Author contributions: S.F., G.C.O., J.K.C., D.H., F.S., G.F.F., D.v.S., and P.W.O. designed research; M.J.C., Y.L., S.L., C.C., J.P.v.P., S.F., J.K.C., and P.W.O. performed research; G.C.O. contributed new reagents/analytic tools; M.J.C., Y.L., S.L., C.C., J.P.v.P., A.M.C.-T., J.P., S.F., D.H., F.S., D.v.S., and P.W.O. analyzed data; and M.J.C., Y.L., D.H., G.F.F., D.v.S., and P.W.O. wrote the paper.

Edited by Todd R. Klaenhammer, North Carolina State University, Raleigh, NC, and approved February 25, 2006

Received 2005 Dec 22

Freely available online through the PNAS open access option.

Abstract

Lactobacillus salivarius subsp. salivarius strain UCC118 is a bacteriocin-producing strain with probiotic characteristics. The 2.13-Mb genome was shown by sequencing to comprise a 1.83 Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids. Megaplasmids previously have not been characterized in lactic acid bacteria or intestinal lactobacilli. Annotation of the genome sequence indicated an intermediate level of auxotrophy compared with other sequenced lactobacilli. No single-copy essential genes were located on the megaplasmid. However, contingency amino acid metabolism genes and carbohydrate utilization genes, including two genes for completion of the pentose phosphate pathway, were megaplasmid encoded. The megaplasmid also harbored genes for the Abp118 bacteriocin, a bile salt hydrolase, a presumptive conjugation locus, and other genes potentially relevant for probiotic properties. Two subspecies of L. salivarius are recognized, salivarius and salicinius, and we detected megaplasmids in both subspecies by pulsed-field gel electrophoresis of sizes ranging from 100 kb to 380 kb. The discovery of megaplasmids of widely varying size in L. salivarius suggests a possible mechanism for genome expansion or contraction to adapt to different environments.

Keywords: megaplasmid, probiotic, heterofermentation

Abstract

Lactobacilli are widely used for fermenting foods products or as adjuncts to foodstuffs. Many Lactobacillus species have been shown to confer probiotic properties, meaning that, upon ingestion, they confer a range of benefits on the host (1). Lactobacilli are part of the normal human gastrointestinal (GI) microbiota, and they may also be found in the GI tracts of other mammalian species (2 –4). Bacteria belonging to the species investigated in this study, Lactobacillus salivarius, have been isolated from the intestinal mucosa of 9% of human subjects examined (5), the tongue and rectum of 12% of healthy adults sampled (6), and feces of infants (7). L. salivarius subsp. salivarius strain UCC118 was isolated from the terminal ileum of a (otherwise healthy) patient undergoing urinary tract reconstructive surgery (8). This strain has been extensively studied for its probiotic properties in human trials and animal models (8 –11). In addition, L. salivarius UCC118 produces a two-component bacteriocin Abp118 (12), which has broad spectrum activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (8).

The application of genomic technologies recently has led to major advances in our understanding of lactobacilli (13, 14), through genome sequence determination for Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, and Lactobacillus sakei (15 –18). L. plantarum has the largest Lactobacillus genome sequenced to date (3.3 Mb) and has a commensurate biochemical complexity. For example, L. plantarum possesses enzymes for the biosynthesis of all amino acids except leucine, isoleucine, and valine, whereas L. johnsonii is predicted to be incapable of synthesizing any amino acids (19). L. sakei, although displaying the highest levels of orthology with L. plantarum, is unable to synthesize 18 amino acids (18). L. johnsonii and L. acidophilus are more related to each other than to L. plantarum (see also below) and have smaller genomes, reflected in nutritional fastidiousness. L. acidophilus appears to be auxotropic for 14 amino acids (17). L. johnsonii lacks the ability for de novo synthesis of purines and cofactors (16, 19). In common with Streptococcusthermophilus (20), which has been passed for centuries in the nutrient-rich medium of milk, L. johnsonii and L. acidophilus appear to have undergone genome reduction in adapting to a lifestyle of close host association.

The genus Lactobacillus is very diverse, which is evident from the lack of long-range synteny observed between the four available complete Lactobacillus genomes and the assembled genome from Lactobacillus gasseri (refs. 13, 14, and 19 and unpublished analyses). The Lactobacillus 16S rRNA gene phylogeny (available as Fig. 4, which is published as supporting information on the PNAS web site) shows that L. salivarius is part of a distinct clade at the periphery of the genus that is not represented by completed or in-progress genome sequence projects. The genome of L. salivarius UCC118 described here reveals the presence of a 242-kb megaplasmid, which, although apparently dispensable for viability based on gene content, confers on the strain a large number of contingency metabolic capabilities and traits directly related to GI tract survival or competitiveness.

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Acknowledgments

We thank numerous Alimentary Pharmabiotic Centre colleagues for help with this work, T. Walunas of Integrated Genomics for assistance with ERGO, Kyoto Encyclopedia of Genes and Genomes/Pathway Solutions for permission to use images of Kyoto Encyclopedia of Genes and Genomes reference pathways, D. Walsh for HPLC analysis, K. Hayes for annotation assistance, and Prof. M. H. Saier, Jr., for making the Transport Classification Database available. This research was supported by Science Foundation Ireland through a Centre for Science, Engineering, and Technology award to the Alimentary Pharmabiotic Centre and by grants from the Higher Education Authority PRTLI1 and PRTLI3 programmes, the Department of Agriculture and Food FIRM 01/R&D/C/159 program, and the Irish Research Council for Science, Engineering, and Technology EMBARK postdoctoral program (to C.C.).

Acknowledgments

Abbreviations

GI	gastrointestinal
IS	insertion sequence
PFGE	pulsed-field gel electrophoresis.

Abbreviations

Footnotes

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. {"type":"entrez-nucleotide","attrs":{"text":"CP000233","term_id":"90820184"}}CP000233 and {"type":"entrez-nucleotide","attrs":{"text":"CP000234","term_id":"90821902"}}CP000234).

Footnotes

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