Identification and analysis of the osmotolerance associated genes in Listeria monocytogenes

Abstract

Listeria monocytogenes, the causative agent of listeriosis, has strong osmotolerance and is able to grow in severe circumstances. Many studies of the mechanisms of listerial osmotolerance have been performed. However, there is much which remains unknown. In previous studies we constructed two kinds of mutant in L. monocytogenes EGD strain to analyse the mechanisms of osmotolerance in L. monocytogenes by molecular genetic methods. In this paper, we summarized the genetical studies of osmotolerance in this bacterium by many researchers and ourselves. First, a transposon-insertional mutant strain was constructed that showed reduced growth in high osmotic agar compared with the parental strain. The results of cloning and sequencing analysis showed that the rel gene, which encodes guanosine tetra- and pentaphosphate synthesis and hydrolysis protein, is involved in osmotolerance in L. monocytogenes. Next, the expression levels of five sigma factor coding genes in L. monocytogenes were examined using real-time polymerase chain reaction (PCR) and it was found that the rpoN gene (the alternative sigma factor RpoN (sigma54)-encoding gene) was activated under high osmotic conditions. A deletion mutant of rpoN was constructed and its response to osmotic stress was analysed. In minimal medium with NaCl and carnitine, an osmoprotectant, the mutant showed deficient growth to that of the parental strain when the starting optical density was high, though the expression level of carnitine transporter operon, opuC, and the rate of carnitine uptake in the mutant was similar to that of EGD. These results suggest that the rpoN mutant may need larger amounts of carnitine which might be needed for its growth under high osmolarity. Through the analysis of these mutants, new insights have been obtained into osmotolerance in L. monocytogenes.

Keywords:

• Listeria monocytogenes
• osmotolerance

Acknowledgement

The works introduced here were supported by grants from Ministry of Health, Labor and Welfare of Japan.