I've been working through the data on regulation of competence in Vibrio cholerae. V. cholerae has most of the same competence genes as H. influenzae, and some of these have been shown to be needed for competence (especially the type IV pilin system and the inner membrane transport protein (Rec2 homolog). Most of these genes are controlled by promoters with sequences resembling the Sxy-dependent CRP-S sites we have characterized in H. influenzae. Sxy is known to be needed for competence development, as is the complex carbohydrate chitin.
Chitin is a polymer of N-acetyl-glucosamine (GlcNac) subunits, and is the main component of the exoskeletons of most arthropods, including the marine crustaceans that V. cholerae forms biofilms on. Because V. cholerae can break down and metabolize chitin, this is thought to be a major nutrient source in biofilms. So how does chitin availability regulate competence? Does it regulate sxy? Does anything else regulate sxy?
A recent paper by Yamamoto et al. (Gene 457:42-49, 2010) investigated transcriptional and translational control of sxy expression in V. cholerae, and I've spent the afternoon coming to grips with what they did and what they concluded. First they showed that the GlcNac dimer induces competence but the monomer does not. But the transformation frequencies are very low by H. influenzae standards, 1.4x10^-8 and 4.4x10-8 for two different wildtype strains, 14-fold and 44-fold above the detection limit of 10^-9. Transformation frequencies were 70 and 136-fold higher with a GlcNac tetramer. Perhaps because of the result in the next paragraph, the authors concluded that the activator was the GlcNac dimer, not the tetramer, and used this for the rest of their experiments.
Expression of a transcriptional fusion to lacZ was induced about 2-fold by both the dimer and the tetramer of GlcNac (2.1x and 2.3x), but expression of a translational fusion was induced 25- and 34-fold. This tells us that chitin's main contribution to the induction of competence is by increasing the translation of sxy mRNA. The GlcNac tetramer wasn't much more effective than the dimer - the difference between its effect on sxy and on competence may mean that it independently regulates another component of competence.
They also mapped the start site of sxy transcription to 104 nt upstream of the GTG start codon (so the V. cholerae sxy mRNA has a long untranslated leader like the H. influenzae and E. coli sxy mRNAs.), They identified various candidate regulatory elements: the -35 and -10 elemments of the promoter, the Shine Dalgarno sequence beside the start codon, and several inverted repeats that they hypothesized had regulatory roles.
They next analyzed a large set of transcriptional and translational fusions of parts of the sxy gene to lacZ. These showed the following:
First, removing the candidate sxy promoter eliminated expression, and replacing it promoter with a Ptac promoter increased expression of all fusions about 10-fold. This tells us that they have correctly identified the sxy promoter, and that it is relatively weak or not fully induced under the conditions used.
With the sxy promoter and the transcriptional fusion, the GlcNac dimer increased expression only 2-fold, but with the translational fusion the dimer increased expression 25-fold. With the Ptac promoter the effects were 0.9-fold and 30-fold. These effects again tell us that chitin's effect is mainly on translation. Nevertheless the authors concluded that chitin dimers act at the promoter to regulate sxy transcription. I think this conclusion is not justified by the small effect seen only with one fusion.
Deletion of only the second inverted repeat had no effect on either kind of fusion. But deletions between this and the third inverted repeat dramatically increased translation in the absence of GlcNac dimers (making translation constitutive), and deletions of coding sequences downstream of the dimer had the opposite effect, eliminating translation entirely.
In their inspection of the sxy sequence for candidate regulatory elements, the authors overlooked a very strong potential CRP-N site 50 nt upstream of the promoter. This suggests that V. cholerae sxy transcription may regulated by CRP/cAMP and thus by the phosphotransferase carbohydrate-utilization system (the PTS). The H. influenzae sxy gene is also regulated by CRP and the PTS, and the E. coli gene has a partial CRP site whose role hasn't been tested yet.
Bottom line: Transcription of V. cholerae sxy is likely regulated by CRP, and translation is tightly regulated by chitin dimers. Chitin tetramers may separately regulate another compoonent of competence. As in E. coli and H. influenzae, CRP activation is likely a signal of nutritional stress (that preferred sugar sources are unavailable). Regulation of competence by chitin may have evolved because of its role as a nutrient , but it may also signal that the cell is in a biofilm, where DNA is usually abundant.
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