Table 1.
Virulence, Invasion, and Intracellular Spread of L. monocytogenes Strains
Figure 1.
Overexpression of Flagellin by MogR-Negative L. monocytogenes at RT Results in a Chaining Phenotype
(A) Western blot analysis of FlaA levels in cellular fractions of wild-type and ΔmogR strains. L. monocytogenes cultures were grown at RT or 37 °C for approximately 24 h. Secreted, surface-localized, and cytoplasmic FlaA protein was detected by Western blot using a FlaA-specific antibody.
(B) L. monocytogenes strains wild-type, ΔflaA, ΔmogR, and ΔmogR ΔflaA were grown at RT or 37 °C for approximately 24 h, stained for flagella, and analyzed by phase-contrast microscopy. Open arrowheads indicate flagella. The percentage of chaining events within a given population is shown below each panel. A chaining event was defined as a chain of three or more bacteria. A total of 300 bacterial events were counted for each population. The mean and standard deviation of three independent experiments are given.
Figure 2.
MogR Primarily Functions as a Transcriptional Repressor at 37 °C
(A) Graphical representation of genes differentially expressed between ΔmogR and wild-type during growth in different conditions as determined by microarray analysis. Only genes whose absolute expression was altered by a minimum of 3.5-fold (p < 0.01) are represented. Shaded bars indicate genes repressed by MogR, and hatched bars represent genes activated by MogR.
(B) Venn diagram representation of MogR-repressed genes. Only genes that were downregulated in wild-type relative to ΔmogR by at least 3.5-fold (p < 0.01) are presented.
(C) Graphical analysis of the fold-repression of genes regulated by MogR during growth in different conditions. Genes were defined as MogR repressed if they exhibited at least a 3.5 fold-change (p < 0.01) between wild-type and ΔmogR. The most highly repressed gene in a given condition was assigned a rank of 1. Gene rankings were plotted against fold-repression. The average fold-repression was 5-, 27-, and 21-fold for growth in BHI at RT, BHI at 37 °C, and J774 host cells at 37 °C, respectively.
Table 2.
Microarray Analysis of Flagellar Motility Gene Expression in MogR-Negative L. monocytogenes Relative to Wild-Type during Growth in Different Conditions
Figure 3.
Purified MogR Is Sufficient to Bind the Promoter Regions of Multiple Flagellar Motility Genes
(A) Gel shift analysis of MogR binding to the flaA promoter region. Radiolabeled flaA promoter DNA spanning region −162 to +8 relative to the transcription start site was incubated with increasing concentrations of purified His6-tagged MogR (lanes 1 to 8) and in the presence of unlabeled competitor DNA (lanes 7 and 8). The binding reactions were analyzed by nondenaturing PAGE. The identity of unlabeled competitor DNA added in 50-fold excess to binding reactions is indicated. Shifted (S), supershifted (SS), and super-supershifted (SSS) DNA complexes are indicated.
(B) Gel shift analysis of MogR binding to flagellar motility gene promoter regions. Increasing amounts of purified His6-tagged MogR were incubated with various radiolabeled DNA probes (lanes 1 to 4) and in the presence of unlabeled competitor probe DNA of the same identity (lane 4). The identity of each probe is indicated. The probe for cheY spans the region from −108 to +74 relative to the transcription start site. The probe for lmo0703 spans the region from −164 to +21 relative to the translation start site. The probes for lmo0723 and lmo1699 span the regions from −123 to +18 relative to the translation start site.
(C) DNase I footprinting analysis of MogR binding to the flaA promoter region. The noncoding strand probe spans the region from −162 to +8 relative to the transcription start site. Negative numbers indicate the approximate distance from the transcriptional start site. The high affinity MogR binding site is indicated by a solid bracket and is denoted as region I. The lower affinity binding sites are marked with dashed brackets and identified as regions II and III.
(D) DNA sequence of the flaA promoter region. The bottom strand is the coding strand and reads 5′ to 3′. The −35 and −10 elements and transcription start site (+1) are indicated in bold and by a solid line above the sequence. Solid bracket indicates the region protected at low MogR concentrations (region I), while the dashed brackets mark the regions protected with higher concentrations of MogR (regions II and III).
Figure 4.
MogR Recognizes a Minimum of Two TTTT-N5-AAAA Sites
(A) Gel shift analysis of MogR binding to the −35 region of the flaA promoter. Radiolabeled flaA probe DNA spanning region −58 to −13 relative to the transcription start site was incubated with increasing concentrations of purified His6-tagged MogR (lanes 2 to 4, 6 to 8, and 10 to 12) and in the presence of unlabeled competitor DNA (lanes 4, 8, and 12). Radiolabeled wild-type (wt) probe was used in lanes 1 to 4; radiolabeled probe harboring mutations in four residues of one binding site (TA) was used in lanes 5 to 8; and a radiolabeled probe harboring mutations in six residues interspersed between the predicted MogR contact sites (N-mut) was used in lanes 9 to 12. Sequences of the DNA probes are given below the panel. Mutations introduced are underlined and in bold. Gray boxes denote putative MogR binding sites. The binding reactions were analyzed by nondenaturing PAGE. The identity of unlabeled competitor DNA added in 50-fold excess to binding reactions is indicated.
(B) Sequences of predicted MogR binding sites within the promoter regions of genes directly regulated by MogR. Gray boxes around text in bold indicates predicted MogR binding sites with italicized text indicating deviations from the consensus TTTT-N5-AAAA binding site. The transcriptional start sites of flaA, cheY, and lmo0675 have been precisely mapped by primer extension and are designated by +1. The approximate transcriptional start regions for lmo0723, lmo0703, and lmo1699 have been identified by primer extension. The predicted −35 and −10 elements are underlined. The distance to the coding region is indicated, and the translation initiation codon is boxed and in bold. Region I, and regions II and III correspond to the high and low affinity binding sites, respectively, mapped by DNase I footprinting. Region I is denoted by a solid bracket. Regions II and III are marked by a dashed bracket.
Figure 5.
MogR Protein Levels Are Temperature Independent
Western blot analysis of MogR protein levels from wild-type L. monocytogenes. Whole cell lysates prepared from 14- to 16-h cultures grown at 37 °C or 30 °C were separated on a 12% SDS-PAGE gel and analyzed by Western blot using a MogR-specific antibody. Number of CFUs of bacteria analyzed and growth temperature is indicated.
Figure 6.
DegU Is Required to Alleviate MogR Repression Activity and for Posttranscriptional Regulation of FlaA Production
(A) Analysis of flaA transcript levels. L. monocytogenes strains wild-type, ΔmogR, ΔdegU, and ΔmogR ΔdegU were grown 14 to 16 h in BHI broth at RT or 37 °C. RNA was harvested and flaA transcript levels were analyzed by primer extension. flaA promoter activity was also determined by β-galactosidase assays using flaA::Tn917 transposon insertion derived strains grown similarly at RT or 37 °C. β-Galactosidase activities represent the means and standard deviations of three independent experiments.
(B) Western blot analysis of FlaA protein levels in the strains used in (A). Whole cell lysates were prepared from bacterial cultures described in (A), separated on a 12% SDS-PAGE gel, and analyzed by Western blot using a FlaA-specific antibody.
(C) Flagellar staining of strains described in (A). Cultures grown at 37 °C or RT were stained for flagella and analyzed by microscopy. Open arrowheads indicate flagella. The percentage of bacteria harboring at least one flagellum is given below.
(D) Motility analysis of strains used in (A). A single colony was inoculated with a straight needle in low-agar (0.375%) BHI plates and incubated at 37 °C or RT for 48 h.
Figure 7.
Model for the Regulation of FlaA Expression at Low Temperatures in L. monocytogenes
(A) Binding of MogR to TTTT-N5-AAAA sites (open boxes) in the flaA promoter region represses flaA transcription by occluding RNAP binding (solid lines over open boxes). The flaA coding region is represented as a filled block arrow; transcription initiating from the flaA promoter is represented as a bent arrow; and the −35 and −10 sites are marked. At low temperatures, MogR repression activity is antagonized by an unknown factor (X). This factor may be DegU, a DegU-interacting factor, and/or a DegU-regulated factor. X may modulate MogR repression activity by either interacting directly with MogR (thick dashed line) or binding to the flaA promoter region (thin dashed line) impeding MogR binding to TTTT-N5-AAAA sites.
(B) DegU regulates FlaA protein levels through a posttranscriptional mechanism. At low temperatures when flaA transcripts are present (wavy lines), DegU functions to maximize FlaA protein production, either by enhancing translation of flaA transcripts or stabilizing FlaA protein.