Leuconostoc bacteriocins and their application in genome editing

Research output: ThesisDoctoral ThesisCollection of Articles

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 to the leucocin C, showing the immunity function of LecI. By cloning the leucocin genes in plasmid vectors, active bacteriocins were produced in heterologous hosts Escherichia coli and Lactococcus lactis. Finally, leucocins A and C were used in a Lc. lactis genome editing method developed in this study. It has been shown that expression of Listerial mptC gene in Lc. lactis renders the host sensitive to class IIa bacteriocins. In this doctoral study, a counterselection method based on bacteriocin sensitivity was developed to select the loss of a plasmid. Listerial mptC gene was cloned in an integration vector aiming at a chromosomal deletion, and the plasmid was transferred into Lc. lactis. When the bacteriocin sensitive integrants were cultured with leucocin A or C, only the cells which had lost the mptC-plasmid through a second homologous recombination could survive. The second recombination may also cause the desired deletion. With this bacteriocin counterselection method, fragments up to 35 kb were deleted from Lc. lactis chromosome.

The results obtained from this doctoral study provide further knowledge of Leuconostoc bacteriocins and their scientific use.
Original languageEnglish
Place of PublicationHelsinki
Publisher
Print ISBNs978-951-51-3203-1
Electronic ISBNs978-951-51-3204-8
Publication statusPublished - 2017
MoE publication typeG5 Doctoral dissertation (article)

Fields of Science

  • 1183 Plant biology, microbiology, virology
  • mikrobiologia
  • elintarvikemikrobiologia
  • maitohappobakteerit
  • Leuconostoc carnosum
  • antimikrobiset yhdisteet
  • geenitekniikka
  • geenit

Cite this

Wan, Xing. / Leuconostoc bacteriocins and their application in genome editing. Helsinki : University of Helsinki, 2017. 53 p.
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title = "Leuconostoc bacteriocins and their application in genome editing",
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 to the leucocin C, showing the immunity function of LecI. By cloning the leucocin genes in plasmid vectors, active bacteriocins were produced in heterologous hosts Escherichia coli and Lactococcus lactis. Finally, leucocins A and C were used in a Lc. lactis genome editing method developed in this study. It has been shown that expression of Listerial mptC gene in Lc. lactis renders the host sensitive to class IIa bacteriocins. In this doctoral study, a counterselection method based on bacteriocin sensitivity was developed to select the loss of a plasmid. Listerial mptC gene was cloned in an integration vector aiming at a chromosomal deletion, and the plasmid was transferred into Lc. lactis. When the bacteriocin sensitive integrants were cultured with leucocin A or C, only the cells which had lost the mptC-plasmid through a second homologous recombination could survive. The second recombination may also cause the desired deletion. With this bacteriocin counterselection method, fragments up to 35 kb were deleted from Lc. lactis chromosome. The results obtained from this doctoral study provide further knowledge of Leuconostoc bacteriocins and their scientific use.",
keywords = "1183 Plant biology, microbiology, virology, mikrobiologia , elintarvikemikrobiologia , maitohappobakteerit , Leuconostoc carnosum , antimikrobiset yhdisteet , geenitekniikka , geenit",
author = "Xing Wan",
year = "2017",
language = "English",
isbn = "978-951-51-3203-1",
series = "Dissertationes Schola Doctoralis Scientiae Circumiectalis, Alimentariae, Biologicae. Universitatis Helsinkiensis",
publisher = "University of Helsinki",
number = "10/2017",
address = "Finland",

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Leuconostoc bacteriocins and their application in genome editing. / Wan, Xing.

Helsinki : University of Helsinki, 2017. 53 p.

Research output: ThesisDoctoral ThesisCollection of Articles

TY - THES

T1 - Leuconostoc bacteriocins and their application in genome editing

AU - Wan, Xing

PY - 2017

Y1 - 2017

N2 - 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 to the leucocin C, showing the immunity function of LecI. By cloning the leucocin genes in plasmid vectors, active bacteriocins were produced in heterologous hosts Escherichia coli and Lactococcus lactis. Finally, leucocins A and C were used in a Lc. lactis genome editing method developed in this study. It has been shown that expression of Listerial mptC gene in Lc. lactis renders the host sensitive to class IIa bacteriocins. In this doctoral study, a counterselection method based on bacteriocin sensitivity was developed to select the loss of a plasmid. Listerial mptC gene was cloned in an integration vector aiming at a chromosomal deletion, and the plasmid was transferred into Lc. lactis. When the bacteriocin sensitive integrants were cultured with leucocin A or C, only the cells which had lost the mptC-plasmid through a second homologous recombination could survive. The second recombination may also cause the desired deletion. With this bacteriocin counterselection method, fragments up to 35 kb were deleted from Lc. lactis chromosome. The results obtained from this doctoral study provide further knowledge of Leuconostoc bacteriocins and their scientific use.

AB - 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 to the leucocin C, showing the immunity function of LecI. By cloning the leucocin genes in plasmid vectors, active bacteriocins were produced in heterologous hosts Escherichia coli and Lactococcus lactis. Finally, leucocins A and C were used in a Lc. lactis genome editing method developed in this study. It has been shown that expression of Listerial mptC gene in Lc. lactis renders the host sensitive to class IIa bacteriocins. In this doctoral study, a counterselection method based on bacteriocin sensitivity was developed to select the loss of a plasmid. Listerial mptC gene was cloned in an integration vector aiming at a chromosomal deletion, and the plasmid was transferred into Lc. lactis. When the bacteriocin sensitive integrants were cultured with leucocin A or C, only the cells which had lost the mptC-plasmid through a second homologous recombination could survive. The second recombination may also cause the desired deletion. With this bacteriocin counterselection method, fragments up to 35 kb were deleted from Lc. lactis chromosome. The results obtained from this doctoral study provide further knowledge of Leuconostoc bacteriocins and their scientific use.

KW - 1183 Plant biology, microbiology, virology

KW - mikrobiologia

KW - elintarvikemikrobiologia

KW - maitohappobakteerit

KW - Leuconostoc carnosum

KW - antimikrobiset yhdisteet

KW - geenitekniikka

KW - geenit

M3 - Doctoral Thesis

SN - 978-951-51-3203-1

T3 - Dissertationes Schola Doctoralis Scientiae Circumiectalis, Alimentariae, Biologicae. Universitatis Helsinkiensis

PB - University of Helsinki

CY - Helsinki

ER -

Wan X. Leuconostoc bacteriocins and their application in genome editing. Helsinki: University of Helsinki, 2017. 53 p. (Dissertationes Schola Doctoralis Scientiae Circumiectalis, Alimentariae, Biologicae. Universitatis Helsinkiensis; 10/2017).