First Report on the Complete Genome Sequence of Lactobacillus crustorum MN047, a Potent Probiotic Strain Isolated from Koumiss in China.

The complete genome sequence of Lactobacillus crustorum deciphered by PacBio RS II and Illumina HiSeq 4000 sequencing was first reported with one chromosome and two plasmids. Sequence analysis of L. crustorum MN047 showed probiotic characteristics.

GENOME ANNOUNCEMENT

Lactobacillus crustorum, one of the less studied lactobacilli, was first isolated from koumiss, which is a traditional mare’s milk product fermented by its own natural microbes, including lactic acid bacteria (LAB) and yeast. For centuries, koumiss has been considered not only a wholesome beverage (1) but also a functional food for medical purposes (2, 3). LAB are recognized to play a major role in the formation of substances beneficial to human health (4). Our previous study found that the L. crustorum MN047 can produce bacteriocin MN047 A (BMA) (5). The BMA had broad spectrum antibacterial activity in both Gram-positive and Gram-negative pathogens, including multidrug-resistant strains. Although two genomic shotgun sequences have been assembled for L. crustorum (https://www.ncbi.nlm.nih.gov/genome/genomes/15930), no complete genome sequence of the species is available. To further investigate L. crustorum MN047 at the genetic level, this strain was subjected to complete-genome sequencing analysis.

The genome of L. crustorum MN047 was extracted at the log phase and constructed de novo by a combined strategy of both PacBio RS II and Illumina HiSeq 4000 sequencing. A 20-kb single-molecule real-time (SMRT) bell library was prepared from sheared genomic DNA using a 20-kb template library preparation workflow (6). SMRT sequencing was conducted on a PacBio RS II platform using the C4 sequencing chemistry and P6 polymerase with a 240-min movie time on one SMRT cell. Raw sequences were filtered, and 766,898,300 bp of clean data, with a total of 123,121 sequences and a mean read length of 6,228 bp, were obtained. The filtered subreads were de novo assembled using RS_HGAP Assembly version 3 in SMRT Analysis version 2.3.0 software (Pacific Biosciences, USA) (7), which yielded three contigs with 331-fold coverage. Correction of the PacBio assembly was performed by soapSNP and soapIndel software using data from the Illumina HiSeq 4000 sequencing. For Illumina HiSeq 4000 sequencing, a 270-bp insert library was constructed with a read length of 2 × 150 bp. After filtering for quality, 623 Mb of clean data were obtained. Finally, a chromosome and two plasmids were achieved with 0 gaps and single-base quality of 0.9951. Gene prediction was managed with Glimmer version 3.02. Annotation was performed using BLAST with the NR, Swiss-Prot, TrEMBL, COG, KEGG, and GO databases.

The genome sequence of L. crustorum MN047 consists of a circular chromosome (2,261,471 bp) and two circular plasmids (46,788 bp and 8,220 bp), with mean G+C contents of 35.02%, 37.91%, and 36.19%, respectively. There are 2,218 protein-coding genes, 55 tRNA genes, 12 rRNA genes, and three sRNA genes. A total of 2,176 genes were assigned a putative function, including acid tolerance, bile salt hydrolysis, cell adhesion, thiamine, riboflavine, cobalamin and folate biosynthesis, antioxidative property, heavy-metal degradation, aromatic-compounds degradation, exopolysaccharide production, and bacteriocin production. Therefore, this genome sequence provides bases for studying the mechanisms for the probiotic properties of L. crustorum MN047 systematically.

Accession number(s).

The complete genome sequence of L. crustorum MN047 was deposited in GenBank under the accession numbers CP017996 (chromosome), CP017997 (plasmid 1), and CP017998 (plasmid 2).