Enterococcus faecalis CECT7121 is a corn silage probiotic bacterium that shows antibacterial activity against Gram-positive pathogens from different origins. Its genome sequence is 2.9 Mb long with a GC content of 37.3%. Genome annotation identified three bacteriocin gene clusters in the genome.Fil: Delpech, Gastón. Universidad Nacional del Centro de la Provincia de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hebert, Elvira Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Sparo, Mónica Delfina. Universidad Nacional del Centro de la Provincia de Buenos Aires; ArgentinaFil: Saavedra, Maria Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentin

Delpech, Gastón

Hebert, Elvira Maria

Saavedra, Maria Lucila

Sparo, Mónica Delfina

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Draft Genome Sequence of Enterococcus faecalis StrainCECT7121, a Corn Silage Isolate with Antibacterial Activityagainst Gram-Positive PathogensGastón Delpech,a Elvira M. Hebert,b Mónica Sparo,a Lucila SaavedrabaMicrobiología Clínica, Medicina, Universidad Nacional del Centro de la Provincia de Buenos Aires, Olavarría, ArgentinabLaboratorio de Genética y Biología Molecular, Centro de Referencia para Lactobacilos (CERELA-CONICET), San Miguel de Tucumán, ArgentinaABSTRACT Enterococcus faecalis CECT7121 is a corn silage probiotic bacterium thatshows antibacterial activity against Gram-positive pathogens from different origins.Its genome sequence is 2.9 Mb long with a GC content of 37.3%. Genome annota-tion identified three bacteriocin gene clusters in the genome.Enterococcus faecalis CECT7121 (formerly MR99) is a probiotic strain isolated fromArgentinean corn silage that has bactericidal activity against Gram-positive bacteria(1, 2). E. faecalis CECT7121 was grown in brain heart infusion broth (LaboratoriosBritania, Buenos Aires, Argentina) at 35  2°C for 18 h (2), and genomic DNA isolationwas performed as described by Brown et al. (3).Genome libraries for DNA sequencing were constructed using a TruSeq DNA PCR-free library preparation kit (Illumina, Inc.) with an insert size of 350 bp. The sequencingprocess was carried out at Macrogen, Inc. (Seoul, Republic of Korea) using an IlluminaHiSeq platform. The paired-end library generated 17,520,654 reads (101-bp paired end).The quality of the raw data was checked with FastQC (4); Trimmomatic was used bothto remove low-quality sequences (Q  20) and to trim adapters (5). Reads wereassembled de novo using SPAdes 3.12.0 (6) and annotated with the NCBI ProkaryoticGenome Annotation Pipeline (7). The draft genome sequence of E. faecalis CECT7121has a length of 2,901,597 bp with a GC content of 37.3% and was assembled into 17contigs with an N50 value of 386,449 bp. This genome contains 2,821 coding genes and69 RNAs.An in silico genomic screening of biotechnological properties evidenced that the E.faecalis CECT7121 genome contains genes involved in bacteriocin production. Thebacteriocin-mining software tool BAGEL4 identified 3 areas of interest (AOI) in contigs12, 9, and 7, corresponding to potential bacteriocin genes in the genome of strainCECT7121. BLAST analysis determined that contig 12.7.AOI_01 contains 2 open readingframes (ORFs) encoding putative enterocin P (E value, 2  1029) and its immunityprotein (O30435_ENTFC; E value, 3  1027). Sequence analysis of this gene clusterrevealed the same genetic organization previously described (8). Contig 9.9.AOI_01revealed an ORF related to enterocin SE-K4 gene cluster. The deduced amino acidsequence of this putative bacteriocin gene exhibited a high degree of identity with ahypothetical protein widely distributed among E. faecalis strains. However, a 5-aminoacid deletion at the C-terminal domain was observed when it was compared toenterocin SE-K4 (ent SE-K4), first described in E. faecalis K4 (UniProtKB identifierQ8GR39_ENTFL). An entAim gene, encoding a putative immunity protein from theenterocin A cluster (E value, 1  1030), was found immediately downstream of thebacteriocin gene. In addition, a putative promoter sequence was predicted upstream ofthe bacteriocin gene. Nevertheless, the presence of two putative rho-independentterminators, just before and after these two gene clusters, suggests that they might notCitation Delpech G, Hebert EM, Sparo M,Saavedra L. 2019. Draft genome sequence ofEnterococcus faecalis strain CECT7121, a cornsilage isolate with antibacterial activity againstGram-positive pathogens. Microbiol ResourAnnounc 8:e00245-19. https://doi.org/10.1128/MRA.00245-19.Editor Irene L. G. Newton, Indiana University,BloomingtonCopyright © 2019 Delpech et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.Address correspondence to Mónica Sparo,monicasparo@gmail.com, or Lucila Saavedra,lucila@cerela.org.ar.Received 5 March 2019Accepted 25 April 2019Published 16 May 2019GENOME SEQUENCEScrossmVolume 8 Issue 20 e00245-19 mra.asm.org 1 on May 21, 2019 by guesthttp://mra.asm.org/Downloaded from be functional. Contig 7.1.AOI_01 exhibits putative enterocin P and enterocin SE-K4clusters as well. A putative enterocin P gene (E value, 2  1025) and its immunityprotein (ORF 00018; E value, 3  1026; UniProtKB identifier O30435_ENTFC) arelocated downstream of a putative ent SE-K4 (E value, 3  1051) and ORF 00015 (withno function determined). No putative promoter sequences were predicted upstream ofthese genes. Rho-independent terminators were predicted downstream of ORF 00015and ORF 00018.Based on the above-described analyses, we might speculate that the functionalbacteriocin gene clusters are those described in contigs 12 (enterocin P) and 7(enterocin SE-K4).Data availability. This whole-genome shotgun project has been deposited atDDBJ/ENA/GenBank under the accession number SIJP00000000. The version describedin this paper is the first version, SIJP01000000. Raw data are available under SRAaccession number PRJNA523127.ACKNOWLEDGMENTThis work was supported by funding from the Centro de Estudios Bioquímicos-Tandil, Argentina.REFERENCES1. Sparo MD, Castro MS, Andino PJ, Lavigne MV, Ceriani C, Gutierrez GL,Fernández MM, De Marzi MC, Malchiodi EL, Manghi MA. 2006. Partialcharacterization of enterocin MR99 from a corn silage isolate of Entero-coccus faecalis. J Appl Microbiol 100:123–134. https://doi.org/10.1111/j.1365-2672.2005.02752.x.2. Delpech G, Bistoletti M, Ceci M, Lissarrague S, Bruni SS, Sparo M. 2017.Bactericidal activity and synergy studies of peptide AP-CECT7121 againstmulti-resistant bacteria isolated from human and animal soft-tissue in-fections. Probiotics Antimicrob Proteins 9:355–362. https://doi.org/10.1007/s12602-017-9289-3.3. Brown L, Villegas JM, Elean M, Fadda S, Mozzi F, Saavedra L, Hebert EM.2017. YebC, a putative transcriptional factor involved in the regulation ofthe proteolytic system of Lactobacillus. Sci Rep 7:8579. https://doi.org/10.1038/s41598-017-09124-1.4. Andrews S. 2010. FastQC: a quality control tool for high throughput se-quence data. http://www.bioinformatics.babraham.ac.uk/projects/fastqc.5. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer forIllumina sequence data. Bioinformatics 30:2114 –2120. https://doi.org/10.1093/bioinformatics/btu170.6. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, LesinVM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N,Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome as-sembly algorithm and its applications to single-cell sequencing. J ComputBiol 19:455– 477. https://doi.org/10.1089/cmb.2012.0021.7. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L,Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI ProkaryoticGenome Annotation Pipeline. Nucleic Acids Res 44:6614–6624. https://doi.org/10.1093/nar/gkw569.8. Cintas LM, Casaus P, Håvarstein LS, Hernández PE, Nes IF. 1997. Biochem-ical and genetic characterization of enterocin P, a novel sec-dependentbacteriocin from Enterococcus faecium P13 with a broad antimicrobialspectrum. Appl Environ Microbiol 63:4321– 4330.Delpech et al.Volume 8 Issue 20 e00245-19 mra.asm.org 2 on May 21, 2019 by guesthttp://mra.asm.org/Downloaded from 

Draft genome sequence of enterococcus faecalis strain CECT7121, a corn silage isolate with antibacterial activity against gram-positive pathogens

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Draft Genome Sequence of Enterococcus faecalis StrainCECT7121, a Corn Silage Isolate with Antibacterial Activityagainst Gram-Positive PathogensGastón Delpech,a Elvira M. Hebert,b Mónica Sparo,a Lucila SaavedrabaMicrobiología Clínica, Medicina, Universidad Nacional del Centro de la Provincia de Buenos Aires, Olavarría, ArgentinabLaboratorio de Genética y Biología Molecular, Centro de Referencia para Lactobacilos (CERELA-CONICET), San Miguel de Tucumán, ArgentinaABSTRACT Enterococcus faecalis CECT7121 is a corn silage probiotic bacterium thatshows antibacterial activity against Gram-positive pathogens from different origins.Its genome sequence is 2.9 Mb long with a GC content of 37.3%. Genome annota-tion identiﬁed three bacteriocin gene clusters in the genome.Enterococcus faecalis CECT7121 (formerly MR99) is a probiotic strain isolated fromArgentinean corn silage that has bactericidal activity against Gram-positive bacteria(1, 2). E. faecalis CECT7121 was grown in brain heart infusion broth (LaboratoriosBritania, Buenos Aires, Argentina) at 35 2°C for 18 h (2), and genomic DNA isolationwas performed as described by Brown et al. (3).Genome libraries for DNA sequencing were constructed using a TruSeq DNA PCR-free library preparation kit (Illumina, Inc.) with an insert size of 350 bp. The sequencingprocess was carried out at Macrogen, Inc. (Seoul, Republic of Korea) using an IlluminaHiSeq platform. The paired-end library generated 17,520,654 reads (101-bp paired end).The quality of the raw data was checked with FastQC (4); Trimmomatic was used bothto remove low-quality sequences (Q 20) and to trim adapters (5). Reads wereassembled de novo using SPAdes 3.12.0 (6) and annotated with the NCBI ProkaryoticGenome Annotation Pipeline (7). The draft genome sequence of E. faecalis CECT7121has a length of 2,901,597 bp with a GC content of 37.3% and was assembled into 17contigs with an N50 value of 386,449 bp. This genome contains 2,821 coding genes and69 RNAs.An in silico genomic screening of biotechnological properties evidenced that the E.faecalis CECT7121 genome contains genes involved in bacteriocin production. Thebacteriocin-mining software tool BAGEL4 identiﬁed 3 areas of interest (AOI) in contigs12, 9, and 7, corresponding to potential bacteriocin genes in the genome of strainCECT7121. BLAST analysis determined that contig 12.7.AOI_01 contains 2 open readingframes (ORFs) encoding putative enterocin P (E value, 2  1029) and its immunityprotein (O30435_ENTFC; E value, 3  1027). Sequence analysis of this gene clusterrevealed the same genetic organization previously described (8). Contig 9.9.AOI_01revealed an ORF related to enterocin SE-K4 gene cluster. The deduced amino acidsequence of this putative bacteriocin gene exhibited a high degree of identity with ahypothetical protein widely distributed among E. faecalis strains. However, a 5-aminoacid deletion at the C-terminal domain was observed when it was compared toenterocin SE-K4 (ent SE-K4), ﬁrst described in E. faecalis K4 (UniProtKB identiﬁerQ8GR39_ENTFL). An entAim gene, encoding a putative immunity protein from theenterocin A cluster (E value, 1  1030), was found immediately downstream of thebacteriocin gene. In addition, a putative promoter sequence was predicted upstream ofthe bacteriocin gene. Nevertheless, the presence of two putative rho-independentterminators, just before and after these two gene clusters, suggests that they might notCitation Delpech G, Hebert EM, Sparo M,Saavedra L. 2019. Draft genome sequence ofEnterococcus faecalis strain CECT7121, a cornsilage isolate with antibacterial activity againstGram-positive pathogens. Microbiol ResourAnnounc 8:e00245-19. https://doi.org/10.1128/MRA.00245-19.Editor Irene L. G. Newton, Indiana University,BloomingtonCopyright © 2019 Delpech et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.Address correspondence to Mónica Sparo,monicasparo@gmail.com, or Lucila Saavedra,lucila@cerela.org.ar.Received 5 March 2019Accepted 25 April 2019Published 16 May 2019GENOME SEQUENCEScrossmVolume 8 Issue 20 e00245-19 mra.asm.org 1 on May 21, 2019 by guesthttp://mra.asm.org/Downloaded from be functional. Contig 7.1.AOI_01 exhibits putative enterocin P and enterocin SE-K4clusters as well. A putative enterocin P gene (E value, 2  1025) and its immunityprotein (ORF 00018; E value, 3  1026; UniProtKB identiﬁer O30435_ENTFC) arelocated downstream of a putative ent SE-K4 (E value, 3  1051) and ORF 00015 (withno function determined). No putative promoter sequences were predicted upstream ofthese genes. Rho-independent terminators were predicted downstream of ORF 00015and ORF 00018.Based on the above-described analyses, we might speculate that the functionalbacteriocin gene clusters are those described in contigs 12 (enterocin P) and 7(enterocin SE-K4).Data availability. This whole-genome shotgun project has been deposited atDDBJ/ENA/GenBank under the accession number SIJP00000000. The version describedin this paper is the ﬁrst version, SIJP01000000. Raw data are available under SRAaccession number PRJNA523127.ACKNOWLEDGMENTThis work was supported by funding from the Centro de Estudios Bioquímicos-Tandil, Argentina.REFERENCES1. Sparo MD, Castro MS, Andino PJ, Lavigne MV, Ceriani C, Gutierrez GL,Fernández MM, De Marzi MC, Malchiodi EL, Manghi MA. 2006. Partialcharacterization of enterocin MR99 from a corn silage isolate of Entero-coccus faecalis. J Appl Microbiol 100:123–134. https://doi.org/10.1111/j.1365-2672.2005.02752.x.2. Delpech G, Bistoletti M, Ceci M, Lissarrague S, Bruni SS, Sparo M. 2017.Bactericidal activity and synergy studies of peptide AP-CECT7121 againstmulti-resistant bacteria isolated from human and animal soft-tissue in-fections. Probiotics Antimicrob Proteins 9:355–362. https://doi.org/10.1007/s12602-017-9289-3.3. Brown L, Villegas JM, Elean M, Fadda S, Mozzi F, Saavedra L, Hebert EM.2017. YebC, a putative transcriptional factor involved in the regulation ofthe proteolytic system of Lactobacillus. Sci Rep 7:8579. https://doi.org/10.1038/s41598-017-09124-1.4. Andrews S. 2010. FastQC: a quality control tool for high throughput se-quence data. http://www.bioinformatics.babraham.ac.uk/projects/fastqc.5. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a ﬂexible trimmer forIllumina sequence data. Bioinformatics 30:2114–2120. https://doi.org/10.1093/bioinformatics/btu170.6. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, LesinVM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N,Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome as-sembly algorithm and its applications to single-cell sequencing. J ComputBiol 19:455–477. https://doi.org/10.1089/cmb.2012.0021.7. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L,Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI ProkaryoticGenome Annotation Pipeline. Nucleic Acids Res 44:6614–6624. https://doi.org/10.1093/nar/gkw569.8. Cintas LM, Casaus P, Håvarstein LS, Hernández PE, Nes IF. 1997. Biochem-ical and genetic characterization of enterocin P, a novel sec-dependentbacteriocin from Enterococcus faecium P13 with a broad antimicrobialspectrum. Appl Environ Microbiol 63:4321–4330.Delpech et al.Volume 8 Issue 20 e00245-19 mra.asm.org 2 on May 21, 2019 by guesthttp://mra.asm.org/Downloaded from 

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Draft genome sequence of enterococcus faecalis strain CECT7121, a corn silage isolate with antibacterial activity against gram-positive pathogens

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