system in E. chrysanthemi pv. zeae 3.1 Introduction Quorum sensing modulates diverse biological functions in a variety of bacteria. The functions which quorum sensing modulates include Ti-plasmid conjugal transfer in Agrobacterium tumefaciens (Zhang, L.H. et al., 1993), induction of bioluminescence in Vibrio fischeri (Engebrecht, J. et al., 1984), control of virulence genes such as EPS in Erwinia stewartii (Beck von Bodman, S. et al., 1995), production of pectate lyase in E. chrysanthemi (Basham, H.G. et al., 1975a; Basham, H.G. et al., 1975b; Jones, L.R., 1909; Collmer, A. et al., 1994) and production of exotoxins in Pseudomonas aeruginosa (West, S.E.H. et al., 1994; Albus, A. et al., 1997). In E. chrysanthemi, three autoinducers have been discovered, namely OHHL, HHL and DHL (Nasser, W. et al., 1998; Whitehead, N.A. et al., 2001; Whitehead, N.A. et al., 2002). The genes coding for OHHL and HHL biosynthesis have been identified to be expI (Nasser, W. et al., 1998). Work has also been done using deletion mutation to identify the biological functions regulated by OHHL and HHL. It was found that deletion of expI resulted in reduced production of two pectate lyases, ie PelA and PelB (Collmer, A. et al., 1994; Nasser, W. et al., 1998). The bacterial isolate, EC1, which causes rice foot rot disease, is believed to belong to a subspecies of E. chrysanthemi, known as E. chrysanthemi pv. zeae (Liu, 40 Q.G. et al., 2004). Our preliminary study showed that it also produces AHL quorum sensing signals. But the gene encoding AHL production, and the biological functions regulated by AHL signals have not been identified and characterized. In this chapter, the close but distinct taxonomical relationship of EC1 with known E. chrysanthemi strains was confirmed by phenotype and 16S rDNA analysis. In addition, the gene encoding AHL biosynthesis was also cloned. It was further shown that the AHL quorum sensing system plays a role in regulation of the virulence of E. chrysanthemi pv. zeae. 3.2 Results 3.2.1 Phenotypic and genetic differences between EC1 and E. chrysanthemi strains The bacterial isolate EC1 which was isolated from rice plants showing typical soft rot symptoms, was provided for by collaborators in China (Liu, Q.G. et. al., 2004). To determine its biochemical and genetic differences with the well-characterized E. chrysanthemi pv. chrysanthemi strains, EC3937 and EC16 (Table 2.1), we compared their ability to use different carbon sources, their 16S rDNA sequence homology and other phenotypes. Biochemical analysis using ApI 20E kit (BIOMERIEUX INDUSTRY) showed that EC1 profile belongs to the Pantoea spp. which is also part of the Erwinia clade according to The International Society for Plant Pathology (http://www.isppweb.org/names_bacterial_pant2005.asp). The 1.4-kb 16S rDNA fragment of EC1 was then sequenced and subjected to web-based similarity searches 41 against the GenBank. The sequence shared 98 % - 99 % homology with 16S rDNA of E. chrysanthemi strains 571, CFBP 2052 and CFBP 1270. A phylogenetic tree was further created to determine the evolutionary position of EC1 with respect to the three bacteria to which it shares high 16S rDNA homology with. The phylogram is shown in Figure 3.1. The results show a relative tight clade among the Erwinia spp. strains. Nevertheless, two distinct clusters were observed, one comprising EC2052, EC1270 and EC571 while the other comprises just EC1, suggesting that EC1 may be a subspecies of E. chrysanthemi. We then further compared the similarities and differences between EC1 and the well characterized E. chrysanthemi strains EC3937 and EC16. One major difference we observed was that EC1 was able to inhibit the growth of the bacteria, E. coli DH5α and C. violaceum and the fungus, C. albicans (Figure 3.2). 42 Figure 3.1 16S rDNA based phylogenetic position of EC1. The length of each pair of branches represents the distance between sequence pair, while the nucleotide substitutions indicate the number of substitution events. EC1 (strain EC1), EC571 (E. chrysanthemi strain 571, NCBI Accession number AF373199), EC2052 (E. chrysanthemi, Accession number AF520711), EC1270 (E. chrysanthemi 1270, Accession number AF520709). _____________________________________________________________________ Figure 3.2 Virulence bioassay. Bioassay against C. albicans (cda), E. coli DH5α (DH5α) and C. violaceum (CV533) using filter-sterilized supernatant of respective bacteria grown to OD600 = 1.5. Wild-type is EC1. Other Erwinia spp. are EC16 and EC3937. The photos were taken 24 h after incubation. _____________________________________________________________________ 3.2.2 Screening and cloning of the genes involved in AHL biosynthesis in EC1 We tested EC1 on an indicator strain, Agrobacterium NTI (CF11), which contains the TraR and a tra gene linked to the lacZ gene that gets activated in the presence of the 43 autoinducer, OHHL or its derivatives (Zhang, L.H. et al., 1993; Piper, K.R. et al., 1993). We found that EC1 was able to activate the lacZ gene of A. tumefaciens NT1, indicating that EC1 produces AHL-type autoinducers. Tn5 transposon mutagenesis was then performed to identify the genes involved in AHL signal production. Mutants showing altered AHL production were selected (Figure 3.3) using the AHL bioassay method described previously (Zhang, L.H. et al., 1993; Dong Y.H. et al., 2002). Out of the 4,500 mutants screened, 8 mutants were defective in production of AHL. The flanking regions of the transposon insertion in 3 mutants, i.e., WM3, WM6 and WM8, were sequenced. Analysis of the sequenced DNA fragments led to the identification of an open reading frame that encodes a peptide showing about 92.5 % and 99 % homology with the AHL synthase ExpI of E. chrysanthemi strains EC3937 (NCBI No. CAA65306; Nasser, W. et al., 1998) and NCPPB 1066 (NCBI No. AAA86841), respectively. This ORF was herewith designated as echIEC1. Sequence analysis also revealed that WM3 and WM8 were most likely siblings as Tn5 was inserted at the same position (172nd nucleotide of the 690-bp echIEC1 coding region), whereas WM6 was a separate mutant with Tn5 inserted at the 265th nucleotide within the coding region (Figure 3.4a). Further sequencing of the downstream region of echIEC1, and subsequent homology search of the sequence revealed the echREC1 gene which showed 90.8 % and 98.0 % homology at the peptide level to the expR gene of E. chrysanthemi strain 3937 (NCBI No. CAD27339) and NCPPB 1066 (NCBI No. AAA86840). The echIEC1 and echREC1 are opposite-oriented with 33-bp a overlap at the 3’-end of the genes (Figure 3.4a, 3.4b). The promoter elements including the -10 and -30 regions, 44 and the ribosome binding site were identified in the corresponding promoter regions of the two genes. A putative lux box, which is the binding site of LuxR-type transcription factors (Stevens A.M. et al., 1997; Castang S. et al., 2006), was found at the region close to the -35 element in the promoter of echREC1 (Figure 3.4b). No lux box was found in the promoter of echIEC1. Instead, two putative lux box sequences were found in the 5’-region of the echIEC1 coding sequence (Figure 3.4b). The wild-type echIEC1 was cloned from the strain EC1 by PCR amplification. For the complementation test, the gene was placed under the control of the pTac promoter in the expression vector pDSK-Genr. The resultant construct was introduced into the mutants WM3, WM6 and WM8 separately. Bioassay results showed that AHL produced in the three mutants was fully restored (Figure 3.3), demonstrating that echIEC1 is the gene responsible for AHL production in strain EC1. 3.2.3 Mutation of echIEC1 did not significantly affect toxin production by E. chrysanthemi pv. zeae Using the stab method of virulence factor bioassay, it was observed that mutation of echIEC1 had no effect on the ability of toxin production when assayed against C. albicans and E.coli DH5α, but the growth inhibition zones of WM3, WM6, and WM8 on C. violaceum CV533 became slightly smaller than the wild-type EC1 (data not shown). 45 Figure 3.3 AHL assay of representative AHL-deficient mutants and complementary strains. EC1 and its derivatives were streaked on the top of the agar bar. The indicator strain, CF11, which was spotted along the length of the MM agar, turns a blue colour in the presence of AHL. The number of spots of blue is indicative of the concentration of AHL that has diffused along the length of the agar. Wild-type is EC1 and mutants are WM3, WM6 and WM8. Complementary strains are WM3-echI, WM6-echI and WM8-echI. Over expression strain is EC1-echI. Other Erwinia spp. are EC16 and EC3937. _____________________________________________________________________ 46 47 Figure 3.4 Physical map and sequence analysis of the DNA fragment containing the genes involved in AHL quorum sensing signal biosynthesis and regulation. (a) Physical map of the 2.3-kb fragment containing the echIEC1 and echREC1 genes. WM3, WM6 and WM8 are the AHL-defective mutants and Tn5 insertion sites are indicated by arrows. (b) The nucleotide sequences of echIEC1 and echREC1 and predicted peptide products. The transcriptional polarities are indicated by the arrows. The predicted ribosomal binding sites (RB) and putative promoter elements are indicated by bold-font and lines. The boxed sequences are the Lux box consensus sequences. * denotes the stop codon. _____________________________________________________________________ 3.2.4 Mutation of echIEC1 resulted in enhanced swimming motility To determine if AHL quorum sensing signal plays a role in the regulation of swimming motility of E. chrysanthemi pv. zeae, a swimming assay was performed as described in materials and methods. X-Gal was added to the motility plates for better visualization of EC1 as it contains a lacZ gene which encodes the enzyme, βgalactosidase. This enzyme then degrades X-Gal to a blue colour product, 4-chloro3-brum indigo that stains the bacterial cells. The echIEC1 transposon mutant WM6 showed a significantly greater swimming diameter compared to the wild-type EC1 (Figure 3.5a). Swimming motility of the mutant was restored to the wild-type EC1 level in the complementary strain WM6-echI expressing the wild type echIEC1 gene (Figure 3.5a). The two reference strains, E. chrysanthemi EC3937 and EC16, showed a contrasting phenotype in swimming motility, with the swimming zone of EC16 being even smaller than EC1 and that of EC3937 comparable to the mutant WM6 (Figure 3.5a). 48 Similarly, the other two echIEC1 mutants, i.e., WM3 and WM8, also showed significantly increased swimming motility which was restored to wild-type level in the corresponding complementary strains (Figure 3.5b). The major AHL signal produced by the ExpI enzyme (a homolog of EchIEC1) of E. chrysanthemi is N-(3-oxo-herxanoyl)-homoserine lactone (OHHL) (Nasser, W., et al., 1998). Due to the great similarity between ExpI and EchIEC1, we speculated that strain EC1 might also mainly produce OHHL, which was confirmed by HPLC analysis (data not shown). In order to determine if OHHL is the contributory factor for modulation of swimming ability, extraneous OHHL was added to the swimming plates and the swimming assay was performed as described previously. The results showed that addition of 20 µM of OHHL was able to decrease the swimming motility of the mutants to the level of wild-type EC1 (Figure 3.6). 3.2.5 Autoinducer mutants showed no significant difference in flagella and LPS production but displayed increased EPS production In order to determine if AHL signal affects other virulence factors such as flagella (Otteman, K.M. et al., 1997), lipopolysaccharides (LPS) (Schoonejans E. et al., 1987) and exopolysaccharides (EPS) (Condemine G. et al., 1999), assays were performed using strain EC1 and its AHL-defective mutants as described in materials and method. The absence or presence of flagella were observed using TEM. All the three mutants, WM3, WM6 and WM8 showed similar number, length and positioning (petrichous) of flagella as that of the wild-type strain EC1 (Figure 3.7). 49 50 Figure 3.5 Mutation of the gene encoding AHL biosynthesis enhanced EC1 swimming motility. (a) Bioassay plates showing the swimming motilities of strains EC1, EC16, EC3937, WM6 and WM6-echI. (b) Quantification analysis of the swimming motility of EC1, its derivatives and reference strains. _____________________________________________________________________ Figure 3.6 OHHL modulates the swimming motility of E. chrysanthemi pv. zeae. Open bar represents the assay performed on the medium without extraneous OHHL and closed bar represents the assay on the medium containing a final concentration of 20 µM OHHL. _____________________________________________________________________ 51 Figure 3.7 Electron micrographs of EC1 and AHL- mutants. Bar ( ) = 200 nm. _____________________________________________________________________ For LPS analysis, samples were prepared from the cell cultures of wild-type strain EC1 and the AHL-deficient mutant WM6 at OD600 of 0.8 and 1.2. After separation by SDS-PAGE electrophoresis, LPS bands were visualized by silver staining. The results showed that AHL quorum sensing signal did not seem to affect the quality and quantity of LPS as EC1 and WM6 displayed same number of LPS bands with similar intensity (Figure 3.8). 52 Figure 3.8 LPS assay using SDS-PAGE and stained with silver solution. LPS samples were prepared at bacterial cell density OD600 = 0.8 (a) and at OD600 = 1.2 (b). _____________________________________________________________________ EPS was analysed using the alcian blue assay. It was observed that the mutants, WM3, WM6 and WM8, produced about 60 % higher relative EPS content than the wild-type EC1. As expected, the complemented strains, i.e., WM3-echI, WM6-echI and WM8-echI, showed similar relative EPS contents to wild-type strain EC1 (Figure 3.9). 53 Figure 3.9 Alcian blue assay. EC1 is the wild-type strain, WM3, WM6 and WM8 are the AHL-deficient mutants, and WM3-echI, WM6-echI and WM8-echI are the corresponding complementary strains. _____________________________________________________________________ 3.2.6 AHL-deficient mutants showed decreased virulence against potato tubers and Chinese cabbage The ability of strain EC1 and the AHL-deficient mutants to cause maceration in potato tubers and Chinese cabbage were investigated. Similar to E. chrysanthemi strains EC3937 and EC16, which were known to cause infections in dicot plants, E. chrysanthemi pv. zeae strain EC1 could also cause soft rot symptoms in potato and Chinese cabbage (Figure 3.10). When inoculated on potato tubers, the mutants caused smaller maceration zones and generated lesser tissue fluid in the vicinity of 54 maceration than the wild-type EC1 (Figure 3.10a). Similar results were obtained when EC1 and the mutants were tested using Chinese cabbage (Figure 3.10b). In both experiments, the virulence was fully restored in the complementary strains WM3-echI, WM6-echI and WM8-echI. 55 Figure 3.10 Pathogenesis assay using (a) potato tubers and (b) Chinese cabbage. The cut plant tissues were inoculated with 2 µl of bacterial cells at OD600 = 1.2. Photographs were taken after incubation for 24 h at 28°C. The experiments were repeated 3 times with similar results. The figures show a set of representative samples. _____________________________________________________________________ 56 3.2.7 AHL-deficient mutants still possessed the ability to inhibit rice seed germination The ability of strain EC1 and the AHL-deficient mutant, WM6, was tested on its capability to inhibit rice seeds germination. Figure 3.11 shows that WM6 had a similar ability as the wild-type EC1 in inhibition of the seeds germination. The complementary strain WM6-echI also showed similar inhibiting properties. In contrast, both E. chrysanthemi strains, EC3937 and EC16, were not able to inhibit rice seeds germination. 3.2.8 AHL-deficient mutants showed no significant difference with the wildtype parental strain in pectate lyase and protease production In order to determine if the reduced virulence of the mutants against potato tubers and Chinese cabbage were attributed to decreased production of certain enzymes implicated in plant tissue maceration, the pectate lyase and protease activities of the strain EC1 and its mutants were determined quantitatively. As figure 3.12a shows, no significant difference in pectate lyase activity was observed among the wild-type EC1, the mutants and the corresponding complementary strains when considering the variations among the repeats. Similarly, abolishing AHL production in strain EC1 seemed to have caused only a minor reduction, if any, of the protease activity (Figure 3.12b) 57 Figure 3.11 E. chrysanthemi pv. zeae inhibited rice seed germination. Rice seeds were added to tubes containing bacterial suspension (about 2 x 1010 cells in LB) or LB (blank) for 6 h, then washed and incubated at 28°C under 16 h light and 8 h dark conditions. _____________________________________________________________________ 58 (a) (b) Figure 3.12 Analysis of exoenzymes produced by E. chrysanthemi pv. zeae. The supernatants of bacterial cultures were collected when OD600 = 1.2. Pectate lyase (a) and protease (b) activity in the supernatants were then determined. 59 3.3 Summary The 16S rDNA analysis showed that the isolate EC1 was distinct from several E. chrysanthemi strains (Figure 3.1). In addition, strain EC1 showed phenotypical differences with the well characterized E. chrysanthemi strains, EC3937 and EC16, including the ability to inhibit the growth of bacteria and fungi (Figure 3.2), and rice seeds germination (Figure 3.11). These results plus the fact that EC1 is able to infect monocot rice, established that strain EC1 belongs to E. chrysanthemi pv. zeae. Mutation of the quorum sensing system using Tn5 mutagenesis in EC1 did not affect the toxin production phenotypes but did affect other phenotypes associated with pathogenesis such as causing an increase in swimming ability (Figure 3.5) and EPS production (Figure 3.9) and a decrease in virulence on potato tubers and Chinese cabbage (Figure 3.10). The gene disrupted by the mutation was identified to be expI, having a 92.5 % peptide homology to EC3937 (NCBI Accession No. CAA65306) and was labelled as echIEC1. Upon further sequencing of the upstream region of echIEC1, another gene called expR was identified that showed a 90.8 % peptide homology to EC3937 (NCBI Accession No. CAD27339). In EC1, this gene was labelled echREC1. These two genes were placed in opposite orientations and contained a putative lux box close to the -35 region of the expREC1 gene (Figure 3.4). Complementation with the echIEC1 gene restored the swimming ability, EPS production and virulence ability of the AHL mutants against potato tubers and Chinese cabbage to that of wild-type EC1 levels. Further, exogeneous addition of autoinducer, OHHL restored the swimming ability of the AHL mutants, supporting 60 that the echIEC1 gene was indeed responsible for the observed phenotypes and that it directs the synthesis of OHHL. 61 CHAPTER 4 Screening of the genes involved in E. chrysanthemi pv. zeae toxin production and regulation 4.1 Introduction The main virulence factors produced by E. chrysanthemi are known to be the pectate lyases. Not surprisingly therefore, most work on gene regulation focused on pectate lyases. It was found that pectin was the main signal recognized by E. chrysanthemi for induction of the genes encoding pectate lyase biosynthesis via the KdgR transcriptional repressor (Hugouvieux-Cotte-Pattat, N. et al., 1992; Whitehead, N.A. et al., 2002; Barras, F. et al., 1994; Robert-Baudouy, J. et al., 2000). In addition to pectin, other physiological and environmental cues were also found to contribute to the regulation of pectate lyase production. These include the ExpI-ExpR quorum sensing system, catabolite repression, temperature, and iron deficiency (Whitehead, N.A. et al., 2002; Barras, F. et al., 1994; Robert-Baudouy, J. et al., 2000; Hugouvieux-Cotte-Pattat, N. et al., 1996). Apart from the main virulence factors, pectate layses, other factors are also known to affect the virulence of E. chrysanthemi. For example, the extracellular proteases and cellulases are implicated in soft rot pathogenesis (Marits, R. et al., 1999; Chatterjee, A. et al., 1995) and siderophore and pigment production are associated with the systemic infection of E. chrysanthemi in host plants (Reverchon, S. et al., 2002; Sauvage, C. et al., 1994; Venkatesh, B. et al., 2006). 62 The findings described in chapter 3 that E. chrysanthemi pv. zeae strain EC1, but not E. chrysanthemi strains EC3937 and EC16, is able to inhibit the growth of bacteria, fungi and rice seeds germination suggest that EC1 may produce an antibiotic(s) or toxin(s). This unique ability may be the key factor as to why this pathovar of E. chrysanthemi can cause infections on monocot plants. Therefore, to elucidate the molecular mechanism of E. chrysanthemi pv. zeae pathogenicity in this chapter, we set to screen for and identify the genes involved in toxin production and regulation. 4.2 Results 4.2.1 Screening of the Tox- mutants of strain EC1 A mini-Tn5 transposon was used to generate a mutant library of strain EC1. The Tn5 mutants were spotted on C. albicans overlay plates to screen for the loss of antibiotics/toxin(s) production activity. The potential tox- candidates were purified by subculture and further confirmed by the same overlay assay using E. coli DH5α. From about 20,000 transposon mutants, we obtained 32 mutants which were not able to inhibit the growth of C. albicans and E. coli DH5α (Figure 4.1). The data suggests that strain EC1 is likely to produce a diffusible antibiotics/toxin(s) that is able to inhibit the growth of both prokaryotes and eukaryotes. As illustrated in the following chapters, these mutants were also attenuated in their ability to inhibit rice seed germination. 63 (a) C. albicans (b) E. coli DH5α 64 Figure 4.1 Examples of growth inhibition assay by strain EC1 and its mutants. Bioassay was conducted against C. albicans (a) and E. coli DH5α (b) using the supernatants of EC1 and mutants, grown in MM medium to OD600 =1.5. Zone of inhibition on the bacterial and fungal growth lawn indicates the presence of antibiotic(s)/toxin(s) produced by strain EC1. _____________________________________________________________________ 4.2.2 Single Tn5 insertion in the genome of EC1 mutants was responsible for the Tox- phenotype To confirm the transposon insertion, we used a Tn5-specific probe for Southern blot analysis. The genomic DNA samples of the mutants were digested by ClaI to completion and separated by gel electrophoresis. Southern blot results showed that all the mutants contained only a single Tn5 insertion in the genome (Figure 4.2a). The data also showed that some mutants shared a same-sized hybridization band suggesting that they were likely to be siblings or contained the transposon insertion at the same DNA fragment. For further confirmation, Southern blotting was repeated but with the EcoRI-digested genomic DNA samples (Figure 4.2b) and the mutants were grouped based on the size and pattern of the hybridization bands in both experiments (Table 4.1). The mutants sharing the same-sized bands in both blots are most likely to contain Tn5 insertion at the same DNA fragment, thus for time efficiency and better use of experimental resources, one mutant from each group was selected for further analysis. 65 (a) (b) Figure 4.2 Southern blot analysis of the Tox- mutants of strain EC1. Total genomic DNA samples were digested to completion by Cla1 (a) and EcoR1 (b), respectively. After separation by gel electrophoresis, hybridization was conducted with a 0.4-kb fragment of a gentamycin gene carried by the mini-Tn5 transposon. Numbers indicate the different mutants, WT is the wild-type EC1 and pTGN is the transposon plasmid. _____________________________________________________________________ 66 Table 4.1 Classification of the Tox- mutants based on the size of hybridization bands Group Southern Blot Band Size (kb) Mutant Number (EM) Cla1 EcoRI A 6.5 23 7, 8,15,17,19,20 B 6.5 18 104 C 6.5 0.5 53 D 5.5 2.6 21,22,23,25,27,30,37,38,42,40,55,36 E 3.8 2.3 11 F 3 2.8 67 G 3 8.5 13 H 1.5 8.5 76 I 1 1 107 J 1 0.5 9,10 K 0.9 23 29,33,43,46,54 _____________________________________________________________________ 4.2.3 Sequence analysis of Tox- mutants revealed the genes implicated in polyketide antibiotics biosynthesis The flanking regions of the Tn5 insertion from these mutants were amplified separately by TAIL-PCR. Subsequent sequence analysis and homology search enabled the putative peptide products of the transposon disrupted genes to be grouped into following functional categories: (1) polyketide biosynthesis (mutants EM13, 67 EM20, EM9, EM11, EM107), (2) peptide biosynthesis (mutant EM104), (3) regulation (mutant EM53), and (4) hypothetical protein (mutant EM40) (Table 4.2). For mutant EM40, Tn5 was found inserted within an open reading frame encoding a peptide showing about 82 % identity to a hypothetical protein of E. carotovora subsp. atroseptica (NCBI Accession No. YP_049272). Further sequencing for the flanking region and subsequent homology search of the sequences obtained (Appendix 1) identified more accurately the Tn5 insertion point in EM40 to be immediately upstream of an ORF that showed 80 % homology to Acyl-CoA thioesterase of E. carotovora (NCBI Accession No. YP_049268). Other ORFs showing similarity to a multidrug efflux pump (80 % homology, NCBI Accession No. ZP_00821286), acriflavin resistance protein A (76 % homology, NCBI Accession No. YP_049276) and an ABC transporter (80 % homology, NCBI Accession No. ZP_01538624) seems to be consistent with the Tox- phenotype of the mutant as the genes encoding transportation and those encoding the biosynthesis of secondary metabolites are commonly linked (Binet, R. et al., 1996; Binet, R. et al., 1997). The flanking sequence of the Tn5 in mutant EM13 showed a 59 % homology to JamP of Stigmatella aurantiaca DW4/3-1 (NCBI Accession No. ZP_01467455). The Tn5 insertion was located at the position corresponding to the 369-aa of the 1171-aa peptide (Table 4.2). JamP has been identified to be the second last module of a 54-kb gene cluster implicated in the synthesis of the jamaicamides A and B toxins of Lyngbya majuscule (Edwards, D.J. et al., 2004). 68 Table 4.2 Sequence analysis of Tox- mutants of the strain EC1 Group (Mutant) Sequence similarity Group D (EM40) 82% homology to: Tn5 insertion point Sequence Appendix 1 Hypothetical protein ECA1166 of Erwinia carotovora subsp. atroseptica SCRI1043 Group G (EM13) 59% homology to: Appendix 2 JamP – containing polyketide synthase and secondary metabolite biosynthesis regions of Stigmatella aurantiaca DW4/3-1 Group A (EM20) 55% homology to: Appendix 3 Beta-ketoacyl synthase of Shewanella baltica OS155 Group J (EM9) Group I (EM107) Group E (EM11) 60% homology to: Appendix 4 polyketide synthase P3-A6-PKS of Chromobacterium violaceum 69 Table 4.2 (Cont’d) Sequence analysis of the Tox- mutants of strain EC1 Mutant Sequence similarity Group B (EM104) 49% homology to: Tn5 insertion point Sequence Appendix 5 Peptide synthetase of gene mcyA of Anabaena sp. 901 Group C (EM53) 92% homology to: Appendix 6 Rap - Regulator of antibiotic and pigment production of Serratia sp. ATCC 39006 The Tn5 insertion in mutant EM20 was identified to be located at a position corresponding to the 210th amino acid of a 1987-aa peptide that shared about 55 % identity to the beta-ketoacyl synthase of Shewanella baltica OS155 (NCBI Accession No. YP_001049810); the enzyme is mainly involved in the biosynthesis of anaerobic unsaturated fatty acid (Mohan, S. et al., 1994; Cronan, J.E.Jr. et al., 1972; Wang, H., et al., 2004). Upon further sequencing of the flanking regions, the Tn5 was found to be inserted just downstream of an ORF which shows a 31 % homology to a multidomain beta-ketoacyl synthase of Shewanella oneidensis MR-1 (Appendix 3; NCBI Accession No. NP_717212). 70 The ORF to which Tn5 was inserted in mutant EM104 encodes a peptide with a 49 % homology to McyA of Anabaena sp. 901 (NCBI Accession No. AA062586) (Table 4.2). MycA is a nonribosomal peptide synthetase, and its coding gene is the first operon of the 55.4-kb microcystin synthetase gene cluster that is involved in the biosynthesis of the toxin hepatotoxic heptapeptides known as microcystins (Rouhiainen, L. et al., 2004). The gene disrupted by Tn5 in the mutant EM53 encodes a close homologue of the transcriptional regulator Rap of Serratia sp. ATCC 39006 (NCBI Accession No. AAF01153), which is involved in regulation of antibiotics and pigment biosynthesis (Table 4.2; Thomson, N.R. et al., 1997). The detailed characterization of EM53 will be described in chapter 6. The other three mutants EM9, EM11 and EM107 had Tn5 insertion respectively at the positions corresponding to 106-aa, 152-aa, and 849-aa of a 1614-aa long peptide which shows about 60 % homology to the polyketide synthase P3-A6PKS of Chromobacterium violaceum (NCBI accession No. ABM65752). More detailed analysis will be described in chapter 5. 4.3 Summary In this chapter, 32 Tox- mutants of strain EC1 were obtained and confirmed to contain a single copy of Tn5 insertion (Figure 4.1, Figure 4.2). Some of these mutants could be siblings or contain the transposon at the same DNA fragment based on the two separate enzyme-digested hybridization patterns. These mutants were grouped together, giving 11 different banding patterns (Table 4.1). The total length of the 71 DNA fragments containing transposon is about 39-90 kb (Table 4.1). After deduction of the length of transposon fragment, which is about 0.88-kb and 0.90-kb in each hybridization band digested by ClaI and EcoRI (Tang, X. et al., 1999), respectively, it is estimated the size of gene cluster involved in toxin production is in the range from 30-kb (ClaI) – 80-kb (EcoRI). The flanking DNA sequences around the Tn5 insertion were obtained by TAIL-PCR from the mutants in groups A-E, G, H-I. Blastx searches revealed homologues to the proteins of following the functional categories: (1) polyketide biosynthesis, (2) peptide biosynthesis, (3) transcriptional regulator, and (4) ABC transporter (Table 4.2). TAIL-PCR of the mutants in groups F, H and K however, resulted in sequences that were too short to produce meaningful homology searches even after repeated attempts. 72 CHAPTER 5 Sequence analysis and characterization of the gene of E. chrysanthemi pv. zeae encoding a novel polyketide synthase 5.1 Introduction E. chrysanthemi pv. zeae is a pathovar of E. chrysanthemi, which is able to cause rice foot rot disease (Liu, Q.G. et al., 2004). The results in chapter 3 showed that E. chrysanthemi strains EC3937 and EC16 were not able to inhibit bacterial and fungal growth, as well as rice seed germination, whereas E. chrysanthemi pv. zeae strain EC1 produced antibiotics/toxin-like inhibitory substances. The toxin production ability of strain EC1 was further confirmed by transposon mutagenesis (Chapter 4). Partial sequencing of the mutated genes revealed significant homologies to those encoding for polyketide synthase, peptide synthase, and transcriptional regulators. This chapter focus on sequencing and analysis of the Tn5 disrupted genes in mutants EM9, EM11 and EM107. The partial sequence data in chapter 4 showed that in these three mutants the transposon insertion disrupted the same ORF but at different positions. Here we completed the sequencing of the ORF and its flanking sequences from both strands, and conducted phenotype analysis of the mutants. 73 5.2 Results 5.2.1 Cloning and sequencing of EC1 chromosomal fragment containing the polyketide synthase gene The preliminary DNA partial sequence analysis of the mutants EM9, EM11 and EM107 revealed a likely polyketide synthase gene (Chapter 4). By using TAIL-PCR, we further extended the flanking sequence to 6998-bp. ORF search using the Clone program identified 2 ORFs (Figure 5.1). A blast search of the peptide sequence from ORF1 showed that it shared about 63 % homology with the polyketide synthase P3A6-PKS of C. violaceum (NCBI Accession No. ABM65752). It showed the highest homology (71 %) to the product of the Yfa gene of Photorhabdus luminescens (NCBI Accession No. AAK16098), but mainly on the N-terminal region. A blast search of ORF2 showed a low 25 % homology to a branched-chain amino acid permease of Lactobacillus delbrueckii subsp. bulgaricus ATCC11842 (NCBI Accession No. YP_618597). 74 Figure 5.1 Generic map of the regions flanking Tn5 insertion sites in mutants EM9, EM11 and EM107. The arrow and the number indicate the Tn5 insertion sites in each mutant. 5.2.2 Domain structure analysis of the polyketide synthase Protein domain analysis of the peptide encoded by ORF1 using the PROSITE (http://au.expasy.org/prosite/) and Pfam (http://www.sanger.ac.uk/Software/Pfam/) programs, revealed the presence of two domains, namely, an N- and C- terminal domain of β-ketoacyl-synthase and an acyltransferase domain. These domains are generally involved in biosynthesis of secondary metabolites and fatty acids (Shen, B., 2003). Among the several homologues, the polyketide synthase P3-A6-PKS from C. violaceum shares the most similar domain structures, except that its peptide is much shorter than the polyketide synthase of strain EC1 (Fig. 5.2a). Yfa of P. luminescens shares the best peptide sequence identity but lacks an acyltransferase domain, which is responsible for transfer of acyl group between substrates and coenzyme A (CoA) (Fig. 5.2b). Based on these domain analysis, we also found the other two homologues that contain both β-ketoacyl-synthase and acyltransferase domains and have a similar length of peptide with the polyketide synthase of strain EC1. But these two homologues share only a moderate homology with their counterparts in EC1 and carry an extra PP domain (Fig. 5.2b). While the catabolic product of the EC1 polyketide synthase remains to be identified, it is interesting to note that the polyketide synthase P3-A6-PKS from C. violaceum is encoded by one of the 14 genes of a 36.4-kb gene cluster that is involved 75 in the biosynthesis of an anticancer agent, FK228 (Cheng, Y.Q. et al., 2007), and the Yfa of Photorhabdus luminescens is a polyketide synthase that may play a role in the synthesis of siderophore, enterobactin (Ciche, T.A. et al., 2001). (a) 76 (b) Domain Function Β-ketoacyl synthase Catalyzes the condensation of malonyl-ACP with a growing fatty acid chain. The N-terminal domain contains most of the structures involved in dimer formation. The active site is located between the N- and C-terminal domains. Acyl transferase Catalyzes the exchange of acyl groups between substrate and coenzyme A (CoA) PP domain Phosphopantetheine prosthetic group acts as a transient attachment site for activated fatty acids and amino-acid groups Figure 5.2 Protein domain analysis. (a) The domain structure of the polyketide synthase of strain EC1 and its homologues. (b) Domain and its function (http://www.sanger.ac.uk/Software/Pfam/; Kauppinen, S. et al., 1988; SiggaardAndersen, M. et al., 1994; Mikkelsen, J. et al., 1985). _____________________________________________________________________ 5.2.3 The polyketide synthase mutants were attenuated in their virulence against potato tubers and Chinese cabbage To determine the role of polyketide toxins in the virulence of strain EC1, we compared the pathogenic ability of the wild-type strain and its three Tox- mutants, EM9, EM11, and EM107, in which the gene encoding for a polyketide synthase were mutated by Tn5 insertion (Table 4.2). The pathogenicity assay showed that the mutants were significantly attenuated in causing local maceration on potato tubers (Figure 5.3a) and Chinese cabbage (Figure 5.3b) one day after inoculation. Even after three days, the symptoms caused by the mutants were less severe than the wildtype EC1 as exemplified by a much smaller diameter of maceration zone. 77 Figure 5.3 Pathogenesis assay using (a) potato tubers and (b) Chinese cabbage. The plant tissues or slices were inoculated with 2 µl of bacterial cells at OD600 = 1.2. Photographs were taken one and three days after incubation at 28°C. _____________________________________________________________________ 78 5.2.4 Polyketide mutants are defective in inhibition of rice seed germination The ability of the polyketide synthase mutants (EM9, EM11, EM107) to inhibit rice seed germination was investigated. In this assay, rice seeds were treated by submerging them in the respective bacterial suspension (OD600 = 1.2) for 6 h, followed by three washes with ddH2O. The seeds were then incubated under sufficient moisture at 28°C under a 16 h light, 8 h dark period and observed daily for germination. Figure 5.4 shows that EC1 caused complete inhibition of rice seed germination, while the seeds treated with the polyketide mutants were germinated and grew normally similar to the blank control (Figure 5.4). Figure 5.4 Pathogenesis assay on rice seed germination. Rice seeds were treated with bacterial cells of OD600 = 1.2 for 6 h, then washed and incubated at 28°C under 16 h light and 8 h dark conditions. Photos were taken 10 days after inoculation. 79 5.2.5 Pigment and siderophore production in the polyketide mutants were affected By performing assays on other factors that are implicated in systemic infection, namely pigment and siderophore, we found that both these phenotypes were affected in all the three polyketide mutants, EM9, EM11 and EM107 (Figure 5.5). (a) (b) Figure 5.5 Pigment and siderophore assays. (a) Pigment assay. Bacteria were streaked on NGM plates. (b) Siderophore assay using the supernatant of bacteria grown to OD600 = 1.2. Yellow colour indicates the presence of siderophores. _____________________________________________________________________ 80 5.3 Summary In this chapter, we focused on the gene encoding a polyketide synthase that shows a high homology (63 % - 71 %) to polyketide synthase P3-A6-PKS of C. violaceum and Yfa of Photorhabdus luminescens, as there were three independent mutants with Tn5 inserted in this gene but at different positions (Table 4.2; Figure 5.1). Phenotypic analysis demonstrated that all the mutants showed similar phenotypes, including attenuated virulence on dicot (Figure 5.3) and monocot (Figure 5.4) plants and reduced production of pigments and siderophores (Figure 5.5). The polyketide synthase of strain EC1 contains two domains, which are the Nterminal β-ketoacyl-synthase domain and the acyltransferase domain in the middle of peptide. Its closest structural homologue is the polyketide synthase P3-A6-PKS from C. violaceum, which is implicated in the synthesis of an anticancer agent, FK228 (Cheng, Y.Q. et al., 2007). 81 CHAPTER 6 A key transcriptional regulator that modulates the toxin production and virulence of E. chrysanthemi pv. zeae 6.1 Introduction Previous studies show that KdgR is the key regulator of pectate lyases, produced by E. chrysanthemi (Robert-Baudouy, J. et al., 2000). In a closely related bacterial species Erwinia carotovora, the production of pectate lyases is modulated by the product encoded by the hor gene, which belongs to the MarR/SlyA family of transcriptional regulators (Thomson, N.R. et al., 1997; Ellison, D.W. et al., 2006; Barnard, A.M. et al., 2007). In addition, Hor also regulates other biological functions of Erwinia carotovora. The Hor- marker exchange mutant is defective in antibiotic production, and attenuated in virulence (Thomson, N.R. et al., 1997; McGowan, S.J. et al., 2005). In Serratia marcescens, Rap, the homologue of Hor, is essential for regulation of antibiotic and pigment production (Thomson, N.R. et al., 1997). The similar functions of Hor and homologues may suggest a common evolutionary origin of genetic regulatory networks in these microorganisms. A weak hor homologue has also been detected in E. chrysanthemi strains by hybridization using the rap gene from S. marcescens as a probe (Thomson N.R. et al., 1997), but its function in this organism has not been reported. In chapter 3, we showed that the Tox- phenotype of mutant EM53 was due to transposon insertion in a 82 hor homologue (horEC1). This chapter describes the sequence analysis of the horEC1 gene and characterization of its biological functions in E. crysanthemi pv. zeae. 6.2 Results 6.2.1 Cloning and sequencing of the DNA fragment containing the hor homologue The flanking regions of Tn5 insertion were cloned using TAIL-PCR. Sequence analysis of the 1711-bp fragment revealed two ORFs (Fig. 6.1). ORF1 encodes a 155-aa long peptide. A blast search found that the peptide shares 89 % and 82 % identity to an outer membrane lipoprotein Pcp of E. carotovora subsp. carotovora (NCBI Accession No. AF168687_1) and Serratia sp. (NCBI Accession No. AF168597), respectively. ORF2 encodes a peptide of 145-aa, and its best homologues include the transcriptional regulator Rap of Serratia sp. (NCBI Accession No. AF168597, 92 % identity), the Hor of E. carotovora subsp. carotovora (NCBI Accession No. AF168687_2, 84 % identity), and the SlyA of Salmonella choleraesuis (NCBI Accession No. P61090, 75 % identity). Figure 6.1 The genome structure of Tn5 inserted chromosomal region of the Toxmutant EM53. 83 The horEC1 gene and the pcp gene are separated by an 456-bp non-coding region and transcribed from the opposite directions. Interestingly, the same gene order was also maintained in other bacterial species, including S. marcescens, E. carotovora, and E. coli (Thomson, N. R. et al., 1997). Tn5 was found inserted at the 198-bp of the horEC1 coding region (Fig. 6.1). Domain analysis, carried out with PROSITE (http://au.expasy.org/prosite/), revealed the presence of a MarR-type HTH domain from the 29th to the 99th amino acid residue (Figure 6.2). The Tn5 transposon was found to be inserted within this domain. This domain was also found to be well-conserved in SlyA of Serratia and Hor of E. carotovora. Sequence alignment of HorEC1 with its homologues from S. marcescens and E. carotovora identified a few variations in this domain but most are the amino acids of similar properties except at the following 3 positions: (1) Position 41 where Hor of E. carotovora contains a polar tyrosine residue in place of a positively charged, hydrophilic residue, histidine which is present in HorEC1 of EC1 and SlyA of Serratia sp. (2) Position 42 where the amino acid in all the three homologues differ, with HorEC1 containing the polar hydrophilic residue glutamine, Hor of E. carotovora containing the positively charged, hydrophilic residue histidine and SlyA of Serratia sp. containing the negatively charged hydrophilic residue glutamic acid (3) Position 95 where the amino acid for Hor of E. carotovora is the hydrophilic serine, which is different to the hydrophobic methionine in HorEC1 and SlyA of Serratia sp. (Figure 6.3). 84 Figure 6.2 Domain analysis of the transcriptional regulator HorEC1 of E. chrysanthemi pv. zeae strain EC1, SlyA of Serratia spp. and Hor of E. carotovora subsp. carotovora. The green box indicates a HTH domain which is involved in DNA binding. This domain is 71-aa long, from position 29 to 99. _____________________________________________________________________ 85 Figure 6.3 Sequence alignment of HorEC1 and homologues. Alignment of the predicted amino acid sequence of Hor of EC1 (HorEC1) with Hor from E. carotovora subsp. carotovora ATCC39048 and SlyA from Serratia sp. ATCC39006. The * indicates the conserved amino acids. 6.2.2 Expression of the wild-type horEC1 gene in EM53 restored its toxin production Complementation experiments were conducted to confirm the role of HorEC1 transcriptional regulator in modulation of toxin production in E. chrysanthemi pv. zeae. The wild-type horEC1 gene was amplified by PCR and cloned under the control of the lac promoter in the expression vector pUC19. After sequence verification, the 86 construct was introduced into the mutant EM53 and EC1 as well. The resultant transformants (Table 6.1) were assayed for the ability to inhibit the growth of C. albicans and E. coli DH5α. The results showed that expression of horEC1 in EM53 partially restored its inhibition ability against C. albicans and E. coli DH5α (Figure 6.4). 6.2.3 HorEC1 played an essential role for infection of E. chrysanthemi pv. zeae on both dicot and monocot plants Pathogenicity assays showed that the virulence ability of mutant EM53 was significantly attenuated on potato tubers (Figure 6.5a) and Chinese cabbage (Figure 6.5b). The two independent complementary strains, EM53H1-4 and EM53H2-1a, showed a partial restoration of the maceration ability of the mutant. The two strains over-expressing the horEC1 gene in wild-type background showed no significant difference with the parental strain EC1 in its ability to macerate potato tubers or Chinese cabbage. The ability of EM53 to inhibit rice seeds germination was investigated and found to be defective compared to wild-type, EC1. From Figure 6.6b, it can be seen that while EC1 inhibited the rice seeds from germination completely, EM53 failed to inhibit the rice seeds germination, and all the EM53-treated seeds were germinated but with a minor growth retardation (~5%) in terms of length of shoots and roots, in comparison with the water control (Figure 6.6a). The complementary strain expressing horEC1 largely restored the inhibitory activity close to that of the wild-type level with an 80 % - 90 % germination inhibition rate (Figure 6.6b). 87 Table 6.1 List of bacterial strains Strain or Plasmid Remarks Reference EC1 An environmental isolate from rice plant belonging to a subspecies of E. chrysanthemi This study EC16 Wild type, Chrysanthemum morifolium isolate Chatterjee, A.K. et al., 1983 EC3937 Wild type, Saintpaulia isolate Lemattre, M. et al., 1972 EM53H1-4 Construct pUC19- horEC1 independent transformant This study EM53H2-1a Construct pUC19- horEC1 independent transformant This study EC1 harbouring the construct EC1H1-2 This study pUC19- horEC1 independent transformant EC1 harbouring the construct EC1H2-8 This study pUC19- horEC1 independent transformant Escherichia coli DH5α supE44 ∆lacU169 ( Φ80lacZ ∆M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 Laboratory Collection DH5α(pUC19) DH5α harbouring the pUC19 plasmid This study Plasmid Yanisch-Perron, C. et al., 1985 r pUC19 Amp pUC19- horEC1 pUC19 harbouring horEC1, Amp This study pTGN pBSL202 carrying a 2-kb XbaI-SmaI fragment containing the gfp-nptII fusion operon Tang, X. et al., 1999 r _____________________________________________________________________ 88 Figure 6.4 Toxin bioassay against C. albicans (a) and (b) E. coli DH5α. Filtersterilized supernatant of bacteria grown to OD600 = 1.5 were added to the wells. Zone of inhibition of fungal and bacterial lawn indicates existence of antibiotics. The photos were taken 24 h after incubation. Strains used are listed in Table 6.1. 89 Figure 6.5 Pathogenesis assay using (a) potato tubers and (b) Chinese cabbage. The slices were inoculated with 2 µl of bacterial cultures at OD600 = 1.2. Photographs were taken after incubation for 24 h or 72 h at 28°C. Strains used are listed in Table 6.1. _____________________________________________________________________ 90 (b) 91 Figure 6.6 Pathogenesis assay on rice seeds germination. Rice seeds were treated for 6 h with bacterial culture (OD600 = 1.2), then washed and incubated at 28°C under 16 h light and 8 h dark conditions. Strains used are listed in Table 6.1. H2O and E. coli DH5α harbouring the pUC19 plasmid was included as a control. _____________________________________________________________________ 6.2.4 HorEC1 did not appear to play a significant role in regulation of pectate lyase and protease production We quantitatively tested the pectate lyase activity of the mutants to determine if its reduced virulence to potato, Chinese cabbage and rice seeds germination was attributed to decreased production of pectate lyases and proteases. However, the results showed that there was no significant reduction in both pectate lyase (Figure 6.7a) and protease (Figure 6.7b) activity in EM53. The wild-type EC1, the horEC1 mutants, the complementary and the over-expression strains showed similar levels of enzyme activities. 6.2.5 Mutation of horEC1 had no effect on production of AHL quorum sensing signals AHL quorum sensing signal production by the HorEC1- mutant, EM53 was compared with the wild-type strain EC1. Diffusion plate assay did not reveal any significant variations among the tested strains, which includes the mutant, wild-type strain, complementary strains and the wild-type over-expressing the horEC1 gene (Figure 6.8). 92 (a) (b) Figure 6.7 Analysis of exoenzymes produced by E. chrysanthemi pv. zeae. The supernatants of bacterial cultures were collected when OD600 = 1.2. Pectate lyase (a) and protease (b) activity in the supernatants were then determined. Strains used are listed in Table 6.1. _____________________________________________________________________ 93 Figure 6.8 AHL signal bioassay. The indicator strain, CF11, which is spotted along the length of the MM agar, turns a blue colour in the presence of AHL signals. The number of spots of blue is indicative of the concentration of AHL as it diffuses along the length of the agar. Strains used are listed in Table 6.1. _____________________________________________________________________ 6.2.6 Mutation of horEC1 affected swimming ability, biofilm formation and pigment production As shown in Figure 6.9, mutation of horEC1 in mutant EM53 enhanced the bacterial swimming ability by about 50 % compared to the wild-type EC1. The phenotype was restored by expression of the wild-type horEC1 gene in mutants. Over-expression of horEC1 in wild-type strain, however, did not affect the swimming ability compared to the wild-type EC1. The biofilm formation of the mutants was, however, diminished compared to the wild-type EC1 (Figure 6.10a). Upon microscopic examination of the biofilm, it 94 can be seen that while the biofilm of the wild-type EC1 is composed of a dense layer of cells, that of EM53 is composed of sporadic cells. The complemented mutants (EM53H1-4 and EM53H2-1a) had this phenotype restored. The over-expression strains EC1H1-2 and EC1H2-8 showed no discernible difference in the thickness of the biofilm produced (Figure 6.10b). Similarly, investigation of the pigment production revealed that it was defective in EM53 as it was white in colour, in contrast to the pinkish brown colour of the wild-type EC1. Further, the complemented mutants were able to partially restore the white colouration of the mutant to that of a beige colour. The overexpression strains showed brown colouration (Figure 6.11). Figure 6.9 Mutation of horEC1 resulted in enhanced bacterial swimming motility. Bacteria were spotted onto swim agar plates and incubated for 8 h at 28°C. Strains used are listed in Table 6.1. _____________________________________________________________________ 95 (a) (b) 96 Figure 6.10 Mutation of horEC1 decreased biofilm formation. (a) Glass slides containing biofilm stained with crystal violet. (b) Micrographs of biofilm on the glass slides. Bacteria were inoculated in SOBG media, incubated at room temperature and observed daily for biofilm formation. Photographs were taken after incubation for 48 h at r.t. Strains used are listed in Table 6.1. _____________________________________________________________________ Figure 6.11 Pigmentation assay. Bacteria were grown on NGM agar plates at 28°C. They were observed daily for pigment production and photographed at day 4 after inoculation. Strains used are listed in Table 6.1. _____________________________________________________________________ 97 6.3 Summary Sequence analysis of the tox- mutant EM53 led to identification of a gene encoding a conserved transcription regulator HorEC1 (Figure 6.1). The regulator shows a high homology to the Rap of Serratia sp. ATCC 39006 (92 % identity), the Hor of E. carotovora subsp. carotovora (84 % identity), and the SlyA of Salmonella choleraesuis (75 % identity). These transcriptional regulators belong to the MarR/SlyA family and contain the MarR HTH domain that is involved in DNA binding. There is strong conservation of this domain among the three bacteria genus, Erwinia, Serratia and Salmonella in terms of location within the peptide as well as the length of the peptides (Figure 6.2 – 6.3). Disruption of this gene in strain EC1 was shown to (1) abolish the inhibitory activity against C. albicans and E. coli DH5α (Figure 6.4), and (2) reduce the virulence against potato tubers, Chinese cabbage and rice seeds (Figure 6.5-6.6). Mutation of horEC1 also resulted in other phenotypic changes, namely (1) enhanced swimming motility (Figure 6.9), (2) abolished biofilm formation (Figure 6.10), and (3) reduced pigment production (Figure 6.11). These altered phenotypes were restored or partially restored by overexpression of the wild-type horEC1 gene in the mutant EM53. 98 CHAPTER 7 E. chrysanthemi pv. zeae produces a toxin(s) that inhibits rice seeds germination 7.1 Introduction Polyketides are a large family of natural products found in bacteria and fungi that are normally produced during stationary growth phase (Shen, B., 2003). These products are normally synthesized from acyl CoA precursors by enzymes known as polyketide synthases (PKSs) in a stepwise and sequential manner, thereby having the capability of generating a vast array of novel compounds that may have biological properties as antibiotics, anticancer substances and other pharmacologically valuable agents (Liou, G.F. et al., 2003). PKSs are thought to evolve from fatty acid synthases since they have related mechanistic and architectural similarities (Liou, G.F. et al., 2003) and therefore may also form the biosynthesis machinery of fatty acids and siderophores. Chapter 3 showed that E. chrysanthemi pv. zeae strain EC1, but not E. chrysanthemi strain EC3937 and EC16, produced clear inhibition zones against E. coli and C. albicans. Moreover, in most Tox- mutants, Tn5 was inserted in the genes encoding PKSs (Chapter 4). Futhermore, mutation of the gene encoding transcriptional regulator HorEC1 abolished antibiotic production and diminished the ability of E. chrysanthemi pv. zeae to inhibit rice seeds germination. These findings suggest that E. chrysanthemi pv. zeae may produce a polyketide toxin(s) with toxic 99 activity against both prokaryotic and eukaryotic organisms. To demonstrate this possibility, we partially purified the toxin and characterized its properties and activities. 7.2 Results 7.2.1 Maximal toxin production occurred at stationary phase in minimal medium Strain EC1 bacterial growth and toxin production in LB rich medium and minimal medium were determined. In rich medium, bacterial growth reached stationary phase after a culture at 28oC with shaking of 180 rpm for 6-6.5 h, when the toxin activity against E. coli and C. albicans was detected (Figure 7.1a). Maximal toxin production was found around 8 h, followed by a reduction in toxin activity being detected after 10 h of culture. Under the same culture conditions, strain EC1 grew slower in minimal medium than in rich medium and reached stationary phase at around 9 h after culture. In contrast to a short period of toxin production in rich medium, toxin production in minimal medium, however, was detected at mid-exponential phase, and it continued to increase through stationary growth (Figure 7.1b) 100 (a) (b) Figure 7.1 Bacterial growth and toxin production in LB medium (a) and minimal medium (b). cda, C. albicans and DH5α, E. coli strain DH5α. Supernatant were collected by centrifugation of 1 ml bacterial culture at each time point and filtersterilized. 15 µl of the filter-sterilized supernatant were then added to the wells in the bioassay plates and incubated at 37 °C, 24 h. The experiment was repeated twice and the data were the means of two repeats. 101 7.2.2 Virulence factor can be extracted using Amberlite XAD7 beads. The toxin(s) were able to be separated from the supernatant by passing the filtersterilized supernatant of bacterial culture through an amberlite XAD7 beads column. The bound toxin(s) could not be washed away using either water or methanol, but could be eluted with acetone (Figure 7.2a). We concentrated the acetone eluate from the supernatant of a three litre EC1 culture to a final volume of 3 ml (dissolved in DMSO), equivalent to a 1000x concentration. Bioassay of the concentrated toxin extract against C. albicans and E. coli DH5α reveals a marked increase in inhibition zones compared to the supernatant (Figure 7.2a), whereas dilution of the concentrated extract by 1000x reduced the size of inhibition zone on C. albicans and E. coli DH5α to the similar level of the filtered supernatant (Figure 7.2b). These findings indicate that Amberlite XAD7 beads can extract the toxin(s) of interest from the bacterial supernatants with a high efficiency. ( 102 (b) Figure 7.2 Bioassay of chromatography fractions and extracted toxin. (a) Bioassay of various fractions after Amberlite XAD7 column chromatography. Supernatant was added to a 10 cm x 20 cm column containing 114 g of XAD7 at a flow rate of 1 ml/min. The column was then washed using 3 L of water, followed by 500 ml of methanol. The bound toxin was eluted with 2 L of acetone. The acetone was evaporated from the eluted solutions by rotary evaporator, and the residues were dissolved in 3 ml of DMSO. For bioassay, 15 µl from each fraction was added to each well of a lawn of C. albicans or E. coli DH5α and incubated at 37°C for 16 h. (b) Dilution analysis of the concentrated toxin extract which was performed under the same conditions as in (a). _____________________________________________________________________ 7.2.3 Toxin was stable under various conditions In order to determine if the toxin(s) has a peptide linkage that is vulnerable to protease, we treated the toxin(s) with proteinase K. We found that treatment of the toxin solution with three different concentrations of proteinase K had no effect on the inhibitory activity of the toxin on C. albicans and E. coli DH5α (Figure 7.3a). 103 We also found that the toxin was heat (Figure 7.3b) and acid stable (Figure 7.4a). The toxin was also stable in alkaline solution, except that at pH 11 the inhibitory activity on both the bacterium and the fungus was partially reduced (Figure 7.4b). Figure 7.3 Bioassay against E. coli DH5α and C. albicans. (a) The toxin(s) was treated with 10 U, 50 U, and 100 U proteinase K (PK) respectively at 37°C for 4 h. (b) The toxin(s) was treated at 121°C for 15 min. _____________________________________________________________________ 104 Figure 7.4 Treatment of toxin in acid (a) and alkaline (b) solution. The extraction toxin was treated for 1 h before bioassay against E. coli DH5α and C. albicans (cda). 10 µl of the toxin was added to PBS solution adjusted to the various pH levels and incubated for 1 h at r.t. 15 µl of the sample were then added to the bioassay plates. Controls were the PBS solutions adjusted to the various pH levels. _____________________________________________________________________ 7.2.4 The toxin extract inhibited the root germination of rice seeds The toxin(s) extract from Amberlite XAD7 were tested for its ability to inhibit rice seeds germination. To 5 ml of water containing 10 rice seeds, 10 to 50 µl of toxin extract was added. The mixture was poured onto a plate containing 3 pieces of filter 105 paper, which was then incubated at 28ºC. Rice seeds were also treated with strain EC1 bacterial culture as described previously, as a positive control. At day 10, no rice seeds germination was noted in samples treated with wildtype EC1, whereas full germination was observed in blank control with either water or DMSO solvent (Figure 7.5). In contrast, toxin treatment resulted in poor or absence of root germination of rice seeds (Figure 7.5). Interestingly, the toxin did not seem to inhibit rice seed shoot germination but significantly affect its growth. The severity of inhibition was related well with the concentration of toxin extract (Figure 7.5). The data suggest that the extracted toxin is a key factor, but may not be the sole factor accounting for the inhibitory effect of E. chrysanthemi pv. zeae on rice seed germination. 106 Figure 7.5 Rice seeds germination. Rice seeds were soaked in ddH2O for 6 h at room temperature. The rice seeds were then placed in Petri dish linked with 3M Whatman paper soaked in 5 ml of EC1 bacteria at OD600 = 1.2, or 5 ml ddH2O containing 10 µl toxin extracts or 50 µl toxin extracts and incubated at 28°C. The controls were 5 ml ddH2O, and the same volume of ddH2O with 10 µl or 50 µl of DMSO. 7.3 Summary Toxin production in rich medium LB was detected after bacterial cells entered stationary phase, whereas in minimal medium it occurred at an earlier growth phase (Figure 7.1). The toxin could be extracted using Amberlite XAD7 beads (Figure 7.2) and could withstand protease treatment, heat treatment (Figure 7.3), and acid and alkaline conditions (Figure 7.4). The results from chapter 4 and chapter 6 showed that mutation of either the polyketide synthesis genes or regulatory gene abolished the inhibitory activity of strain EC1 on rice seeds germination, suggesting that toxin may be a key virulence determinant of E. chrysanthemi pv. zeae on rice. This has been confirmed by the results of this chapter. Addition of toxin extract to rice seeds abolished their root germination and significantly retarded rice shoot growth. It was noted that differences existed between the inhibitory effect of bacterial inoculums and toxin treatment on rice seed germination. While EC1 bacterial treatment completely abolished both the rice shoot and root germination, the toxin fraction exit from Amberlite XAD7 appeared more specifically to inhibit the root germination of rice seeds. 107 CHAPTER 8 General discussion and conclusion 8.1 Summary of major findings In this study, we confirmed that strain EC1, the bacterial isolate from rice plants, to be a pathovar of the bacterial species E. chrysanthemi, that is, E. chrysanthemi pv. zeae, which is known to cause rice foot rot disease (Goto, M., 1979; Liu, Q.G. et al., 2004). This bacterial pathogen is little known in literature, we hence conducted biological, biochemical and 16S rDNA analysis. These results, especially the finding that the pathogen inhibits rice seed germination, convinced us that EC1 should belong to the pathovar of E. chrysanthemi pv. zeae. We cloned and characterized the echIEC1 gene encoding for AHL quorum sensing signal biosynthesis from E. chrysanthemi pv. zeae strain EC1. Mutation of echIEC1 attenuated the virulence of strain EC1 on dicotyledonous plants but did not seem to affect its ability to inhibit rice seeds germination. The data suggest the AHL quorum sensing system is part of the regulatory complexes that determine the overall virulence of the pathogen. To the best of our knowledge, we showed for the first time that E. chrysanthemi pv. zeae strain EC1 produced an antibiotic-like toxin(s). The toxin produced by strain EC1 has a strong inhibitory effect on bacterial and fungal growth, and on rice seeds germination. In contrast, the two closely related strains EC3937 and EC16, which belong to E. chrysanthemi pv. chrysanthemi, did not produce toxin- 108 like substances. Most importantly, we found that the toxin-defective mutants were not able to inhibit rice seeds germination. Given that only the pathovar E. chrysanthemi pv. zeae, but not other E. chrysanthemi pathovars, can infect monocot plants, we propose that the toxin could be a key virulence determinant that enables E. chrysanthemi pv. zeae to infect monocotyledons. We obtained a number of mutants defective in toxin production via screening of a transposon mutant library. Sequence analysis revealed several novel genes encoding for polyketide synthases and a gene which encodes a conserved transcriptional regulator. Consistently, these mutants were unable to inhibit bacterial and fungal growth, and significantly attenuated in their virulence to inhibit rice seeds germination. 8.2 Quorum sensing in Erwinia strains and its role in regulation of bacterial virulence The quorum sensing modulators of E. chrysanthemi pv. zeae strain EC1, namely the proteins encoded by the echIEC1 and echREC1 gene were found to be highly homologous (90.8 % - 92.5 %) to the ExpI-ExpR quorum sensing system of E. chrysanthemi strain 3937 (Figure 3.4; Nasser, W. et al., 1998). While the orientation of the two genes in EC1 genome differs from that of the luxI-luxR operon of marine bacterium Vibro fischeri (NCBI Accession No: Y00509) (Gray, K.M. et al., 2001), it is similar to that of E. chrysanthemi strain EC3937 (NCBI Accession No: X96440) as well as to other Erwinia strains. This high similarity in operon genetic structure between echIEC1-echREC1 and its counterparts is consistent with the close taxonomical 109 relationship between E. chrysanthemi pv. zeae and E. chrysanthemi. Further evidence of similarity is presented by the observation that echIEC1 is likely to direct the synthesis of the autoinducer, OHHL, as exogenous addition of this autoinducer to an echIEC1 mutant of EC1 restored its phenotype (Figure 3.6). We have however yet to identify if E. chrysanthemi pv. zeae also produces the other two autoinducers, HHL and DHL similar to E. chrysanthemi (Nasser, W. et al., 1998; Whitehead, N.A. et al., 2002; Whitehead, N.A. et al., 2001). The quorum sensing system in E. chrysanthemi was reported to be one part of the regulatory complexes that affect the production of the major virulence factors, in this case, the pectinases (Reverchon, S. et al., 1998; Robert-Baudouy, J. et al., 2000). However, this quorum sensing regulation does not seem to play a major role as null mutation of expI only caused a slight decrease in pectinase gene transcription, mainly that of pelA and pelB, but did not change the overall pectinase activity (Nasser, W. et al., 1998). In a similar vein, we also found that the echIEC1 mutant of EC1 showed no significant reduction in the pectate lyase and protease activity (Figure 3.12). Nevertheless, abolishment of AHL production by mutation of the echIEC1 gene resulted in several significant phenotype changes in E. chrysanthemi pv. zeae strain EC1. Firstly, all the three mutants showed significantly increased swimming motility (Figure 3.5). Secondly, EPS production in these mutants was increased (Figure 3.9). Thirdly, the mutants were less virulent than the wild-type strain EC1 when inoculated on potato tubers and on Chinese cabbage leaves (Figure 3.10). Complementation of these mutants by expressing the wild-type echIEC1 gene restored the mutant phenotypes, demonstrating the essential role of AHL quorum sensing system in 110 modulation of these bacterial activities in E. chrysanthemi pv. zeae. The intriguing mechanism with which the AHL quorum sensing system modulates the virulence of E. chrysanthemi pv. zeae on dicot plants remains to be further investigated. However, in contrast to the obvious influence of the AHL quorum sensing system on the pathogenic activity of strain EC1 on dicot plants, mutation of the echIEC1 gene did not seem to affect the ability of the pathogen to inhibit the growth of C. albicans and rice seeds germination (Figure 3.11). These data suggest that the virulence of E. chrysanthemi pv. zeae is constituted of multiple virulence factors as is the case in plant pathogen E. carotovora sup. carotovora, which produces carbapenem antibiotic in addition to exoenzymes (Jones, S. et al., 1993; McGowan, S.J. et al., 2005). It has been documented that bacterial pathogens may resort different signalling and regulation systems to modulate the production of different virulence factors. For example, several quorum sensing signalling systems are found in the human bacterial pathogen Pseudomonas aeruginosa, including the LasI-LasR system (Passador, L. et al., 1993; Pearson, J.P. et al., 1994; Pearson, J.P. et al., 1995), and the RhlI-RhlR system (Brint, J.M. et al., 1995; Latifi, A. et al., 1995), which appears to regulate different sets of virulence genes. 8.3 EC1 is likely to produce polyketide toxins The toxin(s) that seems to affect the virulence of EC1 against potato tubers, Chinese cabbage and rice seeds germination is likely a polyketide as the genes that were disrupted by Tn5 mutagenesis showing decreased toxicity are related to polyketide 111 synthesis, including the genes that encode beta-ketoacyl synthase (EM9, EM11, EM107), polyketide synthase (EM13), and peptide synthase (EM104) (Table 4.2). The polyketide synthesis genes normally occur in an operon or in the same gene cluster (Shen, B., 2003; Edwards, D.J. et al., 2004). Although we have not yet completed the sequencing of all the transposon-tagged toxin biosynthesis genes, the available data showed that the mutated genes in mutants EM13 and EM104 are part of the same operon (Appendix 5, Table 4.2). In E. chrysanthemi, polyketide synthases have been reported to be involved in siderophore (chrysobactin and achromobactin) and pigment (indigoidine) biosynthesis both of which have been implicated in systemic infection of host plants (Persmark, M. et al., 1989; Reverchon, S. et al., 2002; Franza, T. et al., 2001; Franza, T. et al., 2005). Blast searches found that these genes have on average about 24 % sequence identity to the polyketide synthase genes that was disrupted with Tn5 mutagenesis in E. chrysanthemi pv. zeae strain EC1 (That is, mutants EM13, EM20, EM9 ,EM11, EM07, EM104). Given that E. chrysamthemi strains EC3937 and EC16 were not able to produce toxin-like molecules, and that the siderophore and pigment producing ability were both abolished in the polyketide tox- mutants EM9, EM11 and EM107 of strain EC1 (Figure 5.5), we conclude that the toxin produced by E. chrysanthemi pv. zeae strain EC1 is not the pigment or siderophores produced by E. chrysanthemi strains EC3937 and EC16. At this stage, we can not preclude the possibility that the toxin is also a pigment with the siderophore activity, clarification of which awaits further purification of the toxin. 112 8.4 The role of the HorEC1 transcriptional regulator Similar to the pigment indigoidine biosynthesis of E. chrysanthemi, which is regulated by a transcriptional regulator PecS (Reverchon, S. et al., 2002), the toxin production in E. chrysanthemi pv. zeae is also a genetically regulated process. Screening of the tox- mutants led to identification of the horEC1 gene that encodes a conserved transcriptional regulator belonging to the MarR/SlyA family (Table 4.2). The regulator showed a high homology (92 %) to the transcriptional regulator, Rap of Serratia sp. (NCBI Accession No. AF168597) and Hor of E. carotovora subsp. carotovora (NCBI Accession No. AF168687). In addition, both the genome structure and domain organization of HorEC1 were similar to that of other transcriptional regulators, suggesting a similar evolutionary origin of these bacterial pathogens (Figure 6.2-6.3). These data are consistent with the previous southern blot analysis which showed that both the hor and pecS homologues were present in the genome of E. chrysanthemi (Thomson, N.R. et al., 1997). For elucidating the detailed regulatory pathways, it will be useful to determine whether PecS is also involved in regulation of the toxin or pigment production by E. chrysanthemi pv. zeae. HorEC1 appears to be a global regulator modulating a range of biological functions in E. chrysanthemi pv. zeae. In addition to the regulation of the toxin biosynthesis (Figure 6.4), the regulator is also required for biofilm formation (Figure 6.10), swimming motility (Figure 6.9) and virulence on both dicot and monocot plants (Figure 6.5-6.6). 113 8.5 The general characteristics of toxin(s) Among two media tested, minimal medium was superior to LB rich medium for supporting the toxin production by E. chrysanthemi pv. zeae strain EC1 (Figure 7.1). While in rich medium, toxin production occurred after bacterial cells entered stationary phase, minimal medium promoted toxin production at an earlier stage of bacterial growth. Since the toxin produced by E. chrysanthemi pv. zeae is very stable (Figure 7.3-7.4), these findings suggest that the nutrient compositions in the medium could have significant effect on the expression pattern of the toxin biosynthesis genes. Similar effect of medium composition on toxin production has been reported in other bacterial species. For example, production of albicidin, which is also a polyketide toxin (Huang, G. et al., 2001), is promoted by amino acids methionine and glutamine but repressed by peptones and ammonium ions (Zhang, L.H. et al., 1998). More detailed analysis of the factors affecting toxin production by E. chrysanthemi pv. zeae is warranted for developing optimal conditions for toxin purification and for understanding corresponding molecular mechanisms of genetic regulation. Further supporting that the genes involved in polyketide synthesis played a significant role in the inhibition ability of E. chrysanthemi pv. zeae against rice seeds germination, the extracted toxin similarly affected rice seeds germination. In contrast to the bacterial pathogen that stopped both root and shoot germination and elongation (Figure 7.5), it is worthy to note that the toxin extract showed a much stronger inhibitory effect on rice seeds root germination than on shoot development (Figure 7.5). It seems unlikely that this difference is due to the concentration of the toxin 114 extract used, not being as high as that produced by the inoculated EC1 bacterial cells that was about 1 x 1010 c.f.u. per ml, as treatment with a bacterial cell suspension containing only about 100 c.f.u. per ml still provided a complete inhibition of rice seeds germination (Xu, J.L., personal communication). Considering that mutation of the toxin biosynthesis genes abolished the inhibitory activity of strain EC1, two possibilities are worthy of further investigations. Firstly, E. chrysanthemi pv. zeae may produce more than one toxic compounds, which are not all included in the toxin extract due to the limitation of our toxin extraction methods. Secondly, the polyketide toxin may not only specifically inhibit the rice root germination but may also have a synergistic effect to facilitate other virulence factors, for example, the exoenzymes, to damage rice shoot germination. 8.6 Conclusion E. chrysanthemi is widely distributed bacterial pathogen that infects many dicot plants, but little was known previously how its subspecies E. chrysanthemi pv. zeae could cause infections in monocot plants. The results from this study have demonstrated for the first time that the pathogen produced a novel polyketide toxin(s) not present in other Erwinia spp., which plays a key role in inhibition of rice seeds germination. Many polyketide compounds have been used or are known to have promising pharmaceutical potentials as antibiotics against bacterial and fungal pathogens or as therapeutic drugs against cancer diseases and immune disorders (Borchardt, J., 1999; Schneider, G., 2005). Characterization of the E. chrysanthemi pv. zeae toxin and its 115 biosynthesis genes will not only enrich our knowledge on host-pathogen interactions and pathogenecity, but may also provide promising leading compounds and useful gene pool for future drug design and development. 116 BIBLIOGRAPHY 1. Albus, A., Pesci, E., Runyen-Janecky, L., West, S., and Iglewski, B. (1997) Vfr controls quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 179: 3928-3935. 2. Avrova, A.O., Hyman, L.J., Toth, R.L., and Toth, I.K. (2002) Application of amplified fragment length polymorphism fingerprinting for taxonomy and identification of the soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi. Appl. Environ. Microbiol. 68: 1499-1508. 3. Barnard, A.M., and Salmond, G.P. (2007) Quorum sensing in Erwinia species. Anal. Bioanal. Chem. 387: 415-423. 4. Barras, F., Van Gijsegem, F., and Chatterjee, A.K. (1994) Extracellular enzymes and pathogenesis of soft-rot Erwinia. Annu. Rev. Phytopathol. 32: 201-234. 5. Basham, H.G., and Bateman, D.F. (1975a) Killing of plant cells by pectic enzymes: the lack of direct injurious interaction between pectic enzymes or their soluble reaction products and plant cells. Phytopathology. 65: 141-153. 6. Basham, H.G., and Bateman, D.F. (1975b) Relationship of cell death in plant tissue treated with a homogeneous endopectate lyase to cell wall degradation. Physiol. Plant. Pathol. 5: 249-261. 117 7. Beaulieu, C., Boccara, M., and Van Gijsegem, F. (1993) Pathogenic behavior of pectinase-defective Erwinia chrysanthemi mutants on different plants. Mol. Plant-Microb. Interact. 6: 197-202. 8. Beck von Bodman, S., and Farrand, S.K. (1995) Capsular polysaccharide biosynthesis and pathogenicity in Erwinia stewartii require induction by an N-acylhomoserine lactone autoinducer. J. Bacteriol. 177: 5000-5008. 9. Bergey’s Manual of Determinative Bacteriology. Edited. John G. Holt 9th Edition, Baltimore: Williams & Wilkins (c1994) 408-418. 10. Binet, R., Letoffe, S., Ghigo, J.M., Delepelaire, P., and Wandersman, C. (1997) Protein secretion by gram-negative bacterial ABC exporters. Folia Microbiol. (Praha). 42: 179-183. 11. Binet, R., and Wandersman, C. (1996) Cloning of the Serratia marcescens hasF gene encoding the Has ABC exporter outer membrane component: a TolC analogue. Mol. Microbiol. 22: 265-273. 12. Boccara, M., Diolez, A., Rouve, M., and Kotoujansky, A. (1988) The role of individual pectate lyases of Erwinia chrysanthemi strain 3937 in pathogenicity on saintpaulia plants. Physiol. Mol. Plant Pathol. 33: 95104. 13. Borchardt, J. (1999) Combinatorial biosynthesis panning for pharmaceutical gold. Modern Drug Discovery. 2: 22-29. 14. Brint, J.M., and Ohman, D.E. (1995) Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR-RhlI, another set 118 of regulators in strain PAO1 with homology to the autoinducer-responsive LuxR-LuxI family. J. Bacteriol. 177: 7155-7163. 15. CABI and EPPO for the EU under Contract 90/399003. Data Sheets on Quarantine Pests: Erwinia chrysanthemi EPPO quarantine pest. 1-4. 16. Caldas, C., Cherqui, A., Pereira, A., and Simoes, N. (2002) Purification and characterization of an extracellular protease from Xenorhabdus nematophila involved in insect immunosuppression. Appl. Environ. Microbiol. 68: 1297-1304. 17. Castang, S., Reverchon, S., Gouet, P., and Nasser, W. (2006) Direct evidence for the modulation of the activity of the Erwinia chrysanthemi quorum-sensing regulator ExpR by acylhomoserine lactone pheromone. J. Biol. Chem. 281: 29972-29987. 18. Castillo, A., Nasser, W., Codemine, G., and Reverchon, S. (1998) The PecT repressor interacts with different operators of pectate lyase genes in Erwinia chrysanthemi. Biochim Biophys. Acta. 1442: 148-160. 19. Chatterjee, A., Cui, Y., and Chatterjee, A.K. (2002) RsmA and quorum sensing signal, N-[3-oxohexanoyl]-L-homoserine lactone, control the levels of rsmB RNA in Erwinia carotovora subsp. carotovora by affecting its stability. J. Bacteriol. 184: 4089-4095. 20. Chatterjee, A., Cui, Y., Liu, Y., Dumenyo, C.K., and Chatterjee, A. (1995) Inactivation of rsmA leads to overproduction of extracellular pectinases, cellulases, and proteases in Erwinia carotovora subsp. carotovora in the absence of the starvation/cell density-sensing signal, N- 119 (3-oxohexanoyl)-L-homoserine lactone. Appl. Environ. Microbiol. 61: 1959-1967. 21. Chatterjee, A.K., Thurn, K.K., and Feese, D.A. (1983) Tn5 induced mutations in the enterobacterial phytopathogen Erwinia chrysanthemi. Appl. Environ. Microbiol. 45: 644-650. 22. Chatterjee, A.K., Thurn, K.K., and Tyrell, D.J. (1985) Isolation and characterization of Tn5 insertion mutants of Erwinia chrysanthemi that are deficient in polygalacturonate catabolic enzymes oligogalacturonate lyase and 3-deoxy-D-glycero-2,5-hexodiulosonate dehydrogenase. J. Bacteriol. 262: 708-714. 23. Cheng, Y.Q., Yang, M., and Matter, A.M. (2007) Characterization of a gene cluster responsible for the biosynthesis of anticancer agent FK288 in Chromobacterium violaceum No. 968. Appl. Environ. Microbiol. 73: 3460-3469. 24. Choy, W.K., Zhou, L., Syn, C.K.C., Zhang, L.H., and Swarup, S. (2004) MorA defines a new class of regulators affecting flagellar development and biofilm formation in diverse Pseudomonas Species. J. Bacteriol. 186: 7221-7228. 25. Cianciotto, N.P. (2005) Type II secretion: a protein secretion system for all seasons. Trends Microbiol. 13: 581-588. 26. Ciche, T.A., Bintrim, S.B., Horswill, A.R., and Ensign, J.C. (2001) A phosphopantetheinyl transferase homolog is essential for Photorhabdus 120 luminescens to support growth and reproduction of the Entomopathogenic nematode Heterorhabditis bacteriophora. J. Bacteriol. 183: 317-3126. 27. Cohen, M.L. (2000) Changing patterns of infectious disease. Nature. 406: 762-767. 28. Collmer, A., and Bauer D.W. (1994) Erwinia chrysanthemi and Pseudomonas syringae: Plant pathogens trafficking in extracellular virulence proteins. Curr. Top. Microbiol. Immunol. 192: 43-78. 29. Condemine G., Castillo, A., Passeri F., and Enard C. (1999) The PecT repressor coregulates synthesis of exopolysaccharides and virulence factors in Erwinia chrysanthemi. Mol. Plant Microbe. Interact. 12: 45-52. 30. Cronan Jr, J.E., Silbert, D.F., and Wulff, D.L. (1972) Mapping of the fabA locus for unsaturated fatty acid biosynthesis in Escherichia coli. J. Bacteriol. 112: 206-211. 31. Dang, J.L., and Jones, J.D.G. (2001) Plant pathogens and integrated defence responses to infection. Nature. 411: 826-833. 32. de Kievit, T. R., and Iglewski, B.H. (2000) Bacterial quorum sensing in pathogenic relationships. Infect. Immun. 68: 4839-4849. 33. Ditta, G., Stafield, S., Corbin, D., and Helinski, D.R. (1980) Broad host range DNA cloning system for gram-negative bacteria: construction of a gene back of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA. 77: 73477351. 121 34. Dong, Y.H., Gusti, A.R., Zhang, Q., Xu, J.L., and Zhang, L.H. (2002) Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species. Appl. Environ. Microbiol. 68: 1754-1759. 35. Dong, Y.H. Wang, L.H., Xu, J.L., Zhang, H.B., Zhang, X.F., and Zhang, L.H. (2001) Quenching quorum-sensing-dependent bacterial infection by an NN-acyl homoserine lactonase. Nature. 411: 813-817. 36. Dong, Y.H., Xu, J.L., Li, X.Z., and Zhang, L.H. (2000) AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. Proc. Natl. Acad. Sci. USA. 97: 3526-3531. 37. Douet, V., Loiseau, L., Barras, F., and Py, B. (2004) Systematic analysis, by the yeast two-hybrid, of protein interaction between components of the type II sercretory machinery of Erwinia chrysanthemi. Res. Microbiol. 155: 71-75. 38. Edwards D.J., Marquez, B.L., Nogle, L.M., McPhail, K., Goeger, D.E., Roberts, M.A., and Gerwick, W.H. (2004) Structure and biosynthesis of the jamaicamides, new mixed polyketide-peptide neurotoxins from the marine cyanobacterium Lyngbya majuscule. Chem. Biol. 11: 817-833. 39. Ellison, D.W., and Miller, V.L. (2006) Regulation of virulence by members of the MarR/SlyA family. Curr. Opin. Microbiol. 9: 153-159. 40. Enard, C., and Expert, D. (2000) Characterization of a tonB mutation in Erwinia chrysanthemi 3937: TonBEch is a member of the enterobacterial TonB family. Microbiol. 146: 2051-2058. 122 41. Enard, C., Diolez, A., and Expert, D. (1988) Systemic virulence of Erwinia chrysathemi 3937 requires a functional iron assimilation system. J. Bacteriol. 170: 2419-2426. 42. Engebrecht, J., and Silverman, M. (1984) Identification of genes and gene products necessary for bacterial bioluminescence. Proc. Natl. Acad. Sci. USA. 81: 4154-4158. 43. Expert, D., Enard, C., and Masclaux, C. (1996) The role of iron in plant host-pathogen interactions. Trends Microbiol. 4: 232-237. 44. Expert, D., and Toussaint, A. (1985) Bacteriocin-resistant mutants of Erwinia chrysanthemi: possible involvement of iron acquisition in phytopathogenicity. J. Bacteriol. 163: 221-227. 45. Fauci, A.S. (2001) Infectious diseases: Considerations for the 21st century. Clin. Infect. Dis. 32: 675-685. 46. Franza, T., and Expert, D. (1991) The virulence-associated chrysobactin iron uptake system of Erwinia chrysanthemi 3937 involves an operon encoding transport and biosynthetic functions. J. Bacteriol. 173: 68746881. 47. Franza, T., Mahe, B., and Expert, D. (2005) Erwinia chrysanthemi requires a second iron transport route dependent of the siderophore achromobactin for extracellular growth and plant infection. Mol. Microbiol. 55: 261-275. 123 48. Fuqua, W.C., Winans, S.C., and Greenberg, E.P. (1994) Quorum sensing in bacteria: LuxR-LuxI family of cell density – responsive transcriptional regulators. J. Bacteriol. 176: 269-275. 49. Garfinkel, D., and Nester, E. W. (1980) Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism. J. Bacteriol. 144: 732-743. 50. Goto, M. (1979) Bacterial foot rot of rice caused by a strain of Erwinia chrysanthemi. Phytopathology. 69: 213-216. 51. Gray, J.S.S., Brand, J.M., Koerner, T.A.W., and Montgomery, R. (1993) Structure of an extracellular polysaccharide produced by Erwinia chrysanthemi. Carbohydrate Res. 245: 271-387. 52. Gray, K.M., and Garey J.R. (2001) The evolution of bacterial LuxI and LuxR quorum sensing regulators. Microbiol. 147: 2379-2387. 53. Hauben, L., Moore, E.R., Vauterin, L., Steenackers, M., Mergaert, J., Verdonck, L. and Swings, J. (1998) Phylogenetic position of phytopathogens with the Enterobacteriaceae. Syst. Appl. Microbiol. 21: 384-397. 54. Hiroyuki, U., Furuya, N., Iiyama, K., Hidaka, M., Tsuchiya, K., and Matsuyama, N. (2006) Burkholderia gladioli associated with symptoms of bacterial grain rot and leaf-sheath browning of rice plants. J. Gen. Plant. Pathol. 72: 98-103. 124 55. Huang, G., Zhang, L.H., and Birch, R.G. (2001) A multifunctional polyketide-peptide synthetase essential for albicidin biosynthesis in Xanthomonas albilineans. Microbiol. 147: 631-642. 56. Hugouvieux-Cotte-Pattat, N., Condemine, G., Nasser, W., and Reverchon, S. (1996) Regulation of pectinolysis in Erwinia chrysanthemi. Annu. Rev. Micorbiol. 50: 213-257. 57. Hugouvieux-Cotte-Pattat, N., Dominguez, H., and Robert-Baudouy J. (1992) Environmental conditions affect transcription of the pectinase genes of Erwinia chrysanthemi 3937. J. Bacteriol. 174: 7807-7818. 58. Ito, Y., and Shibuya, N. (1999) Receptors for the microbial elicitors of plant defense responses. Tanpakushitsu. Kakusan. Koso. 44: 2322-2330. 59. Jones, L.R. (1909) The bacterial soft rot of certain vegetables. II. Pectinase, the cytological enzyme produced by Bacillus carotovorus and certain other soft-rot organisms. New York (Geneva) Agr. Exp. Sta. Bull. 11: 289-366. 60. Jones, S., Yu, B., Bainton, N.J., Birdsall, M., Bvcroft, B.W., chhabra, S.R., Cox, A.J., Golby, P., Reeves, P.J., Stephes, S. et al (1993) The lux autoinducer regulates the production of exoenzymes virulence determinants in Erwinia carotovora and Pseudomonoas aeruginosa. EMBO J. 12: 2477-2482. 61. Kalia, V., Basandrai, A.K., Thakur, S.K., and Jarial, R.S. (2006) Identification of sources of resistance against Erwinia stalk rot (Erwinia 125 chrysanthemi pv. zeae) among medium maturing inbred lines of maize. Maize Genetics Cooperation Newsletter 80. 62. Kauppinen, S., Siggaard-Andersen, M., von Wettstein-Knowles, P. (1988) beta-ketoacyl-ACP synthase I of Escherichia coli: nucleotide sequence of the fabB gene and identification of the cerulenin binding residue. Carlsberg Res. Commun. 53: 357-370. 63. Keen, N.T., Takami, S., Kobayashi, D., and Trollinger, D. (1998) Improved broad-host range plasmids for DNA cloning in gram-negative bacteria. Gene. 70: 191-197. 64. Kuhn, R., Starr, M.P., Kuhn, D.A., Bauer, H., and Knackmuss, H.J. (1965) Indigoidine and other bacterial pigments related to 3,3’-bipyridyl. Arch. Mikrobiol. 51: 71-84 65. Latifi, A., Winson, M.K., Foglino, M., Bycroft, B.W., Stewart, G.S., Lazdunski, A., Williams, P. (1995) Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1. Mol. Microbiol. 17: 333-343. 66. Larsen, R.A., Wilson, M.M., Guss, A.M., and Metcalf, W.W. (2002) Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch. Microbiol. 178: 193-201. 126 67. Lee, Y.A., and Yu, C.P. (2006) A differential medium for the isolation and rapid identification of a plant soft rot pathogen, Erwinia chrysanthemi. J. Microbiol. Methods. 64: 200-206. 68. Lemattre, M., and Narcy, J.P. (1972) A new bacterial infection of Saintpaulia ionantha by Erwinia chrysanthemi. C. R. Acad. Agric. Fr. 58: 227-231. 69. Lindeberg, M., and Collmer, A. (1992) Analysis of eight out genes in a cluster required for pectic enzyme secretion by Erwinia chrysanthemi: sequence comparison with secretion genes from other gram-negative bacteria. J. Bacteriol. 174: 7385-7397. 70. Lindeberg, M., Boyd, C.M., Keen, N.T., and Collmer, A. (1998) External loops at the C terminus of Erwinia chrysanthemi pectate lyase C are required for species-specific secretion through the out type II pathway. J. Bacteriol. 180: 1431-1437. 71. Liou, G.F., and Khosla, C. (2003) Building-block selectivity of polyketide synthases. Curr. Opin. Chem. Biol. 7: 279-284. 72. Liu, Q.G., and Wang, Z.Z. (2004) Infection characteristics of Erwinia chrysanthemi pv. zeae on rice. J. South China Agricul. University. 25: 5557. 73. Liu, Y., Cui, Y., Mukherjee, A., and Chatterjee, A.K. (1998) Characterization of a novel RNA regulator of Erwinia carotovora ssp. carotovora that controls production of extracellular enzymes and secondary metabolites. Mol. Microbiol. 29: 219-234. 127 74. Liu, Y.G., Mitsukawa, N., Oosumi,T., and Whitier, R.F. (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junction by thermal asymmetric interlaced PCR. Plant J. 8: 457-463. 75. Marits, R., Koiv, V., Laasik,E., and Mae, A. (1999) Isolation of an extracellular protease gene of Erwinia carotovora subsp. carotovora strain SCC3193 by transposon mutagenesis and the role of protease in phytopathogenicity. Microbiol. 145: 1959-1966. 76. McClean, K.H., Winson, M.K., Fish, L., Taylor, A., Chhabra, S.R., Camara, M., Daykin, M., Lamb, J.H., Swift, S., Bycroft, B.W., Stewart, G.S., and Williams, P. (1997) Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiol. 143: 3703-3711. 77. McGowan, S.J., Barnard, A.M.L., Bosgelmez, G., Sebaihia, M., Simpson, N.J.L., Thomson, N.R., Todd, D.E., Welch, M., Whitehead, N.A., and Salmond, G.P.C. (2005) Carbapenem antibiotic biosynthesis in Erwinia carotovora is regulated by physiological and genetic factors modulating the quorum sensing-dependent control pathway. Mol. Microbiol. 55: 526-545. 78. McManus, P.S., Stockwell, V.O., Sundin, G.W., and Jones, A.L. (2002) Antibiotic use in plant agriculture. Annu. Rev. Phytopathol. 40: 443-465. 128 79. Mikkelsen, J., Hojrup, P., Rasmussen, M.M., Roepstorff, P., and Knudsen, J. (1985) Amino acid sequence around the active-site serine residue in the acyltransferase domain of goat mammary fatty acid synthetase. Biochem. J. 227: 21-27. 80. Mohan, S., Kelly, T.M., Eveland, S.S., Raetz, C.R., and Anderson, M.S. (1994) An Escherichia coli gene (fabZ) encoding (3R)hydroxymyristoyl acyl carrier protein dehydrase. Relation to fabA and suppression of mutations in lipid A biosynthesis. J. Biol. Chem. 269: 32896-32903. 81. Monaghan, R.L., and Barrett, J.F. (2006) Antibacterial drug discoverythen, now and the genomics future. Biochem. Pharmacol. 71: 901-909. 82. Munzinger, M., Budzikiewicz, H., Expert, D., Enard, C., and Meyer, J.M. (2000) Achromobactin, a new citrate siderophore of Erwinia chrysanthemi. J. Biosci. 55: 328-332. 83. Nasser, W., Bouillant, M.L., Salmond, G., and Reverchon, S. (1998) Characterization of the Erwinia chrysanthemi expI-expR locus directing the synthesis of two N-acyl-homoserine lactone signal molecules. Mol. Microbiol. 29: 1391-1405. 84. Nasser, W., Robert-Baudouy, J., and Reverchon, S. (1997) Antagonistic effect of CRP and KdgR in the transcription control of the Erwinia chrysanthemi pectionlysis genes. Mol. Microbiol. 26: 1071-1082. 85. Nealson, K.H., and Hastings, J.W. (1979) Bacterial bioluminescence: its control and ecological significance. Microbiol. Rev. 43: 496-518. 129 86. Nealson, K.H., Platt, T., and Hastings, J.W. (1970) Cellular control of the synthesis and activity of the bacterial luminescent system. J. Bacteriol. 104: 313-322. 87. Otteman, K.M., and Miller, J. F. (1997) Roles for motility in bacterialhost interactions. Mol. Microbiol. 24: 1109-1117. 88. Palacio-Bielsa, A., Cambra, M.A., and Lopez, M.M. (2006) Characterization of potato isolates of Dickeya chrysanthemi in Spain by a microtitre system for biovar determination. Annals. Appl. Biol. 148: 157164. 89. Passador, L., Cook, J.M., Gambello, M.J., Rust, L., and Iglewski, B.H. (1993) Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science. 260: 1127-1130. 90. Payne, J.H., Schoedel, C., Keen N.T., and Collmer, A. (1987) Multiplication and virulence in plant tissues of Escherichia coli clones producing pectate lyase isozymes PLb and PLe at high levels and of an Erwinia chrysanthemi mutant deficient in PLe. Appl. Environ. Microbiol. 53: 2315-2320. 91. Pearson, J.P., Gray, K.M., Passador, L., Tucker, K.D., Eberhard, A., Iglewski, B.H., and Greenberg, E.P. (1994) Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc. Natl. Acad. Sci. USA. 91: 197-201. 130 92. Pearson, J.P., Passador, L., Iglewski B.H., and Greenberg, E.P. (1995) A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA. 92: 1490-1494. 93. Persmark, M., Expert, D., and Neilands, J.B. (1989) Isolation, characterization, and synthesis of chrysobactin, a compound with siderophore activity from Erwinia chrysanthemi. J. Biol. Chem. 264: 3187-3193. 94. Piper, K.R., Beck von Bodman, S., and Farrand, S.K. (1993) Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature. 362: 448-450. 95. Py, B., Looiseau, L., and Barras, F. (2001) An inner membrane platform in the type II secretion machinery of gram-negative bacteria. EMBO rep. 2: 244-248 96. Rashid, M.H., and Kornberg, A. (2000) Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA. 97: 4885-4890. 97. Rauscher, L., Expert, D., Matzanke, B.F., and Trautwein, A.X. (2002) Chrysobactin-dependent iron acquisition in Erwinia chrysanthemi. J. Biol. Chem. 277: 2385-2395. 98. Reading, N.C., and Sperandio, V. (2006) Quorum sensing: the many languages of bacteria. FEMS Microbiol. Lett. 254: 1-11. 99. Reddy, K.J., Soper B.W., Tang, J., and Bradley, R.L. (1996) Phenotypic variation in exopolysaccharide production in the marine, 131 aerobic nitrogen-fixing unicellular cyanobacterium Cyanothece sp. World J. Microbiol. Biotechnol. 12: 311-318. 100. Reverchon, S., Bouillant, M.L., Salmond, G., and Nasser, W. (1998) Integration of the quorum-sensing system in the regulatory networks controlling virulence factor synthesis in Erwinia chrysanthemi. Mol. Microbiol. 29: 1407-1418. 101. Reverchon, S., Expert, D. Robert-Baudouy, J., and Nasser, W. (1997) The cyclic AMP receptor protein is the main activator of pectinolysis genes in Erwinia chrysanthemi. J. Bacteriol. 179: 3500-3508. 102. Reverchon, S., Nasser, W., Robert-Baudouy, J. (1994) pecS: a locus controlling pectinase, cellulase and blue pigment production in Erwinia chrysanthemi. Mol. Microbiol. 11: 1127-1139. 103. Reverchon, S., Rouanet, C., Expert, D., and Nasser, W. (2002) Characterization of indigoidine biosynthetic genes in Erwinia chrysanthemi and role of this blue pigment in pathogenicity. J. Bacteriol. 184: 654-665. 104. Robert-Baudouy, J., Nasser, W., Condemine, G., Reverchon, S., Shevchik, V.E., and Hugouvieux-Cotte-Pattat, N. (2000) Pectic enzymes of Erwinia Chrysanthemi, regulation and role in pathogenesis. Plant-Microbe Interact. 5: 221-368. 105. Rouhianinen, L., Vakkilainen, T., Siemer, B.L., Buikema, W., Haselkorn, R., and Sivonen, K. (2004) Genes coding for hepatotoxic 132 heptapeptides (microcystins) in the cyanobacterium anabaena strain 90. Appl. Environ. Microbiol. 70: 686-692. 106. Sabet, A.K. (1954) On the host range and systematic position of the bacteria responsible for the yellow slime diseases of wheat (Triticum vulgare Vill.) and cocksfoot grass (Dactylis glomerata L.). Ann. Appl. Biol. 41: 606-611. 107. Samson, R., Legendre, J.B., Christen, R., Achouak, W., and Gardan, L. (2004) Transfer of Pectobacterium chrysanthemi (Brenner et al. 1973) Hauben et al. 1998 and Brenneria paradisiaca to the genus Dickeya gen. nov. as Dickeya chrysanthemi comb. nov. and Dickeya paradisiaca comb. nov. and delineation of four novel species: Dickeya dadantii sp. nov., Dickeya dianthicola sp. nov., Dickeya dieffenbachiae sp. nov. and Dickeya zeae sp. nov. Int. J. of Syst. Evol. Microbiol. 54: 1-13. 108. Sauvage, C., and Expert, D. (1994) Differential regulation by iron of Erwinia chrysanthemi pectate lyases: pathogenicity of iron transport regulatory (cbr) mutants. Mol. Plant-Microbe. Interact. 7: 71-77. 109. Schneider, G. (2005) Enzymes in the biosynthesis of aromatic polyketide antibiotics. Curr. Opin. Struct. Biol. 15: 629-636. 110. Schoonejans E., Expert D., and Toussaint A. (1987) Characterization and virulence properties of Erwinia chrysanthemi lipopolyasscharidedefective, ΦEC2-resistant mutants. J. Bacteriol. 169: 4011-4017. 111. Schwyn, B., and Neilands, J. B. (1987). Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160: 47–56. 133 112. Shen, B. (2003) Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms. Curr. Opin. Chem. Biol. 7: 285-295. 113. Siggaard-Andersen, M., Wissenbach, M., Chuck, J.A., Svendsen, I., Olsen, J.G., and von Wettstein-Knowles, P. (1994) The fab-J-encoded beta-ketoacyl-[acyl carrier protein] synthase IV from Escherichia coli is sensitive to cerulenin and specific for short-chain substrates. Proc. Natl. Acad. Sci. USA. 91: 11027-11031. 114. Sinha, S.K., and Prasad, M. (1977) Bacterial stalk rot of maize, its symptoms and host-range. Zentralbl. Bakteriol. Parasitenkd Infektionskr Hyg. 132: 81-88. 115. Slade, M.B., and Tiffin, A.I. (1984) Biochemical and serological characterization of Erwinia. Methods Microbiol. 15: 227-293. 116. Starr, M.P., and Chatterjee, A.K. (1972) The genus Erwina: Enterobacteria pathogenic to plants and animals. Annu. Rev. Micorbiol. 26: 389-426. 117. Stevens, A.M., and Greenberg, E.P. (1997) Quorum sensing in Vibrio fischeri: essential elements for activation of the luminescence genes. J. Bacteriol. 179: 557-562. 118. Strange, R.N., and Scott, P.R. (2005) Plant Disease: A threat to global food security. Annu. Rev. Phytopathol. 43: 83-116. 119. Tang, X., Lu, B.F., and Pan, S.Q. (1999) A bifunctional transposon mini-Tn5gfp-km which can be used to select for promoter fusions and 134 report gene expression levels in Agrobacterium tumefaciens. FEMS Microbiol. Lett. 179: 37-42. 120. Thomson, N.R., Cox, A., Bycroft, B.W., Stewart, G.S.A.B., Williams, P., and Salmond, G.P.C. (1997) The Rap and Hor proteins of Erwinia, Serratia and Yersinia: a novel subgroup in a growing superfamily of proteins regulating diverse physiological processes in bacterial pathogens. Mol. Microbiol. 26: 531-544. 121. Venkatesh, B., Babujee, L., Liu, H., Hedley, P., Fujikawa, T., Birch, P., Toth, I., and Tsuyumu, S. (2006). The Erwinia chrysanthemi 3937 PhoQ Sensor Kinase Regulates Several Virulence Determinants. J. Bacteriol. 188: 3088-3098. 122. Waldee, E.L. (1945) Comparative studies of some peritrichous phytopathogenic bacteria. Iowa State Coll. J. Sci. 19: 435-484. 123. Wang, H., and Cronan, J.E. (2004) Functional replacement of the FabA and FabB proteins of Escherichia coli fatty acid synthesis by Enterococcus faecalis FabZ and FabF homologues. J. Bacteriol. Chem. 279: 34489-34495. 124. West, S.E.H., Sample, A.K., and Runyen-Janecky, L.J. (1994) The vfr gene product, required for Pseudomonas aeruginosa exotoxin A and protease production, belongs to the cyclic AMP receptor protein family. J. Bacteriol. 176: 7532-7542. 135 125. Whitehead, N.A., Barnard, A.M.L., Slater, H., Simpson, N.J.L., and Salmond, G.P.C. (2001) Quorum sensing in gram-negative bacteria. FEMS Microbiol. Rev. 25: 365-404. 126. Whitehead, N.A., Byers, J.T., Commander, P., Corbett, M.J., Coulthurst, S.J., Everson, L., Harris, A.K.P., Pemberton, C.L., Simpson, N.J.L., Slater, H., Smith, D.S., Welch, M., Williamson, N., and Salmond, G.P.C. (2002) The regulation of virulence in phytophathogenic Erwinia species: quorum sensing, antibiotics and ecological considerations. Antionie Van Leeuwenhoek. 81: 223-231. 127. Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 33: 103-119. 128. Yap, M.N., Yang, C.H., Barak, J.D., Jahn, C.E., and Charkowski, A.O. (2005) The Erwinia chrysanthemi type III secretion system is required for multicellular behavior. J. Bacteriol. 187: 639-648. 129. Yoneyama, H., and Katsumata, R. (2006) Antibiotic resistance in bacteria and its future for novel antibiotic development. Biosci. Biotechnol. Biochem. 70: 1060-1075. 130. Zhang, H.B., Wang, C., and Zhang, L.H. (2004) The quormone degradation system of Agrobacterium tumefaciens is regulated by starvation signal and stress alarmone (p)ppGpp. Mol. Microbiol. 52: 1389-1401. 136 131. Zhang, L.H. (2003) Quorum quenching and proactive host defense. Trends Plant Sci. 8: 238-244. 132. Zhang, L.H., and Dong, Y.H. (2004) Quorum sensing and signal interference: diverse implications. Mol. Microbiol. 53: 1563-1571. 133. Zhang, L.H., and Kerr, A. (1991) A diffusible compound can enhance conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens. J. Bacteriol. 173: 1867-1872. 134. Zhang, L.H., Murphy, P.J., Kerr, A., and Tate, M. E. (1993) Agrobacterium conjugation and gene regulation by N-acyl-homoserine lactones. Nature. 362: 446-447. 135. Zhang, L.H., Xu, J.L., and Birch, R.G. (1998) Factors affecting biosynthesis by Xanthomonas albilineans of albicidin antibiotics and phytotoxins. J. Appl. Microbiol. 85: 1023-1028. 137 APPENDIX 1 TAIL-PCR sequence of EM40 containing the Tn5 insertion: (Note: the sequences in the red box are the gentamycin ends of the pTGN Tn5 transposon.) Upon further sequence extension using TAIL-PCR, a total of 16.421-kb of sequence was obtained as listed in (b). ORF search from this cluster revealed as listed in (a) the presence of proteins involved in biosynthesis (ORF1), transport (ORF 2-6) and an operon represser (ORF7) which is consistent with the types of genes that are present in a gene cluster that encodes secondary metabolites (Binet, R. et al., 1996; Binet, R. et al., 1997). 138 (a) ORF as predicted using Clone software and its homology based on the nucleotide analysis which was subjected to NCBI BLAST: 139 (b) The following sequences flanking the Tn5 insertion to ORF1 was obtained using TAIL-PCR: 140 ___________________________________________________________________ 141 APPENDIX 2 TAIL-PCR sequence of EM13 containing the Tn5 insertion: (Note: the sequences in the red box are the gentamycin ends of the pTGN Tn5 transposon.) Upon further sequence extension using TAIL-PCR, a total of 24.192-kb of sequence was obtained as listed in (b). Apart from the hypothetical proteins, an ORF search from this cluster revealed as listed in (a) the presence of proteins involved in biosynthesis (ORF1,2,4,12), transport (ORF 10,11,14,16) and regulation (ORF 9) which is consistent with the types of genes that are present in a gene cluster that encodes secondary metabolites (Binet, R. et al., 1996; Binet, R. et al., 1997). 142 (b) ORF as predicted using Clone software and its homology based on the nucleotide analysis which was subjected to NCBI BLAST: 143 144 (b) The following sequences for ORF1 and ORF2 flanking the Tn5 insertion was obtained using TAIL-PCR: 145 146 147 APPENDIX 3 TAIL-PCR sequence of EM20 containing the Tn5 insertion: (Note: the sequences in the red box are the gentamycin ends of the pTGN Tn5 transposon.) _____________________________________________________________________ Upon further sequence extension using TAIL-PCR, a total of 7.361-kb of sequence was obtained as listed in (b). ORF search from this cluster revealed, as listed in (a) the presence of proteins involved in biosynthesis of genes encoding secondary metabolites. (a) ORF as predicted using Clone software and its homology based on the nucleotide analysis which was subjected to NCBI BLAST: 148 (b) Sequence of the flanking region (ORF2) of Tn5 extended using TAIL-PCR: 149 150 APPENDIX 4 TAIL-PCR sequence of EM9 containing the Tn5 insertion: (Note: the sequences in the red box are the gentamycin ends of the pTGN Tn5 transposon.) TAIL-PCR sequence of EM11 containing the Tn5 insertion: 151 TAIL-PCR sequence of EM107 containing the Tn5 insertion: Total sequence of the flanking region of Tn5 extended using TAIL-PCR: 152 153 154 APPENDIX 5 TAIL-PCR sequence of EM104 containing the Tn5 insertion: (Note: the sequences in the red box are the gentamycin ends of the pTGN Tn5 transposon.) The following sequences flanking the Tn5 insertion was obtained using TAILPCR: (As per Appendix 2). 155 APPENDIX 6 TAIL-PCR sequence of EM53 containing the Tn5 insertion: (Note: the sequences in the red box are the gentamycin ends of the pTGN Tn5 transposon.) The following sequences flanking the Tn5 insertion was obtained using TAILPCR: 156 157 [...]... including China, India, Indonesia, Philippines and Korea The disease is caused by the bacterial pathogen Erwinia chrysanthemi The pathovar, E chrysanthemi pv zeae, also causes severe infections in maize (Sinha, S.K et al., 1977) However, in contrast to its closely related pathovar, E chrysanthemi, which infects many crops and plants worldwide, E chrysanthemi pv zeae is much less characterized, in particular,... germination 58 3.12 Analysis of exoenzymes produced by E chrysanthemi pv zeae 59 4.1 Examples of growth inhibition assay by strain EC1 and its mutants 64-65 4.2 Southern blot analysis of the Tox- mutants of strain EC1 66 5.1 Generic map of the regions flanking Tn5 insertion sites in mutants EM9, EM11 and EM107 74-75 5.2 Protein domain analysis 76-77 5.3 Pathogenesis assay using potato tubers and Chinese... Manual, 1994) Using the latest taxonomy list obtained from The International Society for Plant Pathology (http://www.isppweb.org/names_bacterial_pant2005.asp), the genus 4 E chrysanthemi consists of 6 pathovars, namely, E chrysanthemi pv chrysanthemi, E chrysanthemi pv dianthicola, E chrysanthemi pv dieffenbachiae, E chrysanthemi pv paradisiaca, E chrysanthemi pv parthenii and E chrysanthemi pv zeae It is... Mutation of the gene encoding AHL biosynthesis enhanced EC1 swimming motility 50-51 3.6 OHHL modulates the swimming motility of E chrysanthemi pv zeae 51 3.7 Electron micrographs of EC1 and AHL- mutants 52 3.8 LPS assay using SDS-PAGE and stained with silver solution 53 3.9 Alcian blue assay 54 xiii 3.10 Pathogenesis assay using potato tubers and Chinese cabbage 55-56 3.11 E chrysanthemi pv zeae inhibited... obtained three independent E chrysanthemi with Tn5 inserted in various regions of this gene and in all cases, the manifestation of phenotypes due to the Tn5 insertion was similar, including the diminished inhibitory effect on bacterial, fungal growth and rice seeds germination, and the decreased virulence on dicot plants In addition, we identified a transcriptional regulator HorEC1, which is a member of. .. sensing (Fuqua, W.C et al., 1994), to coordinate many important biological activities including expression of virulence genes This promising development illustrates the importance of identification of key bacterial virulence factors and the mechanisms of genetic regulation 2 Bacterial stalk rot is one of the important rice bacterial diseases It occurs in many rice planting countries and regions including... size of hybridization bands 67 4.2 Sequence analysis of Tox- mutants of the strain EC1 69-70 6.1 List of bacterial strains 88 xii LIST OF FIGURES Figure No Title Page 1.1 Pectin catabolism in E chrysanthemi 10 1.2 The Type II secretion system of E chrysanthemi 12 1.3 E chrysanthemi 3937 siderophore and pigment gene clusters 13 1.4 A simplified model for the regulatory network controlling pectinase, indigoidine,... pectinase, indigoidine, achromobactin and chrysobactin synthesis in E chrysanthemi 14 1.5 A quorum-sensing model 19 3.1 16S rDNA based phylogenetic position of EC1 42-43 3.2 Virulence bioassay 43 3.3 AHL assay of representative AHL-deficient mutants and complementary strains 46 3.4 Physical map and sequence analysis of the DNA fragment containing the genes involved in AHL quorum sensing signal biosynthesis... capable of causing systemic disease by spreading through the vascular system of a plant The physiological symptoms of such infection are yellowing of new leaves, wilting and a mushy, foulsmelling stem rot (Slade, M.B et al., 1984) Genetic and physiological studies show that systemic infection of E chrysanthemi is dependent on two abilities, namely iron acquisition and production of the pigment, indigoidine... genes reduces the virulence and maceration capacities of E chrysanthemi (de Kievit, T R et al., 2000; Lindeberg, M et al., 1992) It is also interesting to note that while the Out proteins of E chrysanthemi shows high homology to those of E carotovora, the system is unable to secrete the pectinases of E carotovora and vice-versa, indicating strong species specificity of the Out proteins (Lindeberg, M et .. .GENETIC CONTROL OF VIRULENCE IN ERWINIA CHRYSANTHEMI PV ZEAE MUMTAZ BEGUM BINTE MOHAMED HUSSAIN (B.Sc Hons.) (UNIVERSITY OF LEEDS) A THESIS SUBMITTED FOR THE DEGREE OF MASTERS OF SCIENCE INSTITUTE... 1.7 Erwinia chrysanthemi pv zeae Erwinia chrysanthemi pv zeae was first documented in 1954 to cause soft rot in wheat (Sabet, A.K., 1954) Since then, it has been isolated from a wide variety of. .. regions including China, India, Indonesia, Philippines and Korea The disease is caused by the bacterial pathogen Erwinia chrysanthemi The pathovar, E chrysanthemi pv zeae, also causes severe infections