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Leuconostoc Bacteriocins and Their Application in Genome Editing Leuconostoc bacteriocins and their application in genome editing Xing Wan Division of Microbiology and Biotechnology Department of Food and Environmental Sciences Faculty of Agriculture and Forestry University of Helsinki, Finland Academic Dissertation in Microbiology To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki for public examination in Room 2402, Biocentre 3, Viikinkaari 1, University of Helsinki, on 9th June 2017, at 12 o’clock noon. Helsinki 2017 Supervisor: Dr. Timo Takala Department of Food and Environmental Sciences Faculty of Agriculture and Forestry University of Helsinki, Finland Co-supervisor: Professor Per Saris Department of Food and Environmental Sciences Faculty of Agriculture and Forestry University of Helsinki, Finland Reviewers: Dr. Morten Kjos Faculty of Chemistry, Biotechnology and Food Science Norwegian University of Life Sciences, Norway Dr. Elina Säde Department of Food Hygiene and Environmental Health Faculty of Veterinary Medicine University of Helsinki, Finland Opponent: Professor Mikael Skurnik Department of Bacteriology and Immunology The Haartman Institute University of Helsinki Custos: Professor Per Saris Department of Food and Environmental Sciences Faculty of Agriculture and Forestry University of Helsinki, Finland ISBN 978-951-51-3203-1 (paperback) ISBN 978-951-51-3204-8 (PDF) ISSN 2342-5423 (Print) ISSN 2342-5431 (Online) Cover page photo: Leucocins A, B and C on Leuconostoc pseudomesenteroides CIP103316 indicator lawn (photo by Xing Wan) Hansaprint Oy Turenki 2017 2 TABLE OF CONTENTS List of original publications...........................................................................................4 Abbreviations ...............................................................................................................5 ABSTRACT .................................................................................................................6 TIIVISTELMÄ (abstract in Finnish) .............................................................................7 INTRODUCTION ........................................................................................................8 1 Lactic acid bacteria...........................................................................................8 1.1 The genus of Leuconostoc ...............................................................................8 1.2 Leuconostocs in food production .....................................................................9 2 LAB bacteriocins and how to find them .......................................................... 10 2.1 Classification of bacteriocins......................................................................... 11 2.2 Class II: unmodified bacteriocins .................................................................. 13 2.2.1 Class IIa pediocin-like ........................................................................... 13 2.2.2 Class IIb two-peptide ............................................................................. 16 2.2.3 Class IIc circular .................................................................................... 17 2.2.4 Class IId single, linear, non pediocin-like ............................................... 18 2.2.5 Regulation mechanism ........................................................................... 19 2.2.6 Bacteriocin spontaneous resistance......................................................... 21 2.3 Leuconostoc bacteriocins .............................................................................. 22 3 Heterologous expression of class II bacteriocins from LAB ............................. 25 3.1 Expression of native bacteriocin genes .......................................................... 25 3.2 Optimisation of bacteriocin production .......................................................... 25 4 LAB bacteriocin applications.......................................................................... 26 5 LAB genome editing ...................................................................................... 27 AIMS OF THE STUDY.............................................................................................. 29 MATERIALS AND METHODS ................................................................................. 30 RESULTS AND DISCUSSION .................................................................................. 33 1 Genetic characterisation of leucocins (I, II, unpublished)................................. 33 2 Heterologous expression of functional proteins (I, II, III, unpublished) ............ 35 2.1 Overexpression of leucocins (I, II, unpublished) ............................................ 35 2.2 Heterologous expression of other functional proteins (I, III, unpublished) ...... 37 3 Using bacteriocin targeting mechanism in genome editing (III, unpublished) ... 38 CONCLUSIONS AND FUTURE PROSPECTS.......................................................... 41 Acknowledgements ..................................................................................................... 42 References .................................................................................................................. 43 3 List of original publications I Wan, X., Li, R., Saris, P.E.J. and Takala, T.M., 2013. Genetic characterisation and heterologous expression of leucocin C, a class IIa bacteriocin from Leuconostoc carnosum 4010. Applied Microbiology and Biotechnology, 97:3509-3518. II Wan, X., Saris, P.E.J. and Takala, T.M., 2015. Genetic characterization and expression of leucocin B, a class IId bacteriocin from Leuconostoc carnosum 4010. Research in Microbiology, 166:494-503. III Wan, X., Usvalampi, A.M., Saris, P.E.J. and Takala, T.M., 2016. A counterselection method for Lactococcus lactis genome editing based on class IIa bacteriocin sensitivity. Applied Microbiology and Biotechnology, 100:9661- 9669. The author’s contribution in articles: I Wan, X participated in the design of the study, performed all the experiments together with the co-authors, participated in data analysis and result interpretation, and was responsible for writing the manuscript together with the co-authors. II Wan, X participated in the design of the study, performed all the experiments, participated in data analysis and result interpretation, and was responsible for writing the manuscript together with the co-authors. III Wan, X participated in the design of the study, performed majority of the experiments (except constructing H1-H2-pUCori fragments for integration plasmids), analysed the data, interpreted the results, and wrote the manuscript together with the co-authors. The publications are referred to in the following text by their roman numerals. 4 Abbreviations 3CS three-component regulatory system aa amino acid ABC ATP-binding cassette AMP antimicrobial peptide bp base pair(s) C-terminal carboxyterminal Da Dalton, atomic mass unit DCO double crossover DNA deoxyribonucleic acid e.g. exempli gratia, for example Erm erythromycin et al. et alii, and others FOA 5’-fluoroorotic acid Gram- Gram-negative bacteria Gram+ Gram-positive bacteria GG double-glycine i.e. id est, in other words kb kilobase pair(s) kDa kilodalton LAB lactic acid bacteria Man-PTS mannose-phosphotransferase system MRSA methicillin-resistant Staphylococcus aureus N-terminal aminoterminal o/n overnight ORF open reading frame PCR polymerase chain reaction SCO single crossover SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis SS signal sequence ssp. subspecies 5 ABSTRACT Leuconostocs are lactic acid bacteria used as starters in food fermentations. They can also be spoilage bacteria, especially in vacuum packed meat products. Many Leuconostocs produce antimicrobial peptides, also known as bacteriocins. Leuconostoc bacteriocins are usually small, less than 10 kDa non-modified peptides, which are secreted by dedicated transporters. These bacteriocins kill many different bacteria, including strains of Enterococcus, Listeria, and Staphylococcus genera. Sensitivity to bacteriocins is usually dependent on a specific receptor on the target cell membrane. On Listeria cell membrane, mannose-phosphotransferase system Man-PTS has been shown to be the target for bacteriocins leucocins A and C from Leuconostoc. These class IIa bacteriocins bind to the subunit IIC (MptC) of Man-PTS and open the transporter, which leads to cell leakage and eventually cell death. Bacteriocin producers express specific immunity proteins, which protect the producer cells by blocking the transporter pore. In this doctoral dissertation, the bacteriocins produced by Leuconostoc carnosum 4010 were studied. The strain 4010 is a commercial protective culture for vacuum packaged meat products. The strain produces two antilisterial bacteriocins, leucocins A and C. In this study, the strain was found to produce a third bacteriocin, leucocin B, which is active against some Leuconostoc and Weissella strains. Genes required for the production of the three leucocins were characterised. The leucocin genes were arranged in operons on two native plasmids. A single ABC-transporter was found in the strain 4010 and was believed to carry out the bacteriocin secretion for all three leucocins. Immediately downstream of the leucocin genes lcnA, lebB, and lecC, putative immunity genes lcnB, lebI, and lecI were found. The immunity mechanisms of leucocin C was studied by expressing the lecI gene in L. monocytogenes. LecI producing Listeria was less sensitive
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