Genetic Dissection of a Prevalent Plasmid-Encoded Conjugation System in Lactococcus lactis
Plasmid pNP40, which was first identified nearly 40 years ago in Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, encodes functions such as heavy metal-, bacteriophage-, and nisin-resistance, as well as plasmid transfer ability by conjugation. Here, we report an optimized conjugation prot...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2021-05-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2021.680920/full |
| _version_ | 1818727399533051904 |
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| author | Guillermo Ortiz Charneco Philip Kelleher Andrius Buivydas Hugo Streekstra Emiel Ver Loren van Themaat Paul P. de Waal Jennifer Mahony Douwe van Sinderen |
| author_facet | Guillermo Ortiz Charneco Philip Kelleher Andrius Buivydas Hugo Streekstra Emiel Ver Loren van Themaat Paul P. de Waal Jennifer Mahony Douwe van Sinderen |
| author_sort | Guillermo Ortiz Charneco |
| collection | DOAJ |
| description | Plasmid pNP40, which was first identified nearly 40 years ago in Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, encodes functions such as heavy metal-, bacteriophage-, and nisin-resistance, as well as plasmid transfer ability by conjugation. Here, we report an optimized conjugation protocol for this plasmid, yielding a transfer frequency that is approximately 4,000-fold higher than those previously reported in literature, while we also observed high-frequency plasmid co-mobilization. Individual mutations in 18 genes that encompass the presumed conjugation cluster of pNP40 were generated using ssDNA recombineering to evaluate the role of each gene in the conjugation process. A possible transcriptional repressor of this conjugation cluster, the product of the traR gene, was identified in this manner. This mutational analysis, paired with bioinformatic predictions as based on sequence and structural similarities, allowed us to generate a preliminary model of the pNP40 conjugation machinery. |
| first_indexed | 2024-12-17T22:13:29Z |
| format | Article |
| id | doaj.art-571e0b72f23e4f6f981129c7eba52931 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-17T22:13:29Z |
| publishDate | 2021-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-571e0b72f23e4f6f981129c7eba529312022-12-21T21:30:41ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2021-05-011210.3389/fmicb.2021.680920680920Genetic Dissection of a Prevalent Plasmid-Encoded Conjugation System in Lactococcus lactisGuillermo Ortiz Charneco0Philip Kelleher1Andrius Buivydas2Hugo Streekstra3Emiel Ver Loren van Themaat4Paul P. de Waal5Jennifer Mahony6Douwe van Sinderen7School of Microbiology and APC Microbiome Ireland, University College Cork, Cork, IrelandSchool of Microbiology and APC Microbiome Ireland, University College Cork, Cork, IrelandSchool of Microbiology and APC Microbiome Ireland, University College Cork, Cork, IrelandDSM Biotechnology Center, Delft, NetherlandsDSM Biotechnology Center, Delft, NetherlandsDSM Biotechnology Center, Delft, NetherlandsSchool of Microbiology and APC Microbiome Ireland, University College Cork, Cork, IrelandSchool of Microbiology and APC Microbiome Ireland, University College Cork, Cork, IrelandPlasmid pNP40, which was first identified nearly 40 years ago in Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, encodes functions such as heavy metal-, bacteriophage-, and nisin-resistance, as well as plasmid transfer ability by conjugation. Here, we report an optimized conjugation protocol for this plasmid, yielding a transfer frequency that is approximately 4,000-fold higher than those previously reported in literature, while we also observed high-frequency plasmid co-mobilization. Individual mutations in 18 genes that encompass the presumed conjugation cluster of pNP40 were generated using ssDNA recombineering to evaluate the role of each gene in the conjugation process. A possible transcriptional repressor of this conjugation cluster, the product of the traR gene, was identified in this manner. This mutational analysis, paired with bioinformatic predictions as based on sequence and structural similarities, allowed us to generate a preliminary model of the pNP40 conjugation machinery.https://www.frontiersin.org/articles/10.3389/fmicb.2021.680920/fulllactococcistarter culturedairy fermentationconjugative plasmidrecombineeringmutagenesis |
| spellingShingle | Guillermo Ortiz Charneco Philip Kelleher Andrius Buivydas Hugo Streekstra Emiel Ver Loren van Themaat Paul P. de Waal Jennifer Mahony Douwe van Sinderen Genetic Dissection of a Prevalent Plasmid-Encoded Conjugation System in Lactococcus lactis Frontiers in Microbiology lactococci starter culture dairy fermentation conjugative plasmid recombineering mutagenesis |
| title | Genetic Dissection of a Prevalent Plasmid-Encoded Conjugation System in Lactococcus lactis |
| title_full | Genetic Dissection of a Prevalent Plasmid-Encoded Conjugation System in Lactococcus lactis |
| title_fullStr | Genetic Dissection of a Prevalent Plasmid-Encoded Conjugation System in Lactococcus lactis |
| title_full_unstemmed | Genetic Dissection of a Prevalent Plasmid-Encoded Conjugation System in Lactococcus lactis |
| title_short | Genetic Dissection of a Prevalent Plasmid-Encoded Conjugation System in Lactococcus lactis |
| title_sort | genetic dissection of a prevalent plasmid encoded conjugation system in lactococcus lactis |
| topic | lactococci starter culture dairy fermentation conjugative plasmid recombineering mutagenesis |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2021.680920/full |
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