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 ␤-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.

Stress Physiology of Lactic Acid Bacteria

“…The data described above were generated with simplified laboratory models that do not fully mimic the physicochemical complexity encountered during industrial fermentation and processing (607,608) or the multitude of stresses encountered in the GIT (609). Therefore, in situ transcriptome and proteome profiling studies are valuable, complementary approaches to identify factors contributing to the functionality of LAB under these circumstances.…”

Behavior of the Meat-Borne Bacterium Lactobacillus sakei during Its Transit through the Gastrointestinal Tracts of Axenic and Conventional Mice

“…Purification of genomic DNA to be used in PCR experiments was performed as described in reference 4. DNA of small or mediumsized plasmids (Ͻ6 kb) was isolated from L. sakei by using the GenElute plasmid miniprep kit (Sigma-Aldrich) according to the recommendations of the manufacturer and following the modifications suggested by Hüfner et al (27). Standard methods were used for electrophoresis and PCR DNA fragment purification (36).…”

Characterization of the CtsR Stress Response Regulon in Lactobacillus plantarum

“…This is a novel, unexpected finding, since CtsR inactivation generally causes derepression of heat stress genes, thus rendering cells more prepared to face stressful conditions. Indeed, in most Grampositive bacteria analyzed so far, ctsR mutations increased heat resistance and/or general stress tolerance (4,16,17,18,24,36). The cell morphology of wild-type L. plantarum and ⌬ctsR mutant strains was analyzed before and after heat stress exposure by scanning electron microscopy (SEM) (Fig.…”