A bifunctional O-GlcNAc transferase governs flagellar motility through anti-repression

Abstract

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°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°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 ␤-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. Flagellar motility is a fundamental mechanism by which bacteria acquire nutrients, colonize surfaces, and establish infections. Although flagellar motility confers a growth advantage in many environments, production of flagella is a complex, energy-demanding developmental process and thus is exquisitely regulated in response to many environmental cues (Aldridge and Hughes 2002;Macnab 2003). For example, flagella can enhance adherence and invasion in the early stages of host infection, yet continuous production of flagella during infection can stimulate innate immune responses (Hayashi et al. 2001;Molofsky et al. 2006;Ren et al. 2006) or impede subsequent colonization events (for review, see Ramos et al. 2004). Thus, many facultative bacterial pathogens down-regulate production of flagella shortly after infection (Akerley et al. 1995;Hughes and Galan 2002). A primary environmental cue that initiates repression of flagellar gene transcription during infection is physiological temperature (37°C) (Ott et al. 1991;Akerley and Miller 1993;Kapatral et al. 1996).Listeria...

O-Glycosylation of the N-terminal Region of the Serine-rich Adhesin Srr1 of Streptococcus agalactiae Explored by Mass Spectrometry

“…In L. monocytogenes, the flagellin monomer is modified with N-acetylglucosamine (GlcNAc) residues at up to six different sites by GmaR, a cytoplasmic O-GlcNAc-transferase (OGT) homologous to eukaryotic OGTs (18). GmaR was the first prokaryotic enzyme of this type to be characterized (20). The type of sugar added to flagellins and the number of modified sites is different between species and is even straindependent in Clostridium difficile (13).…”

“…The glycosylation of flagellins has been demonstrated in Clostridium spp. and in Listeria monocytogenes (13,18); the modification is required for flagella assembly in C. difficile (13) and motility in L. monocytogenes (19,20). In L. monocytogenes, the flagellin monomer is modified with N-acetylglucosamine (GlcNAc) residues at up to six different sites by GmaR, a cytoplasmic O-GlcNAc-transferase (OGT) homologous to eukaryotic OGTs (18).…”

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

“…While O-GlcNAc appears more common in metazoans, there is evidence for this PTM in simple eukaryotes such as Aspergillus niger (Machida and Jigami 1994) and in some prokaryotes (Shen et al 2006). O-GlcNAc is cycled on and off serine (Ser) and/or threonine (Thr) residues by just two enzymes: the O-GlcNAc transferase (OGT) that catalyzes the addition of O-GlcNAc and the O-GlcNAcase that removes O-GlcNAc (Fig.…”

Thermosensing to Adjust Bacterial Virulence in a Fluctuating Environment

“…Transcriptional activator of virulence genes; binds to DNA as homodimer at 20-25°C, defolds partially at higher temperatures and looses its DNA binding capacity; multisensor: salicylate prevents DNA-binding of RovA [137,139,142] Listeria monocytogenes MogR:GmaR MogR binds to flagella genes and represses transcription at 37°C; anti-repressor GmaR binds MogR at 30°C; in complex no repression; GmaR misfolds at 37°C and MogR:GmaR complex disassembles [150,152,153,155] Agrobacterium tumefaciens VirA Two-component system VirAG activates transcription of vir genes only below 32°C; sensor kinase VirA changes conformation at high temperatures and is inactive [4] Pseudomonas syringae CorS Two-component system CorSR activates transcripttion of phytotoxin biogenesis genes at 18°C; at 28°C: rearrangements of the transmembrane domains of the sensor kinase CorS prevent autophosphorylation and/or phosphotransfer to response regulator [177,178] Streptococci M-like proteins Class C proteins bind to plasma proteins low temperatures as coiled coils; homodimers disrupt at 37°C; C-repeats seem to be thermosensitive Cedervall 1995Cedervall 1997 …”

“…3) [150][151][152]. MogR binds to the promoter of the flagellar motility genes and prevents their expression at physiological temperatures [152][153][154]. Below 37°C…”