Screening of chitinolytic bacteria from freshwater lake and analysis of chitinase system of the isolated bacteria

SCREENING OF CHITINOLYTIC BACTERIA FROM FRESHWATER LAKE AND ANALYSIS OF CHITINASE SYSTEM OF THE ISOLATED BACTERIA DINH MINH TRAN SCREENING OF CHITINOLYTIC BACTERIA FROM FRESHWATER LAKE AND ANALYSIS OF CHITINASE SYSTEM OF THE ISOLATED BACTERIA DINH MINH TRAN Doctoral Program in Life and Food Sciences, Graduate School of Science and Technology, NIIGATA UNIVERSITY, 2018 CONTENTS Abbreviations Summary Chapter General introduction 1 Background of research 1.2 Occurrence, form, and application of chitin 10 1.3 Chitinases, classification, and application of bacterial chitinases 13 1.4 Bacterial AA10 proteins and potential application 15 1.5 Chitinolytic bacteria and application in biocontrol 16 1.6 Biofilm-forming bacteria and potential application in agriculture 17 1.7 Aeromonas species and their chitinases 18 1.8 Aeromonas salmonicida subsp salmonicida 20 1.9 Sakata, a sand dune lake 21 1.10 The aim of this study 21 Chapter Screening and characterization of chitinolytic bacteria from a freshwater lake 22 2.1 Abstract 22 2.2 Introduction 23 2.3 Materials and Methods 26 2.4 Results 36 2.5 Discussion 60 2.6 Conclusion 66 Chapter Identification and sequence analysis of chitinase genes from Aeromonas salmonicida subsp salmonicida SWSY-1.411 67 3.1 Abstract 67 3.2 Introduction 68 3.3 Materials and Methods 71 3.4 Results 81 3.5 Discussion 121 3.6 Conclusion 134 References 135 List of tables 159 List of figures 160 Acknowledgments 163 ABBREVIATIONS GlcNAc: N-acetylglucosamine GH: Glycoside hydrolase YEM medium: Yeast extract-supplemented minimal medium LB medium: Luria-Bertani medium PCR: Polymerase chain reaction SDS–PAGE: Sodium dodecyl sulfate–polyacrylamide gel electrophoresis PDA: Potato dextrose agar DDBJ: The DNA Data Bank of Japan AA10: Auxiliary activities family 10 CBM: Carbohydrate-binding module OD: Optical density ORF: Open reading frame IPTG: Isopropyl β-D-thiogalactopyranoside aa: Amino acid bp: Base pair kDa: Kilodalton CBP: Chitin-binding protein ChBD: Chitin-binding domain kb: Kilobase CAZy: Carbohydrate-active enzymes database TIM: Triosephosphate isomerase SUMMARY To develop a novel type of biocontrol agent, this study was focused on bacteria that are characterized by both high chitinase activity and high biofilm development because such bacteria are thought to be secreted and concentrated chitinases in biofilms when adhered to chitin in fungal cell walls Samples, sediments and chitin flakes immersed in the water were collected from a sand dune lake, Sakata, in Niigata, Japan Chitin flakes are thought to be useful for collecting bacteria that have high chitinase activity and also form biofilms from freshwater environments Chitinolytic bacteria were isolated from the sediments and immersed chitin flakes using solid media containing colloidal chitin with or without subsequent subculturing in fresh liquid medium containing chitin flakes Thirty-one isolates from more than 5,100 isolated strains were selected to examine chitinase activity and biofilm formation Phylogenetic analysis of these isolates based on the 16S rRNA gene sequences revealed that most isolates belonged to the family Aeromonadaceae, followed by the families Paenibacillaceae, Enterobacteriaceae, and Neisseriaceae Based on the chitinase activity, biofilm formation, and phylogenetic analysis, four strains, one each of Serratia and Andreprevotia and two strains of Aeromonas, were selected for further investigation Total chitinase activity of each strain in a medium containing chitin powder was lower than that of a reference, Serratia marcescens 2170 However, the specific activity of chitinases of each strain was higher than that of the reference The molecular size of one chitinase produced by Andreprevotia (~121 kDa) was greater than that of typical bacterial chitinases implying a possibly new type of bacterial chitinases In addition, the dialyzed crude proteins containing chitinases of each isolate suppressed the hyphal growth of Trichoderma reesei These results indicate that these four strains are good candidates for biocontrol agents A nearly full-length segment of 16S rRNA gene nucleotides of isolate, A salmonicida SWSY 1.411 shows 100% identity to that of A salmonicida A449 (CP000644) available in the CAZy database In the database, A salmonicida A449 (CP000644) is shown to be possessed one GH18 chitinase, two GH19 chitinases, and one AA10 protein Based on the nucleotide sequence of each gene and the surrounding regions of that gene in A salmonicida A449 genome, primers were designed for the identifying and cloning of chitinase genes in our isolate, A salmonicida SWSY 1.411 Three genes involved in chitin-degradation were identified in the genomic DNA of A salmonicida SWSY 1.411 by the polymerase chain reaction Among them, one gene encodes a GH18 chitinase, one gene encodes a GH19 chitinase, and one gene encodes an AA10 protein These genes were then cloned, sequenced, and analyzed deduced amino acid sequences Primary structures of all deduced enzymes contain a number of functional domains; among them, one chitin-binding domain belongs to a recently classified family of carbohydrate-binding modules, CBM73 ChiA contains a CBM5, a CBM73, and belongs to subfamily A of GH18 chitinases The catalytic domain of ChiB belongs to GH19 chitinases; ChiB contains a CBM5, a CBM73, and a PKD domain In addition, various works have been reported that bacterial GH18 chitinases commonly play high chitinase activity toward insoluble chitins, bacterial GH19 chitinases are primary enzymes involved in the antifungal activity, and bacterial AA10 proteins in combination with chitinases play an important role on hydrolysis of chitin These analyses indicate that the chitinase system of A salmonicida SWSY 1.411 possibly plays an important role on chitin-degradation and inhibition of hyphal growth of fungi CHAPTER GENERAL INTRODUCTION 1 Background of research Chitin is an insoluble linear β-1,4-linked homopolymer of N-acetylglucosamine (GlcNAc) and is widely distributed in nature such as a constituent of insect exoskeletons, shells of crustaceans, cell walls of fungi Chitinases (EC 3.2.1.14) are enzymes that hydrolyze chitin by hydrolyzing β-1,4-glycosidic linkages These enzymes are found in a variety of organisms, for instance, bacteria, fungi, insects, plants, and animals Chitinase genes from a large number of chitinolytic bacteria have been cloned, analyzed, and their biochemical properties have been examined (Watanabe et al 1997, 1997; Tanaka et al 1999; Hashimoto et al 2000) So far, it is clear that various bacterial chitinases play a critical role in the digestion of chitin in fungal cell walls Among the chitinases, the enzymes belonging to family 19 of glycoside hydrolases (GH) have been shown to be primary enzymes involved in the antifungal activity (Ohno et al 1996; Watanabe et al 1999; Tsujibo et al 2000; Kawase et al 2006) These chitinases are mainly distributed in plant and a number of 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rRNA genes from the isolated strains 49 Table 2.4 The growth inhibition of T reesei by using the crude proteins 59 Table 3.1 Primers used in this study 72 Table 3.2 Abbreviation of enzyme sources used for phylogenetic analysis in Figure 3.5 94 Table 3.3 Abbreviation of enzyme sources used for phylogenetic analysis shown in Figure 3.8 104 159 LIST OF FIGURES Figure 1.1 Structure of chitin 10 Figure 1.2 Forms of chitin 11 Figure 2.1 Procedure for sampling and isolation of microorganisms that produce chitinases and form biofilms 27 Figure 2.2 The clearing zone formed by the isolates and references on the YEM agar plates containing colloidal chitin 37 Figure 2.3 Chitinase activity and biofilm formation of the isolates 45 Figure 2.4 Phylogenetic analysis of the isolates (filled rectangle) based on the 16S rRNA gene sequences 47 Figure 2.5 Time course of chitinase production in the culture supernatant 53 Figure 2.6 SDS–PAGE and zymography analyses of the chitinase production in the culture supernatant 54 Figure 2.7 The antagonistic ability of the isolates against the hyphal growth of T reesei using the dual-culture assay 56 Figure 2.8 The inhibition of hyphal growth of T reesei treated by the crude proteins 58 Figure 3.1 Effects of temperatures and pHs on the growth and carbon sources on the chitinase production of A salmonicida SWSY-1.411 83 Figure 3.2 Schematic presentation of gene identification and result of gene identification 85 160 Figure 3.3 Nucleotide sequence of the chiA gene, deduced amino acid sequence of ChiA, and domain structure of ChiA 87 Figure 3.4 Alignment of amino acids in the catalytic domains of ChiA and those of other bacterial GH18 chitinases 91 Figure 3.5 Phylogenetic relationships among family 18 chitinases 93 Figure 3.6 Nucleotide sequence of the chiB gene, deduced amino acid sequence of ChiB, and domain structure of ChiB 98 Figure 3.7 Alignment of amino acids in the catalytic domain of ChiB and those of other GH19 chitinases 100 Figure 3.8 Phylogenetic relationships among family 19 chitinases 103 Figure 3.9 Analysis of the loop structure of ChiB 110 Figure 3.10 Schematic representation of the loop structure of the GH19 chitinases shown in Figure 3.9 111 Figure 3.11 Nucleotide sequence of the aslpmo10A gene, deduced amino acid sequence of AsLPMO10A, and domain structure of AsLPMO10A 113 Figure 3.12 Alignment of amino acids in the catalytic domain of AsLPMO10A and those of other bacterial AA10 proteins 116 Figure 3.13 SDS–PAGE and zymography analyses of proteins 118 Figure 3.14 Antifungal activity of purified rChiB against the hyphal growth of T reerei 120 161 Figure 3.15 Alignment of amino acid sequences of chitin-binding domains belonging to CBM5 and CBM12 123 Figure 3.16 Alignment of amino acid sequences of chitin-binding domains belonging to CBM5 and CBM73 125 Figure 3.17 Schematic representation of domains structure of characterized AA10 proteins 130 162 ACKNOWLEDGMENTS My heartfelt thanks go first to my academic advisor, Professor Kazushi Suzuki, who has supported me both in the field of science and living throughout the duration of my study in Japan, for his encouragement, guidance, patience, and knowledge I am heartily thankful for my co-advisors, Professor Naoki Harada and Associate Professor Hayuki Sugimoto for their strong support and scientific guidance I would like to express my special and heartfelt thanks to Professor Takeshi Watanabe, who gave me an opportunity to study in Japan and was the former advisor of my study, for his encouragement, guidance, patience, and knowledge I would also like to express my special thanks to Professor Dzung Anh Nguyen, my boss, my former supervisor in Institute of Biotechnology and Environment, Tay Nguyen University, Vietnam for his supporting and giving me an opportunity to study in Japan I would like to thank Tay Nguyen University, Vietnam for giving me an opportunity and supporting me during the study in Japan I would also like to thank Associate Professor Tsutomu Sato for valuable suggestions in the final defense and in the revision of my thesis I would like to thank the Japanese Government scholarship (Monbukagakusho: MEXT) for the financial support during my study in Japan I am also thankful to Lab of Applied Microbiology, Faculty of Agriculture and Niigata University for supporting chemical fees and encouraging me during the study I would like to express my warm thanks to Graduate School of Science and Technology, Niigata University for their 163 supporting and encouraging me during the study I wish to thank Sakata Waterfowl and Wetland Center, Niigata, Japan for generous assistance in the sample collection I would like to express my warm thanks to students who have studied and are studying in the Lab of Applied Microbiology, Faculty of Agriculture, Niigata University for helping me so much in my research and in my living in Japan as well I would also like to thank Dr Md Muzahid E Rahman (Bangladesh Agriculture Research Institute, Bangladesh) for his guidance on the use of the MEGA 6.0 software This achievement would not have been possible without the support of my lovely family I cannot find any suitable words to express my gratitude but they are very important in my study, as the motivation for this achievement Finally, I would like to express my gratitude regards and blessings to people who have supported me in any respect to finish this thesis Niigata City, August 24, 2018 Dinh Minh TRAN 164 .. .SCREENING OF CHITINOLYTIC BACTERIA FROM FRESHWATER LAKE AND ANALYSIS OF CHITINASE SYSTEM OF THE ISOLATED BACTERIA DINH MINH TRAN Doctoral Program in Life and Food Sciences, Graduate School of. .. describe the screening of freshwater bacteria for those that possess high chitinase activity and generate biofilms from Sakata, a sand dune lake, in Niigata, Japan, and we analyzed the chitinase and. .. generate biofilms for biocontrol agent 2) Analyze chitinase and antifungal activities of the selected bacteria 3) Identify and analyze the chitinase system from candidates 21 CHAPTER SCREENING AND CHARACTERIZATION