A procedure for the rapid isolation of plasmid DNA larger than 30 megadaltons from lactic streptococci is described. This protocol can be used on a preparative scale to isolate sufficient quantities of plasmid DNA required for restriction analysis, cloning, or transformation experiments. A scaled-down protocol is very useful for rapidly screening the plasmid content of streptococcal strains. With this methodology, previously undetected large plasmids were observed.

Lactobacillus reuteri is a heterofermentative lactic acid bacterium that naturally inhabits the gut of humans and other animals. The probiotic effects of L. reuteri have been proposed to be largely associated with the production of the broad-spectrum antimicrobial compound reuterin during anaerobic metabolism of glycerol. We determined the complete genome sequences of the reuterin-producing L. reuteri JCM 1112T and its closely related species Lactobacillus fermentum IFO 3956. Both are in the same phylogenetic group within the genus Lactobacillus. Comparative genome analysis revealed that L. reuteri JCM 1112T has a unique cluster of 58 genes for the biosynthesis of reuterin and cobalamin (vitamin B12). The 58-gene cluster has a lower GC content and is apparently inserted into the conserved region, suggesting that the cluster represents a genomic island acquired from an anomalous source. Two-dimensional nuclear magnetic resonance (2D-NMR) with 13C3-glycerol demonstrated that L. reuteri JCM 1112T could convert glycerol to reuterin in vivo, substantiating the potential of L. reuteri JCM 1112T to produce reuterin in the intestine. Given that glycerol is shown to be naturally present in feces, the acquired ability to produce reuterin and cobalamin is an adaptive evolutionary response that likely contributes to the probiotic properties of L. reuteri.

Analysis of a region involved in the conjugative transfer of the lactococcal conjugative element pRS01 has revealed a bacterial group II intron. Splicing of this lactococcal intron (designated Ll.ltrB) in vivo resulted in the ligation of two exon messages (ltrBE1 and ltrBE2) which encoded a putative conjugative relaxase essential for the transfer of pRS01. Like many group II introns, the Ll.ltrB intron possessed an open reading frame (ltrA) with homology to reverse transcriptases. Remarkably, sequence analysis of ltrA suggested a greater similarity to open reading frames encoded by eukaryotic mitochondrial group II introns than to those identified to date from other bacteria. Several insertional mutations within ltrA resulted in plasmids exhibiting a conjugative transfer-deficient phenotype. These results provide the first direct evidence for splicing of a prokaryotic group II intron in vivo and suggest that conjugative transfer is a mechanism for group II intron dissemination in bacteria.Conjugation is an important mode of genetic exchange in bacteria. The specific mechanism of DNA transfer comprises two distinct functions, one being the enzymatic preparation of the plasmid DNA prior to replicative transfer and another involving formation of the mating channel through which DNA is transferred into a recipient cell. Initiation of plasmid transfer requires a single-stranded cleavage at a specific origin of transfer (oriT) produced by the action of a specialized nucleoprotein complex called a relaxosome (53). In many conjugative systems, transfer origins are flanked by genes involved in the formation of a functional relaxosome complex. One key feature of an oriT region is the ability of the region to confer mobilization in cis, provided the remaining transfer functions are present in trans.The conjugative element pRS01 from Lactococcus lactis subsp. lactis ML3 and the sex factor from L. lactis subsp. lactis 712 are prototypical mobile elements in lactococci (15, 50). Both elements have been shown to mediate high-frequency transfer of genes encoding lactose utilization (Lac ϩ ) by insertion sequence-directed conintegration with nonconjugative Lac ϩ plasmids (19,40). In addition, both elements confer a cell aggregation (Clu) phenotype (16, 52) associated with highfrequency conjugative transfer. Previous genetic analysis of these elements has identified the gene responsible for the aggregation phenotype (clu) associated within an inversion region (2, 20, 21). To date, however, the oriT and gene(s) encoding relaxosome components of pRS01 have not been localized within either element. In an effort to exploit conjugation as a means of lactococcal strain development, we have characterized the transfer regions of pRS01 by insertional mutagenesis via IS946-mediated cointegration with the 11-kb plasmid pTRK28 (43) (for a representation of pTRK28:: pRS01 cointegration, see Fig. 1 in reference 33). Analysis of the insertion site junctions of pRS01::pTRK28 cointegrate plasmids identified four distinct regions of pRS01 involved...

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