Draft Genome Sequence of a New Homofermentative, Lactic Acid-Producing Enterococcus faecalis Isolate, CBRD01.

We report here the draft genome sequence of the novel homofermentative Enterococcus faecalis isolate CBRD01, which is capable of high lactic acid productivity and yields, with minimal nutritional requirements. The genome is 2.8 Mbp, with 37% G+C, and contains genes for two lactate dehydrogenase (LDH) enzymes found in related organisms.

GENOME ANNOUNCEMENT

Enterococcus faecalis, a facultative Gram-positive anaerobe, is commonly found in the mammalian intestinal tract, human feces, dairy products, plants, and insects (1–4). It can survive in different environments at a wide range of temperatures, pH levels, and salinity (2, 5). E. faecalis secretes bacteriocins with bactericidal effects (6, 7), and it is used as food preservative and a probiotic to improve intestinal microbial balance (8–10). Because E. faecalis is also increasingly recognized as a multiresistant nosocomial pathogen, there is tremendous interest in E. faecalis genomes (11). There are currently >50 publicly available draft genomes (http://www.ncbi.nlm.nih.gov/genomeprj/20875), but few have been fully closed. Here, we report the draft genome sequence of a novel, nonfastidious, homolactic acid strain, E. faecalis CBRD01, isolated from municipal solid waste at the Rapid City Materials Recovery Facility. CBRD01 shows great promise in homofermentative lactic acid production at high yields, production rates, and purity on relatively simple fermentation medium, thereby minimizing production costs and pollution (L. P. Christopher, M. R. Subramanian, and S. Talluri, U.S. patent application 14/109,587). E. faecalis CBRD01 was deposited with the ATCC, under the patent deposition designation PTA-12846.

To understand the metabolic and pathogenic properties of E. faecalis CBRD01, the genome was sequenced and analyzed using the ERGO platform (12). Using Ion Torrent PGM technology (13), three sequencing runs were performed on a 316 Chip, yielding ~300 Mb of raw reads. De novo- and template-based assemblies were performed using E. faecalis OG1RF (accession no. CP002621). The genome of CBRD01 is 2,813,167 bp, with an average G+C content of 37%, assembled into 140 contigs. A total of 2,949 open reading frames (ORFs) (protein coding and RNA), including 40 tRNA species and three rRNA operons, were identified (14). Seventy-one percent of the ORFs have a functional annotation (12); approximately 20% utilize the alternate start codon TTG, compared to 8% in E. faecalis D32, V583, or OG1RF.

Like other E. faecalis strains, CBRD01 has two distinct ORFs for the key enzyme lactate dehydrogenase (LDH). ORF NHR02052 has a predicted length of 317 amino acids (aa), identical to orthologs in E. faecalis D32 and V583. ORF NHR01261 is predicted to be 403 aa, identical to E. faecalis OG1RF. This protein appears to utilize TTG rather than the standard ATG start codon. Unlike E. faecalis V583, there were no ORFs associated with xylose utilization; however, there are ORFs for glycerol, galactose, arabinose, sucrose, fructose, and maltose utilization.

We found one complete ORF (NHR01192) for pyruvate ferredoxin oxidoreductase (EC 1.2.7.1) and a second ORF that appears to have a frameshift (NHR01082 and NHR01932). We did not identify either pyruvate formate lyase or formate hydrogen lyase.

E. faecalis is resistant to ampicillin and vancomycin (15). CBRD01 also contains a factor for methicillin resistance (NHR01325), a multidrug resistance protein B, an efflux transporter regulator, and ORFs conferring resistance to β-lactams and aminoglycosides. We found only three transposase ORFs, similar to E. faecalis OG1RF (one transposase) but lower than the 33 in E. faecalis D32. Seventy-seven insertion sequence (IS) elements were identified, compared to 29 in E. faecalis OG1RF and 91 in E. faecalis D32, as well as at least three prophage sequences.

Nucleotide sequence accession number.

The genome sequence of E. faecalis isolate CBRD01 has been deposited in GenBank under the accession no. AWYG01000000.