Development of a novel selection / counter‑selection system for chromosomal gene integrations and deletions in lactic acid bacteria

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dc.contributor.authorVan Zyl, Winschau F.en_ZA
dc.contributor.authorDicks, Leon M. T.en_ZA
dc.contributor.authorDeane, Shelly M.en_ZA
dc.date.accessioned2021-07-29T10:59:07Z
dc.date.available2021-07-29T10:59:07Z
dc.date.issued2019-03-29
dc.descriptionCITATION: Van Zyl, W., Dicks, L. M. T. & Deane, S. M. 2019. Development of a novel selection / counter‑selection system for chromosomal gene integrations and deletions in lactic acid bacteria. BMC Molecular Biology, 20:10, doi:10.1186/s12867-019-0127-x.
dc.descriptionThe original publication is available at https://bmcmolbiol.biomedcentral.com
dc.description.abstractBackground: The underlying mechanisms by which probiotic lactic acid bacteria (LAB) enhance the health of the consumer have not been fully elucidated. Verification of probiotic modes of action can be achieved by using singleor multiple-gene knockout analyses of bacterial mutants in in vitro or in vivo models. We developed a novel system based on an inducible toxin counter-selection system, allowing for rapid and efficient isolation of LAB integration or deletion mutants. The Lactococcus lactis nisin A inducible promoter was used for expression of the Escherichia coli mazF toxin gene as counter-selectable marker. Results: The flippase (FLP)/flippase recognition target (FRT) recombination system and an antisense RNA transcript were used to create markerless chromosomal gene integrations/deletions in LAB. Expression of NisR and NisK signalling proteins generated stable DNA integrations and deletions. Large sequences could be inserted or deleted in a series of steps, as demonstrated by insertion of the firefly bioluminescence gene and erythromycin resistance marker into the bacteriocin operons or adhesion genes of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA. Conclusions: The system was useful in the construction of L. plantarum 423 and E. mundtii ST4SA bacteriocin and adhesion gene mutants. This provides the unique opportunity to study the role of specific probiotic LAB genes in complex environments using reverse genetics analysis. Although this work focuses on two probiotic LAB strains, L. plantarum 423 and E. mundtii ST4SA, the system developed could be adapted to most, if not all, LAB species.en_ZA
dc.description.urihttps://bmcmolbiol.biomedcentral.com/articles/10.1186/s12867-019-0127-x
dc.description.versionPublisher's version
dc.format.extent16 pages : illustrationsen_ZA
dc.identifier.citationVan Zyl, W., Dicks, L. M. T. & Deane, S. M. 2019. Development of a novel selection / counter‑selection system for chromosomal gene integrations and deletions in lactic acid bacteria. BMC Molecular Biology, 20:10, doi:10.1186/s12867-019-0127-x
dc.identifier.issn1471-2199 (online)
dc.identifier.otherdoi:10.1186/s12867-019-0127-x
dc.identifier.urihttp://hdl.handle.net/10019.1/110788
dc.language.isoen_ZAen_ZA
dc.publisherBMC (part of Springer Nature)
dc.rights.holderAuthors retain copyright
dc.subjectHomologous recombinationen_ZA
dc.subjectLactic acid bacteria -- Geneticsen_ZA
dc.subjectProbioticsen_ZA
dc.subjectMazF toxinen_ZA
dc.subjectGene insertion/deletionen_ZA
dc.subjectInsertional mutagenesisen_ZA
dc.subjectAntibioticsen_ZA
dc.titleDevelopment of a novel selection / counter‑selection system for chromosomal gene integrations and deletions in lactic acid bacteriaen_ZA
dc.typeArticleen_ZA
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