Lactobacillus rhamnosus LOCK908, a patented probiotic strain (Polish patent no. 209987), was isolated from the feces of a healthy 6-year-old girl. Here, we present the complete genome sequence of LOCK908 and identify genes likely to be involved in the biosynthesis of exopolysaccharides (EPSs)

Aleksandrzak-Piekarczyk, Tamara

Bardowski, Jacek K.

Koryszewska-Bagińska, Anna

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Genome Sequence of the Probiotic Strain Lactobacillus rhamnosus(Formerly Lactobacillus casei) LOCK908Anna Koryszewska-Bagin´ska, Jacek Bardowski, Tamara Aleksandrzak-PiekarczykInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, PolandLactobacillus rhamnosus LOCK908, a patented probiotic strain (Polish patent no. 209987), was isolated from the feces of ahealthy 6-year-old girl. Here, we present the complete genome sequence of LOCK908 and identify genes likely to be involved inthe biosynthesis of exopolysaccharides (EPSs).Received 3 February 2014 Accepted 3 February 2014 Published 20 February 2014Citation Koryszewska-Bagin´ska A, Bardowski J, Aleksandrzak-Piekarczyk T. 2014. Genome sequence of the probiotic strain Lactobacillus rhamnosus (formerly Lactobacillus casei)LOCK908. Genome Announc. 2(1):e00120-14. doi:10.1128/genomeA.00120-14.Copyright © 2014 Koryszewska-Bagin´ska et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.Address correspondence to Tamara Aleksandrzak-Piekarczyk, tamara@ibb.waw.pl.Lactobacillus rhamnosus LOCK908 (formerly Lactobacillus caseiLOCK908) was obtained from the Pure Culture Collection ofthe Technical University of Lódz (Lódz, Poland). To verify theidentity of strain LOCK908, sequence analysis and comparisons ofthe 16S rRNA, rpoA, and pheS genes were performed, which indi-cated that LOCK908 belongs to the L. rhamnosus species (1).Previous in vitro tests conducted on L. rhamnosus LOCK908revealed the strain to be resistant to gastric acids and bile salts,have antagonistic activity against pathogenic bacteria (2, 3), andhave an ability to adhere to the Caco-2 epithelial cell line (4).Moreover, it has been proven by in vivo experiments that the mix-ture of L. rhamnosus LOCK908, L. rhamnosus LOCK900 (5), andL. casei LOCK919 (6) strains modulates the immune system byinducing Th1 and regulatory cytokine production and by sup-pressing the proallergic response (7).The genomic DNA was extracted using a genomic mini puri-fication kit (A&A Biotechnology). Genomic libraries containingshotgun and 8-kb inserts were constructed, and high-quality readstotaling 155 Mbp were generated by using the GS FLX Titaniumpyrosequencing system (Roche). Sequence assembly was per-formed using Newbler Assembler version 2.4 software (Roche).Contig alignment was performed using SeqMan software from theLasergene package (DNAStar). To further improve consensusquality, a sequencing run on the Illumina system HiScanSQ wasperformed, giving 177-fold final coverage of the genome. Func-tional annotation of genes and rRNA was performed using theRAST annotation server (http:/rast.nmpdr.org/) (8) and checkedby BLAST analysis (9) when needed. tRNAs were identified withtRNAscan-SE (10).The complete genome of L. rhamnosus LOCK908 contains asingle circular chromosome of 2,990,900 bp, with an overall GCcontent of 46.8%. No plasmids were detected in the sequencedDNA of LOCK908. There are 2,931 coding sequences (CDSs) and60 tRNAs. Ribosomal operons coding for 5S, 16S, and 23S rRNAare present in 5 copies each. The genome sequence of LOCK908contains 313 subsystems, according to the RAST server. Approx-imately 32% of the total CDSs were annotated as encoding hypo-thetical proteins with unknown function.A comparison of the LOCK908 genome with that of LOCK900(5), another genome of L. rhamnosus sequenced by our group,reveals the former to be larger and therefore to contain an appro-priately larger number of protein-coding genes. In particular, thenumbers of genes distributed in the categories of cell wall andcapsule, DNA metabolism and phages, prophages, and transpos-able elements are increased in the LOCK908 genome compared tothat of LOCK900.The most significant feature of LOCK908 is its high exopoly-saccharide (EPS) productivity, which is relatively common forprobiotic L. rhamnosus strains (11) , and it has also been observedin our research (our unpublished data). Indeed, EPS-related genesinvolved in the pathway of capsular and extracellular polysaccha-rides were identified from genome information. In particular, an18-kb gene cluster (LOCK908_2101 to LOCK908_2117), whichcontains 17 EPS-related genes, with a genetic organization andstructure typical in the biosynthesis of EPS, was found in the chro-mosome. In addition, several genes from another cluster(LOCK908_2053 to LOCK908_2071) might be involved in theregulation, chain length determination, biosynthesis of the re-peating unit, polymerization, and export of the EPS (12).Nucleotide sequence accession number. The genome infor-mation for the chromosome of L. rhamnosus LOCK908 has beendeposited in the GenBank database with the accession no.CP005485.ACKNOWLEDGMENTSGenome sequencing was done at the Institute of Biochemistry and Bio-physics, Polish Academy of Sciences. This research was supported by agrant from the National Centre for Research and Development, no. 12/P01/2010/10, awarded to the Technical University of Lódz.REFERENCES1. Naser SM, Dawyndt P, Hoste B, Gevers D, Vandemeulebroecke K,Cleenwerck I, Vancanneyt M, Swings J. 2007. Identification of lactoba-cilli by pheS and rpoA gene sequence analyses. Int. J. Syst. Evol. Microbiol.57:2777–2789. http://dx.doi.org/10.1099/ijs.0.64711-0.2. Cukrowska B, Motyl I, Kozáková H, Schwarzer M, Górecki RK, Kle-wicka E, S´liz˙ewska K, Libudzisz Z. 2009. Probiotic Lactobacillus strains:Genome AnnouncementsJanuary/February 2014 Volume 2 Issue 1 e00120-14 genomea.asm.org 1in vitro and in vivo studies. Folia Microbiol. (Praha) 54:533–537. http://dx.doi.org/10.1007/s12223-009-0077-7.3. Motyl I. 2002. Ph.D. thesis.Technical University of Lódz, Lódz, Poland.4. Marewicz E, Moneta J, Motyl I, Heczko P, Libudzisz Z. 2000. Compar-ison of adherence to Caco-2 human epithelial cell line of Lactobacillus andBifidobacterium strains of various origin: human, animal and vegetal.Med. Sci. Prompt. 54:34.5. Aleksandrzak-Piekarczyk T, Koryszewska-Baginska A, Bardowski J.2013. Genome sequence of the probiotic strain Lactobacillus rhamnosus(formerly Lactobacillus casei) LOCK900. Genome Announc. 1(4):e00640-13. http://dx.doi.org/10.1128/genomeA.00640-13.6. Koryszewska-Baginska A, Aleksandrzak-Piekarczyk T, Bardowski J.2013. Complete genome sequence of the probiotic strain Lactobacilluscasei (formerly Lactobacillus paracasei) LOCK919. Genome Announc.1(5):e00758-13. http://dx.doi.org/10.1128/genomeA.00758-13.7. Cukrowska B, Rosiak I, Klewicka E, Motyl I, Schwarzer M, Libudzisz Z,Kozakova H. 2010. Impact of heat-inactivated Lactobacillus casei andLactobacillus paracasei strains on cytokine responses in whole blood cellcultures of children with atopic dermatitis. Folia Microbiol. 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Adv. 29:54 – 66. http://dx.doi.org/10.1016/j.biotechadv.2010.08.011.12. Jolly L, Stingele F. 2001. Molecular organization and functionality ofexopolysaccharide gene clusters from lactic acid bacteria. Int. Dairy J.11:733–745. http://dx.doi.org/10.1016/S0958-6946(01)00117-0.Koryszewska-Bagin´ska et al.Genome Announcements2 genomea.asm.org January/February 2014 Volume 2 Issue 1 e00120-14

Genome Sequence of the Probiotic Strain Lactobacillus rhamnosus (Formerly Lactobacillus casei) LOCK908.

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Genome Sequence of the Probiotic Strain Lactobacillus rhamnosus (Formerly Lactobacillus casei) LOCK908.

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