SigmaB activation under environmental and energy stress conditions in Listeria monocytogenes

Abstract

To measure sigmaB activation in Listeria monocytogenes under environmental or energy stress conditions, quantitative reverse transcriptase PCR (TaqMan) was used to determine the levels of transcripts for the sigmaB -dependent opuCA and clpC genes in strains having null mutations in genes encoding regulator of sigma B proteins (rsbT and rsbV) and sigma B (sigB) and in the L. monocytogenes wild-type 10403S strain under different stress conditions. The DeltasigB, DeltarsbT, and DeltarsbV strains previously exhibited increased hemolytic activities compared to the hemolytic activity of the wild-type strain; therefore, transcript levels for hly were also determined. RsbT, RsbV, and sigmaB were all required for opuCA expression during growth under carbon-limiting conditions or following exposure to pH 4.5, salt, ethanol, or the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Expression of clpC was RsbT, RsbV, and sigmaB dependent in the presence of CCCP but not under the other conditions. hly expression was not RsbT, RsbV, or sigmaB dependent in the presence of either CCCP or salt. opuCA transcript levels did not increase in the presence of rapidly lethal stresses (i.e., pH 2.5 or 13 mM cumene hydroperoxide) despite the enhanced survival of the wild type compared with the survival of the mutant strains under these conditions. These findings highlight the importance of complementing phenotypic characterizations with gene expression studies to identify direct and indirect effects of null mutations in regulatory genes, such as sigB. Overall, our data show that while sigmaB activation occurs through a single pathway under both environmental and energy stress conditions, regulation of expression of some stress response and virulence genes in the sigmaB regulon (e.g., clpC) appears to require networks involving multiple transcriptional regulators.

FIG. 1.

Relative levels of cDNA for opuCA, expressed as log (opuCA mRNA copy number/mean housekeeping gene [HKG] copy number) for L. monocytogenes wild-type strain 10403S (solid bars), ΔsigB (shaded bars), ΔrsbT (cross-hatched bars), and ΔrsbV (open bars) exposed to salt stress (BHI broth containing 0.3 M NaCl). Following addition of the NaCl, cells were collected by centrifugation (5 min, 2,520 × g) either immediately (“no incubation”) or following exposure to NaCl for 5 min. RNAprotect was added to stop transcription and stabilize the RNA following centrifugation. The values are the means from three independent experiments. Comparisons of the four strains under each condition (e.g., normalized opuCA transcript levels in BHI broth with no incubation for the wild-type and three mutant strains) using Fisher's LSD resulted in identification of strains whose opuCA transcript levels differed; the bars for strains whose opuCA transcript levels differed (within a given condition) are labeled with different letters.

FIG. 2.

Relative levels of cDNA for opuCA, expressed as log (opuCA mRNA copy number/mean housekeeping gene [HKG] copy number) under ethanol stress conditions (BHI broth containing 16.5% [vol/vol] ethanol) (A) or under acid stress conditions (BHI broth containing HCl, pH 4.5) (B). RNAprotect was added to stop transcription and stabilize the RNA, and then cells were collected by centrifugation (5 min, 2,520 × g) either immediately following addition of each stressor (“no incubation”) or following exposure to stress for 5 min. The strains used were L. monocytogenes wild-type strain 10403S (solid bars) and the ΔsigB (shaded bars), ΔrsbT (cross-hatched bars), and ΔrsbV (open bars) strains. The values are means from three independent experiments. Comparisons of the four strains under each condition (e.g., normalized opuCA transcript levels in BHI broth with no incubation for the wild-type and three mutant strains) using Fisher's LSD resulted in identification of strains whose opuCA transcript levels differed; the bars for strains whose opuCA transcript levels differed (within a given condition) are labeled with different letters. EtOH, ethanol.

FIG. 3.

Relative cDNA copy numbers for opuCA, expressed as log (opuCA mRNA copy number/mean housekeeping gene [HKG] copy number) under energy stress conditions (intracellular ATP depletion [BHI broth containing 32 μg/ml CCCP]). Following addition of the CCCP, cells were collected by centrifugation and treated as described in the legend to Fig. 1. The strains used were L. monocytogenes wild-type strain 10403S (solid bars) and the ΔsigB (shaded bars), ΔrsbT (cross-hatched bars), and ΔrsbV (open bars) strains. Comparisons of the four strains under each condition with Fisher's LSD resulted in identification of strains whose opuCA transcript levels differed; the bars for strains whose opuCA transcript levels differed (within a given condition) are labeled with different letters.

FIG. 4.

Relative cDNA copy numbers for opuCA, expressed as log (opuCA mRNA copy number/mean housekeeping gene [HKG] copy number) under energy stress conditions (carbon starvation; growth in glucose-limiting [0.04%, wt/vol] defined media). Culture aliquots were removed at 6, 12, 18, and 24 h postinoculation. RNAprotect was added to each aliquot to stop transcription and to stabilize the RNA, and then cells were collected by centrifugation (5 min, 2,520 × g). As a consequence of the different patterns of housekeeping gene expression in the wild-type and mutant strains at 18 and 24 h, only data for 6 and 12 h were used to quantify the relative expression patterns of opuCA in the different strains. The strains used were L. monocytogenes wild-type strain 10403S (solid bars) and the ΔsigB (shaded bars), ΔrsbT (cross-hatched bars), and ΔrsbV (open bars) strains. Comparisons of the four strains under each condition (e.g., normalized opuCA transcript levels at 6 h for the wild-type and three mutant strains) using Fisher's LSD resulted in identification of strains whose opuCA transcript levels differed; the bars for strains whose opuCA transcript levels differed at either 6 or 12 h are labeled with different letters.

FIG. 5.

Bacterial growth and expression of opuCA, gap, and rpoB in glucose-limiting defined medium. The strains used were L. monocytogenes wild-type strain 10403S (⧫) and the ΔsigB (□), ΔrsbT (▴), and ΔrsbV (○) strains. (A) Bacterial growth, expressed in units of absorbance at 600 nm, after 6, 12, 18, and 24 h of incubation in glucose-limiting DM. (Adapted from reference .) (B) Log copy numbers of opuCA transcripts. (C) Log copy numbers of rpoB transcripts. (D) Log copy numbers of gap transcripts. The values are means from three independent experiments.

FIG. 6.

Relative cDNA copy numbers for clpC, expressed as log (clpC mRNA copy number/mean housekeeping gene [HKG] copy number) following exposure to CCCP. Following addition of the CCCP, cells were collected by centrifugation and treated as described in the legend to Fig. 1. The strains used were L. monocytogenes wild-type strain 10403S (solid bars) and the ΔsigB (shaded bars), ΔrsbT (cross-hatched bars), and ΔrsbV (open bars) strains. Comparisons of the four strains under each condition with Fisher's LSD resulted in identification of strains whose clpC transcript levels differed; the bars for strains whose clpC transcript levels differed (within a given condition) are labeled with different letters.