Journal of Biotechnology 194 (2015) 37–38
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcements
Complete genome sequence of Lactobacillus helveticus H9, a probiotic strain originated from kurut Yongfu Chen 1 , Wenyi Zhang 1 , Zhihong Sun, Bilighe Meng, Heping Zhang ∗ Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot 010018, China
a r t i c l e
i n f o
Article history: Received 17 November 2014 Accepted 27 November 2014 Available online 10 December 2014 Keywords: Lactobacillus helveticus Antihypertensive peptides Proteolytic system
a b s t r a c t Lactobacillus helveticus H9 is a probiotic strain that is able to produce antihypertensive peptides during milk fermentation. Its genome has a circular 1.87 Mb chromosome. Comparative genomic analysis revealed that the component of proteinases, peptide transport systems and peptidases in L. helveticus appeared to be strain-specific. Such information may help us to understand how the proteolytic system is related to its probiotic properties. © 2014 Published by Elsevier B.V.
Lactobacillus helveticus, commonly known as homofermentative, thermophilic lactic acid bacterium, is widely used in the manufacture process of dairy products (Beganovic et al., 2013). Proteolytic activity of this species is of importance in the respects of its high population number in dairy products and reduction of cheese bitterness (Griffiths and Tellez, 2013). In fact, the proteolytic activity coding by the proteolytic system of L. helveticus strains is considered to be an important source of antihypertensive peptides (Wakai et al., 2012). More recently, taking the advantage of the proteome and transcriptome analysis of L. helveticus H9 during milk fermentation, we proved that certain peptidases and peptide transport systems likely served as key factors of producing such peptides (Chen et al., 2014b; Zhang et al., 2014). However, due to a lack of clear genetic background regarding the proteolytic system-related proteins coded by this strain, the mechanism remains obscure. In the present study, L. helveticus H9, a novel probiotic strain isolated from kurut in Tibet of China (Chen et al., 2014a), was further sequenced by a combined use of 454 sequencing and illumina paired-end sequencing technology. Genomic libraries containing 3-kb inserts were constructed, and 137,143 paired-end reads and 34,068 single-end reads were generated using the GS FLX system, giving 25.4-fold coverage of the genome. The majority (92.54%) of reads were assembled into 11
∗ 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). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jbiotec.2014.11.038 0168-1656/© 2014 Published by Elsevier B.V.
Table 1 General genome features of Lactobacillus helveticus H9. Attributes
Value
Genome size (bp) GC content (%) rRNAs tRNA s Pseudogenes Total predicted CDSs
1.87 Mb 37% 12 60 120 1887
large scaffolds, including 54 non-redundant contigs, using the 454 Newbler assembler (454 Life Sciences, Branford, CT). A total of 6,621,398 reads (2.5-kb library) were generated to reach a depth of 265-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. helveticus H9 has a circular 1.87 Mb chromosome with a GC content of 37% (Table 1). There are 1887 coding genes, 4 rRNA operons, 60 tRNAs, and 120 pseudogenes in the chromosome. Comparing the genomes of L. helveticus H10 (Zhao et al., 2011) and L. helveticus H9 revealed that the proteolytic system of these strains are highly similar except for slight differences in the number and the component of proteinases, peptide transport systems and peptidases. In L. helveticus H10, homologs of a proteinase (LBHH 0440), an aminopeptidase (LBHH 0527) and an endopeptidase (LBHH 2039), are annotated as pseudogenes.
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Y. Chen et al. / Journal of Biotechnology 194 (2015) 37–38
Similarly, four genes, namely an oligopeptide ABC transporter solute-binding component (LBH 1145), a di-tripeptide transporter (LBH 1838) and two dipeptidases (LBH 1471 and LBH 0028), are present as pseudogenes in L. helveticus H9. Previously, it has been demonstrated by an in vitro analysis that L. helveticus H9 had a better ability to produce antihypertensive peptides than L. helveticus H10 (Chen et al., 2014a). However, the exact reason leading to the different capacity in the release of antihypertensive peptides needs to be further explored. GenBank accession number: The genome information of L. helveticus H9 is available in GenBank database under the accession number of CP002427.1. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC No. 4811). Acknowledgements This research was supported by the National Natural Science Foundation of China (Grant Nos. 31101315 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), 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 Beganovic, J., Kos, B., Lebos Pavunc, A., Uroic, K., Dzidara, P., Suskovic, J., 2013. Proteolytic activity of probiotic strain Lactobacillus helveticus M92. Anaerobe 20, 58–64. Chen, Y., Liu, W., Xue, J., Yang, J., Chen, X., Shao, Y., Kwok, L.Y., Bilige, M., Mang, L., Zhang, H., 2014a. Angiotensin-converting enzyme inhibitory activity of Lactobacillus helveticus strains from traditional fermented dairy foods and antihypertensive effect of fermented milk of strain H9. J. Dairy Sci., http://dx.doi.org/10.3168/jds.2014-7962. Chen, Y.F., Zhao, W.J., Wu, R.N., Sun, Z.H., Zhang, W.Y., Wang, J.C., Bilige, M., Zhang, H.P., 2014b. Proteome analysis of Lactobacillus helveticus H9 during growth in skim milk. J. Dairy Sci., http://dx.doi.org/10.3168/jds.2014-8520. 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. Griffiths, M.W., Tellez, A.M., 2013. Lactobacillus helveticus: the proteolytic system. Front. Microbiol., http://dx.doi.org/10.3389/fmicb.2013.00030. Li, H., Durbin, R., 2009. Fast and accurate short read alignment with BurrowsWheeler transform. Bioinformatics 25, 1754–1760. Wakai, T., Yamaguchi, N., Hatanaka, M., Nakamura, Y., Yamamoto, N., 2012. Repressive processing of antihypertensive peptides, Val-Pro-Pro and Ile-Pro-Pro, in Lactobacillus helveticus fermented milk by added peptides. J. Biosci. Bioeng. 114, 133–137. Zhang, W.Y., Chen, Y.F., Zhao, W.J., Kwok, L.Y., Zhang, H.P., 2014. Gene expression of proteolytic system of Lactobacillus helveticus H9 during milk fermentation. Ann. Microbiol., http://dx.doi.org/10.1007/s13213-13014-10902-13213. Zhao, W., Chen, Y., Sun, Z., Wang, J., Zhou, Z., Sun, T., Wang, L., Chen, W., Zhang, H., 2011. Complete genome sequence of Lactobacillus helveticus H10. J. Bacteriol. 193, 2666–2667.
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcements
Complete genome sequence of Lactobacillus helveticus H9, a probiotic strain originated from kurut Yongfu Chen 1 , Wenyi Zhang 1 , Zhihong Sun, Bilighe Meng, Heping Zhang ∗ Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot 010018, China
a r t i c l e
i n f o
Article history: Received 17 November 2014 Accepted 27 November 2014 Available online 10 December 2014 Keywords: Lactobacillus helveticus Antihypertensive peptides Proteolytic system
a b s t r a c t Lactobacillus helveticus H9 is a probiotic strain that is able to produce antihypertensive peptides during milk fermentation. Its genome has a circular 1.87 Mb chromosome. Comparative genomic analysis revealed that the component of proteinases, peptide transport systems and peptidases in L. helveticus appeared to be strain-specific. Such information may help us to understand how the proteolytic system is related to its probiotic properties. © 2014 Published by Elsevier B.V.
Lactobacillus helveticus, commonly known as homofermentative, thermophilic lactic acid bacterium, is widely used in the manufacture process of dairy products (Beganovic et al., 2013). Proteolytic activity of this species is of importance in the respects of its high population number in dairy products and reduction of cheese bitterness (Griffiths and Tellez, 2013). In fact, the proteolytic activity coding by the proteolytic system of L. helveticus strains is considered to be an important source of antihypertensive peptides (Wakai et al., 2012). More recently, taking the advantage of the proteome and transcriptome analysis of L. helveticus H9 during milk fermentation, we proved that certain peptidases and peptide transport systems likely served as key factors of producing such peptides (Chen et al., 2014b; Zhang et al., 2014). However, due to a lack of clear genetic background regarding the proteolytic system-related proteins coded by this strain, the mechanism remains obscure. In the present study, L. helveticus H9, a novel probiotic strain isolated from kurut in Tibet of China (Chen et al., 2014a), was further sequenced by a combined use of 454 sequencing and illumina paired-end sequencing technology. Genomic libraries containing 3-kb inserts were constructed, and 137,143 paired-end reads and 34,068 single-end reads were generated using the GS FLX system, giving 25.4-fold coverage of the genome. The majority (92.54%) of reads were assembled into 11
∗ 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). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jbiotec.2014.11.038 0168-1656/© 2014 Published by Elsevier B.V.
Table 1 General genome features of Lactobacillus helveticus H9. Attributes
Value
Genome size (bp) GC content (%) rRNAs tRNA s Pseudogenes Total predicted CDSs
1.87 Mb 37% 12 60 120 1887
large scaffolds, including 54 non-redundant contigs, using the 454 Newbler assembler (454 Life Sciences, Branford, CT). A total of 6,621,398 reads (2.5-kb library) were generated to reach a depth of 265-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. helveticus H9 has a circular 1.87 Mb chromosome with a GC content of 37% (Table 1). There are 1887 coding genes, 4 rRNA operons, 60 tRNAs, and 120 pseudogenes in the chromosome. Comparing the genomes of L. helveticus H10 (Zhao et al., 2011) and L. helveticus H9 revealed that the proteolytic system of these strains are highly similar except for slight differences in the number and the component of proteinases, peptide transport systems and peptidases. In L. helveticus H10, homologs of a proteinase (LBHH 0440), an aminopeptidase (LBHH 0527) and an endopeptidase (LBHH 2039), are annotated as pseudogenes.
38
Y. Chen et al. / Journal of Biotechnology 194 (2015) 37–38
Similarly, four genes, namely an oligopeptide ABC transporter solute-binding component (LBH 1145), a di-tripeptide transporter (LBH 1838) and two dipeptidases (LBH 1471 and LBH 0028), are present as pseudogenes in L. helveticus H9. Previously, it has been demonstrated by an in vitro analysis that L. helveticus H9 had a better ability to produce antihypertensive peptides than L. helveticus H10 (Chen et al., 2014a). However, the exact reason leading to the different capacity in the release of antihypertensive peptides needs to be further explored. GenBank accession number: The genome information of L. helveticus H9 is available in GenBank database under the accession number of CP002427.1. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC No. 4811). Acknowledgements This research was supported by the National Natural Science Foundation of China (Grant Nos. 31101315 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), 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 Beganovic, J., Kos, B., Lebos Pavunc, A., Uroic, K., Dzidara, P., Suskovic, J., 2013. Proteolytic activity of probiotic strain Lactobacillus helveticus M92. Anaerobe 20, 58–64. Chen, Y., Liu, W., Xue, J., Yang, J., Chen, X., Shao, Y., Kwok, L.Y., Bilige, M., Mang, L., Zhang, H., 2014a. Angiotensin-converting enzyme inhibitory activity of Lactobacillus helveticus strains from traditional fermented dairy foods and antihypertensive effect of fermented milk of strain H9. J. Dairy Sci., http://dx.doi.org/10.3168/jds.2014-7962. Chen, Y.F., Zhao, W.J., Wu, R.N., Sun, Z.H., Zhang, W.Y., Wang, J.C., Bilige, M., Zhang, H.P., 2014b. Proteome analysis of Lactobacillus helveticus H9 during growth in skim milk. J. Dairy Sci., http://dx.doi.org/10.3168/jds.2014-8520. 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. Griffiths, M.W., Tellez, A.M., 2013. Lactobacillus helveticus: the proteolytic system. Front. Microbiol., http://dx.doi.org/10.3389/fmicb.2013.00030. Li, H., Durbin, R., 2009. Fast and accurate short read alignment with BurrowsWheeler transform. Bioinformatics 25, 1754–1760. Wakai, T., Yamaguchi, N., Hatanaka, M., Nakamura, Y., Yamamoto, N., 2012. Repressive processing of antihypertensive peptides, Val-Pro-Pro and Ile-Pro-Pro, in Lactobacillus helveticus fermented milk by added peptides. J. Biosci. Bioeng. 114, 133–137. Zhang, W.Y., Chen, Y.F., Zhao, W.J., Kwok, L.Y., Zhang, H.P., 2014. Gene expression of proteolytic system of Lactobacillus helveticus H9 during milk fermentation. Ann. Microbiol., http://dx.doi.org/10.1007/s13213-13014-10902-13213. Zhao, W., Chen, Y., Sun, Z., Wang, J., Zhou, Z., Sun, T., Wang, L., Chen, W., Zhang, H., 2011. Complete genome sequence of Lactobacillus helveticus H10. J. Bacteriol. 193, 2666–2667.
