MASARYK UNIVERSITY faculty of medicine department of biology BACTERIOCINOGENY OF YERSINIAE: MOLECULAR INTERACTIONS OF COLICIN FY WITH A SUSCEPTIBLE BACTERIAL CELL Brno, 2013 Juraj Bosák BIBLIOGRAPHIC IDENTIFICATION Name and Surname: Juraj Bosák Title 0f Thesis: Bacteriocinogeny of yersiniae: Molecular interactions of colicin FY with a susceptible bacterial cell Study Programme: Medical Biology 5103V022 Supervisor: doc. MUDr. David Šmajs, Ph.D. Defended in: 2013 Keywords: Antibacterial toxin, Bacteriocin, Colicin, Yersinia, Y. enterocolitica, Escherichia, Yersiniosis, Probiotics 6 ABSTRACT There are numerous antimicrobial agents that are produced by bacteria. The role of these substances is to improve fitness of the producer in a daily fight for survival. Bacteriocins are substances that inhibit growth of other bacteria. One important subgroup of bacteriocins is represented by colicins, antimicrobial agents produced by colicinogenic strains of Escherichia coli and other related species of the family Enterobacteriaceae. These toxins specifically inhibit closely related bacteria based on the presence of a specific receptor on the cell surface. Genus Yersinia comprises important human pathogens such as Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. Growth inhibition between various strains of yersiniae is quite common. Out of numerous identified bacteriocin-like substances produced by yersiniae, only two, pesticin I and enterocoliticin, were characterized in detail. In fact, these are the only two characterized bacteriocins known to attack yersiniae. Therefore, a search for fully characterized bacteriocins that act specifically against yersiniae has been demanded. In this study, we describe a novel colicin - colicin FY - isolated from a colicinogenic strain of Ye- rsinia frederiksenii; we show its complete plasmid sequence (pYF27601), mechanism of its toxicity, corresponding receptor (YiuR), and translocation routes into a susceptible bacterium. This is the first well characterized colicin, which is active mainly against strains of Y. enterocolitica. The universal susceptibility of Y. enterocolitica to colicin FY together with absence of activity on strains outside the genus Yersinia suggests potential therapeutic applications of colicin FY. 7 ABSTRAKT Bakterie produkují různé antimikrobiální látky, které je zvýhodňují vůči jiným bakteriím v každo- denním boji o přežití. Koliciny jsou významnou skupinou bakteriocinů; jsou produkovány některý- mi kmeny druhu Escherichia coli a příbuznými druhy čeledě Enterobacteriaceae. Koliciny se vyzna- čují inhibičním účinkem vůči příbuzným bakteriím, který je dán přítomností specifického receptoru na povrchu citlivé bakterie. U rodu Yersinia, zahrnující patogenní druhy Y. pestis, Y. pseudotuberculosis a Y. enterocolitica, byla popsána produkce různých antibakteriálních substancí, avšak pouze dvě – pesticin I a enterokoliticin - byly detailně charakterizovány. V současnosti jsou to jediné dva detailně charakterizované bakteri- ociny, které účinkují vůči yersiniím. Tato práce se zaměřila na charakterizaci nově objeveného antimikrobiálního agens produko- vaného nepatogenním kmenem Yersinia frederiksenii. Jedná se o nový typ kolicinu, který byl poj- menován kolicin FY. Byla získána kompletní sekvence plazmidu produkujícího kolicin FY a současně byl popsán mechanizmus účinku kolicinu FY. Protein YiuR byl identifikován jako receptor kolicinu FY. Kolicin FY se vyznačuje univerzálním účinkem vůči kmenům Y. enterocolitica. Současně neby- la pozorována inhibice jiných bakterií mimo rod Yersinia, což naznačuje potenciální terapeutické využití kolicinu FY. DECLARATION I hereby declare that I have worked independently, using only the primary and secondary sources listed in the bibliography, under the supervision of doc. MUDr. David Šmajs, Ph.D. .......................................................................... In Brno Mgr. Juraj Bosák ACKNOWLEDGEMENTS I would like to thank my supervisor doc. MUDr. David Šmajs, Ph.D. for his ideas, support, and pa- tience. Special thank belongs to Petra Laiblová (Kotrsalová). She started this project and in fact discovered the new bacteriocin. My objective has been characterization of her discovery. Many thanks also belong to my colleagues from the Department of Biology, namely Lenka Micen- ková, Lenka Mikalová, Michal Strouhal, and Marie Zobaniková for always being here for me. Míša, thank you for your love and understanding during the years spent at The University. Your sup- port allowed finishing my studies. This study was supported by a grants from the Ministry of Health of the Czech Republic (NS9665-4/2008, NT13413-4/2012) and by institutional support from the Czech Republic (MSM0021622415) CONTENTS I. THEORETICAL PART .........................................................17 1. Theoretical background ........................................................19 1.1. Bacteriocins ................................................................19 1.2. Colicins ...................................................................20 1.2.1. Classification .................................................................20 1.2.2. Genetic organization ..........................................................22 1.2.3. Release from the producing cell .................................................24 1.2.4. Structure .....................................................................24 1.2.5. Mechanism of action ..........................................................27 1.2.5.1. Colicin interactions with the outer membrane ...................................27 1.2.5.1.1. Interaction partners in the outer membrane ...................................27 TonB-dependent transporters (TBDTs) ......................................27 Outer membrane porins ...................................................28 1.2.5.1.2. Interaction of colicins with primary receptors ..................................29 1.2.5.1.3. Interaction of colicins with secondary receptors (translocators) ...................31 Translocon of Tol-dependent colicins ........................................31 Translocon of TonB-dependent colicins ......................................32 1.2.5.2. Colicin interactions in the periplasmic space ....................................33 1.2.5.2.1. Interaction partners in the periplasmic space ..................................33 1.2.5.2.2. Interaction of colicins with the translocation system ............................34 1.2.5.3. Colicin interactions with the inner membrane ...................................35 1.2.5.3.1. Interaction of pore-forming colicins with the inner membrane ...................35 1.2.5.3.2. Interaction of enzymatic colicins with the inne membrane .......................36 1.2.5.4. Colicin interactions in the cytoplasm ...........................................37 II. EXPERIMENTAL PART ......................................................39 2. Specific aims .................................................................41 3. Materials and methods .........................................................43 3.1. Bacterial strains ................................................................43 3.2. Culture media .................................................................43 3.3. Detection of the colicin production ...............................................43 3.4. Preparation of crude colicin extracts ..............................................45 3.5. Colicin activity assay ............................................................45 3.6. Isolation of plasmids ............................................................45 3.7. PCR product purification ........................................................45 3.8. Preparation of chemocompetent cells and transformation ............................45 3.9. Preparation of electrocompetent cells and transformation ............................45 3.10. Plasmid in vitro mutagenesis and construction of plasmid library ....................46 3.11. DNA sequencing and sequence analysis. .46 3.12. Construction of the genome library ..............................................47 3.13. Chromosomal mutagenesis in vivo ...............................................47 3.14. Identification of receptor for colicin FY ...........................................47 3.15. DNA cloning. .47 3.16. Colicin purification and immunoblot analysis .....................................48 3.17. Channel-forming assay .........................................................48 3.18. 16S rRNA analysis .............................................................48 3.19. Bioserotype classification .......................................................49 3.20. Pulsed field gel electrophoresis ..................................................49 3.21. Detection of virulence factors ...................................................49 3.22. Antibiotic susceptibility assay ...................................................50 4. Results and discussion .........................................................51 4.1. Bacteriocin production in the genus Yersinia .......................................51 4.2. Activity spectrum of the bacteriocin of Y. frederiksenii 27601 .........................53 4.3. Isolation and sequencing of the colicinogenic plasmid YF27601 .......................54 4.4. Analysis of the colicinogenic region on pYF27601 ...................................56 4.5. Sequence analysis of colicin FY on the protein level ..................................57 4.6. Cloning, purification,