Identification of the gene encoding the alternative sigma factor sigmaB 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 several rsb genes from L. monocytogenes, encoding homologs of the alternative sigma factor sigmaB and the RsbUVWX proteins, which govern transcription of a general stress regulon in the related bacterium Bacillus subtilis. The L. monocytogenes and B. subtilis sigB and rsb genes are similar in sequence and physical organization; however, we observed that the activity of sigmaB 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 sigmaB in coordinating osmotic responses in L. monocytogenes. A null mutation in the sigB gene led to substantial defects in the ability of L. monocytogenes to use betaine and carnitine as osmoprotectants. Subsequent measurements of betaine transport confirmed that the absence of sigmaB reduced the ability of the cells to accumulate betaine. Thus, sigmaB 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.

FIG. 1

Similarity of the structure and organization of RsbU, RsbV, RsbW, ς^B, and RsbX proteins with their B. subtilis and S. aureus homologs. The percent similarity scores of pairwise alignments of the primary amino acid sequences of the L. monocytogenes proteins with the B. subtilis and S. aureus proteins are shown between the blocks representing the respective genes. Coding regions that overlap are indicated by overlapping of the respective blocks representing each gene. The ς^B-dependent promoters (P_B) identified upstream of rsbV in B. subtilis (28) and in L. monocytogenes (this report) are indicated by arrows.

FIG. 2

Multiple sequence alignments of the RsbU (A), RsbV (B), RsbW (C), ς^B (D), and RsbX (E) proteins with their homologs from B. subtilis (Bsu) and S. aureus (Sau). Lm, L. monocytogenes. (A through D) Alignments were derived by using the PILEUP program of the Genetics Computer Group. (B) The serine residue of SpoIIAA that is phosphorylated by SpoIIAB and is believed to be the site of RsbW-dependent phosphorylation of RsbV is indicated by an arrowhead. Highly conserved domains are shaded. (C) Alignment of the RsbW proteins and the SpoIIAB homolog from B. subtilis. Domains showing homology with regions I and II of the histidine protein kinase family of two-component system proteins are shaded, and residues within these domains that are identical in every homolog are boldfaced. (D) The conserved subregions of sigma factors that were identified previously by Helman and Chamberlin (27) are underlined with arrows and numbered. (E) Pairwise alignment of the B. subtilis and L. monocytogenes RsbX proteins was performed with BESTFIT. Vertical lines indicate identical residues, and the periods and colons indicate semiconservative and conservative changes, respectively.

FIG. 3

Primer extension analyses of transcripts originating upstream of rsbV in L. monocytogenes. In each panel, 50 μg of each RNA sample was used as a template for extension of the labeled VPROM2 primer. (A) RNA was derived from logarithmically growing cells before (lane 1) and after the addition of 4% NaCl (lane 2), entrance into stationary phase (lane 3), or acidification of the medium to pH 5.3 with glacial acetic acid (lane 4). The sequence ladder was derived from sequencing reactions using the VPROM2 primer. (B and C) Bands are shown at the same position relative to the sequencing ladder in panel A. (B) RNA samples were derived from logarithmically growing cells (25°C) of LO4035 (wild type) (lanes 1, 3, 5, 7, and 9) and the isogenic sigB::km mutant LMA2B (lanes 2, 4, 6, 8, and 10) before stress (lanes 1 and 2) and 20 min after the addition of 4% NaCl (lanes 3 and 4), acidification to pH 5.3 with glacial acetic acid (lanes 5 and 6), a temperature upshift to 48°C (lanes 7 and 8), and entrance into stationary phase (lanes 9 and 10). (C) RNA samples were derived from LO4035 (wild type) during logarithmic growth at 25°C (lane 1) and 20 min after the addition of 4% NaCl (lane 2), 2% ethanol (lane 3), 1 mM EDTA (lane 4), 0.15% H₂O₂ (lane 5), or glacial acetic acid to pH 5.3 (lane 6), a temperature upshift to 48°C (lane 7), a downshift to 4°C (lane 8), or entrance into stationary phase (lane 9).

FIG. 4

Alignment of known ς^B-dependent promoters. The sequences immediately upstream of the ς^B-dependent transcription start site of the L. monocytogenes rsbV gene are aligned with known ς^B-dependent promoters from B. subtilis. The most highly conserved residues, centering around the −10 and −35 regions, are shaded. The alignment of B. subtilis promoters was derived from von Blohn et al. (53). Bs, B. subtilis; Lm, L. monocytogenes.

FIG. 5

Growth of wild-type and sigB mutant strains of L. monocytogenes after osmotic upshift in BHI (A) or DM (B and C). (A) LO4035 (wild type) and LMA2B (sigB::km) were grown in BHI, and the cultures were divided into two equal portions at 425 min (arrow). Sodium chloride was added to one portion to a final concentration of 6%, and both portions were further incubated. Symbols: ■, LO4035 without NaCl; □, LO4035 with 6% NaCl; •, LMA2B without NaCl; ○, LMA2B with 6% NaCl. (B) Equivalent volumes of 18-h cultures of LO4035 and LMA2B in DM were inoculated into DM without NaCl (▴, LO4035; ■, LMA2B), DM with 3% NaCl (✕⃒, LO4035; ×, LMA2B), and DM with 3% NaCl supplemented with 1 mM betaine (▵, LO4035; □, LMA2B. (C) Equivalent volumes of 18-h cultures of LO4035 and LMA2B in DM were inoculated into fresh DM (▴, LO4035; ■, LMA2B), DM with 3% NaCl (✕⃒, LO4035; ×, LMA2B), and DM with 3% NaCl supplemented with 1 mM carnitine (▵, LO4035; □, LMA2B).

FIG. 5

Growth of wild-type and sigB mutant strains of L. monocytogenes after osmotic upshift in BHI (A) or DM (B and C). (A) LO4035 (wild type) and LMA2B (sigB::km) were grown in BHI, and the cultures were divided into two equal portions at 425 min (arrow). Sodium chloride was added to one portion to a final concentration of 6%, and both portions were further incubated. Symbols: ■, LO4035 without NaCl; □, LO4035 with 6% NaCl; •, LMA2B without NaCl; ○, LMA2B with 6% NaCl. (B) Equivalent volumes of 18-h cultures of LO4035 and LMA2B in DM were inoculated into DM without NaCl (▴, LO4035; ■, LMA2B), DM with 3% NaCl (✕⃒, LO4035; ×, LMA2B), and DM with 3% NaCl supplemented with 1 mM betaine (▵, LO4035; □, LMA2B. (C) Equivalent volumes of 18-h cultures of LO4035 and LMA2B in DM were inoculated into fresh DM (▴, LO4035; ■, LMA2B), DM with 3% NaCl (✕⃒, LO4035; ×, LMA2B), and DM with 3% NaCl supplemented with 1 mM carnitine (▵, LO4035; □, LMA2B).

FIG. 6

Betaine accumulation in LO4035 and LMA2B. Cells in the mid-logarithmic growth phase (OD₆₀₀, ∼0.4) were harvested, washed, and resuspended in potassium phosphate buffer. The washed cell suspension from each strain was energized by the addition of glucose and then divided into two equal volumes, and sodium chloride to a final concentration of 3% was added to one of the samples. After 20 min of induction, ¹⁴C-labeled betaine was added to each sample, and aliquots were removed over time, harvested by centrifugation through oil, and counted by scintillation counting. Symbols: □, LO4035; ■, LO4035 plus 3% NaCl; ▴, LMA2B; ▵, LMA2B plus 3% NaCl.