The molecular mechanism for carbon catabolite repression of the chitin response in Vibrio cholerae.

Vibrio cholerae is a facultative pathogen that primarily occupies marine environments. In this niche, V. cholerae commonly interacts with the chitinous shells of crustacean zooplankton. As a chitinolytic microbe, V. cholerae degrades insoluble chitin into soluble oligosaccharides. Chitin oligosaccharides serve as both a nutrient source and an environmental cue that induces a strong transcriptional response in V. cholerae. Namely, these oligosaccharides induce the chitin sensor, ChiS, to activate the genes required for chitin utilization and horizontal gene transfer by natural transformation. Thus, interactions with chitin impact the survival of V. cholerae in marine environments. Chitin is a complex carbon source for V. cholerae to degrade and consume, and the presence of more energetically favorable carbon sources can inhibit chitin utilization. This phenomenon, known as carbon catabolite repression (CCR), is mediated by the glucose-specific Enzyme IIA (EIIAGlc) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). In the presence of glucose, EIIAGlc becomes dephosphorylated, which inhibits ChiS transcriptional activity by an unknown mechanism. Here, we show that dephosphorylated EIIAGlc interacts with ChiS. We also isolate ChiS suppressor mutants that evade EIIAGlc-dependent repression and demonstrate that these alleles no longer interact with EIIAGlc. These findings suggest that EIIAGlc must interact with ChiS to exert its repressive effect. Importantly, the ChiS suppressor mutations we isolated also relieve repression of chitin utilization and natural transformation by EIIAGlc, suggesting that CCR of these behaviors is primarily regulated through ChiS. Together, our results reveal how nutrient conditions impact the fitness of an important human pathogen in its environmental reservoir

The nucleoid occlusion protein SlmA is a direct transcriptional activator of chitobiose utilization in <i>Vibrio cholerae</i>

<div><p>Chitin utilization by the cholera pathogen <i>Vibrio cholerae</i> is required for its persistence and evolution via horizontal gene transfer in the marine environment. Genes involved in the uptake and catabolism of the chitin disaccharide chitobiose are encoded by the <i>chb</i> operon. The orphan sensor kinase ChiS is critical for regulation of this locus, however, the mechanisms downstream of ChiS activation that result in expression of the <i>chb</i> operon are poorly understood. Using an unbiased transposon mutant screen, we uncover that the nucleoid occlusion protein SlmA is a regulator of the <i>chb</i> operon. SlmA has not previously been implicated in gene regulation. Also, SlmA is a member of the TetR family of proteins, which are generally transcriptional repressors. <i>In vitro</i>, we find that SlmA binds directly to the <i>chb</i> operon promoter, and <i>in vivo</i>, we show that this interaction is required for transcriptional activation of this locus and for chitobiose utilization. Using point mutations that disrupt distinct functions of SlmA, we find that DNA-binding, but not nucleoid occlusion, is critical for transcriptional activation. This study identifies a novel role for SlmA as a transcriptional regulator in <i>V</i>. <i>cholerae</i> in addition to its established role as a cell division licensing factor.</p></div

Sequence specificity between the SBS and induced promoter for P_<i>chb</i> activation.

<p>A 112 bp fragment of P_<i>chb</i>-<i>gfp</i> (from -162 to -50 bp) was swapped out with a fragment of equal size from the <i>lacZ</i> gene and activation was determined by assessing fluorescence. All data are shown as the mean ± SD and are from at least three independent biological replicates. *** = <u><i>p</i></u><0.001.</p

SlmA is a direct transcriptional activator of P_<i>chb</i>.

<p>(<b>A</b>) Promoter map of P_<i>chb</i> showing the SBS and the TSSs mapped in the presence (induced) and absence (uninduced) of chitin. “WT SBS” represents the native P_<i>chb</i> sequence, “<i>E</i>. <i>coli</i> SBS” indicates that the native SBS was swapped for the consensus SBS from <i>E</i>. <i>coli</i>, and “mutated SBS” indicates that the 6 highly conserved residues in the SBS were mutated. SBS sequences for the indicated mutants are shown in bold text and the 6 most highly conserved residues of the SBS are underlined. GFP fluorescence was measured in strains harboring the indicated mutations in the P_<i>chb</i>-<i>gfp</i> transcriptional reporter. The <i>cbp</i> status of each strain is indicated by a “+” (<i>cbp</i> intact) or a “Δ” (Δ<i>cbp</i> strain background). Data are shown as the mean ± SD and are from at least three independent biological replicates. (<b>B</b>) EMSAs performed with purified SlmA and the indicated promoter probes. The two P_<i>chb</i> probes were incubated with (from left to right) 0 nM, 7.5 nM, 15 nM, 30 nM, 60 nM, 120 nM, 240 nM, or 480 nM SlmA. The P_<i>nanH</i> probe was incubated with 0 nM or 480 nM SlmA. (<b>C</b>) Growth curves of the indicated strains in M9 minimal medium containing 0.5% chitobiose as the sole carbon source. Data from <b>B</b> and <b>C</b> are representative of at least 2 independent experiments. *** = <i>p</i><0.001, * = p<0.05, NS = not significant.</p

Model for transcriptional activation of P_<i>chb</i>.

<p>(<b>A</b>) In the absence of chitin, CBP inhibits ChiS from activating expression of P_<i>chb</i>. (<b>B</b>) In the presence of chitin, CBP binds chitin oligomers, which relieves repression of ChiS. We hypothesize that ChiS interacts with or activates another currently unidentified protein, which recruits RNA Polymerase to activate transcription of P_<i>chb</i> in a SlmA-dependent manner. SlmA may help to recruit the putative factor to the P_<i>chb</i> promoter or is an otherwise required coactivator of this locus. Upon expression of the <i>chb</i> operon, proteins involved in chitin uptake and utilization of chitobiose, including CBP and the chitobiose ABC permease, are synthesized.</p

Acinetobacter baylyi regulates type IV pilus synthesis by employing two extension motors and a motor protein inhibitor

Type IV pili (T4P) are retractile appendages used by bacteria for DNA uptake and other purposes. T4P extension is thought to occur through the action of a single motor protein, PilB. Here, Ellison et al. show that T4P synthesis in Acinetobacter baylyi depends not only on PilB but also on an additional, distinct motor, TfpB