Probiotic Strain Isolated from the Vagina of Healthy Women

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Nathan Malone
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1 JB Accepts, published online ahead of print on 1 April 2011 J. Bacteriol. doi: /jb Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved Complete Genome Sequence of Lactococcus lactis subsp. lactis CV56, a Probiotic Strain Isolated from the Vagina of Healthy Women Yong Gao 1, Ying Lu 1, Kun-Ling Teng 1, Mei-Ling Chen 1, Hua-Jun Zheng 2, Yong-Qiang Zhu 2, Jin Zhong 1 * 1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing , P.R. China 2 Chinese National Human Genome Center at Shanghai,Shanghai , P.R. China *Corresponding author. Mailing address: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, NO.1 Beichen West Road, Chaoyang District, Beijing , P.R. China. Phone/Fax: (86) zhongj@sun.im.ac.cn. 1

2 Abstract: Lactic acid bacteria existed in urinogenital system play an important role in maintaining the health of host. Here, we report the finished and annotated genome of a Lactococcus strain isolated from the vagina of healthy women, which shows probiotic properties including nisina production and adhesion to vaginal epithelial cells. The vast diversity of lactic acid bacteria (LAB) allows them to occupy different kinds of ecological niches such as dairy products, meats, gastrointestinal tract and vagina (5). As the dominant bacteria in the vaginal microflora of healthy women, Lactobacillus play an important role in maintaining the natural balance of microflora (2). So far, only a few probitoic Lactococcus strains like L. lactis ssp. lactis HV219 have been found in 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 nisina but also shows higher adhesion ability 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, probiotic and adaptation 2

3 properties of Lactococcus species in different environment. The whole genome was sequenced by using Roche GS FLX system. A total of 233,256 reads, counting up to 87,594,199 bases were obtained, providing a 36 fold coverage. Assembly was performed by Newbler ( and produced 77 contigs. The contig order was first determined through BLASTN against the reference strain L. lactis IL1403 and then confirmed by Polymerase Chain Reaction (PCR). Relationship of contigs that can t be ordered by reference was determined by multiplexa PCR. Gaps were closed by sequencing PCR fragments from the genomic DNA template using ABI Phred, Phrap and Consed software package ( was used for finally 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, no-redundant protein database (nr, NCBI) and 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 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 3

4 chromosome (2,399,458 bp, % GC content) and five plasmids: 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 2352 predicated protein-coding genes, 6 rrna operons and 62 trna genes, while the five plasmids contain 116 protein-coding genes. Of all the 2468 protein-coding genes, we assign 1812(73.42 % ) with biological function, 494(20.02 % )as conserved hypothetical protein, 102(4.13 %)as novel hypothetical protein and 60(2.43 %) as pseudogenes. Genes involved in nisina biosynthesis and cell adhesion are revealed on the chromosome. Besides, the five plasmids contain protein-coding genes involved in antibiotic resistance, bacteriophage resistance, cation transport and stress response. Nucleotide sequence accession numbers. The genome sequence of L. lactis CV56 has been deposited in GenBank under the accession numbers between CP and CP This research was supported by Beijing Natural Science Foundation ( ), the National Natural Science Foundation of China ( ) and Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-EW-G-14, KSCX2-EW-J-6, KSCX2-EW-Q-14). 4

5 Delcher, A. L., D. Harmon, S. Kasif, O. White, and S. L. Salzberg Improved microbial gene identification with GLIMMER. Nucleic Acids Res 27: Donati, L., A. Di Vico, M. Nucci, L. Quagliozzi, T. Spagnuolo, A. Labianca, M. Bracaglia, F. Ianniello, A. Caruso, and G. Paradisi Vaginal microbial flora and outcome of pregnancy. Arch Gynecol Obstet 281: Lagesen, K., P. Hallin, E. A. Rodland, H. H. Staerfeldt, T. Rognes, and D. W. Ussery RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 35: Lowe, T. M., and S. R. Eddy trnascan-se: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25: Pfeiler, E. A., and T. R. Klaenhammer The genomics of lactic acid bacteria. Trends in Microbiology 15: Todorov, S. D., M. Botes, S. T. Danova, and L. M. Dicks Probiotic properties of Lactococcus lactis ssp. lactis HV219, isolated from human vaginal secretions. J Appl Microbiol 103:

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Complete genome sequence of Clostridium acetobutylicum. DSM 1731, a solvent producing strain with multi-replicon JB Accepts, published online ahead of print on 8 July 2011 J. Bacteriol. doi:10.1128/jb.05596-11 Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.