Regulation of Transcription of Compatible Solute Transporters by the General Stress Sigma Factor, σ^B, inListeria monocytogenes

Abstract

Listeria monocytogenes is well known for its durable physiological characteristics, which allow the organism to grow at low temperature and pH and high osmolarity. Growth under high osmolarity depends on the accumulation of compatible solutes, among which glycine betaine and carnitine are the preferred solutes for this organism. Three different transport systems, Gbu, BetL, and OpuC, have been identified in L. monocytogenes which serve to scavenge the preferred compatible solutes. The general stress response regulator B has been shown to play an important role in osmotic adaptation in L. monocytogenes, presumably by directing transcription from one or more of the solute transport genes. In the studies presented here, we have used primer extension analyses to identify the promoter elements responsible for transcription of the opuC, gbuA, and betL genes. All three genes are osmotically inducible to some degree. betL is transcribed from a Bindependent promoter, while gbuA is transcribed from dual promoters, one of which is B dependent. opuC is transcribed exclusively from a B -dependent promoter. The betL promoter is similar in sequence to the B -independent gbuAP1 promoter. Kinetic analysis of transcript accumulation after osmotic upshift demonstrated that B -dependent transcripts from gbuAP2 and sigB accumulate for an extended period after upshift, suggesting that B activity may provide a mechanism for sustained high-level expression during osmotic challenge. In contrast to osmotic upshift, expression from the B -dependent opuC and gbuAP2 promoters after temperature upshift and ethanol stress was minimal, suggesting that additional mechanisms may also participate in regulating transcription from these B -dependent promoters.

Specific Osmolyte Transporters Mediate Bile Tolerance in Listeria monocytogenes

“…Significantly, the BilE bile exclusion system shares significant homologies with osmolyte uptake systems in gram-positive organisms (34) and was originally annotated as a glycine betaine/ carnitine/choline ABC transporter (11). In addition, BilE is coregulated with osmolyte uptake systems OpuC, BetL, and Gbu by the alternative sigma factor B (4,13,28,31,33). However, the system does not appear to play a role in compatible solute uptake or osmotolerance and may represent an evolutionary adaptation of an osmolyte transporter toward a more defined role in bile tolerance.…”

σBActivation under Environmental and Energy Stress Conditions inListeria monocytogenes

“…As B can be present in either an active or inactive state (2), measurement of transcript levels for B -dependent genes was used to indirectly quantify the activity of this protein. To do this, transcript levels were determined for opuCA, which encodes a glycine/betaine/carnitine/choline ABC transporter (6,19,28,47,48), and for clpC, which encodes endopeptidase Clp ATP-binding chain C, a stress response protein that contributes to L. monocytogenes phagosomal escape (30,44,45), in the L. monocytogenes wild-type, ⌬sigB, ⌬rsbT, and ⌬rsbV strains under different stress conditions. The previously observed higher hemolytic activities in the ⌬sigB, ⌬rsbT, and ⌬rsbV strains than in the wild-type strain (9) suggested that hly might be expressed in a B -dependent manner.…”

Functional Consequences of Genome Evolution inListeria monocytogenes: the lmo0423 and lmo0422 Genes Encode σCand LstR, a Lineage II-Specific Heat Shock System

“…The role of the general stress regulator sigma B in facilitating growth under stress conditions and in facilitating virulence has recently been of interest with L. monocytogenes (2,3,11,34,41,56,63). In addition to sigma B, there are four other putative alternative sigma subunits in the L. monocytogenes EGDe genome sequence (21).…”

“…Once confirmed, the ⌬sigC allele was excised as a BamHI-PstI fragment, and the ⌬lmo0422 and ⌬lmo0421 alleles were excised as EcoRI-HindIII fragments from the respective pCR4-TOPO vectors and cloned into the temperature-sensitive pKSV-7 shuttle vector (54) to generate plasmids pKSV7⌬421, pKSV7⌬422, and pKSV7⌬sigC. To recombine ⌬sigC, ⌬lmo0422, and ⌬lmo0421 deletions onto the L. monocytogenes chromosome, the pKSV7⌬421, pKSV7⌬422, and pKSV7⌬sigC plasmids were electroporated into L. monocytogenes 10403S as previously described (2,11). Transformants were selected on BHI plates containing 10 g of chloramphenicol/ml.…”