Identification of the Gene Encoding the Alternative Sigma Factor ς ^B from Listeria monocytogenes and Its Role in Osmotolerance

Abstract

Listeria monocytogenes is well known for its robust physiology, which permits growth at low temperatures under conditions of high osmolarity and low pH. Although studies have provided insight into the mechanisms used by L. monocytogenes to allay the physiological consequences of these adverse environments, little is known about how these responses are coordinated. In the studies presented here, we have cloned the sigB gene and severalrsb genes from L. monocytogenes, encoding homologs of the alternative sigma factor ςB and the RsbUVWX proteins, which govern transcription of a general stress regulon in the related bacterium Bacillus subtilis. TheL. monocytogenes and B. subtilis sigB andrsb genes are similar in sequence and physical organization; however, we observed that the activity of ςB in L. monocytogenes was uniquely responsive to osmotic upshifting, temperature downshifting, and the presence of EDTA in the growth medium. The magnitude of the response was greatest after an osmotic upshift, suggesting a role for ςB in coordinating osmotic responses in L. monocytogenes. A null mutation in the sigB gene led to substantial defects in the ability of L. monocytogenesto use betaine and carnitine as osmoprotectants. Subsequent measurements of betaine transport confirmed that the absence of ςB reduced the ability of the cells to accumulate betaine. Thus, ςB coordinates responses to a variety of physical and chemical signals, and its function facilitates the growth of L. monocytogenes under conditions of high osmotic strength.

Identification and transcriptional characterization of the gene encoding the stress-response σ factor σH in Streptomyces coelicolor A3(2)

“…Analysis of the sequence upstream of sigH revealed an ORF terminating 20 bp before the potential initiation codon of sigH. Comparison of the ORF with databases has revealed signi¢cant similarity with anti-c factor RsbW for general stress-response c B of Bacillus subtilis [14], Staphylococcus aureus [4], and Listeria monocytogenes [5], and putative anti-c factor UsfX of stationary-phase stress-response c F of Mycobacterium tuberculosis [15] (Fig. 3B).…”

“…Relevant restriction sites are indicated. B: Comparison of organization and structure of the S. coelicolor sigH operon with other known stress-response operons; the M. tuberculosis sigF operon [15], the B. subtilis sigB operon [7,18], the S. aureus sigB operon [4], and the L. monocytogenes sigB operon [5]. The proposed or identi¢ed promoters are indicated by bent arrows, P B corresponds to c B -dependent promoter, and P A to c B -independent c A -like promoter.…”

“…The six residues in these regions marked by ¢lled circles correspond to the amino acids directly involved in 310 and 335 recognition in various c factors [8]. B: The comparison of the S. coelicolor UshX protein (USHXSC) with potential anti-c factor UsfX of M. tuberculosis c F (USHXMT) [15], and RsbW anti-c B factor from B. subtilis (RSBWBS) [14], S. aureus (RSBWSA) [4], and L. monocytogenes (RSBWLM) [5]. Identical residues are highlighted in black.…”

“…The sigB operon is regulated similarly in L. monocytogenes and S. aureus by the c B -dependent promoter located upstream of rsbV. In L. monocytogenes, the promoter is induced by entry into stationary phase and several stress conditions, with the highest degree of stimulation for osmotic stress and heat shock [5]. The sigB operon in S. aureus is regulated by a putative c A -like promoter upstream of rsbU, and putative internal c B -dependent promoter located upstream of rsbV.…”

“…The sigH-P2 might be recognized by S. coelicolor c H , and the operon autocatalytically activated like other sigB operons [4,5,7]. Thus, the c H might have a principal role in regulation of osmotic response in S. coelicolor, situation similar for c B in L. monocytogenes [5]. We are currently inactivating the sigH gene in S. coelicolor in order to test this hypothesis.…”