Identification of Lactobacillus sakei genes induced during meat fermentation and their role in survival and growth

Abstract

Lactobacillus sakei is a lactic acid bacterium that is ubiquitous in the food environment and is one of the most important constituents of commercial meat starter cultures. In this study, in vivo expression technology (IVET) was applied to investigate gene expression of L. sakei 23K during meat fermentation. The IVET vector used (pEH100) contained promoterless and transcriptionally fused reporter genes mediating beta-glucuronidase activity and erythromycin resistance. A genomic library of L. sakei 23K was established, and the clones were subjected to fermentation in a raw-sausage model. Fifteen in carne-induced fusions were identified. Several genes encoded proteins which are likely to contribute to stress-related functions. One of these genes was involved in acquisition of ammonia from amino acids, and the remaining either were part of functionally unrelated pathways or encoded hypothetical proteins. The construction and use of isogenic mutants in the sausage model suggested that four genes have an impact on the performance of L. sakei during raw-sausage fermentation. Inactivation of the heat shock regulator gene ctsR resulted in increased growth, whereas knockout of the genes asnA2, LSA1065, and LSA1194 resulted in attenuated performance compared to the wild-type strain. The results of our study are the first to provide an insight into the transcriptional response of L. sakei when growing in the meat environment. In addition, this study establishes a molecular basis which allows investigation of bacterial properties that are likely to contribute to the ecological performance of the organism and to influence the final outcome of sausage fermentation.

FIG. 1.

Schematic illustration showing the cloning strategy for construction of the IVET vector pEH100. The IVET cassette containing promoterless reporter genes ′gusA and ′ermGT was designed based on the previously published IVET vector pJW100 (65). ORFs are represented as dark gray arrows (genes of the IVET cassette) or light gray arrows (other genes). Abbreviations: MCS, multiple cloning site; tt, transcriptional terminator rrnT1T2 from E. coli; ColE1, E. coli replication region; repA, Lactobacillus replication gene; ′bglM, promoterless β-glucanase gene.

FIG. 2.

Lactobacillus populations during raw-sausage fermentation in the presence of erythromycin. Batches were inoculated with the negative-control strain EH100 (empty bars) or White1 (diagonally striped bars) or the positive-control strain EH200 (black bars) or Blue1 (gray bars) or the IVET library (horizontally striped bars). Bacterial cell counts were determined at the beginning of fermentation (0 h incubation), after 24 h (24 h incubation), and then 24 h after the first back-slopping (48 h incubation) and 24 h after the second back-slopping (72 h incubation) by plating on mMRS agar supplemented with chloramphenicol.

FIG. 3.

Schematic representation of ici fragment localizations (dark boxes) in the genome of L. sakei 23K. ici genes and ORFs located downstream that might be part of a putative operon are displayed as gray arrows, adjacent ORFs as white arrows. Vertical black lines denote transcriptional terminators according to the genome annotation (GenBank accession no. NC_007576).

FIG. 4.

Growth of L. sakei strains 23K(pLPV111) (•), RVASP (○), RVCTSR (▾), RVCLPC (▿), RVNOD (▪), RVBETA (□), RVTERC (♦), and RVRR3 (⋄) in mMRS medium (A) and during raw-sausage fermentation (B). Bacterial cell counts were determined by plating on mMRS agar supplemented with erythromycin. Values are means of two independent experiments.