In order to measure the transcript levels of proteins identified in this study, seven genes out of 18 were selected for RT-PCR in the wild-type L. monocytogenes and ΔsigB mutant and the relative mRNA levels were measured. To quantify mRNA levels, the band density of each PCR product was measured after agarose gel electrophoresis. As shown in Fig. 2, the transcript levels are consistent with the proteomic data. Moreover, transcription of the identified genes, including gyrB, lmo1374, ftsA Copanlisib chemical structure and lmo2779, are directly or indirectly under the control of σB. No RT-PCR products were observed in the negative
control (data not shown). Caspase activation The role of the L. monocytogenesσB under stress conditions has been studied intensively, especially under osmotic, cold, acid, high hydrostatic pressure or oxidative stress (Cole et al.,
1990; Sleator et al., 2001; Wemekamp-Kamphuis et al., 2004). Recently, the relationship between alternative sigma factor and antimicrobial resistance has been reported in various bacteria. In B. subtilis, sigma factors σM, σW and σX contribute to resistance to various cell envelope-targeting antibiotics (Mascher et al., 2007). In addition, σB contributes to the upregulation of its own regulon upon exposure to bacitracin or vancomycin (Mascher et al., 2003). In L. monocytogenes, σB is important for growth and survival upon treatment with bacteriocin (lacticin 3147 and nisin) or antibiotics (penicillin G and ampicillin) (Begley et al., 2006). According to a recent report, both σB and σL contribute to tolerance DOCK10 to the antimicrobial peptide SdpC and the bacteriocin nisin in L. monocytogenes (Palmer et al., 2009). Antibiotic-induced cell wall stress is known to induce the expression of many genes. In our two independent proteomic analyses, 18 vancomycin-inducible proteins were identified with minimum twofold upregulation in
the wild-type L. monocytogenes compared with the ΔsigB mutant. Among these proteins, Lmo0539, Lmo0524, Lmo2085, Lmo2114, Pgm, InlD, Lmo1027 and Lmo0079 were already confirmed to be σB-dependent proteins induced under salt or stationary-phase stress (Kazmierczak et al., 2003; Raengpradub et al., 2008; Oliver et al., 2009). Among the three transporter proteins identified under vancomycin stress, Lmo0524 and Lmo1431 were induced only in wild-type L. monocytogenes, whereas Lmo2114 showed a 3.5-fold increase in the wild-type strain (Table 2). Indeed, bacitracin treatment highly stimulated the bceAB gene, which encodes a putative ABC transporter in B.