GENOME ANNOUNCEMENT
Genome Sequence of Lactobacillus rhamnosus ATCC 8530 Vanessa Pittet, Emily Ewen, Barry R. Bushell, and Barry Ziola Department of Pathology and Laboratory Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Lactobacillus rhamnosus is found in the human gastrointestinal tract and is important for probiotics. We became interested in L. rhamnosus isolate ATCC 8530 in relation to beer spoilage and hops resistance. We report here the genome sequence of this isolate, along with a brief comparison to other available L. rhamnosus genome sequences.
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actobacillus rhamnosus is an organism of interest for human health and probiotics. In our case, we became interested in a particular L. rhamnosus isolate due to its ability to spoil beer. Among five L. rhamnosus isolates tested, only one (ATCC 8530) was able to grow in beer (1). We therefore performed further analyses on this isolate and found that it was among a few lactic acid bacteria (LAB) that showed increased resistance to hops in the presence of ethanol (2). This finding was particularly intriguing considering the antimicrobial properties of both compounds and because hops has been suggested as a replacement for antibiotics to control LAB contamination in the fuel ethanol industry (6). To get a better understanding of how L. rhamnosus ATCC 8530 grows in beer and resists hops, we sequenced its genome for comparative analysis with other sequenced LAB. Sequencing was done using the Roche 454 Genome Sequence FLX platform. Paired and unpaired reads were obtained during two different runs, giving a final coverage of ⬃30⫻. A total of 520,000 reads were assembled using Newbler GS De Novo assembler, giving 34 contigs split among six scaffolds. The scaffolds were then ordered according to the L. rhamnosus Lc 705 genome, and gaps were closed by sequencing PCR amplicons via the ABI 3700xl platform. The final circularized genome is 2,960,339 bp with a G⫹C content of 46.8%. Genome annotation was done via the Institute for Genome Sciences Annotation Engine, which predicted 2,904 coding sequences (CDS), 60 tRNA, and five rRNA operons. Of the predicted CDS, ⬃700 are hypothetical proteins with unknown function. Unlike L. rhamnosus Lc 705 and HN001, no plasmids are found in this isolate. Comparative analysis with L. rhamnosus ATCC 8530 and the four other currently available L. rhamnosus genome sequences (3, 4) showed that L. rhamnosus ATCC 8530 is least similar to the probiotic isolates GG and HN001. In contrast, the intestinal isolate L. rhamnosus LMS2-1 is very similar to ATCC 8530, with most (⬎90%) predicted proteins having ⱖ99% identity. Preliminary analyses indicate that only four regions are different between L. rhamnosus ATCC 8530 and LMS2-1, all related to mobile elements (L. rhamnosus ATCC 8530 has no predicted bacteriophagerelated genes). The majority of L. rhamnosus ATCC 8530 was also conserved with the dairy industry isolate L. rhamnosus Lc 705, except for one distinct location missing in the former isolate and several bacteriophage-related regions not found in the latter isolate, respectively. Considering the high degree of similarity between L. rhamno-
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sus ATCC 8530 and LMS2-1, further analyses will be done to determine if the latter isolate can grow in beer, as well as to show a combinatorial growth effect with hops and ethanol. Comparative genomic analyses will also be done between L. rhamnosus ATCC 8530 and other beer-spoilage LAB isolates that we are currently sequencing to elucidate the mechanisms these organisms use to grow in the harsh environment found in beer (5). Nucleotide sequence accession number. The genome sequence for L. rhamnosus ATCC 8530 is available in GenBank (accession number CP003094). ACKNOWLEDGEMENTS Genome sequencing was done at Plant Biotechnology Institute, National Research Council of Canada, Saskatoon SK. The Institute for Genome Sciences at the University of Maryland School of Medicine performed the annotation via their Annotation Engine Pipeline. V.P. held a Natural Sciences and Engineering Research Council of Canada (NSERC) Alexander Graham Bell Canada Graduate Scholarship, as well as the Anheuser-Busch InBev, Brian Williams, and MillerCoors Graduate Student Scholarships from the American Society of Brewing Chemists Foundation. E.E. was the holder of a NSERC Undergraduate Student Research Award. This research was supported by MillerCoors Brewing Company, Golden, CO, and NSERC Discovery Grant 24067.
REFERENCES 1. Haakensen M, Schubert A, Ziola B. 2008. Multiplex PCR for putative Lactobacillus and Pediococcus beer-spoilage genes and ability of gene presence to predict growth in beer. J. Am. Soc. Brewing Chem. 66:63–70. 2. Haakensen M, Schubert A, Ziola B. 2009. Broth and agar hop-gradient plates used to evaluate the beer-spoilage potential of Lactobacillus and Pediococcus isolates. Int. J. Food Microbiol. 130:56 – 60. 3. Kankainen M, et al. 2009. Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human-mucus binding protein. Proc. Natl. Acad. Sci. U. S. A. 106:17193–17198. 4. Morita H, et al. 2009. Complete genome sequence of the probiotic Lactobacillus rhamnosus ATCC 53103. J. Bacteriol. 191:7630 –7631. 5. Pittet V, Morrow K, Ziola B. 2011. Ethanol tolerance of lactic acid bacteria, including relevance of the exopolysaccharide gene gtf. J. Am. Soc. Brewing Chem. 69:57– 61. 6. Rückle L, Senn T. 2006. Hop acids can efficiently replace antibiotics in ethanol production. Int. Sugar J. 108:139 –147.
Received 26 October 2011 Accepted 7 November 2011 Address correspondence to Barry Ziola,
[email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JB.06430-11
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