Abstract
Enterococcus faecium M3K31 is a bacteriocinogenic lactic acid bacterium (LAB) isolated from griffon vulture (Gyps fulvus subsp. fulvus) feces. The draft genome sequence of this strain provides genetic data that support its biotechnological potential.
GENOME ANNOUNCEMENT
The enterococci are a diverse group of Gram-positive gastrointestinal (GI) tract colonizers recognized as microorganisms of applied, regulatory, and biotechnological interest (1–3). Enterococcus faecium M3K31 is a bacteriocinogenic lactic acid bacterium (LAB) isolated from the feces of griffon vultures (Gyps fulvus subsp. fulvus), with high antimicrobial activity against Listeria spp., and it is the producer of the bacteriocin enterocin HF (EntHF) (4). Genomic DNA from E. faecium M3K31 was purified using the DNeasy blood and tissue kit (Qiagen, Valencia, CA, USA) and sequenced by using a MiSeq platform (Illumina, Inc., San Diego, CA, USA) at the DNA High-Throughput Sequencing and Genotypic Unit of the University of Illinois at Urbana-Champaign (IL). The shotgun DNAseq library was prepared with the Kapa library preparation kit (Kapa Biosystems, Inc., Wilmington, MA). The library was quantitated by quantitative PCR (qPCR) and sequenced for 251 cycles using a MiSeq sequencing kit version 2. Reads were quality filtered using Nesoni (version 0.130; P. Harrison, 2015) and de novo assembled using SPAdes (version 3.5) (5). Contigs <1,000 bp and with coverage <5-fold were removed. Coding DNA sequences (CDSs) were predicted and annotated using the RAST (http://rast.nmpdr.org/) server (6).
The draft genome of E. faecium M3K31 consists of 70 contigs, for a total of 2,722,557 bp, with a G+C content of 38.1%. The largest contig was 245,388 bp, and the smallest contig was 1,062 bp. The total number of CDSs was 2,687, and the number of RNAs was 79. In silico analysis of the draft genome sequence with the BAGEL3 software (http://bagel2.molgenrug.nl/) (7) confirmed the presence of the EntHF biosynthetic cluster (GenBank accession numbers P86183 and KJ442693), the enterocin P structural and immunity genes (8), and the gene encoding a bacteriocin (GenBank accession no. ELB21426), which resembles (85% identity) a putative peptide named SRCAM 602 (GenBank accession no. P86393) proposed to be produced (9) but not encoded by Paenibacillus polymyxa NRRL-30509 (10). The structural genes related to each bacteriocin were found in three different contigs. The rifampin resistance (rpoB) gene was detected, but no other relevant antibiotic resistance genes or genes associated with virulence factors related to invasiveness and disease severity were identified (11). Screening of the whole genome of E. faecium M3K31 confirmed the absence of the following virulence or virulence-associated factors: insertion sequence (IS16), the enterococcal surface protein (esp), and the putative glycosyl hydrolase (hyl) virulence genes, hence meeting the European Food Safety Authority (EFSA) requirements for the potential safe use of this enterococcal strain as a feed additive (12). One clustered regularly interspaced short palindromic repeat (CRISPR) array, considered a barrier to foreign DNA uptake, was identified using CRISPRfinder (13). The availability of this draft genome may strengthen the value of the bacteriocin-producing E. faecium M3K31 as a potentially useful natural food preservative and therapeutic for human and veterinary applications, and as a potential probiotic for use in animal nutrition.
Nucleotide sequence accession numbers.
This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. LAXK00000000. The version described in this paper is the first version, LAXK01000000.
ACKNOWLEDGMENTS
We thank the Ministerio de Economía y Competitividad (MINECO) (Spain) for the Fellowship (FPI) awarded to S. Arbulu.
Funding Statement
This work was supported by funding from project AGL2012-34829 from the Ministerio de Economía y Competitividad (MINECO), by grant S2013/ABI-2747 from the Comunidad de Madrid (CAM), by the EEA grant NILS Science and Sustainability Coordinated Mobility of Researchers (017-ABEL-CM-2013) (Spain), and by the Natural Sciences and Engineering Research Council of Canada (NRSC) and the Canada Research Chair in Bioinorganic and Medicinal Chemistry (Canada).
Footnotes
Citation Arbulu S, Frantzen C, Lohans CT, Cintas LM, Herranz C, Holo H, Diep DB, Vederas JC, Hernández PE. 2016. Draft genome sequence of the bacteriocin-producing strain Enterococcus faecium M3K31, isolated from griffon vultures (Gyps fulvus subsp. fulvus). Genome Announc 4(2):e00055-16. doi:10.1128/genomeA.00055-16.
REFERENCES
- 1.Franz CM, 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. doi: 10.1016/j.ijfoodmicro.2011.08.014. [DOI] [PubMed] [Google Scholar]
- 2.Palmer KL, Godfrey P, Griggs A, Kos VN, Zucker J, Desjardins C, Cerqueira G, Gevers D, Walker S, Wortman J, Feldgarden M, Haas B, Birren B, Gilmore MS. 2012. Comparative genomics of enterococci: variation in Enterococcus faecalis, clade structure in E. faecium, and defining characteristics of E. gallinarum and E. casseliflavus. mBio 3(1):e00318-11. doi: 10.1128/mBio.00318-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Nes IF, Diep DB, Ike Y. 2014. Enterococcal bacteriocins and antimicrobial proteins that contribute to niche control, p 1–24. In Gilmore MS, Clewell DB, Ike Y, Shankar N (ed), Enterococci: from commensals to leading causes of drug resistant infection. Massachusetts Eye and Ear Infirmary, Boston, MA. [PubMed] [Google Scholar]
- 4.Arbulu S, Lohans CT, van Belkum MJ, Cintas LM, Herranz C, Vederas JC, Hernández PE. 2015. Solution structure of enterocin HF, an antilisterial bacteriocin produced by Enterococcus faecium M3K31. J Agric Food Chem 63:10689–10695. doi: 10.1021/acs.jafc.5b03882. [DOI] [PubMed] [Google Scholar]
- 5.Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. doi: 10.1089/cmb.2012.0021. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O. 2008. The RAST server: Rapid Annotations using Subsystems Technology. BMC Genomics 9:75. doi: 10.1186/1471-2164-9-75. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Van Heel AJ, de Jong A, Montalbán-López M, Kok J, Kuipers OP. 2013. BAGEL3: automated identification of genes encoding bacteriocins and (non-)bactericidal posttranslationally modified peptides. Nucleic Acids Res 41:W448–W453. doi: 10.1093/nar/gkt391. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Cintas LM, Casaus P, Håvarstein LS, Hernández PE, Nes IF. 1997. Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum. Appl Environ Microbiol 63:4321–4330. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Svetoch EA, Stern NJ, Eruslanov BV, Kovalev YN, Volodina LI, Perelygin VV, Mitsevich EV, Mitsevich IP, Pokhilenko VD, Borzenkov VN, Levchuk VP, Svetoch OE, Kudriatseva TY. 2005. Isolation of Bacillus circulans and Paenibacillus polymyxa strains inhibitory to Campylobacter jejuni and characterization of associated bacteriocins. J Food Prot 68:11–17. [DOI] [PubMed] [Google Scholar]
- 10.Lohans CT, Huang Z, van Belkum MJ, Giroud M, Sit CS, Steels EM, Zheng J, Whittal RM, McMullen LM, Vederas JC. 2012. Structural characterization of the highly cyclized lantibiotic paenicidin A via a partial desulfurizatio/reduction strategy. J Am Chem Soc 134:19540–19543. doi: 10.1021/ja3089229. [DOI] [PubMed] [Google Scholar]
- 11.Sánchez J, Basanta A, Gómez-Sala B, Herranz C, Cintas LM, Hernández PE. 2007. Antimicrobial and safety aspects, and biotechnological potential of bacteriocinogenic enterococci isolated from mallard ducks (Anas platyrhynchos). Int J Food Microbiol 117:295–305. doi: 10.1016/j.ijfoodmicro.2007.04.012. [DOI] [PubMed] [Google Scholar]
- 12.EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2012. Guidance on the safety assessment of Enterococcus faecium in animal nutrition. EFSA J 10:2682. doi: 10.2903/j.efsa.2012.2682. [DOI] [Google Scholar]
- 13.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. doi: 10.1093/nar/gkm360. [DOI] [PMC free article] [PubMed] [Google Scholar]