σB-Dependent and σB-Independent Mechanisms Contribute to Transcription ofListeria monocytogenesCold Stress Genes during Cold Shock and Cold Growth

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