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 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.

Figure 1.

Lmo0688 is required to alleviate MogR repression of flaA transcription at low temperatures and to glycosylate FlaA. (A) Motility analysis of L. monocytogenes strains. A single colony of wild type (wt), Δ688, Δ688/c688, ΔmogR Δ688, and ΔmogR was inoculated in low-agar (0.375%) BHI plates and incubated for 48 h at room temperature. Strain Δ688/c688 contains a complementing lmo0688 expressed from a heterologous promoter in an ectopic chromosomal locus. (B) Analysis of FlaA protein in whole-cell lysates of strains used in A. L. monocytogenes cultures were grown in BHI broth for 20 h at room temperature. Lysates were separated by SDS-PAGE and analyzed by Western blot using a FlaA-specific antibody. (C) Northern blot analysis of flaA transcripts in selected strains used in A. L. monocytogenes cultures were grown in BHI broth at room temperature or 37°C, and RNA was harvested after 20 h. (D) Analysis of β-O-linked glycosylation in cell-wall-associated fractions of ΔmogR Δ688 and ΔmogR. L. monocytogenes cultures were grown in BHI broth for 20 h at room temperature. Cell-wall-associated FlaA was resolved by SDS-PAGE and detected by Coomassie stain (left panel) and Western blot using a β-O-linked GlcNAc-specific antibody (right panel). Differential mobility of FlaA by SDS-PAGE is noted (arrows).

Figure 2.

DegU and Lmo0688 have similar roles in antagonizing MogR repression of flagellar motility gene expression. Cluster display of microarray analyses of Δ688 (Lmo0688 regulon), ΔdegU (DegU regulon), and ΔmogR (MogR regulon) compared with wild type grown in BHI broth at room temperature or 37°C. (Far left) Relationships among genes are represented by a dendrogram, where the branch lengths reflect the degree of similarity between gene expression patterns. The color scale ranges from saturated green for fold ratios 5.0 and below to saturated red for fold ratios 5.0 and above. Green represents genes that are activated in wild type relative to the mutant, red represents genes that are repressed in wild type relative to the mutant, and black represents genes for which no difference in expression was observed between the mutant and wild type. Gray indicates genes for which the hybridization data was too poor to be included in the analysis.

Figure 3.

Lmo0688 is the DegU-regulated factor that antagonizes MogR repression activity. (A) Northern blot analysis of flaA transcript levels. RNA was harvested from L. monocytogenes strains wild type (wt), ΔdegU, ΔdegU/c688, ΔmogR, ΔmogR Δ688, and Δ688 following growth for 20 h in BHI broth at room temperature. (B) Western blot analysis of FlaA protein levels. Cultures were grown 6 h in BHI broth at room temperature. Whole-cell lysates were analyzed using a FlaA-specific antibody. (C) Motility analysis of strains used in B. A single colony was inoculated in 0.375% BHI agar and incubated for 48 h at room temperature.

Figure 4.

The glycosyltransferase activity of Lmo0688 is dispensable for FlaA production. (A) Schematic of Lmo0688 protein. The predicted glycosyltransferase domain (amino acids 6–103) is shown in red, and the DxD active site motif (amino acids 83–85) is represented as a black star. Three TPR domains (amino acids 165–350) are represented as a hatched box. The C-terminal domain lacks homology with known proteins. (B) Partial multiple sequence alignment of a subset of Lmo0688 homologs. Completely conserved identical residues are blocked in blue, conserved identical residues are blocked in green, and conserved similar residues are blocked in gray. The DxD glycosyltransferase motif is marked above the alignment, and the active site mutations in L. monocytogenes Lmo0688 (D83N D85N) are noted below the alignment. (C) In vitro glycosylation assay. L. monocytogenes strains ΔmogR Δ688 ΔflaA and ΔmogR Δ688 were grown in BHI broth for 4 h at 28°C and mechanically lysed. Purified His₆-tagged Lmo0688 (wild type or active site mutant, D83N D85N) was incubated with the indicated whole-cell lysates in the presence of [¹⁴C]-UDP-GlcNAc. O-GlcNAcylation was visualized by autoradiography. (D) Motility analysis of wild type (wt) and 688*. A single colony was inoculated in 0.375% BHI agar and incubated for 48 h at room temperature. 688* carries the active site mutations (D83N D85N) that disrupt glycosyltransferase activity. (E) Analysis of FlaA glycosylation in strains used in D. L. monocytogenes strains were grown in BHI broth for 20 h at room temperature. FlaA protein levels in the cell-wall-associated fraction were resolved by SDS-PAGE and examined by Coomassie stain (left panel) and Western blot analysis using an β-O-linked GlcNAc-specific antibody (right panel).

Figure 5.

Lmo0688 removes MogR bound to flaA promoter region DNA by protein–protein interaction. (A) Gel shift analysis of MogR and Lmo0688 binding to flaA promoter region DNA. Radiolabeled flaA promoter region DNA spanning −162 to +8 relative to the transcriptional start site was incubated with a constant amount (40 nM) of purified His₆-tagged MogR to which increasing concentrations of His₆-tagged Lmo0688 (lanes 2–6) or 240 nM His₆-tagged DegU (lane 7) was added. (Lanes 8–11) Increasing concentrations of His₆-tagged Lmo0688 alone was incubated with radiolabeled flaA promoter region DNA. The binding reactions were separated by nondenaturing PAGE and detected by autoradiography. Shifted (S), supershifted (SS), and supersupershifted (SSS) DNA complexes are indicated. (B) Pull-down assay of MogR by Ni²⁺ affinity purification of His₆-tagged Lmo0688. Purified His₆-tagged Lmo0688 was incubated with cell lysates prepared from L. monocytogenes strains wild type (wt), imogR, and ΔmogR. His₆-tagged Lmo0688 and interacting proteins were isolated using Ni-NTA agarose beads. Proteins isolated in the pull-down assay were separated on a 10% SDS-PAGE gel and analyzed by Western blot using either a MogR- or Lmo0688-specific antibody.

Figure 6.

Temperature-dependent expression of Lmo0688 confers temperature specificity to flagellar motility gene transcription. (A) Western blot analysis of Lmo0688 in whole-cell lysates using an Lmo0688-specific antibody. L. monocytogenes strains wild type (wt), ΔdegU, ΔdegU/c688, Δ688/c688, Δ688, ΔmogR, and ΔmogR ΔdegU were grown for 6 h at room temperature or 37°C in BHI broth. Fivefold more sample was loaded for ΔmogR ΔdegU. (B) Northern blot analysis of lmo0688 transcript levels. RNA was harvested from strains grown in A. Blots were overexposed to detect the presence of lmo0688 transcript in the wild-type sample at room temperature. (C) Analysis of flaA promoter activity determined by β-galactosidase assays. flaA∷Tn917 transposon insertion-derived strains were grown for 18–20 h at room temperature or 37°C in BHI broth. β-Galactosidase activities represent the means and standard deviations of three independent experiments.

Figure 7.

Working model for temperature-dependent regulation of flagellar motility gene expression in L. monocytogenes. At 37°C and above, MogR represses transcription by binding to target sequences in promoter region DNA (flaA and gmaR shown). As temperature decreases, DegU activates transcription of gmaR through an unknown mechanism (lightning bolt). GmaR removes MogR bound to target sequences by protein–protein interaction, alleviating repression of flagellar motility gene promoters. Anti-repression results in the production of the flagellin monomer (FlaA). GmaR glycosylates FlaA with β-O-linked GlcNAc (stars). The gmaR coding region is represented by an open arrow, and transcription initiating from an uncharacterized gmaR promoter is indicated by a dashed bent arrow. The flaA coding region is represented by a gray arrow, and transcription initiating from the flaA promoter is indicated by a solid bent arrow. A solid line indicates repression occurring by MogR binding to target sequences (open boxes). This model is representative of events occurring at all flagellar motility gene promoter regions.