RPOE KNOCK-OUT MUTANT OF LACTOBACILLUS FERMENTUM MS79 IS MORE SENSITIVE TO OSMOTIC STRESS COMPARED TO THE WILD TYPE STRAIN

초록

Bifidobacteria are Gram-positive, anaerobic branched or pleomorphic rods, and one of the normal flora in the large intestine of humans and animals. They play an important role in maintaining health by producing antimicrobial substances to inhibit undesirable pathogens. In the food industry, bifidobacteria are widely used as food additives. During production of viable cells bifidobacteria can be exposed to various stresses such as low temperature, osmotic and oxidative stress. Therefore studies on the response of Bifidobacterium spp. to various stresses including cold shock are required. In this study, cspA gene was isolated from B. breve KB 69 by PCR using degenerate primers designed based on the two conserved regions of cold shock domain among previously known csp genes of other bacterial species. Subsequently, we performed the whole cspA gene sequencing by single primer PCR technique. The cspA gene encodes 240 bp, which displays 99% identity with cspA gene of B. breve UCC 2003. B. breve KB 69 cspA have two RNA-binding motifs that are highly conserved amino acids among other bacterial CspA/CspB and eukaryotic Y-box factors. An inverted repeat sequence followed by a string of T residues was located 27 bp downstream from the stop codon which might act as an ρ independent transcriptional termination signal. The regulation of cspA gene was investigated under various stresses (cold shock, osmotic, oxidative, and ethanol) at transcriptional level. The amount of cspA mRNA was measured by using reverse transcriptase polymerase chain reaction (RT-PCR). The amount of cspA transcript was maximal during early exponential phase but was hardly detectable during stationary phase, indicating cspA expression might be dependent on growth phase. Moreover, the level of cspA mRNA was increased by cold shock, and the expression was transiently triggered under other stresses. Further studies are underway to find cis-acting elements and corresponding trans-acting factors responsible