Complete Genome Sequence of Lactococcus lactis subsp. lactis CV56, a Probiotic Strain Isolated from the Vaginas of Healthy Women

Abstract

Lactic acid bacteria that exist in the urinogenital system play an important role in maintaining the health of the host. Here, we report the finished and annotated genome of a Lactococcus strain that was isolated from the vaginas of healthy women and shows probiotic properties, including nisin A production and adhesion to vaginal epithelial cells.

TEXT

The vast diversity of lactic acid bacteria (LAB) allows them to occupy different kinds of ecological niches, such as dairy products, meats, the gastrointestinal tract, and the vagina (5). As the dominant bacteria in the vaginal microflora of healthy women, Lactobacillus spp. play an important role in maintaining the natural balance of microflora (2). So far, only a few probiotic Lactococcus strains, like L. lactis subsp. lactis HV219, have been found in the vaginal microflora (6). In this study, we identified a Lactococcus strain named L. lactis subsp. lactis CV56 from vaginal secretions of healthy women. This strain not only exhibits strong antimicrobial activity by producing bacteriocin nisin A but also shows a greater ability to adhere to vaginal epithelial cells than other Lactococcus strains, such as L. lactis MG1363. The whole-genome sequencing of L. lactis CV56 will help us identify the genomic diversity, metabolic diversity, and probiotic and adaptation properties of Lactococcus species in different environments.

The whole genome was sequenced by using the Roche GS FLX system. A total of 233,256 reads, counting up to 87,594,199 bases, were obtained, providing 36-fold coverage. Assembly was performed by use of Newbler software (454 Sequencing) and produced 77 contigs. The contig order was first determined through a BLASTN search against the reference strain L. lactis IL1403 and then confirmed by PCR. The relationships of contigs that cannot be ordered by reference were determined by multiplex PCR. Gaps were closed by sequencing PCR fragments from the genomic DNA template by using ABI 3730. The Phred/Phrap/Consed software package (2a, 2b) was used for final assembly and edition, and low-quality regions of the genome were resequenced.

Putative protein-coding sequences were identified by Glimmer3 (1), and those shorter than 90 bp were eliminated. All predicted proteins were searched against KEGG databases, the NCBI nonredundant protein database (nr), and the Conserved Domain Database (CDD), using BLASTP or RPSBLAST. The tRNA genes were predicted by tRNA-scan SE1.21 (4), and the rRNA genes were detected using the RNAmmer 1.2 server (3). Functional classification of proteins was conducted using NCBI clusters of orthologous groups (COG).

The complete genome of L. lactis CV56 consists of a single, circular chromosome (2,399,458 bp, 35.24% GC content) and five plasmids, namely, pCV56A (44,098 bp, 32.08% GC content), pCV56B (35,934 bp, 34.54% GC content), pCV56C (31,442 bp, 32.49% GC content), pCV56D (5,543 bp, 32.24% GC content), and pCV56E (2,262 bp, 33.82% GC content). The chromosome of L. lactis CV56 contains 2,352 predicted protein-coding genes, 6 rRNA operons, and 62 tRNA genes, while the five plasmids contain 116 protein-coding genes. Of all the 2,468 protein-coding genes, we annotated 1,812 (73.42%) as having biological functions, 494 (20.02%) as encoding conserved hypothetical proteins, 102 (4.13%) as encoding novel hypothetical proteins, and 60 (2.43%) as pseudogenes. Genes involved in nisin A biosynthesis and cell adhesion were revealed on the chromosome. As well, the five plasmids contain protein-coding genes involved in antibiotic resistance, bacteriophage resistance, cation transport, and stress response.

Nucleotide sequence accession numbers.

The genome sequences of L. lactis CV56 have been deposited in GenBank under the accession numbers CP002365 to CP002370.