Journal of Biotechnology 179 (2014) 63–64
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcement
Complete genome sequence of Lactobacillus reuteri I5007, a probiotic strain isolated from healthy piglet Chengli Hou 1 , Qingwei Wang 1 , Xiangfang Zeng, Fengjuan Yang, Jiang Zhang, Hong Liu, Xi Ma, Shiyan Qiao ∗ State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing 100193, China
a r t i c l e
i n f o
Article history: Received 9 March 2014 Accepted 11 March 2014 Available online 28 March 2014
a b s t r a c t Lactobacillus reuteri I5007 is a well-characterized probiotic strain isolated from the colonic mucosa of healthy weaning piglets. Here, we present the complete genome sequence of this strain, which consists of a circular chromosome and six distinct plasmids. © 2014 Elsevier B.V. All rights reserved.
Keywords: Lactobacillus reuteri Genome sequence Probiotic Piglet
Lactobacillus reuteri is a resident of the human and animal intestinal tracts (Lee et al., 2008). Comparative genomic analysis revealed that the evolution of L. reuteri with vertebrates resulted in the emergence of host specialization (Frese et al., 2011). Lactobacillus reuteri I5007, initially known as Lactobacillus fermentum I5007, was isolated from the colonic mucosa of healthy weaning piglets (Huang et al., 2004). Subsequent studies showed that I5007 has several important characteristics, including: (1) strong adhesion to Caco-2 cells and porcine intestinal mucus and competitiveness against Salmonella typhimurium and Escherichia coli (Li et al., 2008; Yang et al., 2007); (2) positive regulation of immune function in piglets (Wang et al., 2009a); (3) improvement in antioxidant status of pigs (Wang et al., 2013, 2009b). Moreover, I5007 has an interaction with Caco-2 cells (Yang et al., 2007) and can affect the small intestinal proteomes of weaning piglets (Wang et al., 2012). In addition, L. reuteri I5007 has strong adhesion to other host cells, including IPEC-J2, and IEC-6, and can produce large amounts of exopolysaccharides (unpublished data). To gain insight into its genetic and physiological properties, the whole genome sequence of I5007 was determined. L. reuteri I5007 genome sequence was determined using a Roche Genome Sequencer FLX system and PCR-based gap filling
∗ Corresponding author at: Department of Animal Science and Technology, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, China. Tel.: +86 10 62731456; fax: +86 10 62733688. E-mail address: qiaoshy@mafic.ac.cn (S. Qiao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jbiotec.2014.03.019 0168-1656/© 2014 Elsevier B.V. All rights reserved.
strategies (Hao et al., 2011). A total of 87,911 reads totaling 57,014,356 bases was obtained, resulting in 22-fold genome coverage. De novo genome assembly of the resulting reads was performed using the Newbler v2.3 assembly software package, 110 contigs ranging from 500 bp to 141,014 bp were obtained with total size of 198,716 bp (the N50 contig size is 39,886 bp). All the relationships between the contigs were determined by multiplex PCR, while the gaps between the contigs were filled in by PCR reactions. The regions with low quality were re-sequenced to be precise, and the final sequencing accuracy was 99.9988%. Putative protein-coding sequences (ORFs) were identified with Glimmer 3 (Azcarate-Peril et al., 2004). tRNA genes were annotated by tRNAscan-SE (Lowe and Eddy, 1997), while rRNA genes were predicted by RNAmmer (Lagesen et al., 2007). Functional annotation of CDSs was performed through blastp searches against non-redundant protein database/NCBI, followed by manual inspection. CRISPR finder tool was used to determine clustered regularly interspaced short palindromic repeat (CRISPR) (Grissa et al., 2007). The complete genome of L. reuteri I5007 contained a single circular chromosome of 1,947,706 bp and six indigenous plasmids with lengths ranging from 6499 to 53,021 bp (Table 1). The chromosome contained 1891 protein-coding genes with a GC content of 38.99%, and six plasmids together contained 163 protein-coding genes. The genome contained six rRNAs gene, 69 tRNAs gene, and a CRISPR (868,686–868,801). L. reuteri I5007 genome sequence revealed some of the genes that were known to be involved in the resistance to low pH, bile salt, heat stress, and heavy metal. L. reuteri I5007 encoded most of the known adhesion factors, including fibronectin-binding
64
C. Hou et al. / Journal of Biotechnology 179 (2014) 63–64
Table 1 General features of L. reuteri I5007 genome. Chromosome
Size (bp) GC content (%) Protein-coding genes Assigned function Conserved hypothetical Unknown function rRNA operons tRNA genes
1,947,706 38.99 1891 1713 44 134 6 69
Plasmids pLRI01
pLRI02
pLRI03
pLRI04
pLRI05
pLRI06
53,021 36.47 54 22 13 19 0 0
15,577 36.94 19 19 0 0 0 0
40,038 38.62 53 35 3 15 0 0
16,384 39.05 17 12 0 5 0 0
14,050 42.95 12 7 0 5 0 0
6499 38.96 8 1 0 7 0 0
domain protein (LRI 1035), mucus-binding protein (LRI 1680), dlt cluster (LRI 1701–LRI 1711), and sortase family protein (LRI 1722). It contained various antioxidant enzyme encoding genes such as thioredoxin reductases (LRI 0332, LRI 0841, LRI 1564), NADH oxidases (LRI 0078, LRI 1663, LRI 1664, LRI 1675), peptide methionine sulfoxide reductases (LRI 0773, LRI 1772), and alkyl hydroperoxide reductase (LRI 0840). Genomic analysis revealed that L. reuteri I5007 has the capacity to de novo synthesize l-lysine and folic acid. Interestingly, the genome encoded two gene clusters (LRI 0601–LRI 0620, LRI 0873–LRI 0891) for exopolysaccharide biosynthesis, and most genes were not found in other L. reuteri strains. To our knowledge, this is the first report of the complete genome sequence of the porcine L. reuteri. The results supported and extended previous studies about I5007, more importantly, provided some previously undescribed physiological characteristics. Nucleotide sequence accession number The complete genome sequence of L. reuteri I5007 has been deposited in GenBank under accession number CP006011. The accession numbers for the plasmids pLRI01, pLRI02, pLRI03, pLRI04, pLRI05, pLRI06 are CP006012, CP006013, CP006014, CP006015, CP006016 and CP006017, respectively. The strain is available from the corresponding author upon request. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30930066). We thank the Shanghai-MOT Key Laboratory of Health and Disease Genomics at the Chinese National Human Genome Center in Shanghai for providing technical assistance with this study. We thank Suzhou BioNovoGene Company (http://www.bionovogene.com) for assistance with bioinformatics analysis.
References Azcarate-Peril, M.A., Altermann, E., Hoover-Fitzula, R.L., Cano, R.J., Klaenhammer, T.R., 2004. Identification and inactivation of genetic loci involved with Lactobacillus acidophilus acid tolerance. Appl. Environ. Microbiol. 70, 5315–5322. Frese, S.A., Benson, A.K., Tannock, G.W., Loach, D.M., Kim, J., Zhang, M., Oh, P.L., Heng, N.C., Patil, P.B., Juge, N., Mackenzie, D.A., Pearson, B.M., Lapidus, A., Dalin, E., Tice, H., Goltsman, E., Land, M., Hauser, L., Ivanova, N., Kyrpides, N.C., Walter, J., 2011. The evolution of host specialization in the vertebrate gut symbiont Lactobacillus reuteri. PLoS Genet. 7, e1001314. Grissa, I., Vergnaud, G., Pourcel, C., 2007. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 35, W52–W57. Hao, P., Zheng, H., Yu, Y., Ding, G., Gu, W., Chen, S., Yu, Z., Ren, S., Oda, M., Konno, T., Wang, S., Li, X., Ji, Z.S., Zhao, G., 2011. Complete sequencing and pan-genomic analysis of Lactobacillus delbrueckii subsp. bulgaricus reveal its genetic basis for industrial yogurt production. PLoS ONE 6, e15964. Huang, C.H., Qiao, S.Y., Li, D.F., Piao, X.S., Ren, J.P., 2004. Effects of Lactobacillus on the performance, diarrhea incidence, VFA concentration and gastrointestinal microbial flora of weaning pigs. Asian-Aust. J. Anim. Sci. 17, 401–409. Lagesen, K., Hallin, P., Rodland, E.A., Staerfeldt, H.H., Rognes, T., Ussery, D.W., 2007. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 35, 3100–3108. Lee, K., Lee, H.G., Choi, Y.J., 2008. Proteomic analysis of the effect of bile salts on the intestinal and probiotic bacterium Lactobacillus reuteri. J. Biotechnol. 137, 14–19. Li, X.J., Yue, L.Y., Guan, X.F., Qiao, S.Y., 2008. The adhesion of putative probiotic lactobacilli to cultured epithelial cells and porcine intestinal mucus. J. Appl. Microbiol. 104, 1082–1091. Lowe, T.M., Eddy, S.R., 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25, 955–964. Wang, A., Yu, H., Gao, X., Li, X., Qiao, S., 2009a. Influence of Lactobacillus fermentum I5007 on the intestinal and systemic immune responses of healthy and E. coli challenged piglets. Antonie van Leeuwenhoek 96, 89–98. Wang, A.N., Cai, C.J., Zeng, X.F., Zhang, F.R., Zhang, G.L., Thacker, P.A., Wang, J.J., Qiao, S.Y., 2013. Dietary supplementation with Lactobacillus fermentum I5007 improves the anti-oxidative activity of weanling piglets challenged with diquat. J. Appl. Microbiol. 114, 1582–1591. Wang, A.N., Yi, X.W., Yu, H.F., Dong, B., Qiao, S.Y., 2009b. Free radical scavenging activity of Lactobacillus fermentum in vitro and its antioxidative effect on growing-finishing pigs. J. Appl. Microbiol. 107, 1140–1148. Wang, X., Yang, F., Liu, C., Zhou, H., Wu, G., Qiao, S., Li, D., Wang, J., 2012. Dietary supplementation with the probiotic Lactobacillus fermentum I5007 and the antibiotic aureomycin differentially affects the small intestinal proteomes of weanling piglets. J. Nutr. 142, 7–13. Yang, F., Wang, J., Li, X., Ying, T., Qiao, S., Li, D., Wu, G., 2007. 2-DE and MS analysis of interactions between Lactobacillus fermentum I5007 and intestinal epithelial cells. Electrophoresis 28, 4330–4339.
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcement
Complete genome sequence of Lactobacillus reuteri I5007, a probiotic strain isolated from healthy piglet Chengli Hou 1 , Qingwei Wang 1 , Xiangfang Zeng, Fengjuan Yang, Jiang Zhang, Hong Liu, Xi Ma, Shiyan Qiao ∗ State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing 100193, China
a r t i c l e
i n f o
Article history: Received 9 March 2014 Accepted 11 March 2014 Available online 28 March 2014
a b s t r a c t Lactobacillus reuteri I5007 is a well-characterized probiotic strain isolated from the colonic mucosa of healthy weaning piglets. Here, we present the complete genome sequence of this strain, which consists of a circular chromosome and six distinct plasmids. © 2014 Elsevier B.V. All rights reserved.
Keywords: Lactobacillus reuteri Genome sequence Probiotic Piglet
Lactobacillus reuteri is a resident of the human and animal intestinal tracts (Lee et al., 2008). Comparative genomic analysis revealed that the evolution of L. reuteri with vertebrates resulted in the emergence of host specialization (Frese et al., 2011). Lactobacillus reuteri I5007, initially known as Lactobacillus fermentum I5007, was isolated from the colonic mucosa of healthy weaning piglets (Huang et al., 2004). Subsequent studies showed that I5007 has several important characteristics, including: (1) strong adhesion to Caco-2 cells and porcine intestinal mucus and competitiveness against Salmonella typhimurium and Escherichia coli (Li et al., 2008; Yang et al., 2007); (2) positive regulation of immune function in piglets (Wang et al., 2009a); (3) improvement in antioxidant status of pigs (Wang et al., 2013, 2009b). Moreover, I5007 has an interaction with Caco-2 cells (Yang et al., 2007) and can affect the small intestinal proteomes of weaning piglets (Wang et al., 2012). In addition, L. reuteri I5007 has strong adhesion to other host cells, including IPEC-J2, and IEC-6, and can produce large amounts of exopolysaccharides (unpublished data). To gain insight into its genetic and physiological properties, the whole genome sequence of I5007 was determined. L. reuteri I5007 genome sequence was determined using a Roche Genome Sequencer FLX system and PCR-based gap filling
∗ Corresponding author at: Department of Animal Science and Technology, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, China. Tel.: +86 10 62731456; fax: +86 10 62733688. E-mail address: qiaoshy@mafic.ac.cn (S. Qiao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jbiotec.2014.03.019 0168-1656/© 2014 Elsevier B.V. All rights reserved.
strategies (Hao et al., 2011). A total of 87,911 reads totaling 57,014,356 bases was obtained, resulting in 22-fold genome coverage. De novo genome assembly of the resulting reads was performed using the Newbler v2.3 assembly software package, 110 contigs ranging from 500 bp to 141,014 bp were obtained with total size of 198,716 bp (the N50 contig size is 39,886 bp). All the relationships between the contigs were determined by multiplex PCR, while the gaps between the contigs were filled in by PCR reactions. The regions with low quality were re-sequenced to be precise, and the final sequencing accuracy was 99.9988%. Putative protein-coding sequences (ORFs) were identified with Glimmer 3 (Azcarate-Peril et al., 2004). tRNA genes were annotated by tRNAscan-SE (Lowe and Eddy, 1997), while rRNA genes were predicted by RNAmmer (Lagesen et al., 2007). Functional annotation of CDSs was performed through blastp searches against non-redundant protein database/NCBI, followed by manual inspection. CRISPR finder tool was used to determine clustered regularly interspaced short palindromic repeat (CRISPR) (Grissa et al., 2007). The complete genome of L. reuteri I5007 contained a single circular chromosome of 1,947,706 bp and six indigenous plasmids with lengths ranging from 6499 to 53,021 bp (Table 1). The chromosome contained 1891 protein-coding genes with a GC content of 38.99%, and six plasmids together contained 163 protein-coding genes. The genome contained six rRNAs gene, 69 tRNAs gene, and a CRISPR (868,686–868,801). L. reuteri I5007 genome sequence revealed some of the genes that were known to be involved in the resistance to low pH, bile salt, heat stress, and heavy metal. L. reuteri I5007 encoded most of the known adhesion factors, including fibronectin-binding
64
C. Hou et al. / Journal of Biotechnology 179 (2014) 63–64
Table 1 General features of L. reuteri I5007 genome. Chromosome
Size (bp) GC content (%) Protein-coding genes Assigned function Conserved hypothetical Unknown function rRNA operons tRNA genes
1,947,706 38.99 1891 1713 44 134 6 69
Plasmids pLRI01
pLRI02
pLRI03
pLRI04
pLRI05
pLRI06
53,021 36.47 54 22 13 19 0 0
15,577 36.94 19 19 0 0 0 0
40,038 38.62 53 35 3 15 0 0
16,384 39.05 17 12 0 5 0 0
14,050 42.95 12 7 0 5 0 0
6499 38.96 8 1 0 7 0 0
domain protein (LRI 1035), mucus-binding protein (LRI 1680), dlt cluster (LRI 1701–LRI 1711), and sortase family protein (LRI 1722). It contained various antioxidant enzyme encoding genes such as thioredoxin reductases (LRI 0332, LRI 0841, LRI 1564), NADH oxidases (LRI 0078, LRI 1663, LRI 1664, LRI 1675), peptide methionine sulfoxide reductases (LRI 0773, LRI 1772), and alkyl hydroperoxide reductase (LRI 0840). Genomic analysis revealed that L. reuteri I5007 has the capacity to de novo synthesize l-lysine and folic acid. Interestingly, the genome encoded two gene clusters (LRI 0601–LRI 0620, LRI 0873–LRI 0891) for exopolysaccharide biosynthesis, and most genes were not found in other L. reuteri strains. To our knowledge, this is the first report of the complete genome sequence of the porcine L. reuteri. The results supported and extended previous studies about I5007, more importantly, provided some previously undescribed physiological characteristics. Nucleotide sequence accession number The complete genome sequence of L. reuteri I5007 has been deposited in GenBank under accession number CP006011. The accession numbers for the plasmids pLRI01, pLRI02, pLRI03, pLRI04, pLRI05, pLRI06 are CP006012, CP006013, CP006014, CP006015, CP006016 and CP006017, respectively. The strain is available from the corresponding author upon request. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30930066). We thank the Shanghai-MOT Key Laboratory of Health and Disease Genomics at the Chinese National Human Genome Center in Shanghai for providing technical assistance with this study. We thank Suzhou BioNovoGene Company (http://www.bionovogene.com) for assistance with bioinformatics analysis.
References Azcarate-Peril, M.A., Altermann, E., Hoover-Fitzula, R.L., Cano, R.J., Klaenhammer, T.R., 2004. Identification and inactivation of genetic loci involved with Lactobacillus acidophilus acid tolerance. Appl. Environ. Microbiol. 70, 5315–5322. Frese, S.A., Benson, A.K., Tannock, G.W., Loach, D.M., Kim, J., Zhang, M., Oh, P.L., Heng, N.C., Patil, P.B., Juge, N., Mackenzie, D.A., Pearson, B.M., Lapidus, A., Dalin, E., Tice, H., Goltsman, E., Land, M., Hauser, L., Ivanova, N., Kyrpides, N.C., Walter, J., 2011. The evolution of host specialization in the vertebrate gut symbiont Lactobacillus reuteri. PLoS Genet. 7, e1001314. Grissa, I., Vergnaud, G., Pourcel, C., 2007. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 35, W52–W57. Hao, P., Zheng, H., Yu, Y., Ding, G., Gu, W., Chen, S., Yu, Z., Ren, S., Oda, M., Konno, T., Wang, S., Li, X., Ji, Z.S., Zhao, G., 2011. Complete sequencing and pan-genomic analysis of Lactobacillus delbrueckii subsp. bulgaricus reveal its genetic basis for industrial yogurt production. PLoS ONE 6, e15964. Huang, C.H., Qiao, S.Y., Li, D.F., Piao, X.S., Ren, J.P., 2004. Effects of Lactobacillus on the performance, diarrhea incidence, VFA concentration and gastrointestinal microbial flora of weaning pigs. Asian-Aust. J. Anim. Sci. 17, 401–409. Lagesen, K., Hallin, P., Rodland, E.A., Staerfeldt, H.H., Rognes, T., Ussery, D.W., 2007. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 35, 3100–3108. Lee, K., Lee, H.G., Choi, Y.J., 2008. Proteomic analysis of the effect of bile salts on the intestinal and probiotic bacterium Lactobacillus reuteri. J. Biotechnol. 137, 14–19. Li, X.J., Yue, L.Y., Guan, X.F., Qiao, S.Y., 2008. The adhesion of putative probiotic lactobacilli to cultured epithelial cells and porcine intestinal mucus. J. Appl. Microbiol. 104, 1082–1091. Lowe, T.M., Eddy, S.R., 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25, 955–964. Wang, A., Yu, H., Gao, X., Li, X., Qiao, S., 2009a. Influence of Lactobacillus fermentum I5007 on the intestinal and systemic immune responses of healthy and E. coli challenged piglets. Antonie van Leeuwenhoek 96, 89–98. Wang, A.N., Cai, C.J., Zeng, X.F., Zhang, F.R., Zhang, G.L., Thacker, P.A., Wang, J.J., Qiao, S.Y., 2013. Dietary supplementation with Lactobacillus fermentum I5007 improves the anti-oxidative activity of weanling piglets challenged with diquat. J. Appl. Microbiol. 114, 1582–1591. Wang, A.N., Yi, X.W., Yu, H.F., Dong, B., Qiao, S.Y., 2009b. Free radical scavenging activity of Lactobacillus fermentum in vitro and its antioxidative effect on growing-finishing pigs. J. Appl. Microbiol. 107, 1140–1148. Wang, X., Yang, F., Liu, C., Zhou, H., Wu, G., Qiao, S., Li, D., Wang, J., 2012. Dietary supplementation with the probiotic Lactobacillus fermentum I5007 and the antibiotic aureomycin differentially affects the small intestinal proteomes of weanling piglets. J. Nutr. 142, 7–13. Yang, F., Wang, J., Li, X., Ying, T., Qiao, S., Li, D., Wu, G., 2007. 2-DE and MS analysis of interactions between Lactobacillus fermentum I5007 and intestinal epithelial cells. Electrophoresis 28, 4330–4339.
