SigmaB-dependent and sigmaB-independent mechanisms contribute to transcription of Listeria monocytogenes cold stress genes during cold shock and cold growth

Abstract

The role of the stress response regulator sigma(B) (encoded by sigB) in directing the expression of selected putative and confirmed cold response genes was evaluated using Listeria monocytogenes 10403S and an isogenic DeltasigB mutant, which were either cold shocked at 4 degrees C in brain heart infusion (BHI) broth for up to 30 min or grown at 4 degrees C in BHI for 12 days. Transcript levels of the housekeeping genes rpoB and gap, the sigma(B)-dependent genes opuCA and bsh, and the cold stress genes ltrC, oppA, and fri were measured using quantitative reverse transcriptase PCR. Transcriptional start sites for ltrC, oppA, and fri were determined using rapid amplification of cDNA ends PCR. Centrifugation was found to rapidly induce sigma(B)-dependent transcription, which necessitated the use of centrifugation-independent protocols to evaluate the contributions of sigma(B) to transcription during cold shock. Our data confirmed that transcription of the cold stress genes ltrC and fri is at least partially sigma(B) dependent and experimentally identified a sigma(B)-dependent ltrC promoter. In addition, our data indicate that (i) while sigma(B) activity is induced during 30 min of cold shock, this cold shock does not induce the transcription of sigma(B)-dependent or -independent cold shock genes; (ii) sigma(B) is not required for L. monocytogenes growth at 4 degrees C in BHI; and (iii) transcription of the putative cold stress genes opuCA, fri, and oppA is sigma(B) independent during growth at 4 degrees C, while both bsh and ltrC show growth phase and sigma(B)-dependent transcription during growth at 4 degrees C. We conclude that sigma(B)-dependent and sigma(B)-independent mechanisms contribute to the ability of L. monocytogenes to survive and grow at low temperatures.

FIG. 1.

Log-transformed absolute transcript levels for selected genes before and after centrifugation of L. monocytogenes. Transcript levels were determined by qRT-PCR analysis of RNA isolated from (i) log-phase L. monocytogenes parent strain cells grown at 37°C prior to centrifugation, (ii) log-phase L. monocytogenes parent strain cells centrifuged and immediately exposed for <30 s to fresh BHI prewarmed to 37°C, and (iii) log-phase L. monocytogenes parent strain cells centrifuged and immediately exposed to fresh BHI prechilled to 4°C for <30 s. Values shown represent the averages of results from qRT-PCR assays performed on two independent RNA collections. Error bars indicate the data range. NC, no centrifugation; C, centrifugation.

FIG. 2.

Normalized, log-transformed transcript levels for opuCA, bsh, ltrC, oppA, and fri during the cold shock at 4°C and during control exposure to 37°C of the L. monocytogenes parent strain and a ΔsigB mutant. Transcript levels were determined by qRT-PCR analysis of RNA isolated from the (i) log-phase L. monocytogenes parent strain (10403S) and ΔsigB strain before exposure (directly from the culture [DFC]); (ii) parent strain and ΔsigB strain cold shocked at 4°C in prechilled stainless steel pans for <30 s (0 min), 15 min, and 30 min; and (iii) parent strain and ΔsigB mutant exposed to 37°C in prewarmed stainless steel pans for <30 s (0 min), 15 min, and 30 min. Transcript levels for each gene were log transformed and normalized to the geometric mean of the transcript levels for the housekeeping genes rpoB and gap. Values shown represent the averages of results from qRT-PCR assays performed on three independent RNA collections; error bars show standard deviations. Results of statistical analyses using ANOVA are detailed in the text and shown in Table S1 in the supplemental material.

FIG. 3.

Growth of the L. monocytogenes parent strain 10403S and the ΔsigB strain at 4°C. The OD₆₀₀s of the L. monocytogenes parent strain 10403S and ΔsigB mutant were measured during growth at 4°C for 12 days. Data on the left y axis show the average OD₆₀₀ values from five independent experiments; error bars indicate standard deviations. Data on the right y axis show the average cell counts for L. monocytogenes strain 10403S and the ΔsigB mutant determined in one experiment.

FIG. 4.

Normalized, log-transformed transcript levels of rpoB, gap, opuCA, bsh, ltrC, oppA, and fri for the L. monocytogenes parent strain and the ΔsigB strain grown at 4°C. Transcript levels were determined by qRT-PCR analysis of RNA isolated from the L. monocytogenes parent strain 10403S and the ΔsigB strain grown in BHI at 4°C for 3, 6, 9, and 12 days. Transcript levels for each gene were log transformed and normalized to the geometric mean of the transcript levels for the housekeeping genes rpoB and gap. Values shown represent the averages of results of qRT-PCR assays performed on three independent RNA collections; error bars show standard deviations. Results of statistical analyses using ANOVA are detailed in the text and shown in Table S2 in the supplemental material.

FIG. 5.

RACE-PCR and promoter sequences of ltrC and fri. RACE-PCR was performed using total RNA collected from L. monocytogenes parent strain (10403S) and ΔsigB cells cold shocked at 4°C for 30 min. (A) Agarose gel electrophoresis of RACE-PCR products for ltrC (lanes 2 to 6) and fri (lanes 8 to 12). Lanes 1 and 7, pGEM DNA size marker (Promega, Madison, WI); lanes 2 and 8, PCR of untailed L. monocytogenes parent strain cDNA (negative control); lanes 3 and 9, PCR of poly(dC)-tailed parent strain cDNA; lanes 4 and 10, PCR of untailed L. monocytogenes ΔsigB cDNA (negative control); lanes 5 and 11, PCR of poly(dC)-tailed ΔsigB cDNA; lanes 6 and 12, negative-control PCR with no cDNA. Arrows indicate σ^B-dependent transcripts (i.e., transcripts that were detected in RNA from the parent strain but not in RNA from the ΔsigB strain). The contrast of the ltrC gel picture was enhanced to visualize DNA fragments. (B) σ^B promoter sequence for ltrC determined by RACE-PCR. The −35 and −10 regions are underlined. The triangle indicates the transcriptional start site determined by RACE-PCR.

FIG. 6.

RACE-PCR and promoter sequence of opuCA. RACE-PCR was performed using total RNA collected from the L. monocytogenes parent strain and ΔsigB strain after growth to stationary phase (day 9) at 4°C or after growth to stationary phase (OD₆₀₀ = 2.0) at 37°C and previously described opuCA-specific primers (25). (A) Agarose gel electrophoresis of RACE-PCR products for opuCA in L. monocytogenes grown to stationary phase in BHI at 37°C (lanes 2 to 5) or at 4°C (lanes 7 to 10). Lanes 1 and 6, pGEM DNA size marker; lanes 2 and 7, PCR of untailed L. monocytogenes parent strain (10403S) cDNA (negative control); lanes 3 and 8, PCR of poly(dC)-tailed parent strain cDNA; lanes 4 and 9, PCR of untailed ΔsigB strain cDNA (negative control); lanes 5 and 10, PCR of poly(dC)-tailed ΔsigB cDNA; lane 11, negative-control PCR with no cDNA. (B) σ^B and σ^A promoter sequences for opuCA as determined by RACE-PCR. The −35 and −10 regions for the σ^B- and σ^A-dependent promoters are double and single underlined, respectively. Triangles indicate transcriptional start sites for opuCA determined by RACE-PCR in the L. monocytogenes parent strain (▵) and ΔsigB mutant () grown at 37°C and for the parent strain and ΔsigB mutant grown at 4°C (▴).