Mutagenesis studies of the pseudomonas global regulator, mora, and cloning of its signaling pathway member, adenylate cyclase

MUTAGENESIS STUDIES OF THE PSEUDOMONAS GLOBAL REGULATOR, MorA, AND CLONING OF ITS SIGNALLING PATHWAY MEMBER, ADENYLATE CYCLASE T JYOTHILAKSHMI MENON B.Sc (Hons) Botany, P G Diploma in Biochem Tech, M.Sc (Plant Molecular Biology), University of Delhi, India A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS I am indebted to my supervisor, A/P Sanjay Swarup for his unstinting motivation, patience and support throughout my candidature My thanks to Dr Sivaraman for allowing me to some part of my work in his lab I am deeply grateful to my family for their constant support Thanks also to the DBS staff for their helpful and cooperative attitude throughout which made studying at NUS an unforgettable and pleasurable experience I am very grateful to Dennis and Wei Ling for their help at various times in lab without which I would not have been able to complete my work I am also grateful to all my labmates for the congenial atmosphere in my lab Thanks to all my friends at NUS for their support and their patience in bearing with me A special thanks to Asha M.B., Sheela Reuben, Alex Shapeev and Jessie for being very supportive mentors and friends I would like to thank Shu Shinla, for help with some part of my biochemical work, as part of his project assignment Last but not the least, I am indebted to NUS for providing me with the Research Scholarship without which I could not have completed my studies i TABLE OF CONTENTS ACKNOWLEDGEMENTS………………………………………………………………… i SUMMARY………………………………………………………………………… ……… iii LIST OF TABLES.……………………………………………………………… iv LIST OF FIGURES ……………………………………………………………………… v LIST OF ABBREVIATIONS…………………………………………………………….… vi CHAPTER INTRODUCTION A) BACTERIAL MOTILITY.….………………………………………………………… B) BIOFILM FORMATION… C) TO STICK OR NOT TO STICK? D) C-DI-GMP SIGNALING IN BACTERIA.…… …………………………………………8 E) PRESENT WORK: MORA AS A GLOBAL REGULATOR OF MOTILITY AND BIOFILM FORMATION IN PSEUDOMONAS…………………………………………………………17 CHAPTER MATERIALS AND METHODS A) MUTAGENESIS STUDIES OF MORA 2.1 Bacterial strains and cultivation…………………………………………… 20 2.2 Preparation of competent cells …………………………………………… 20 2.3 Site-directed mutagenesis of MorA-GE and MorA full length domain…… 21 2.4 Transformation of the mutated plasmids…………………………………….22 2.5 Analysis of mutant colonies ….…… …………………………………… 23 2.6 Expression and Purification of the MorA mutant GE-N* …………………23 2.7 Resolubilization of MorA-GE-N* …………………………………………24 2.8 Affinity purification and on column cleavage of MorA-GE… ……………25 B) CLONING AND SEQUENCING OF ADENYLATE CYCLASE FROM P PUTIDA 2.1 Bacterial strains and cultivation… …………………………………………26 2.2 Preparation of competent cells… ………………………………………… 27 2.3 DNA Sequencing…………… …………………………………………… 27 2.4 DNA manipulations and analysis… ……………………………………… 27 CHAPTER RESULTS A) MUTAGENESIS STUDIES OF PSEUDOMONAS MORA………………………………….33 B) CLONING, SEQUENCING AND BIOINFORMATIC ANALYSES OF PPAC………………………………………………………… 36 CHAPTER DISCUSSION A) MUTAGENESIS STUDIES OF PSEUDOMONAS MORA……….……………………… 41 B) CLONING, SEQUENCING AND BIOINFORMATIC ANALYSES OF PPAC……………………………………………………………………………… …43 BIBLIOGRAPHY …………………………………………………………………………….50 APPENDICES ………………………………………………………………………………….60 ii SUMMARY Bacteria can exist in free living state (planktonic state) or as part of surface associated multicellular communities called biofilms Their ability to shift between these two states is determined by various environmental factors including nutrient levels, moisture etc Regulation of flagella formation is critical for both swimming in liquid and viscous media, swarming along surfaces as well as biofilm formation Many factors play a role in this regulation including local and global regulators and quorum sensing MorA is one such global regulator of flagellar development in Pseudomonas, whose mutation results in derepression of flagellar development that leads to enhancement of motility and chemotaxis Our laboratory is working extensively at uncovering the various aspects of MorA mediated signaling pathways In this aspect, previous workers had screened a set of hypermotile morA revertant mutants and identified a few genes downstream of morA One of these genes, an Adenylate cyclase (PpAC) has been cloned and sequenced fully in this study Possible roles in the morA signaling pathway are discussed In addition, some site directed mutants of morA were created and protein expression studies were carried out A comprehensive account of flagellar biosynthesis, motility, and role of various regulators (including MorA) is also discussed here iii LIST OF TABLES Table 2.1 Mutagenesis reactions for site-directed mutagenesis…………… 2b Table 2.2 Primer sequences for mutagenesis and sequencing of mutants……2b Table 2.3 List of primer sequences used for cloning of PpAC and verifying the insert orientation……………………………………………….2e Table 2.4 TOPO™ reaction components…………………………………… 2e Table 2.5 Primers used for gene walking…………………………………… 2f Table 3.1 Physical and chemical properties of MorA-GE and MorA-GE-N* proteins as computed by ProtParam (www expasy ch)………… 3c Table 3.2 List of PpAC homologues in Pseudomonas sp used for multiple sequence analysis and phylogenetic analysis through tree construction…………………………………………………………… 3t Table 3.3 List of Adenylate cyclases from other bacterial species used for alignment and tree construction studies……………………………3t iv LIST OF FIGURES FIG 1.1 Flagellum and its components (Source: Flagellar assemblyPseudomonas fluorescens Pfo-1- Kegg pathway-http://www genome.jp/dbget-bin/show_pathway?pfo02040+Pfl_1501)…………………1a FIG 1.2 Structure and morphogenesis of the bacterial flagellum (from Mc Carter, 2006)……………………………………………………… 1b FIG 1.3 Bacterial small molecule signalling molecules………………………………1c FIG 1.4 Domain structure of GGDEF and EAL family (Romling and Amikam, 2006)….……… .……………………………….1d FIG 1.5a Structure of PleD (from Chan et al.2004)… ……………………………….1e FIG 1.5b Mechanistic model of PleD action (Christen et al 2004)……….…… 1f FIG 1.6a C-di-GMP role in regulation of sessility and motility (from Romling and Amikam, 2006)……… ……………………………………………… 1g FIG 1.6b C-di-GMP regulatory mechanism at the individual cell level (from Romling and Amikam, 2006)…………… ……………………………… 1g v FIG 1.7a C-di-GMP regulates biofilm formation and virulence in an inverse fashion in V cholerae ( Romling and Amikam,2006)………………………………… 1h FIG 1.8 Comparison of various PAS folds (from Vreede et al 2003)………………….1i FIG 1.9 SMART predicted domain structure of MorA (from Choy et al 2004)…… .1j FIG 2.1 Overview of site–directed mutagenesis procedure (Adapted from Quik Change® II XL Site-Directed Mutagenesis Kit Technical Manual, Stratagene)……………………………………………………………………2a FIG 2.2 BSA Standard Curve for Bradford Assay…………………………………… 2c FIG 2.3 Schematic representation of the Pfl_5493 (Adenylate cyclase) locus and flanking genes in P fluorescens PfO-1 …………………………………… 2d FIG 3.1a) PCR products of the mutated plasmids; control pWhitescript™ (4.5kb), pGE-N* and pGE-D* …………………………………………………3a FIG 3.1b) Restriction analyses of the clones obtained following mutagenesis and transformation of mutant plasmids………………………………………….3a FIG 3.2 DNA sequencing chromatogram showing confirmation of mutation in morA GE-N* (Chromas) The orange box indicates the desired mutation (GCA to AAC; Asparagine to Alanine)………………………………………3b FIG 3.3 SDS-PAGE analyses of pre-induction and post-induction samples of pGEX-6P-1 and MorA GE-N*………………………………………………3d vi FIG 3.4 Denaturing SDS-PAGE analyses of post-sonication pellets and supernatants of pGEX-6P-1 and MorA GE-N*…………………………………………….3e FIG 3.5 SDS-PAGE showing GE-N* fusion protein from insoluble bodies following urea treatment and subsequent resolubilization by rapid dilution or dialysis…………………………………………………………………… 3f FIG 3.6 PCR amplification of a 3.75kb fragment from P putida PNL -MK25……………………………………………………………………… 3g FIG 3.7 Uncut plasmids isolated from clones obtained following TA Cloning……… 3h FIG 3.8 Restriction Analysis of clones with EcoRI…………………………………… 3h FIG 3.9a Gene walking strategy used to sequence the 3.75 kb Adenylyl Cyclase locus from P putida………………………………………………………… 3i FIG 3.9b Positions of PpAC homologue locus, Pfl_5493 in P fluorescens on both strands………………………………………………………………… 3i FIG 3.10 Part of assembled multiple sequence alignment of PpAC DNA se quences from various clones………………………………………………….3j FIG 3.11 Clustal W alignment of PpAC and D4-GFP sequence………………………3k FIG 3.12 Final annotated consensus sequence of PpAC, along with predicted aminoacid sequence obtained through alignment and assembly of the sequence data network of Pfl_5493 from P fluorescens………………………3l vii FIG 3.13 Genomic context of PpAC in Pseudomonas in comparison to other Adenylate cyclases from Pseudomonas sp………………………………….3o FIG 3.14a Predicted ORFs of PpAc using Frame Plot……………………………… 3p FIG 3.14b Predicted ORFs of PpAC using ORFinder……………………………… 3p FIG 3.15a Results of InterProScan Analyses of PpAC predicted protein sequence ( www.ebi.ac.uk/cgi-bin/prscan)………………………………………….3q FIG 3.15b SMART predicted domain structure of PpAC…………………………… 3q FIG 3.15c TFSitescan predicted ExsA binding site upstream of PpAC …………… 3q FIG 3.16 TMPred prediction of the topology of PpAC………………………………… 3r FIG 3.17 Results of FUGUE analyses of PpAC………………………………………… 3s FIG 3.18 CLUSTAL W alignment of PpAC and its homologues in Pseudomo nas sp…………………………………………………………………… 3u FIG 3.19a Unrooted tree of PpAC and other known Adenylyl Cyclases from Pseud omonas sp………………………………………………………………… 3x FIG 3.19b Cladogram of PpAC and other Adenylate cyclases from Pseudomo nas sp……………………………………………………………………….3x viii FIG 3.19c Cladogram of PpAC and Adenylate cyclases from other bacterial species generated using Clustal W and Tree View……………………………… 3y FIG 3.19d Phylogram of PpAC and Adenylate cyclases from other bacterial species generated using Clustal W and Tree View…………………………………3y FIG 4.1 Model for PpAC in MorA mediated signalling pathway……………… 4a FIG 4.2 String predicted interaction network of Pfl_5493 from P fluorescens…… 4b ix FIG 4.2 Predicted Interaction Network for Pfl_5493 (http://www.String.embl.de) 4b This would imply a possible cross talk between c-di-GMP and cAMP wherein each may exert an antagonistic effect on each other In Escherichia coli and Salmonella enterica serovar Typhimurium (S typhimurium), the cAMP circuit consists of one adenylate cyclase, a cAMP receptor protein and a phosphodiesterase respectively (Imamura et al.,1996, Lee et al, 2005) It would be interesting to isolate similar cAMP receptor proteins and see whether they are upregulated or down regulated during the course of MorA signaling ( in morA mutants and overexpressing strains) and to discover a similar circuit There may be targets common to both these signaling molecules, through which they can exert their influences PpAC may be part of a network similar to that predicted for Pfl_5493 in Figure 4.2.(http://string.embl.de) String is a database of known and predicted proteinprotein interactions (von Mering et al 2007).The Pfl_5493 network in P fluorescens displays partners predominantly belonging to nucleotide metabolism family including NDKinase genes, pyruvate kinase and RNA polymerases This is in line with the earlier mentioned importance of the rnk gene next to PpAC and its probable importance in the cAMP signaling pathway It would also be interesting to study the interaction of cAMP with the components of the flagellar pathway, effect on virulence and biofilm formation Generation of mutants of this gene and further molecular and biochemical analyses would help in further establishing the connection between these pathways 49 BIBLIOGRAPHY Adaikkalam, V and Swarup, S 2002 Molecular 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Apweiler, R 2001 InterProScan - an integration platform for the signature-recognition methods in InterPro Bioinformatics, 17, 847-848 Zhang, L H 2003 Quorum quenching and proactive host defense Trends in Plant Sci., 8, 238-244 Zhulin, I B., Taylor, B L , and Dixon, R 1997 PAS domain S-boxes in Archaea, bacteria and sensors for oxygen and redox Trends Biochem Sci., 22, 331– 333 58 APPENDIX Generuler kb Range DNA ladder: 14 fragments (in bp): 10000, 8000, 6000, 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 750, 500, 250 The ladders are composed of chromatography-purified individual DNA fragments 59 APPENDIX 60 APPENDIX Procedure for performing DNA sequencing 1.Prepared high quality plasmid DNA for sequencing by Wizard Prep plasmid isolation kit Set up sequencing reaction as follows For µl reaction, 200 to 500 ng of double stranded plasmid, 0.8 pm of primer, 2µl of Big Dye and rest de ionized water was added to make up the volume 3.Performed the cycle sequencing as follows: 96 C for one minute, 25 cycles of 96 C for 30 seconds, 50 C for 15 seconds and 60 C for four minutes with intermediate ramping at the rates of 1.1C/s Then final ramping to 4C The samples were spun down and purified by Ethanol/Sodium acetate precipitation The dried pellets were redissolved in formamide, loaded into the sample trays, and then denatured at 95 C for two minutes Then the samples were cooled on ice 7.The sample trays were sealed with a septum and placed in the autosampler 8.Capillary electrophoresis of the samples were carried according to the manufacturer’s instructions 9.The sequence data was analysed using Chromas software 61 ... factors affect the process of biofilm formation including the type of species, the nature of the environment, the gene products and the surface composition of the bacterium The stages of biofilm formation... maturation of the biofilm Finally, a dynamic equilibrium is established between the biofilm and the environment with the cells in the outermost layer of the biofilm sloughing off and escaping... consisting of a dgc and pde, which were named as dgc1-3 and pde 1-3 They studied the activities of DGC and PDE from the cellular extracts of mutants and confirmed the association of these genes