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Transcriptional and post-transcriptional regulation of the GmaR antirepressor governs temperature-dependent control of flagellar motility in Listeria monocytogenes.

ABSTRACT: Flagellar motility in Listeria monocytogenes (Lm) is restricted to temperatures below 37 degrees C due to the opposing activities of the MogR transcriptional repressor and the GmaR antirepressor. Previous studies have suggested that both the DegU response regulator and MogR regulate expression of GmaR. In this report, we further define the role of DegU for GmaR production and flagellar motility. We demonstrate that deletion of the receiver domain of DegU has no effect on flagellar motility in Lm. Using transcriptional reporter fusions, we determined that gmaR is cotranscribed within an operon initiating with fliN. Furthermore, the fliN-gmaR promoter (p(fliN-gmaR)) is transcriptionally activated by DegU and is also MogR-repressed. DNA affinity purification, gel mobility shift and footprinting analyses revealed that both DegU and MogR directly bind fliN-gmaR promoter region DNA and that the binding sites do not overlap. Quantitative analysis of gmaR transcripts in Delta mogR bacteria indicated that transcriptional activation of p(fliN-gmaR) by DegU is not inherently temperature-dependent. However, GmaR protein was not detectable at 37 degrees C in Delta mogR bacteria, indicating that a temperature-dependent, post-transcriptional mechanism limits GmaR production to temperatures below 37 degrees C. Our findings reveal that flagellar motility in Lm is governed by both temperature-dependent transcriptional and post-transcriptional regulation of the GmaR antirepressor.

SUBMITTER: Kamp HD

PROVIDER: S-EPMC2807129 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Transcriptional and post-transcriptional regulation of the GmaR antirepressor governs temperature-dependent control of flagellar motility in Listeria monocytogenes.

Kamp Heather D HD Higgins Darren E DE

Molecular microbiology 20091001 2

Flagellar motility in Listeria monocytogenes (Lm) is restricted to temperatures below 37 degrees C due to the opposing activities of the MogR transcriptional repressor and the GmaR antirepressor. Previous studies have suggested that both the DegU response regulator and MogR regulate expression of GmaR. In this report, we further define the role of DegU for GmaR production and flagellar motility. We demonstrate that deletion of the receiver domain of DegU has no effect on flagellar motility in Lm ...[more]

PMID: 19796338

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Project description:Facultative bacterial pathogens must adapt to multiple stimuli to persist in the environment or establish infection within a host. Temperature is often utilized as a signal to control expression of virulence genes necessary for infection or genes required for persistence in the environment. However, very little is known about the molecular mechanisms that allow bacteria to adapt and respond to temperature fluctuations. Listeria monocytogenes (Lm) is a food-borne, facultative intracellular pathogen that uses flagellar motility to survive in the extracellular environment and to enhance initial invasion of host cells during infection. Upon entering the host, Lm represses transcription of flagellar motility genes in response to mammalian physiological temperature (37°C) with a concomitant temperature-dependent up-regulation of virulence genes. We previously determined that down-regulation of flagellar motility is required for virulence and is governed by the reciprocal activities of the MogR transcriptional repressor and the bifunctional flagellar anti-repressor/glycosyltransferase, GmaR. In this study, we determined that GmaR is also a protein thermometer that controls temperature-dependent transcription of flagellar motility genes. Two-hybrid and gel mobility shift analyses indicated that the interaction between MogR and GmaR is temperature sensitive. Using circular dichroism and limited proteolysis, we determined that GmaR undergoes a temperature-dependent conformational change as temperature is elevated. Quantitative analysis of GmaR in Lm revealed that GmaR is degraded in the absence of MogR and at 37°C (when the MogR:GmaR complex is less stable). Since MogR represses transcription of all flagellar motility genes, including transcription of gmaR, changes in the stability of the MogR:GmaR anti-repression complex, due to conformational changes in GmaR, mediates repression or de-repression of flagellar motility genes in Lm. Thus, GmaR functions as a thermo-sensing anti-repressor that incorporates temperature signals into transcriptional control of flagellar motility. To our knowledge, this is the first example of a protein thermometer that functions as an anti-repressor to control a developmental process in bacteria.

Project description:Flagellar motility is an essential mechanism by which bacteria adapt to and survive in diverse environments. Although flagella confer an advantage to many bacterial pathogens for colonization during infection, bacterial flagellins also stimulate host innate immune responses. Consequently, many bacterial pathogens down-regulate flagella production following initial infection. Listeria monocytogenes is a facultative intracellular pathogen that represses transcription of flagellar motility genes at physiological temperatures (37 degrees C and above). Temperature-dependent expression of flagellar motility genes is mediated by the opposing activities of MogR, a DNA-binding transcriptional repressor, and DegU, a response regulator that functions as an indirect antagonist of MogR. In this study, we identify an additional component of the molecular circuitry governing temperature-dependent flagellar gene expression. At low temperatures (30 degrees C and below), MogR repression activity is specifically inhibited by an anti-repressor, GmaR. We demonstrate that GmaR forms a stable complex with MogR, preventing MogR from binding its DNA target sites. GmaR anti-repression activity is temperature dependent due to DegU-dependent transcriptional activation of gmaR at low temperatures. Thus, GmaR production represents the first committed step for flagella production in L. monocytogenes. Interestingly, GmaR also functions as a glycosyltransferase exhibiting O-linked N-acetylglucosamine transferase (OGT) activity for flagellin (FlaA). GmaR is the first OGT to be identified and characterized in prokaryotes that specifically beta-O-GlcNAcylates a prokaryotic protein. Unlike the well-characterized, highly conserved OGT regulatory protein in eukaryotes, the catalytic activity of GmaR is functionally separable from its anti-repression function. These results establish GmaR as the first known example of a bifunctional protein that transcriptionally regulates expression of its enzymatic substrate.

Project description:Bacterial lipoproteins are membrane-associated proteins with a characteristic acylated N-terminal cysteine residue anchoring C-terminal globular domains to the membrane surface. While all lipoproteins are modified with acyl chains, the number, length, and position can vary depending on host. The acylation pattern also alters ligand recognition by the Toll-like receptor 2 (TLR2) protein family, a signaling system that is central to bacterial surveillance and innate immunity. In select Listeria monocytogenes isolates carrying certain plasmids, copper exposure converts the lipoprotein chemotype into a weak TLR2 ligand through expression of the enzyme lipoprotein intramolecular acyltransferase (Lit). In this study, we identify the response regulator (CopR) from a heavy metal-sensing two-component system as the transcription factor that integrates external copper levels with lipoprotein structural modifications. We show that phosphorylated CopR controls the expression of three distinct transcripts within the plasmid cassette encoding Lit2, prolipoprotein diacylglyceryl transferase (Lgt2), putative copper resistance determinants, and itself (the CopRS two-component system). CopR recognizes a direct repeat half-site consensus motif (TCTACACA) separated by 3 bp that overlaps the -35 promoter element. Target gene expression and lipoprotein conversion were not observed in the absence of the response regulator, indicating that CopR phosphorylation is the dominant mechanism of regulation. IMPORTANCE Copper is a frontline antimicrobial used to limit bacterial growth in multiple settings. Here, we demonstrate how the response regulator CopR from a plasmid-borne two-component system in the opportunistic pathogen L. monocytogenes directly induces lipoprotein remodeling in tandem with copper resistance genes due to extracellular copper stress. Activation of CopR by phosphorylation converts the lipoprotein chemotype from a high- to low-immunostimulatory TLR2 ligand. The two-component system-mediated coregulation of copper resistance determinants, in tandem with lipoprotein biosynthesis demonstrated here in L. monocytogenes, may be a common feature of transmissible copper resistance cassettes found in other Firmicutes.

Dataset Information

Transcriptional and post-transcriptional regulation of the GmaR antirepressor governs temperature-dependent control of flagellar motility in Listeria monocytogenes.

Publications

Transcriptional and post-transcriptional regulation of the GmaR antirepressor governs temperature-dependent control of flagellar motility in Listeria monocytogenes.

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