Genome Sequence and Transcriptome Analysis of Meat-Spoilage-Associated Lactic Acid Bacterium Lactococcus piscium MKFS47

Abstract

Lactococcus piscium is a psychrotrophic lactic acid bacterium and is known to be one of the predominant species within spoilage microbial communities in cold-stored packaged foods, particularly in meat products. Its presence in such products has been associated with the formation of buttery and sour off-odors. Nevertheless, the spoilage potential of L. piscium varies dramatically depending on the strain and growth conditions. Additional knowledge about the genome is required to explain such variation, understand its phylogeny, and study gene functions. Here, we present the complete and annotated genomic sequence of L. piscium MKFS47, combined with a time course analysis of the glucose catabolism-based transcriptome. In addition, a comparative analysis of gene contents was done for L. piscium MKFS47 and 29 other lactococci, revealing three distinct clades within the genus. The genome of L. piscium MKFS47 consists of one chromosome, carrying 2,289 genes, and two plasmids. A wide range of carbohydrates was predicted to be fermented, and growth on glycerol was observed. Both carbohydrate and glycerol catabolic pathways were significantly upregulated in the course of time as a result of glucose exhaustion. At the same time, differential expression of the pyruvate utilization pathways, implicated in the formation of spoilage substances, switched the metabolism toward a heterofermentative mode. In agreement with data from previous inoculation studies, L. piscium MKFS47 was identified as an efficient producer of buttery-odor compounds under aerobic conditions. Finally, genes and pathways that may contribute to increased survival in meat environments were considered.

FIG 1

(A) Main L. piscium genome features. tmRNA, transfer-messenger RNA; ncRNAs, noncoding RNAs. (B) Genome map of L. piscium. Genes are colored according to their COG functional annotations. Moving inwards, the tracks represent the following features: genes on the forward strand, genes on the reverse strand, pseudogenes, prophages (green), rRNA genes (red), tRNA genes (blue), GC plot (cyan), and GC skew (purple and yellow). (C) Prophage regions detected by using PHAST (26).

FIG 2

Pangenome tree of the Lactococcus genus, constructed based on information on the presence or absence of orthologs. Abbreviations: L.g, L. garvieae; L.l, L. lactis subsp. lactis; L.c, L. lactis subsp. cremoris; L. chungang, L. chungangensis; L. raffinol, L. raffinolactis.

FIG 3

Venn diagram of the distribution of the orthologous gene groups among the three closely related species L. piscium, L. raffinolactis, and L. chungangensis.

FIG 4

Expression levels of the four pyruvate-dissipating enzymes (A) and two acetyl-CoA-dissipating enzymes (B). RPKM, reads per kilobase of a gene per million mapped reads, averaged across all replicates. Designations: pdh, pyruvate dehydrogenase complex; ldh, lactate dehydrogenase; pfl, pyruvate-formate lyase; alsS, acetolactate synthase; adhE, aldehyde-alcohol dehydrogenase; pta, phosphate acetyltransferase.

FIG 5

Differential expression of glycerol catabolism genes. Designations: glpF, glycerol permease; glpK, glycerol kinase; glpO, alpha-glycerophosphate oxidase; gpsA, glycerol-3-phosphate dehydrogenase; dhaMLK, PTS-dependent dihydroxyacetone kinase; yloV, putative dihydroxyacetone kinase. Abbreviations: DHA, dihydroxyacetone; out, outside the cell; in, inside the cell. The direction of the arrows adjacent to the enzyme names indicates up- or downregulation.