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Molecular Analysis of Microcin 24: Genetics, Secretion and Mode of Action of a Novel Microcin

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Molecular Analysis of Microcin 24: Genetics, Secretion and Mode of Action of a Novel Microcin MOLECULAR ANALYSIS OF MICROCIN 24: GENETICS, SECRETION AND MODE OF ACTION OF A NOVEL MICROCIN. A thesis submitted in fhlfilment of the requirements for the degree of Doctor of Philosophy in Cellular and Molecular Biology in the University of Canterbury. GRAHAM JAMES O'BRIEN '/ 1996 DEDICATION. To Lee-ann. Romans16: 27. "to the only wise God be glo;y for ever through Jesus Christ! Amen. 11 iii ACKNOWLEDGMENTS. I would like to thank Assoc. Prof. H.Khris Mahanty for his supervision of this project and his many ideas and enthusiasm which has guided this work. To both Khris and Abanti, many thanks for their hospitality and friendship. Many thanks to Beth Robson and Sam Court for their technical assistance during this research and to my associate supervisor Dr. lA Heinemann for his comments on this work. Thanks also to Dr. F.Y.T Sin, Dr. M.H. Turnbull and Dr. lD. Klena for reading the draft. A note of special appreciation goes to all the students in the Molecular Genetics Laboratory both past and present who have made my time there a great experience. My thanks to the staff of the Plant and Microbial Sciences Department who have been willing to help with equipment and other cries for help and especially Dougal Holmes for his photographic expertise, I would like to acknowledge the assistance of Dr. M. Kennedy with the DNA sequencing, Dr. A.P. Pugsley for supplying the colicin reference, Dr. LL. Lamont for supplying the Fur strains and Prof. R.E. Wooley for performing the embryo lethality assay. Many thanks to Dr. S.T. Chambers and Dr. B. Peddie from Christchurch Hospital for their initial and continued interest in this project. The [mandaI assistance provided by the University of Canterbury through the Doctoral Scholarship scheme and the Health Research Council of New Zealand who awarded me a Postgraduate Scholarship (94/142) for the duration of this work is gratefully acknowledged. Thanks to my parents Dennis and Margaret, for their support and encouragement over the years and especially for the use of their computer. Thanks also to Mr D.M. Petrie for proof reading parts of this work. Lastly, I would like to thank my wife Lee-arm who has supported me through the good and the bad. I could not have done this without her love and understanding. We can now look forward to the new life ahead of us. iv Colic ins and microcins are proteinaceous antimicrobial agents produced by members of the Enterobacteriaceae which are active against other members of this family. Colicin 24 is a novel bacteriocin produced by a uropathogenic strain of Escherichia coli isolated at Christchurch Hospital. Through detailed genetic analysis of the DNA encoding this toxin and assaying the toxic activity, colicin 24 was re-classified as microcin 24 and has been shown to have a similar genetic organisation to that of colicin V and a novel mode of activity. The region of DNA encoding microcin 24 was subcloned from pGOB34 into pBR322 generating pGOB18 (5.44kb). Mutagenesis, DNA sequencing and trans­ complementation identified two regions with high sequence similarity and functional homology to the ColV transporters CvaA and CvaB. The insert DNA of pGOB 18 was sequenced in both directions and has been found to contain 5267bp encoding five open reading frames, mdbA, mtft, mtjS, mtfA and mtjB, forming three operons mdbA, mtftlmtjS and mtfAlmtjB all of which were transcribed in the same direction. The predicted protein products of all the open reading frames except mtjB were confirmed by expressing the genes in minicells. Further mutagenesis and trans-complementation has identified mdbA as a cis acting positive regulatory gene with sequence similarity to the histone-like proteins. The mtft and mtjS genes were confirmed as the Mcc24 immunity gene and the Mcc24 structural gene respectively. The genes mtfA and mtjB were found to encode the transport proteins homologous to CvaA and CvaB respectively, with mtjB encoding a protein which is a member of the ABC family of bacterial transporters. Transport also requires the TolC outer membrane protein. Analysis of the mtjS DNA sequence has identified a double glycine leader sequence, making MtfS the second microcin after ColV to belong to this class of peptide antibiotics. Experimental evidence suggested that unlike CoIV, Mcc24 is inactive within the producing cell, however both toxins require the ABC transporter for post-translational modification of the pre-peptide. The regulation ofMcc24 synthesis is controlled by the interaction between (i, Fur, and MdbA, encoded by the mdbA gene. Analysis of the promoter sequences has identified putative regions of DNA bending which might facilitate the binding of as and v MdbA. A Fur-box with good sequence similarity to the consensus Fur-box has been identified in the mtjI/mtjS promoter and is the proposed site for Fur binding. The activity spectrum of Mcc24 is restricted to enteric bacteria and SernA, the MccE492 receptor, is also required as the receptor for Mcc24. Extracts of Mcc24 have been found to degrade both linearised and covalently closed circular DNA in vitro. The activity is absent in extracts from mtjS - strains, suggesting that Mcc24 inhibits the growth of sensitive cells by degrading DNA. The effect of Mcc24 expression on the virulence of E. coli was tested using the embryo lethality assay, however unlike ColV which increases the virulence of strains, the expression of Mcc24 did not appear to have a significant effect on E. coli virulence in this system. VI TABLE OF CONTENTS. CHAPTER. PAGE Acknowledgments. ............................................................................................... 111 Abstract. ................................................................................................................. iv List of Abbreviations. ......................................................................................... viii List of Figures ......................................................................................................... x List of Tables ....................................................................................................... xiii CHAPTER 1. INTRODUCTION ...................................................................... 1 1.1 Bacteriocins Produced by Escherichia coli.. ............................... 2 1.2 ABC Transporters: Structure and Role in Bacteriocin Transport .................................................................................... 28 1.3 Regulation of Bacteriocin Production ....................................... 33 1.4 Microcin 24 ............................................................................... 40 1.5 Aims of this Study ..................................................................... 42 CHAPTER 2. MATERIALS AND METHODS ............................................. 43 2.1 Bacterial strains, Bacteriophages and Plasmids. .. .................... 43 2.2 Buffers and Media .................................................................... 43 2.3 Bacteriological Methods ........................................................... 48 2.4 DNA Manipulation and Cloning Techniques ........................... 51 2.5 Mutagenesis .............................................................................. 58 2.6 DNA Sequencing of Double Stranded Templates .................... 60 2.7 Protein Analysis Using SDS-PAGE ......................................... 62 2.8 Expression of Plasmid Encoded Proteins ................................. 64 2.9 Microcin Extraction Techniques. .. ........................................... 67 2.1 0 Microcin 24 Activity Techniques ............................................. 70 CHAPTER 3. RESUL TS. .. .............................................................................. 74 3.1 Analysis of the Microcin 24 Encoding Genetic Region ............ 74 3.2 Dideoxynucleotide DNA Sequencing ...................................... 87 3.3 Functional Analysis ofmtjS, mtjI and mdbA . ......................... 108 vii CHAPTER (Continued). PAGE CHAPTER 3. RESULTS (Continued). 3.4 Analysis of Microcin 24 ......................................................... 116 3.5 Proteins Involved in the Expression, Regulation and Activity of Microcin 24 .......................................................... 125 3.6 The Role of Microcin 24 in Determining the Virulence of E. coli . ................................................................ 129 CHAPTER 4. DISCUSSION. ....................................................................... 133 4.1 Establishment of Colicin 24 as a True Microcin, Microcin 24 ............................................................................ 133 4.2 Genetic Organisation of the Microcin 24 Encoding Region .................................................................... 135 4.3 Microcin 24Productlon, Export and Activity ........................ 138 4.4 Significance of Microcin 24 Production ................................. 154 4.5 Conclusion .............................................................................. 156 BIBLIOGRAPHy ................................................................................................ 158 APPEl\TDICES. .. ............................................................................................... 179 APPENDIX 1. BUFFERS AND SOLUTIONS .............................................. 179 APPENDIX 2. MEDIA ................................................................................... 192 APPENDIX 3. PRIMERS USED FOR DIDEOXYNUCLEOTIDE SEQUENCING ......................................................................
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