Journal of Biotechnology 193 (2015) 41–42
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Complete genome sequence of probiotic Lactobacillus plantarum P-8 with antibacterial activity Wenyi Zhang, Zhihong Sun, Menghe Bilige, Heping Zhang ∗ Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot 010018, China
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Article history: Received 12 November 2014 Accepted 12 November 2014 Available online 22 November 2014 Keywords: Lactobacillus plantarum Probiotics Antibacterial activity
a b s t r a c t Lactobacillus plantarum P-8 is a probiotic bacterium, which shows high antibacterial activity. The genome consists of a circular 3,033,693-bp chromosome and six plasmids. Bioinformatics inspection of the genome revealed a gene cluster relating to bacteriocin production. Genome information has provided the basis for understanding the potential molecular mechanism behind the bacteriocin production. © 2014 Published by Elsevier B.V.
Lactobacillus plantarum is a facultative heterofermentative lactic acid bacterium often detected in a broad range of ecological niches (Kleerebezem et al., 2003). Being members in the “Generally Recognized As Safe” list, strains of this species have been found with potential health-promoting effects to human beings, which have been given the “Qualiﬁed Presumption of Safety” status by the European Food Safety Authority (Kanmani et al., 2013). Over the past decades, there has been a growing interest towards L. plantarum, partly due to their signiﬁcant contribution to the intestinal health (Shen et al., 2014). Our new evidence from intestinal research has demonstrated that L. plantarum could improve human gastrointestinal health, while the desirable effects are most likely age-related (Wang et al., 2014). One possible attribute of such effects would be the antibacterial activity possessed by orally administrating the strain, and such activity has indeed been demonstrated by an in vitro analysis performed previously (Bao et al., 2012b). To identify the genetic basis of the antibacterial activity of L. plantarum P-8, a probiotic strain originally isolated from traditional fermented cow milk in Inner Mongolia of China, it was subjected to further genome sequencing (Bao et al., 2012a). A combined strategy of 454 sequencing and illumina paired-end sequencing technology was used to sequence the whole-genome of L. plantarum P-8 (Bentley et al., 2008; Margulies et al., 2005). Genomic libraries containing 3-kb inserts were constructed, and
∗ Corresponding author at: Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, 306 Zhaowuda Street, Hohhot 010018, China. Tel.: +86 471 4319940; fax: +86 471 4305357. E-mail address: [email protected]
(H. Zhang). http://dx.doi.org/10.1016/j.jbiotec.2014.11.011 0168-1656/© 2014 Published by Elsevier B.V.
327,486 paired-end reads and 114,311 single-end reads were generated using the GS FLX system, giving 37.9-fold coverage of the genome. The majority (93.11%) of reads were assembled into 37 large scaffolds, including 94 non-redundant contigs using the 454 Newbler assembler (454 Life Sciences, Branford, CT). A total of 14,382,180 reads (2.5-kb library) was generated to reach a depth of 332-fold coverage with an Illumina Solexa GA IIx (Illumina, San Diego, CA), and mapped to the scaffolds using Burrows-Wheeler alignment (Li and Durbin, 2009). The gaps between scaffolds were ﬁlled by sequencing PCR products using an ABI 3730 capillary sequencer. The genome analysis was performed as described previously (Feng et al., 2008; Ferenci et al., 2009). The complete genome sequence of L. plantarum P-8 contains a circular 3,033,693-bp chromosome and six plasmids named LBPp1, LBPp2, LBPp3, LBPp4, LBPp5, LBPp6, with GC contents of 44.8%, 39.4%, 42.2%, 42.3%, 39.8%, 42.1% and 36.0% (Table 1). There are 2892 coding genes, 5 rRNA operons and 65 tRNAs in the chromosome, as well as 73, 54, 47, 36, 26 and 12 coding genes in the plasmids, respectively. Detailed inspection of the genome revealed a gene cluster involving in bacteriocin production,
Table 1 General genome features of Lactobacillus plantarum P-8. Attributes
Genome size (bp) GC content (%) Plasmid rRNAs tRNA s Total predicted CDSs
3,033,693-bp 44.8% 6 15 65 3140
W. Zhang et al. / Journal of Biotechnology 193 (2015) 41–42
which is composed of 10 genes including two genes coding for bacteriocin peptide (LBP cg0325) and its cognate immunity protein (LBP cg0324), two genes coding for ABC-transporter system (LBP cg0326 and LBP cg0327), four genes coding for type II CAAX proteases (LBP cg0328 and LBP cg0331) and two genes for bacteriocin biosynthesis (LBP cg0332 and LBP cg0333). Additionally, two orphan bacteriocin-related genes (LBP cg1338 and LBP cg2603) out of the cluster that code for immunity proteins were also identiﬁed in the chromosome. The available genome information of L. plantarum P-8 will facilitate the understanding of the regulatory mechanism of the detected bacteriocin regulon and its probiotic properties. Nucleotide sequence accession numbers: Genome information for the chromosome and the six plasmids of L. plantarum P-8 is available in the GenBank database with accession numbers CP005942.1 (chromosome) and CP005943.1–CP005948.1 (plasmids). The strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC No. 6312). Acknowledgements This research was supported by the National Natural Science Foundation of China (Grant Nos. 31430066 and 31201396), HiTech Research and Development Program of China (863 Planning, Grant No. 2011AA100902), International S&T Cooperation Program of China (ISTCP, Grant No. 2014DFR31150), and the China Agriculture Research System (Grant No. CARS-37), and the Excellent Young Scientist Foundation of Inner Mongolia Agricultural University of China (Grant No. 2014XYQ-16). We would like to thank Prof. Kwok Lai Yu for her critical reading of the manuscript. References Bao, Y., Wang, Z., Zhang, Y., Zhang, J., Wang, L., Dong, X., Su, F., Yao, G., Wang, S., Zhang, H., 2012a. Effect of Lactobacillus plantarum P-8 on lipid metabolism in hyperlipidemic rat model. Eur. J. Lipid Sci. Technol. 114, 1230–1236. Bao, Y., Zhang, Y., Li, H., Li, Y., Wang, S., Dong, X., Su, F., Yao, G., Sun, T., Zhang, H., 2012b. In vitro screen of Lactobacillus plantarum as probiotic bacteria and their fermented characteristics in soymilk. Ann. Microbiol. 62, 1311–1320. Bentley, D.R., Balasubramanian, S., Swerdlow, H.P., Smith, G.P., Milton, J., Brown, C.G., Hall, K.P., Evers, D.J., Barnes, C.L., Bignell, H.R., Boutell, J.M., Bryant, J., Carter, R.J., Keira Cheetham, R., Cox, A.J., Ellis, D.J., Flatbush, M.R., Gormley, N.A., Humphray, S.J., Irving, L.J., Karbelashvili, M.S., Kirk, S.M., Li, H., Liu, X., Maisinger, K.S., Murray, L.J., Obradovic, B., Ost, T., Parkinson, M.L., Pratt, M.R., Rasolonjatovo, I.M., Reed, M.T., Rigatti, R., Rodighiero, C., Ross, M.T., Sabot, A., Sankar, S.V., Scally, A., Schroth, G.P., Smith, M.E., Smith, V.P., Spiridou, A., Torrance, P.E., Tzonev, S.S., Vermaas, E.H., Walter, K., Wu, X., Zhang, L., Alam, M.D., Anastasi, C., Aniebo, I.C., Bailey, D.M., Bancarz, I.R., Banerjee, S., Barbour, S.G., Baybayan, P.A., Benoit, V.A., Benson, K.F., Bevis, C., Black, P.J., Boodhun, A., Brennan, J.S., Bridgham, J.A.,
Brown, R.C., Brown, A.A., Buermann, D.H., Bundu, A.A., Burrows, J.C., Carter, N.P., Castillo, N., Chiara, E.C.M., Chang, S., Neil Cooley, R., Crake, N.R., Dada, O.O., Diakoumakos, K.D., Dominguez-Fernandez, B., Earnshaw, D.J., Egbujor, U.C., Elmore, D.W., Etchin, S.S., Ewan, M.R., Fedurco, M., Fraser, L.J., Fuentes Fajardo, K.V., Scott Furey, W., George, D., Gietzen, K.J., Goddard, C.P., Golda, G.S., Granieri, P.A., Green, D.E., Gustafson, D.L., Hansen, N.F., Harnish, K., Haudenschild, C.D., Heyer, N.I., Hims, M.M., Ho, J.T., Horgan, A.M., Hoschler, K., Hurwitz, S., Ivanov, D.V., Johnson, M.Q., James, T., Huw Jones, T.A., Kang, G.D., Kerelska, T.H., Kersey, A.D., Khrebtukova, I., Kindwall, A.P., Kingsbury, Z., Kokko-Gonzales, P.I., Kumar, A., Laurent, M.A., Lawley, C.T., Lee, S.E., Lee, X., Liao, A.K., Loch, J.A., Lok, M., Luo, S., Mammen, R.M., Martin, J.W., McCauley, P.G., McNitt, P., Mehta, P., Moon, K.W., Mullens, J.W., Newington, T., Ning, Z., Ling Ng, B., Novo, S.M., O‘Neill, M.J., Osborne, M.A., Osnowski, A., Ostadan, O., Paraschos, L.L., Pickering, L., Pike, A.C., Pike, A.C., Chris Pinkard, D., Pliskin, D.P., Podhasky, J., Quijano, V.J., Raczy, C., Rae, V.H., Rawlings, S.R., Chiva Rodriguez, A., Roe, P.M., Rogers, J., Rogert Bacigalupo, M.C., Romanov, N., Romieu, A., Roth, R.K., Rourke, N.J., Ruediger, S.T., Rusman, E., Sanches-Kuiper, R.M., Schenker, M.R., Seoane, J.M., Shaw, R.J., Shiver, M.K., Short, S.W., Sizto, N.L., Sluis, J.P., Smith, M.A., Ernest Sohna Sohna, J., Spence, E.J., Stevens, K., Sutton, N., Szajkowski, L., Tregidgo, C.L., Turcatti, G., Vandevondele, S., Verhovsky, Y., Virk, S.M., Wakelin, S., Walcott, G.C., Wang, J., Worsley, G.J., Yan, J., Yau, L., Zuerlein, M., Rogers, J., Mullikin, J.C., Hurles, M.E., McCooke, N.J., West, J.S., Oaks, F.L., Lundberg, P.L., Klenerman, D., Durbin, R., Smith, A.J., 2008. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456, 53–59. Feng, L., Reeves, P.R., Lan, R., Ren, Y., Gao, C., Zhou, Z., Ren, Y., Cheng, J., Wang, W., Wang, J., Qian, W., Li, D., Wang, L., 2008. A recalibrated molecular clock and independent origins for the cholera pandemic clones. PLOS ONE 3, e4053. Ferenci, T., Zhou, Z., Betteridge, T., Ren, Y., Liu, Y., Feng, L., Reeves, P.R., Wang, L., 2009. Genomic sequencing reveals regulatory mutations and recombinational events in the widely used MC4100 lineage of Escherichia coli K-12. J. Bacteriol. 191, 4025–4029. Kanmani, P., Satish Kumar, R., Yuvaraj, N., Paari, K.A., Pattukumar, V., Arul, V., 2013. Probiotics and its functionally valuable products – a review. Crit. Rev. Food Sci. Nutr. 53, 641–658. Kleerebezem, M., Boekhorst, J., van Kranenburg, R., Molenaar, D., Kuipers, O.P., Leer, R., Tarchini, R., Peters, S.A., Sandbrink, H.M., Fiers, M.W., Stiekema, W., Lankhorst, R.M., Bron, P.A., Hoffer, S.M., Groot, M.N., Kerkhoven, R., de Vries, M., Ursing, B., de Vos, W.M., Siezen, R.J., 2003. Complete genome sequence of Lactobacillus plantarum WCFS1. Proc. Natl. Acad. Sci. U. S. A. 100, 1990–1995. Li, H., Durbin, R., 2009. Fast and accurate short read alignment with BurrowsWheeler transform. Bioinformatics 25, 1754–1760. Margulies, M., Egholm, M., Altman, W.E., Attiya, S., Bader, J.S., Bemben, L.A., Berka, J., Braverman, M.S., Chen, Y.J., Chen, Z., Dewell, S.B., Du, L., Fierro, J.M., Gomes, X.V., Godwin, B.C., He, W., Helgesen, S., Ho, C.H., Irzyk, G.P., Jando, S.C., Alenquer, M.L., Jarvie, T.P., Jirage, K.B., Kim, J.B., Knight, J.R., Lanza, J.R., Leamon, J.H., Lefkowitz, S.M., Lei, M., Li, J., Lohman, K.L., Lu, H., Makhijani, V.B., McDade, K.E., McKenna, M.P., Myers, E.W., Nickerson, E., Nobile, J.R., Plant, R., Puc, B.P., Ronan, M.T., Roth, G.T., Sarkis, G.J., Simons, J.F., Simpson, J.W., Srinivasan, M., Tartaro, K.R., Tomasz, A., Vogt, K.A., Volkmer, G.A., Wang, S.H., Wang, Y., Weiner, M.P., Yu, P., Begley, R.F., Rothberg, J.M., 2005. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380. Shen, X., Yi, D., Ni, X., Zeng, D., Jing, B., Lei, M., Bian, Z., Zeng, Y., Li, T., Xin, J., 2014. Effects of Lactobacillus plantarum on production performance, immune characteristics, antioxidant status, and intestinal microﬂora of bursin-immunized broilers. Can. J. Microbiol. 60, 193–202. Wang, L., Zhang, J., Guo, Z., Kwok, L., Ma, C., Zhang, W., Lv, Q., Huang, W., Zhang, H., 2014. Effect of oral consumption of probiotic Lactobacillus planatarum P-8 on fecal microbiota, SIgA SCFAs, and TBAs of adults of different ages. Nutrition 30, 776–783, e771.