Accepted Manuscript Title: Complete genome sequence of Lactobacillus helveticus MB2-1, a probiotic bacterium producing exopolysaccharides Author: Wei Li Xiudong Xia Xiaohong Chen Xin Rui Mei Jiang Qiuqin Zhang Jianzhong Zhou Mingsheng Dong PII: DOI: Reference:
S0168-1656(15)30005-5 http://dx.doi.org/doi:10.1016/j.jbiotec.2015.05.021 BIOTEC 7125
To appear in:
Journal of Biotechnology
Received date: Accepted date:
21-5-2015 28-5-2015
Please cite this article as: Li, Wei, Xia, Xiudong, Chen, Xiaohong, Rui, Xin, Jiang, Mei, Zhang, Qiuqin, Zhou, Jianzhong, Dong, Mingsheng, Complete genome sequence of Lactobacillus helveticus MB2-1, a probiotic bacterium producing exopolysaccharides.Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2015.05.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Complete genome sequence of Lactobacillus helveticus MB2-1, a probiotic bacterium producing exopolysaccharides Wei Lia, Xiudong Xiab†, Xiaohong Chena, Xin Ruia, Mei Jianga, Qiuqin Zhang, Jianzhong Zhoub, Mingsheng Donga,* aCollege
of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095,
P.R. China. bInstitute
of Agro-Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu
210095, P.R. China.
†Xiudong Xia is the co-first author *Corresponding author: Dr Mingsheng Dong* College of Food Science and Technology Nanjing Agricultural University 1 Weigang Road, Nanjing, Jiangsu, P.R. China Tel: +86 25 84396989 Fax: +86 25 84399090 E-mail address: [email protected] ABSTRACT Lactobacillus helveticus MB2-1 is a probiotic bacterium producing exopolysaccharides (EPS), which was isolated from traditional Sayram ropy fermented milk in southern Xinjiang, China. The genome consists of a circular 2,084,058 bp chromosome with no plasmid. The genome sequence indicated that this strain includes a 15.20 kb gene cluster involved in EPS biosynthesis. Genome sequencing information has provided the basis for understanding the potential molecular mechanism behind the EPS production. Keywords: Lactobacillus helveticus; Genome; Probiotic; Exopolysaccharides (EPS). Sayram ropy fermented milk (SRFM) is a type of domestic fermented milk in southern Xinjiang province. The history of making and drinking SRFM dates back to several thousand years ago. It is
1
usually made from local yellow cattle’s milk fermented by its normal microbiota. SRFM is made by a set-style fermentation process and is consumed directly without maturation. It has a stringy texture, a good flavor, and a pleasant taste. The most notable characteristic of SRFM is its high viscosity. Using a scoop or glass rod, SRFM would generate a very long filamentous viscosity material that could reach 0.2–0.3 m (Li et al., 2012). SRFM is believed to be beneficial in the cure of digestives diseases, including diarrhoea, gastrohelcosis and chronic gastroenteritis. The main beneficial effects are believed to have derived from bacterially produced exopolysaccharides (EPS) (Li et al., 2014b). Lactobacillus helveticus MB2-1 is a novel probiotic strain identified by screening of lactic acid bacteria isolated from SRFM samples collected in southern Xinjiang, China, and exhibits high-level resistance to acid and bile stresses, as well as antibacterial, antioxidative and anticancer properties (Li et al., 2014a). Among many outstanding characteristics, it exhibits a strong ability to produce EPS (Li et al., 2014b). In order to interrogate the genome sequence of L. helveticus MB2-1 with regard to its probiotic properties. Whole-genome sequencing of strain MB2-1 was carried out using the PacBio RS II (Pacific Biosciences, Menlo Park, CA) platform with a 10-kb insert library and P4/C2 chemistry and sequenced on 2 single-molecule real-time (SMRT) cells. De novo assembly was conducted using the hierarchical genome assembly process (HGAP) workflow, including consensus polishing with Quiver. This workflow resulted in a single-contig complete closed genome. Comparative genomic analysis was performed with the published genomes of L. helveticus strains CNRZ32 (GenBank accession no. NC_021744.1) (Broadbent et al., 2013), MTCC5463 (GenBank accession no. AEYL01000001.1) (Prajapati et al., 2011), DPC4571 (GenBank accession no. NC_010080.1) (Callanan et al., 2008), R0052 (GenBank accession no. NC_018528.1) (Tompkins et al., 2012), H10 (GenBank accession no. NC_017467.1) (Zhao et al., 2011), H9 (GenBank accession no. CP002427) (Chen et al., 2015) and DSM20075 (GenBank accession no. NZ_ACLM00000000) (Cremonesi et al., 2012). The complete genome of L. helveticus MB2-1 is composed of a single, circular chromosome of 2,084,058 bp. The average G + C content of the chromosome is 36.93% (Table 1). There are 2,167 genes in total, including 1736 coding genes, 12 rRNA operons and 63 tRNAs in the chromosome. This genome size is smaller than that of L. helveticus CNRZ32 (2.226 M), L. helveticus H10 (2.146 M) and L. helveticus R0052 (2.129 M) but slightly larger than the complete genomes of L. helveticus
2
DPC4571 (2.080 M), L. helveticus MTCC5463 (1.911 M), L. helveticus H9 (1.871 M) and L. helveticus DSM20075 (1.809 M). Comparative genome analysis showed that MB2-1 carries most of the core genes of L. helveticus, with no known pathogenic genes identified. Among the eight genomes, MB2-1 and H9 show the highest similarity with respect to gene order and genome structure. There are total of 1,232 coding genes (70.97%) in L. helveticus MB2-1 with predicted functions and 504 coding genes (29.03%) with no predicted functions. A total of 1, 232 coding genes can be associated with clusters of orthologous genes (COGs) functions belonging to 19 COGs. In summary, there are 208 genes for replication, recombination and repair, 129 genes for translation, ribosomal structure and biogenesis, 73 genes for amino acid transport and metabolism, 70 genes for carbohydrate transport and metabolism, 69 genes for translation, 68 genes involved in cell wall/membrane/envelope biogenesis, 67 genes for nucleotide transport and metabolism, 50 genes for inorganic ion transport and metabolism, 41 genes for energy production and conversion, 39 genes for posttranslational modification, protein turnover and chaperones, 36 genes for defense mechanisms, 32 genes for lipid transport and metabolism, 31 genes for coenzyme transport and metabolism, 20 genes for signal transduction mechanisms, 17 genes involved in cell cycle control, cell division, chromosome partitioning, 9 genes for intracellular trafficking, secretion and vesicular transport, and 4 genes involved in secondary metabolites biosynthesis, transport and catabolism. As mentioned above, one of the most significant features of MB2-1 is the ability to produce high viscosity and yield of EPS. The MB2-1 genome carries a 15.20 kb EPS gene cluster (Contig3orf02320 to Contig3-orf02340), which contains 21 EPS-related genes. These genes showed high similarity to those involved in the regulation, polymerization (polymerase)-chain length determination and polymerization-export of the EPS. Eleven of these genes have some homologies with other strains of L. helveticus, including DPC4571, H9, H10 and R0052, etc. (Cremonesi et al., 2012). The remaining 10 genes in the EPS gene clusters are uniquely present in the MB2-1 and regarded as the key enzymes to determine the formation of unique EPS (Lebeer et al., 2009; Jolly et al., 2002). In conclusion, the genome sequence of L. helveticus MB2-1 proves its safety and stability in commercial use and reveals many potential properties for further elucidation. In addition, comparative genomics analysis and functional genomics analysis could also be carried out to trace the origin and evolution of this bacterium.
3
Nucleotide sequence accession numbers: Genome information for the chromosome of L. helveticus MB2-1 has been deposited in the GenBank database with accession number
CP011386. The strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC NO.5729). Acknowledgements This work was co-supported by Key Projects in the National Science & Technology Pillar Program (No. 2013BAD18B01-4), High-Tech Research and Development Program of China (No. 2011AA100903), National Natural Science Foundation of China (No. 31371807 and 31201422 ) and was also supported by the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Broadbent, J.R., Hughes, J.E., Welker, D.L., Tompkins, T.A., Steele, J.L., 2013. Complete genome sequence for Lactobacillus helveticus CNRZ 32, an industrial cheese starter and cheese flavor adjunct. Genome Announc. 1, e00376-13. Callanan, M., Kaleta, P., O’Callaghan, J., O’Sullivan, O., Jordan, K., McAuliffe, O., SangradorVegas, A., Slattery, L., Fitzgerald, G.F., Beresford, T., Ross, R.P., 2008. Genome sequence of Lactobacillus helveticus, an organism distinguished by selective gene loss and insertion sequence element expansion. J. Bacteriol. 190, 727–735. Chen, Y.F., Zhang, W.Y., Sun, Z.H., Meng, B.L.G., Zhang, H.P., 2015. Complete genome sequence of Lactobacillus helveticus H9, a probiotic strain originated from kurut. J. Biotechnol. 194, 37– 38. Cremonesi, P., Chessa, S., Castiglioni, B., 2012. Genome sequence and analysis of Lactobacillus helveticus. Front. Microbiol. 3, 1–13. Lebeer, S., Verhoeven, T.L.A., Francius, G., Schoofs, G., Lambrichts, I., Dufrêne, Y., Vanderleyden, J., De Keersmaecker, S.C.J., 2009. Identification of a gene cluster for the biosynthesis of a long, galactose-rich exopolysaccharide in Lactobacillus rhamnosus GG and functional analysis of the priming glycosyltransferase. J. Bacteriol. 75, 3554–3563. Jolly, L., Newell, J., Porcelli, I., Vincent, S.J.F., Stingele, F., 2002. Lactobalillus helveticus glycosyltransferases: from genes to carbohydrate synthesis. Glycobiology. 12, 319–327.
Li, W., Mutuvulla, M., Chen, X.H, Jiang, M., Dong, M.S., 2012. Isolation and identification of
4
high viscosity-producing lactic acid bacteria from a traditional fermented milk in Xinjiang and its role in fermentation process. Eur. Food Res. Technol. 235, 497–505. Li, W., Ji, J., Chen, X.H, Jiang, M., Rui, X., Dong, M.S., 2014a. Structural elucidation and antioxidant activities of exopolysaccharides from Lactobacillus helveticus MB2-1. Carbohyd. Polym. 102, 351–359.
Li, W., Ji, J., Rui, X., Yu, J.J, Tang, W.Z, Chen, X.H, Jiang, M., Dong, M.S., 2014b. Production of exopolysaccharides by Lactobacillus helveticus MB2-1 and its functional characteristics in vitro. LWT – Food Sci. Technol. 59, 732–739. Prajapati, B., Khedkar, C.D., Chitra, J., Suja, S., Mishra, V., Sreeja, V., Patel, R.K., Ahir, V.B., Bhatt, V.D., Sajnani, M.R., Jakhesara, S.J., Koringa, P.G., Joshi, C.G., 2011. Whole-Genome shotgun sequencing of an indian-origin Lactobacillus helveticus strain, MTCC 5463, with probiotic potential. J. Bacteriol. 193, 4282–4283. Tompkins, T.A., Barreau, G., Broadbent, J.R., 2012. Complete genome sequence of Lactobacillus helveticus R0052, a commercial probiotic strain. J. Bacteriol. 194, 6349. Zhao, W.J., Chen, Y.F., Sun, Z.H., Wang, J.C., Zhou, Z.M., Sun, T.S., Wang, L., Chen, W., Zhang, H.P., 2011. Complete genome sequence of Lactobacillus helveticus H10. J. Bacteriol. 193, 2666– 2667. Table 1 General genome features of Lactobacillus helveticus MB2-1. Attributes Genome size (bp) Total number of contigs GC content (%) Plasmids rRNAs tRNAs Predicted genes
Values 2,084,058 1 36.93 0 12 63 1736
5
S0168-1656(15)30005-5 http://dx.doi.org/doi:10.1016/j.jbiotec.2015.05.021 BIOTEC 7125
To appear in:
Journal of Biotechnology
Received date: Accepted date:
21-5-2015 28-5-2015
Please cite this article as: Li, Wei, Xia, Xiudong, Chen, Xiaohong, Rui, Xin, Jiang, Mei, Zhang, Qiuqin, Zhou, Jianzhong, Dong, Mingsheng, Complete genome sequence of Lactobacillus helveticus MB2-1, a probiotic bacterium producing exopolysaccharides.Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2015.05.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Complete genome sequence of Lactobacillus helveticus MB2-1, a probiotic bacterium producing exopolysaccharides Wei Lia, Xiudong Xiab†, Xiaohong Chena, Xin Ruia, Mei Jianga, Qiuqin Zhang, Jianzhong Zhoub, Mingsheng Donga,* aCollege
of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095,
P.R. China. bInstitute
of Agro-Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu
210095, P.R. China.
†Xiudong Xia is the co-first author *Corresponding author: Dr Mingsheng Dong* College of Food Science and Technology Nanjing Agricultural University 1 Weigang Road, Nanjing, Jiangsu, P.R. China Tel: +86 25 84396989 Fax: +86 25 84399090 E-mail address: [email protected] ABSTRACT Lactobacillus helveticus MB2-1 is a probiotic bacterium producing exopolysaccharides (EPS), which was isolated from traditional Sayram ropy fermented milk in southern Xinjiang, China. The genome consists of a circular 2,084,058 bp chromosome with no plasmid. The genome sequence indicated that this strain includes a 15.20 kb gene cluster involved in EPS biosynthesis. Genome sequencing information has provided the basis for understanding the potential molecular mechanism behind the EPS production. Keywords: Lactobacillus helveticus; Genome; Probiotic; Exopolysaccharides (EPS). Sayram ropy fermented milk (SRFM) is a type of domestic fermented milk in southern Xinjiang province. The history of making and drinking SRFM dates back to several thousand years ago. It is
1
usually made from local yellow cattle’s milk fermented by its normal microbiota. SRFM is made by a set-style fermentation process and is consumed directly without maturation. It has a stringy texture, a good flavor, and a pleasant taste. The most notable characteristic of SRFM is its high viscosity. Using a scoop or glass rod, SRFM would generate a very long filamentous viscosity material that could reach 0.2–0.3 m (Li et al., 2012). SRFM is believed to be beneficial in the cure of digestives diseases, including diarrhoea, gastrohelcosis and chronic gastroenteritis. The main beneficial effects are believed to have derived from bacterially produced exopolysaccharides (EPS) (Li et al., 2014b). Lactobacillus helveticus MB2-1 is a novel probiotic strain identified by screening of lactic acid bacteria isolated from SRFM samples collected in southern Xinjiang, China, and exhibits high-level resistance to acid and bile stresses, as well as antibacterial, antioxidative and anticancer properties (Li et al., 2014a). Among many outstanding characteristics, it exhibits a strong ability to produce EPS (Li et al., 2014b). In order to interrogate the genome sequence of L. helveticus MB2-1 with regard to its probiotic properties. Whole-genome sequencing of strain MB2-1 was carried out using the PacBio RS II (Pacific Biosciences, Menlo Park, CA) platform with a 10-kb insert library and P4/C2 chemistry and sequenced on 2 single-molecule real-time (SMRT) cells. De novo assembly was conducted using the hierarchical genome assembly process (HGAP) workflow, including consensus polishing with Quiver. This workflow resulted in a single-contig complete closed genome. Comparative genomic analysis was performed with the published genomes of L. helveticus strains CNRZ32 (GenBank accession no. NC_021744.1) (Broadbent et al., 2013), MTCC5463 (GenBank accession no. AEYL01000001.1) (Prajapati et al., 2011), DPC4571 (GenBank accession no. NC_010080.1) (Callanan et al., 2008), R0052 (GenBank accession no. NC_018528.1) (Tompkins et al., 2012), H10 (GenBank accession no. NC_017467.1) (Zhao et al., 2011), H9 (GenBank accession no. CP002427) (Chen et al., 2015) and DSM20075 (GenBank accession no. NZ_ACLM00000000) (Cremonesi et al., 2012). The complete genome of L. helveticus MB2-1 is composed of a single, circular chromosome of 2,084,058 bp. The average G + C content of the chromosome is 36.93% (Table 1). There are 2,167 genes in total, including 1736 coding genes, 12 rRNA operons and 63 tRNAs in the chromosome. This genome size is smaller than that of L. helveticus CNRZ32 (2.226 M), L. helveticus H10 (2.146 M) and L. helveticus R0052 (2.129 M) but slightly larger than the complete genomes of L. helveticus
2
DPC4571 (2.080 M), L. helveticus MTCC5463 (1.911 M), L. helveticus H9 (1.871 M) and L. helveticus DSM20075 (1.809 M). Comparative genome analysis showed that MB2-1 carries most of the core genes of L. helveticus, with no known pathogenic genes identified. Among the eight genomes, MB2-1 and H9 show the highest similarity with respect to gene order and genome structure. There are total of 1,232 coding genes (70.97%) in L. helveticus MB2-1 with predicted functions and 504 coding genes (29.03%) with no predicted functions. A total of 1, 232 coding genes can be associated with clusters of orthologous genes (COGs) functions belonging to 19 COGs. In summary, there are 208 genes for replication, recombination and repair, 129 genes for translation, ribosomal structure and biogenesis, 73 genes for amino acid transport and metabolism, 70 genes for carbohydrate transport and metabolism, 69 genes for translation, 68 genes involved in cell wall/membrane/envelope biogenesis, 67 genes for nucleotide transport and metabolism, 50 genes for inorganic ion transport and metabolism, 41 genes for energy production and conversion, 39 genes for posttranslational modification, protein turnover and chaperones, 36 genes for defense mechanisms, 32 genes for lipid transport and metabolism, 31 genes for coenzyme transport and metabolism, 20 genes for signal transduction mechanisms, 17 genes involved in cell cycle control, cell division, chromosome partitioning, 9 genes for intracellular trafficking, secretion and vesicular transport, and 4 genes involved in secondary metabolites biosynthesis, transport and catabolism. As mentioned above, one of the most significant features of MB2-1 is the ability to produce high viscosity and yield of EPS. The MB2-1 genome carries a 15.20 kb EPS gene cluster (Contig3orf02320 to Contig3-orf02340), which contains 21 EPS-related genes. These genes showed high similarity to those involved in the regulation, polymerization (polymerase)-chain length determination and polymerization-export of the EPS. Eleven of these genes have some homologies with other strains of L. helveticus, including DPC4571, H9, H10 and R0052, etc. (Cremonesi et al., 2012). The remaining 10 genes in the EPS gene clusters are uniquely present in the MB2-1 and regarded as the key enzymes to determine the formation of unique EPS (Lebeer et al., 2009; Jolly et al., 2002). In conclusion, the genome sequence of L. helveticus MB2-1 proves its safety and stability in commercial use and reveals many potential properties for further elucidation. In addition, comparative genomics analysis and functional genomics analysis could also be carried out to trace the origin and evolution of this bacterium.
3
Nucleotide sequence accession numbers: Genome information for the chromosome of L. helveticus MB2-1 has been deposited in the GenBank database with accession number
CP011386. The strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC NO.5729). Acknowledgements This work was co-supported by Key Projects in the National Science & Technology Pillar Program (No. 2013BAD18B01-4), High-Tech Research and Development Program of China (No. 2011AA100903), National Natural Science Foundation of China (No. 31371807 and 31201422 ) and was also supported by the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Broadbent, J.R., Hughes, J.E., Welker, D.L., Tompkins, T.A., Steele, J.L., 2013. Complete genome sequence for Lactobacillus helveticus CNRZ 32, an industrial cheese starter and cheese flavor adjunct. Genome Announc. 1, e00376-13. Callanan, M., Kaleta, P., O’Callaghan, J., O’Sullivan, O., Jordan, K., McAuliffe, O., SangradorVegas, A., Slattery, L., Fitzgerald, G.F., Beresford, T., Ross, R.P., 2008. Genome sequence of Lactobacillus helveticus, an organism distinguished by selective gene loss and insertion sequence element expansion. J. Bacteriol. 190, 727–735. Chen, Y.F., Zhang, W.Y., Sun, Z.H., Meng, B.L.G., Zhang, H.P., 2015. Complete genome sequence of Lactobacillus helveticus H9, a probiotic strain originated from kurut. J. Biotechnol. 194, 37– 38. Cremonesi, P., Chessa, S., Castiglioni, B., 2012. Genome sequence and analysis of Lactobacillus helveticus. Front. Microbiol. 3, 1–13. Lebeer, S., Verhoeven, T.L.A., Francius, G., Schoofs, G., Lambrichts, I., Dufrêne, Y., Vanderleyden, J., De Keersmaecker, S.C.J., 2009. Identification of a gene cluster for the biosynthesis of a long, galactose-rich exopolysaccharide in Lactobacillus rhamnosus GG and functional analysis of the priming glycosyltransferase. J. Bacteriol. 75, 3554–3563. Jolly, L., Newell, J., Porcelli, I., Vincent, S.J.F., Stingele, F., 2002. Lactobalillus helveticus glycosyltransferases: from genes to carbohydrate synthesis. Glycobiology. 12, 319–327.
Li, W., Mutuvulla, M., Chen, X.H, Jiang, M., Dong, M.S., 2012. Isolation and identification of
4
high viscosity-producing lactic acid bacteria from a traditional fermented milk in Xinjiang and its role in fermentation process. Eur. Food Res. Technol. 235, 497–505. Li, W., Ji, J., Chen, X.H, Jiang, M., Rui, X., Dong, M.S., 2014a. Structural elucidation and antioxidant activities of exopolysaccharides from Lactobacillus helveticus MB2-1. Carbohyd. Polym. 102, 351–359.
Li, W., Ji, J., Rui, X., Yu, J.J, Tang, W.Z, Chen, X.H, Jiang, M., Dong, M.S., 2014b. Production of exopolysaccharides by Lactobacillus helveticus MB2-1 and its functional characteristics in vitro. LWT – Food Sci. Technol. 59, 732–739. Prajapati, B., Khedkar, C.D., Chitra, J., Suja, S., Mishra, V., Sreeja, V., Patel, R.K., Ahir, V.B., Bhatt, V.D., Sajnani, M.R., Jakhesara, S.J., Koringa, P.G., Joshi, C.G., 2011. Whole-Genome shotgun sequencing of an indian-origin Lactobacillus helveticus strain, MTCC 5463, with probiotic potential. J. Bacteriol. 193, 4282–4283. Tompkins, T.A., Barreau, G., Broadbent, J.R., 2012. Complete genome sequence of Lactobacillus helveticus R0052, a commercial probiotic strain. J. Bacteriol. 194, 6349. Zhao, W.J., Chen, Y.F., Sun, Z.H., Wang, J.C., Zhou, Z.M., Sun, T.S., Wang, L., Chen, W., Zhang, H.P., 2011. Complete genome sequence of Lactobacillus helveticus H10. J. Bacteriol. 193, 2666– 2667. Table 1 General genome features of Lactobacillus helveticus MB2-1. Attributes Genome size (bp) Total number of contigs GC content (%) Plasmids rRNAs tRNAs Predicted genes
Values 2,084,058 1 36.93 0 12 63 1736
5
