PROKARYOTES
crossm Draft Genome Sequence of Enterococcus canintestini 49, a Potential Probiotic That Produces Multiple Bacteriocins Jeella Z. Acedo,a Cherry Ibarra Romero,a Sarah T. Miyata,b Alysson H. Blaine,b Lynn M. McMullen,c John C. Vederas,a Marco J. van Belkuma Department of Chemistry, University of Alberta, Edmonton, Alberta, Canadaa; CanBiocin Inc., Edmonton, Alberta, Canadab; Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canadac
Enterococcus canintestini 49, isolated from dog feces, is active against Clostridium perfringens, vancomycin-resistant enterococci, and Listeria monocytogenes. Its draft genome sequence reported herein contains a gene cluster encoding multiple bacteriocins and indicates the absence of genes for virulence factors. These characteristics signify the strain’s potential for use as a probiotic. ABSTRACT
E
nterococci are promising sources of bacteriocins, which are ribosomally synthesized antimicrobial peptides that can be used in food biopreservation (1). In addition, some enterococci are also used as probiotics to promote human and animal health (2). However, many enterococcal strains produce virulence factors, which calls for a careful screening of strains prior to their use in biotechnological applications. Enterococcus sp. strain 49 was isolated from dog feces and found to inhibit the growth of Clostridium perfringens, vancomycin-resistant enterococci, and Listeria monocytogenes (data not shown). Its 16S rRNA sequence was found to be 99.9% identical to that of Enterococcus canintestini LMG 13590, which was also isolated from dog feces (3). Genomic DNA of E. canintestini 49 was isolated using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s instructions. The sequencing library was prepared using the Nextera XT DNA library preparation kit (Illumina), and the whole genome was sequenced using an Illumina MiSeq kit v3 (2 ⫻ 300 cycles) and MiSeq sequencing technology. The reads were assembled into contiguous sequences (contigs) using the CLC Genomics Workbench v.8 (CLC bio, Aarhus, Denmark). A sequencing coverage of 200⫻ was attained. The assembled draft genome contains 24 contigs and 2,734,830 bases, with 36.2% GC content. The genome was annotated using the NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP), which predicted 2,580 genes, including 2,454 coding sequences (CDS), 7 rRNAs, and 55 tRNAs. The identity of the species was determined based on the average nucleotide identity (ANI) with previously sequenced genomes in the GenBank database as calculated by the JSpecies software (4). E. canintestini 49 exhibits 98.23% ANI with the E. canintestini DSM 21207 reference genome. BAGEL3 (5) and antiSMASH v.3 (6) analyses of the E. canintestini 49 draft genome revealed the presence of a gene cluster with 96.7% nucleotide sequence identity with a bacteriocin gene cluster in Enterococcus faecium NKR-5-3, encoding the genes involved in the biosynthesis of enterocins NKR-5-3A, -C, -D, and -Z. Enterocins NKR-5-3A and -Z comprise a class IIb two-peptide bacteriocin, enterocin NKR-5-3C is a class IIa bacteriocin that contains a YGNGL motif, and enterocin NKR-5-3D is a bacteriocin that also acts as a bacteriocin induction peptide (7, 8). Similarly, the bacteriocin gene cluster of E. canintestini 49 encodes enterocins NKR-5-3A, -Z, and -D and a homolog of enterocin NKR-5-3C that differs by 1 amino acid. BAGEL3 predicted two additional Volume 5 Issue 40 e01131-17
Received 8 September 2017 Accepted 15 September 2017 Published 5 October 2017 Citation Acedo JZ, Ibarra Romero C, Miyata ST, Blaine AH, McMullen LM, Vederas JC, van Belkum MJ. 2017. Draft genome sequence of Enterococcus canintestini 49, a potential probiotic that produces multiple bacteriocins. Genome Announc 5:e01131-17. https://doi .org/10.1128/genomeA.01131-17. Copyright © 2017 Acedo et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Marco J. van Belkum, [email protected].
genomea.asm.org 1
Acedo et al.
putative bacteriocin genes adjacent to the enterocin NKR-5-3ACDZ homolog gene cluster in E. canintestini 49. Production of enterocins NKR-5-3D and -Z, and one of the additional bacteriocins predicted by BAGEL3, was detected by tandem mass spectrometry (MS/MS) sequencing of several inhibitory compounds that were isolated (data not shown). Aside from the ability of E. canintestini 49 to produce multiple bacteriocins, its genome sequence does not contain genes that encode virulence factors, such as surface aggregating protein, gelatinase, enterococcal surface protein, collagen-binding adhesin, and hyalurodinase. These findings highlight the potential use of E. canintestini 49 as a probiotic. Accession number(s). This whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under the accession number LHUG00000000. The version described in this paper is version LHUG01000000. ACKNOWLEDGMENTS This work was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC), Alberta Innovates Health Solutions (AIHS), and the Industrial Research Assistance Program (IRAP). S.T.M. was supported by the R&D Associates Fellowship from Alberta Innovates Technology Futures (AITF).
REFERENCES 1. Franz CMAP, van Belkum MJ, Holzapfel WH, Abriouel H, Gálvez A. 2007. Diversity of enterococcal bacteriocins and their grouping in a new classification scheme. FEMS Microbiol Rev 31:293–310. https://doi.org/10 .1111/j.1574-6976.2007.00064.x. 2. Franz CMAP, Huch M, Abriouel H, Holzapfel W, Gálvez A. 2011. Enterococci as probiotics and their implications in food safety. Int J Food Microbiol 151:125–140. https://doi.org/10.1016/j.ijfoodmicro.2011.08.014. 3. Naser SM, Vancanneyt M, De Graef E, Devriese LA, Snauwaert C, Lefebvre K, Hoste B, Svec P, Decostere A, Haesebrouck F, Swings J. 2005. Enterococcus canintestini sp. nov. from faecal samples of healthy dogs. Int J Syst Evol Microbiol 55:2177–2182. https://doi.org/10.1099/ijs.0.63752-0. 4. Richter M, Rosselló-Móra R. 2009. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci U S A 106: 19126 –19131. https://doi.org/10.1073/pnas.0906412106. 5. van Heel AJ, de Jong A, Montalbán-López M, Kok J, Kuipers OP. 2013. BAGEL3: automated identification of genes encoding bacteriocins and
Volume 5 Issue 40 e01131-17
(non-)bactericidal posttranslationally modified peptides. Nucleic Acids Res 41:W448 –W453. https://doi.org/10.1093/nar/gkt391. 6. Weber T, Blin K, Duddela S, Krug D, Kim HU, Bruccoleri R, Lee SY, Fischbach MA, Müller R, Wohlleben W, Breitling R, Takano E, Medema MH. 2015. antiSMASH 3.0 —a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res 43:W237–W243. https:// doi.org/10.1093/nar/gkv437. 7. Ishibashi N, Himeno K, Fujita K, Masuda Y, Perez RH, Zendo T, Wilaipun P, Leelawatcharamas V, Nakayama J, Sonomoto K. 2012. Purification and characterization of multiple bacteriocins and an inducing peptide produced by Enterococcus faecium NKR-5-3 from Thai fermented fish. Biosci Biotechnol Biochem 76:947–953. https://doi.org/10.1271/bbb.110972. 8. Ishibashi N, Himeno K, Masuda Y, Perez RH, Iwatani S, Zendo T, Wilaipun P, Leelawatcharamas V, Nakayama J, Sonomoto K. 2014. Gene cluster responsible for secretion of and immunity to multiple bacteriocins, the NKR-5-3 enterocins. Appl Environ Microbiol 80:6647–6655. https://doi.org/10.1128/AEM.02312-14.
genomea.asm.org 2
crossm Draft Genome Sequence of Enterococcus canintestini 49, a Potential Probiotic That Produces Multiple Bacteriocins Jeella Z. Acedo,a Cherry Ibarra Romero,a Sarah T. Miyata,b Alysson H. Blaine,b Lynn M. McMullen,c John C. Vederas,a Marco J. van Belkuma Department of Chemistry, University of Alberta, Edmonton, Alberta, Canadaa; CanBiocin Inc., Edmonton, Alberta, Canadab; Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canadac
Enterococcus canintestini 49, isolated from dog feces, is active against Clostridium perfringens, vancomycin-resistant enterococci, and Listeria monocytogenes. Its draft genome sequence reported herein contains a gene cluster encoding multiple bacteriocins and indicates the absence of genes for virulence factors. These characteristics signify the strain’s potential for use as a probiotic. ABSTRACT
E
nterococci are promising sources of bacteriocins, which are ribosomally synthesized antimicrobial peptides that can be used in food biopreservation (1). In addition, some enterococci are also used as probiotics to promote human and animal health (2). However, many enterococcal strains produce virulence factors, which calls for a careful screening of strains prior to their use in biotechnological applications. Enterococcus sp. strain 49 was isolated from dog feces and found to inhibit the growth of Clostridium perfringens, vancomycin-resistant enterococci, and Listeria monocytogenes (data not shown). Its 16S rRNA sequence was found to be 99.9% identical to that of Enterococcus canintestini LMG 13590, which was also isolated from dog feces (3). Genomic DNA of E. canintestini 49 was isolated using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s instructions. The sequencing library was prepared using the Nextera XT DNA library preparation kit (Illumina), and the whole genome was sequenced using an Illumina MiSeq kit v3 (2 ⫻ 300 cycles) and MiSeq sequencing technology. The reads were assembled into contiguous sequences (contigs) using the CLC Genomics Workbench v.8 (CLC bio, Aarhus, Denmark). A sequencing coverage of 200⫻ was attained. The assembled draft genome contains 24 contigs and 2,734,830 bases, with 36.2% GC content. The genome was annotated using the NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP), which predicted 2,580 genes, including 2,454 coding sequences (CDS), 7 rRNAs, and 55 tRNAs. The identity of the species was determined based on the average nucleotide identity (ANI) with previously sequenced genomes in the GenBank database as calculated by the JSpecies software (4). E. canintestini 49 exhibits 98.23% ANI with the E. canintestini DSM 21207 reference genome. BAGEL3 (5) and antiSMASH v.3 (6) analyses of the E. canintestini 49 draft genome revealed the presence of a gene cluster with 96.7% nucleotide sequence identity with a bacteriocin gene cluster in Enterococcus faecium NKR-5-3, encoding the genes involved in the biosynthesis of enterocins NKR-5-3A, -C, -D, and -Z. Enterocins NKR-5-3A and -Z comprise a class IIb two-peptide bacteriocin, enterocin NKR-5-3C is a class IIa bacteriocin that contains a YGNGL motif, and enterocin NKR-5-3D is a bacteriocin that also acts as a bacteriocin induction peptide (7, 8). Similarly, the bacteriocin gene cluster of E. canintestini 49 encodes enterocins NKR-5-3A, -Z, and -D and a homolog of enterocin NKR-5-3C that differs by 1 amino acid. BAGEL3 predicted two additional Volume 5 Issue 40 e01131-17
Received 8 September 2017 Accepted 15 September 2017 Published 5 October 2017 Citation Acedo JZ, Ibarra Romero C, Miyata ST, Blaine AH, McMullen LM, Vederas JC, van Belkum MJ. 2017. Draft genome sequence of Enterococcus canintestini 49, a potential probiotic that produces multiple bacteriocins. Genome Announc 5:e01131-17. https://doi .org/10.1128/genomeA.01131-17. Copyright © 2017 Acedo et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Marco J. van Belkum, [email protected].
genomea.asm.org 1
Acedo et al.
putative bacteriocin genes adjacent to the enterocin NKR-5-3ACDZ homolog gene cluster in E. canintestini 49. Production of enterocins NKR-5-3D and -Z, and one of the additional bacteriocins predicted by BAGEL3, was detected by tandem mass spectrometry (MS/MS) sequencing of several inhibitory compounds that were isolated (data not shown). Aside from the ability of E. canintestini 49 to produce multiple bacteriocins, its genome sequence does not contain genes that encode virulence factors, such as surface aggregating protein, gelatinase, enterococcal surface protein, collagen-binding adhesin, and hyalurodinase. These findings highlight the potential use of E. canintestini 49 as a probiotic. Accession number(s). This whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under the accession number LHUG00000000. The version described in this paper is version LHUG01000000. ACKNOWLEDGMENTS This work was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC), Alberta Innovates Health Solutions (AIHS), and the Industrial Research Assistance Program (IRAP). S.T.M. was supported by the R&D Associates Fellowship from Alberta Innovates Technology Futures (AITF).
REFERENCES 1. Franz CMAP, van Belkum MJ, Holzapfel WH, Abriouel H, Gálvez A. 2007. Diversity of enterococcal bacteriocins and their grouping in a new classification scheme. FEMS Microbiol Rev 31:293–310. https://doi.org/10 .1111/j.1574-6976.2007.00064.x. 2. Franz CMAP, Huch M, Abriouel H, Holzapfel W, Gálvez A. 2011. Enterococci as probiotics and their implications in food safety. Int J Food Microbiol 151:125–140. https://doi.org/10.1016/j.ijfoodmicro.2011.08.014. 3. Naser SM, Vancanneyt M, De Graef E, Devriese LA, Snauwaert C, Lefebvre K, Hoste B, Svec P, Decostere A, Haesebrouck F, Swings J. 2005. Enterococcus canintestini sp. nov. from faecal samples of healthy dogs. Int J Syst Evol Microbiol 55:2177–2182. https://doi.org/10.1099/ijs.0.63752-0. 4. Richter M, Rosselló-Móra R. 2009. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci U S A 106: 19126 –19131. https://doi.org/10.1073/pnas.0906412106. 5. van Heel AJ, de Jong A, Montalbán-López M, Kok J, Kuipers OP. 2013. BAGEL3: automated identification of genes encoding bacteriocins and
Volume 5 Issue 40 e01131-17
(non-)bactericidal posttranslationally modified peptides. Nucleic Acids Res 41:W448 –W453. https://doi.org/10.1093/nar/gkt391. 6. Weber T, Blin K, Duddela S, Krug D, Kim HU, Bruccoleri R, Lee SY, Fischbach MA, Müller R, Wohlleben W, Breitling R, Takano E, Medema MH. 2015. antiSMASH 3.0 —a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res 43:W237–W243. https:// doi.org/10.1093/nar/gkv437. 7. Ishibashi N, Himeno K, Fujita K, Masuda Y, Perez RH, Zendo T, Wilaipun P, Leelawatcharamas V, Nakayama J, Sonomoto K. 2012. Purification and characterization of multiple bacteriocins and an inducing peptide produced by Enterococcus faecium NKR-5-3 from Thai fermented fish. Biosci Biotechnol Biochem 76:947–953. https://doi.org/10.1271/bbb.110972. 8. Ishibashi N, Himeno K, Masuda Y, Perez RH, Iwatani S, Zendo T, Wilaipun P, Leelawatcharamas V, Nakayama J, Sonomoto K. 2014. Gene cluster responsible for secretion of and immunity to multiple bacteriocins, the NKR-5-3 enterocins. Appl Environ Microbiol 80:6647–6655. https://doi.org/10.1128/AEM.02312-14.
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