Draft Genome Sequence of D-Branched-Chain Amino Acid Producer Lactobacillus otakiensis JCM 15040T, Isolated from a Traditional Japanese Pickle.

Lactobacillus otakiensis strain JCM 15040(T) was isolated from an unsalted pickling solution used in the production of sunki, a traditional Japanese pickle. Here, we prepared a draft genome sequence for this strain consisting of 40 contigs containing a total of 2,347,132 bp, 2,310 predicted coding sequences, and a G+C content of 42.4%.

GENOME ANNOUNCEMENT

The genus Lactobacillus consists of Gram-positive lactic acid bacteria (LAB). These non-spore-forming rods are catalase negative, nonmotile, and oxidase negative (1). In recent years, LAB have also attracted attention related to their ability to produce d-amino acids (2, 3). Among the LABs we examined, Lactobacillus otakiensis produces d-branched-chain amino acids, such as d-leucine (d-Leu), d-allo-isoleucine (d-allo-Ile), and d-valine (d-Val) (Y. Mutaguchi, T. Ohmori, H. Akano, K. Doi, and T. Oshima, submitted for publication). The N-terminal amino acid sequence of the enzyme that apparently is responsible for the production of d-branched-chain amino acids in L. otakiensis is completely identical to those deduced from the 4-aminobutyrate aminotransferase genes of Lactobacillus buchneri (Lbuc_2316 and LBUCD034_2418 of L. buchneri strain NRRL B-30929 and L. buchneri strain CD034, respectively) (Y. Mutaguchi, T. Ohmori, T. Wakamatsu, K. Doi, and T. Ohshima, submitted for publication).

L. otakiensis JCM 15040T was isolated from an unsalted pickling solution used in the production of sunki, a traditional Japanese pickle (4). Although L. otakiensis is closely related to Lactobacillus kisonensis and Lactobacillus rapi, which belong to the L. buchneri group, L. kisonensis and L. rapi do not produce d-amino acids. It has been suggested that the 4-aminobutyrate aminotransferase genes Lbuc_2316 and LBUCD034_2418 function in the synthesis of d-branched-chain amino acids in L. buchneri NRRL B-30929 and L. buchneri CD034, respectively. Comparative genomics of strains from the L. buchneri group might shed light on the metabolism of d-branched-chain amino acids and facilitate in their practical application.

The sample was prepared for sequencing by growing L. otakiensis JCM 15040T anaerobically overnight at 30°C in MRS broth (Oxoid). The genomic DNA was then extracted and purified as we described previously (5). The quantity and quality of the DNA were assessed using e-Spect, after which the prepared genome was sequenced using a 454 GS FLX system (Roche) and assembled using Newbler assembler version 2.7 software.

The genomic DNA, which includes a total of 2,347,132 bp, was sequenced using a whole-genome shotgun strategy that generated 467,404 reads and achieved approximately 62-fold coverage. Assembly of all the reads resulted in 40 contigs (>100 bp), with an N50 contig size of 244,550 bp. Genome annotation of the obtained scaffolds was performed using Glimmer 3.02 software and BLAST searches against a nonredundant protein sequence database. The genome of strain JCM 15040T has a G+C content of 42.4%, and annotation using the GTPS (6), RDP, and Silva databases with tRNAscan-SE v.1.21 (7) and additional manual inspection revealed 2,310 predicted coding regions, 56 tRNA genes, and 5 rRNA genes.

By comparing the genome sequences of L. otakiensis and L. buchneri NRRL B-30929 using the bidirectional best hit (BBH) method with BLAST, we found that there are 1,801 orthologs with 83.82% identity. A gene encoding 4-aminobutyrate aminotransferase was located on contig 00006 as LOT_1784, which shows 97.5% identity with Lbuc_2316 of L. buchneri NRRL B-30929 and LBUCD034_2418 of L. buchneri CD034. Three other genes, LOT_0196, LOT_0493, and LOT_2079, encode racemases that function in the synthesis of d-amino acids, with d-Asp, d-Glu, and d-Ala being annotated.

Nucleotide sequence accession numbers.

The L. otakiensis JCM 15040T genome sequence and annotation data have been deposited in DDBJ/EMBL/GenBank under accession no. BASH01000001 to BASH01000040.