Insights Into the Streptomycete Conjugation Mechanism and Interstrain Inhibitor Production by the Sweet Potato Pathogen Streptom
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Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2009 Insights into the Streptomycete conjugation mechanism and interstrain inhibitor production by the sweet potato pathogen Streptomyces ipomoeae Jing Wang Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Recommended Citation Wang, Jing, "Insights into the Streptomycete conjugation mechanism and interstrain inhibitor production by the sweet potato pathogen Streptomyces ipomoeae" (2009). LSU Doctoral Dissertations. 2597. https://digitalcommons.lsu.edu/gradschool_dissertations/2597 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. INSIGHTS INTO THE STREPTOMYCETE CONJUGATION MECHANISM AND INTERSTRAIN INHIBITOR PRODUCTION BY THE SWEET POTATO PATHOGEN STREPTOMYCES IPOMOEAE A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Biological Sciences by Jing Wang B.S., Wuhan University, 1999 May 2009 I dedicate my dissertation work to three people who are most important in my life. To my loving parents, Mr. Chidan Xia and Mrs. Yanqin Wang, who have been proud of me, and who have been giving their endless encouragement and patience to me. To my wife Xue Bai, who always loves and supports me unconditionally, who shared with me many challenges and sacrifices. ii ACKNOWLEDGEMENTS This is a great opportunity to express my gratefulness to all those who gave me the possibility to complete this dissertation. I would like to sincerely thank my advisor and mentor, Dr. Gregg Pettis, for guidance with his expertise, precious time and patience throughout the entire process. I would like to thank my advisory committee members, Drs. John Battista, Tin-wein Yu, Christopher Clark, and Ronald Thune, and former committee member Dr. Ding Shih, for agreeing to serve on my committee, for their valuable advice and accessibility, as well as Dr. Eric Achberger for generous assistance. I would also like to thank all of the members of the Pettis lab for their friendship and collaboration throughout my doctorate program, especially Matthew Ducote who constructed several plasmids used in these studies discussed in Charter Two, Kevin Schully who developed the antiserum against ipomicin protein and designed the ipoA primers detailed in Charter Three and Four, and Dongli Guan for helpful discussions. Additionally, I am grateful for the friendship of many peer graduate students in Department of Biological Sciences over the years, especially Binbin Ren, Fang He, Dahui You, and Steve Pollock. They were always by my side and made the completion of the dissertation work an enjoyable process. Finally, I would like to thank my wife Xue Bai, for preparation of most figures in this dissertation. iii TABLE OF CONTENTS DEDICATION………………………………………………………………………….……...ii ACKNOWLEDGEMENTS…………………………………………………………………..iii LIST OF TABLES……………………………………………………………………………..v LIST OF FIGURES…………………………………………………………………………...vi ABSTRACT……………………………………………………………………....................viii CHAPER ONE: INTRODUCTION…………………………………………………………...1 CHAPTER TWO: THE TRANSFER GENE OF A CIRCULAR STREPTOMYCES PLASMID SHOWS SPECIFICITY FOR THE CIRCULAR CONFIGURATION……...…..20 CHAPTER THREE: GROWTH-REGULATED EXPRESSION OF A BACTERIOCIN, PRODUCED BY THE SWEET POTATO PATHOGEN STREPTOMYCES IPOMOEAE, THAT EXHIBITS INTERSTRAIN INHIBITION…………………………………………...59 CHAPTER FOUR: PRELIMINARY ANALYSIS OF PRODUCTION OF IPOMICIN IN STREPTOMYCES IPOMOEAE SUSCEPTIBLE STRAINS AND EXPRESSION CHARACTERISTICS OF THE GROUP II INHIBITOR…………………..81 CHAPTER FIVE: SUMMARY AND FUTURE DIRECTIONS…………………………...101 APPENDIX A: SUPPLEMENTARY DATA………………………………………………..112 APPENDIX B: LETTER OF PERMISSION……………………………………………….119 VITA………………………………………………………………………………………...125 iv LIST OF TABLES 2.1 Bacterial strains and plasmids used in this study……………………………………….24 2.2 The mutagenic primers used in this study………………………………………………33 2.3 Transfer frequencies of 54-bp minimal clt alleles mutated in the IR region…………....37 2.4 Transfer frequencies of linker scanner mutants in the IR region of pIJ101 clt locus…………………………………………………………………………………40 2.5 Transfer frequencies of linear versus circular pQC179 and pSCON223 from S. lividans strains………………………………………………………………………..41 2.6 Transfer Frequencyies of linear pSCON335 and pSCON346 from TtrA defective strains…………………………………………………………………………………...47 2.7 Transfer frequencies of clt mutants in circular versus linear plasmids…………………48 2.8 Transfer Frequencies of linear pSCON223 and pSCON346 from pIJ101 Tra+ versus Tra- strains……………………………………………………………………….49 4.1 Inhibition groups of strains of S. ipomoeae…………………………………………….85 A.1 Transfer of plasmids harboring chromosome telomere from S. lividan TK23.42 to TK23………………………………………………………………………………..113 A.2 Transfer of linear pSCON346 from S. lividans 98-50 to TK23 containing pHYG1 versus pOS948………………………………………………………………..114 v LIST OF FIGURES 1.1 The model for circular conjugative plasmid transfer in Streptomyces…………….……..7 2.1 pIJ101 clt sequence map……………………………………………….……………….22 2.2 Physical maps of pSCON223 in circular and linear forms, and pSCON346 in linear form………………………………………………………….………………...31 2.3 Agarose gel electrophoresis analysis of total DNA in donors and transconjugants…………………………………………………………………….……42 2.4 Configuration (i.e., linear, circular, or recombination with SLP2) of pQC179 versus pSCON223 in JW1 transconjugants………………………………………………...….45 2.5 Evidence of linear pSCON346 transfer from sttrain 98-50…………………………….50 3.1 Growth-regulated appearance of ipomicin during culturing of S. ipomoeae strain 91-03 as determined by plate bioassay and Western blotting……………………68 3.2 Northern blot of the ipoA message in S. ipomoeae 91-03…………………….………...71 3.3 Temporal appearance of ipomicin protein as produced by S. coelicolor strains M600 and M600∆bldA containing the cloned ipoA gene……………….………73 3.4 Western blot of ipomicin protein and concomitant Northern blot of ipoA message for S. coelicolor M600∆bldA containing plasmid pSIP40 versus pSIP42…..………….75 3.5 Ipomicin stability assay…………………………………………………………….…...77 4.1 Representatives of all three inhibition groups of S. ipomoeae express the ipoA gene….83 4.2 Western blots of ipomicin protein………………………………………………………87 4.3 Detection of the ipoA gene in S. ipomoeae strains 91-01, 92-03 and B12321 by PCR amplification………………………………………………………………………90 4.4 Nucleotide sequence alignment of the ipoA gene in S. ipomoeae strains 91-03, 91-01, 92-03, and B12321………………………………………………………………91 4.5 Inhibition phenotypes of 92-03 determined by plate bioassays………………………...94 4.6 Temporal release of active form of S. ipomoeae group II inhibitor determined by plate bioassay…………………………………………………………………………...96 4.7 Growth-regulated appearance of active group II inhibitor during culturing of S. ipomoeae strain 88-35 as determined by plate bioassay……………………..………....98 5.1 Model for pIJ101-mediated conjugation of the linear Streptomyces chromosome……106 A.1 Growth-regulated appearance of ipomicin during culturing of S. ipomoeae strain 88-29 as determined by plate bioassay and Western blotting……………………..….115 vi A.2 Temporal appearance of ipomicin protein as produced by S. coelicolor strains M600 and M600∆bldA containing pSIP40 versus pSIP42………………………...…116 A.3 TCA-precipitated S. ipomoeae group II inhibitor still retains inhibitory activity…….117 vii ABSTRACT Conjugation in Streptomyces invokes a unique mechanism involving few plasmid-encoded loci. Transfer of circular plasmid pIJ101 requires only a tra gene, and a cis-acting locus clt. Here, I investigated whether the transfer genes of pIJ101 could promote transfer of a linear plasmid. While pIJ101 Tra could transfer circularized versions of the linear plasmid containing clt, the linear plasmid itself could not be transferred efficiently by Tra. All plasmids isolated from transconjugants appeared to be circular regardless of the configuration of the plasmid in the initial donor cells. However the linear plasmid could be transferred in linear form from JW1, which contains the conjugative linear plasmid SLP2. Evidence that linear plasmids transferred from strains containing only SLP2 strongly suggest that TraSLP2 mediated the transfer of heterologous linear plasmid and pIJ101, and Tra is not responsible for transfer of the linear DNA molecules from JW1.These results indicate that Tra of pIJ101 shows specificity for the circular configuration and imply that efficient transfer of linear plasmids may require additional functions compared with circular plasmids. Certain strains of the bacterial sweet potato pathogen Streptomyces ipomoeae produce the bacteriocin ipomicin, which inhibits other sensitive strains of the same species. Here, I show that group III inhibitor ipomicin stably accumulates in culture supernatants of S. ipomoeae in a growth-regulated manner that does not coincide with the pattern of expression of the ipomicin structural gene ipoA. Similar growth-regulated production of ipomicin in Streptomyces coelicolor containing the cloned ipoA gene was found to be directly dependent on translation of the TTA codon in