Comparative genome analysis of Lactobacillus reuteri and Lactobacillus fermentum reveal a genomic island for reuterin and cobalamin production.

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 1112(T) 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 1112(T) has a unique cluster of 58 genes for the biosynthesis of reuterin and cobalamin (vitamin B(12)). 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 (13)C(3)-glycerol demonstrated that L. reuteri JCM 1112(T) could convert glycerol to reuterin in vivo, substantiating the potential of L. reuteri JCM 1112(T) 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.

Quantitative analysis of group II intron expression and splicing in Lactococcus lactis.

The group II intron Ll.ltrB is found within the ltrB relaxase gene of the conjugative element pRS01 in Lactococcus lactis. Precise splicing of the intron is essential for pRS01 transfer. The transcription regulation and in vivo splicing activity of Ll.ltrB have not been investigated thoroughly in L. lactis in the natural pRS01 context. We developed absolute quantitative real-time reverse transcription-PCR assays to quantify RNA levels of the 5' exon (ltrBE1) and the spliced relaxase (ltrB) and intron-encoded protein (ltrA) genes, as well as Ll.ltrB splicing activity under different physiological conditions. The mRNA levels for the ATP-binding protein OppD were assayed for comparison to the ltrB transcripts. The oppD mRNA ranged from 10- to 10,000-fold higher than ltrB region genes. ltrBE1 expression was growth-phase dependent. The mRNA level of ltrA was almost constant during all growth phases and in all media tested. Ll.ltrB in vivo splicing activity ranged from (6.5 +/- 2.1)% to (22.1 +/- 8.0)%. Acid challenge significantly decreased both ltrB region mRNA levels and intron splicing activity. The presence of recipient cells, different mating environments, and temperature stress had no significant effects on expression and splicing. Western blotting showed that the level of LtrB protein expressed from an intronless ltrB gene was much higher (about 20-fold) than the level of protein expressed from an intron-containing construct. Interestingly, LtrB protein showed a tendency to function in cis on its oriT target. The low level of ltrB transcript and relatively inefficient splicing of the intron may limit Ll.ltrB mobility and dissemination in nature.

Identification of bacilysin, chlorotetaine, and iturin a produced by Bacillus sp. strain CS93 isolated from pozol, a Mexican fermented maize dough.

Three antimicrobial compounds produced by Bacillus sp. strain CS93 isolated from pozol were identified by using high-performance liquid chromatography and mass spectrometry. The three compounds were iturin, bacilysin, and chlorotetaine. Production of these compounds by CS93 could account for the medicinal properties attributed to pozol.

Isolation and Plasmid Characterization of a Lactobacillus Species Involved in the Manufacture of Fermented Sausage 1.

Isolation and characterization of a Lactobacillus species capable of proper acid production in a sausage environment is described. The isolate from sausage, categorized as a lactobacillus in the subgenus Streptobacterium , was designated Lactobacillus sp. DR1. Growth occurred at 5 and 42°C but not at 45°C. Fructose, galactose, glucose, mannose, melibiose, N-acetylglucosamine, ribose, sucrose and trehalose were fermented. Gas production from glucose was not observed. In MRS glucose broth, D(-) and L(+) lactic acid were produced. Lactobacillus sp. DR1 contained a single cryptic plasmid of approximately 30 megadaltons (Mdal). In sausage fermentation trials, both Lactobacillus sp. DR1 and plasmid-free derivative DR1C lowered the pH to below 5.3 after 8 h in the smokehouse. Conjugation was demonstrated through the transfer of plasmid pAMß1, which encodes erythromycin resistance, from Streptococcus lactis 2301ß to Lactobacillus sp. DR1. Mutanolysin-generated protoplasts could be regenerated using 0.5 M ammonium chloride, lactose, maltose or sucrose as osmotic stabilizers. Regeneration frequencies ranged from less than 1.0% up to 35%; however, transformation of Lactobacillus sp. DR1 protoplasts by plasmid DNA in the presence of polyethylene glycol (PEG) was unsuccessful.