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Exploitation of plasmid pMRC01 to direct transfer of mobilizable plasmids into commercial lactococcal starter strains.

ABSTRACT: Genetic analysis of the 60.2-kb lactococcal plasmid pMRC01 revealed a 19.6-kb region which includes putative genes for conjugal transfer of the plasmid and a sequence resembling an origin of transfer (oriT). This oriT-like sequence was amplified and cloned on a 312-bp segment into pCI372, allowing the resultant plasmid, pRH001, to be mobilized at a frequency of 3.4 x 10(-4) transconjugants/donor cell from an MG1363 (recA mutant) host containing pMRC01. All of the resultant chloramphenicol-resistant transconjugants contained both pRH001 and genetic determinants responsible for bacteriocin production and immunity of pMRC01. This result is expected, given that transconjugants lacking the lacticin 3147 immunity determinants (on pMRC01) would be killed by bacteriocin produced by the donor cells. Indeed, incorporation of proteinase K in the mating mixture resulted in the isolation of transformants, of which 47% were bacteriocin deficient. Using such an approach, the oriT-containing fragment was exploited to mobilize pRH001 alone to a number of lactococcal hosts. These results demonstrate that oriT of pMRC01 has the potential to be used in the development of mobilizable food-grade vectors for the genetic enhancement of lactococcal starter strains, some of which may be difficult to transform.

SUBMITTER: Hickey RM

PROVIDER: S-EPMC92951 | biostudies-literature | 2001 Jun

REPOSITORIES: biostudies-literature

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Exploitation of plasmid pMRC01 to direct transfer of mobilizable plasmids into commercial lactococcal starter strains.

Hickey R M RM Twomey D P DP Ross R P RP Hill C C

Applied and environmental microbiology 20010601 6

Genetic analysis of the 60.2-kb lactococcal plasmid pMRC01 revealed a 19.6-kb region which includes putative genes for conjugal transfer of the plasmid and a sequence resembling an origin of transfer (oriT). This oriT-like sequence was amplified and cloned on a 312-bp segment into pCI372, allowing the resultant plasmid, pRH001, to be mobilized at a frequency of 3.4 x 10(-4) transconjugants/donor cell from an MG1363 (recA mutant) host containing pMRC01. All of the resultant chloramphenicol-resist ...[more]

PMID: 11375207

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Project description:BackgroundThe spread of antibiotic resistance genes (ARGs) has been of global concern as one of the greatest environmental threats. The gut microbiome of animals has been found to be a large reservoir of ARGs, which is also an indicator of the environmental antibiotic spectrum. The conserved microbiota makes the honeybee a tractable and confined ecosystem for studying the maintenance and transfer of ARGs across gut bacteria. Although it has been found that honeybee gut bacteria harbor diverse sets of ARGs, the influences of environmental variables and the mechanism driving their distribution remain unclear.ResultsWe characterized the gut resistome of two closely related honeybee species, Apis cerana and Apis mellifera, domesticated in 14 geographic locations across China. The composition of the ARGs was more associated with host species rather than with geographical distribution, and A. mellifera had a higher content of ARGs in the gut. There was a moderate geographic pattern of resistome distribution, and several core ARG groups were found to be prevalent among A. cerana samples. These shared genes were mainly carried by the honeybee-specific gut members Gilliamella and Snodgrassella. Transferrable ARGs were frequently detected in honeybee guts, and the load was much higher in A. mellifera samples. Genomic loci of the bee gut symbionts containing a streptomycin resistance gene cluster were nearly identical to those of the broad-host-range IncQ plasmid, a proficient DNA delivery system in the environment. By in vitro conjugation experiments, we confirmed that the mobilizable plasmids could be transferred between honeybee gut symbionts by conjugation. Moreover, "satellite plasmids" with fragmented genes were identified in the integrated regions of different symbionts from multiple areas.ConclusionsOur study illustrates that the gut microbiota of different honeybee hosts varied in their antibiotic resistance structure, highlighting the role of the bee microbiome as a potential bioindicator and disseminator of antibiotic resistance. The difference in domestication history is highly influential in the structuring of the bee gut resistome. Notably, the evolution of plasmid-mediated antibiotic resistance is likely to promote the probability of its persistence and dissemination. Video Abstract.

Dataset Information

Exploitation of plasmid pMRC01 to direct transfer of mobilizable plasmids into commercial lactococcal starter strains.

Publications

Exploitation of plasmid pMRC01 to direct transfer of mobilizable plasmids into commercial lactococcal starter strains.

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