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

Hidetoshi Morita, Hidehiro Toh, Shinji Fukuda, Hiroshi Horikawa, Kenshiro Oshima, Takehito Suzuki, Masaru Murakami, Shin Hisamatsu, Yukio Kato, Tatsuya Takizawa, Hideo Fukuoka, Tetsuhiko Yoshimura, Kikuji Itoh, Daniel J O'Sullivan, Larry L McKay, Hiroshi Ohno, Jun Kikuchi, Toshio Masaoka, Masahira Hattori, Hidetoshi Morita, Hidehiro Toh, Shinji Fukuda, Hiroshi Horikawa, Kenshiro Oshima, Takehito Suzuki, Masaru Murakami, Shin Hisamatsu, Yukio Kato, Tatsuya Takizawa, Hideo Fukuoka, Tetsuhiko Yoshimura, Kikuji Itoh, Daniel J O'Sullivan, Larry L McKay, Hiroshi Ohno, Jun Kikuchi, Toshio Masaoka, Masahira Hattori

Abstract

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.

Figures

Figure 1
Figure 1
Genome-based phylogenetic analysis of L. reuteri JCM 1112T and L. fermentum IFO 3956. (A) Synteny between the L. reuteri JCM 1112T and L. fermentum IFO 3956 chromosomes. Each dot represents an orthologous gene that was defined by bidirectional best hits based on BLASTP comparisons. Genome numbering was initiated at dnaA in both chromosomes. The shaded region indicates the location of the pdu-cbi-cob-hem cluster in the L. reuteri JCM 1112T genome. (B) Phylogenetic relationships between the genomes of sequenced Lactobacillus species and other lactic acid bacteria, including Lactococcus lactis, inferred from 34 concatenated ribosomal protein amino acid sequences. The scale bar represents an evolutionary distance. Sequences were aligned with ClustalW with a bootstrap trial of 1000 and bootstrap values (%) are indicated at the nodes. An unrooted tree was generated using NJplot.
Figure 2
Figure 2
Proposed glycerol and glucose metabolic pathways in L. reuteri JCM 1112T. The pathways are color coded as follows: blue, glycerol metabolism; green, biosynthesis of reuterin; red, glucose metabolism. Dashed lines indicate an unidentified enzyme in L. reuteri JCM 1112T. The bottom part of the figure shows the structure of the pdu-cbi-cob-hem gene cluster in L. reuteri JCM 1112T. Genes are depicted with arrows indicating the transcription direction with the following colors: yellow, pdu including gupCDE genes; pink, cbi genes; orange, cob genes; blue, hem genes; red, pocR; green, eut; sky blue, transposase gene; and white, other genes. Several lines connect corresponding genes between the pathway and the cluster (enzymes, red; transporters, blue).
Figure 3
Figure 3
(A) A comparison of the genomic location that contains the pdu-cbi-cob-hem gene cluster of L. reuteri JCM 1112T (center) with the corresponding location of L. fermentum IFO 3956 (upper) and L. plantarum WCFS1 (lower). Genes in the pdu-cbi-cob-hem gene cluster are depicted by arrows indicating the transcription direction with the same color codes as described in the Fig. 2. Genes conserved between the three genomes are colored gray and light blue bars indicate orthologous regions. The GC content at the third codon position of the ORFs in L. reuteri JCM 1112T is indicated under each ORF. Red lines represent the GC content at the third codon position of the ORFs in the pdu-cbi-cob-hem cluster (LAR_1583-1640). The blue horizontal line indicates the average GC content (32%) at the third codon position of the remaining ORFs in the L. reuteri JCM 1112T genome excluding the pdu-cbi-cob-hem gene cluster. (B) The pdu-cbi-cob gene cluster arrangement in L. reuteri JCM 1112T, S. typhimurium LT2, L. monocytogenes EGD-e, Y. enterocolitica subsp. enterocolitica 8081, and S. sanguinis SK36 are shown using the same color coding as described in (A).
Figure 4
Figure 4
In vivo detection of 3-HPA-hydrate derived from 13C3-glycerol in the cecal contents of mice colonized by wild-type L. reuteri JCM 1112T (A) and its mutant derivative, LRΔgupCDE (B), as detected by 2D 1H, 13C-HSQC NMR. δ1H (ppm) and δ13C (ppm) of 3-HPA-hydrate 2 (Hh-2) and 3-HPA-hydrate 3 (Hh-3) were observed at 2.43 and 42.6, and 3.79 and 61.6 at pH 7.0, respectively. Although the spot derived from 3-HPA-hydrate 1 (Hh-1) in 1H chemical shift was detected, the spot was not shown in (A), because the range of 1H chemical shift showed between 4.0 to 1.5 ppm. See Supplementary Figure S5 for details.

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