Monday, 17 February 2014

A new regulatory link between chitin attachment and natural competence in Vibrio cholera

We know that Vibrio species have an affinity for chitin. We know that chitin induces competence in Vibrio. But the cellular mechanisms linking these two events elude us. Some definitions; competence is a bacterium’s ability to uptake DNA from the environment and chitin is an unimaginably common biopolymer, consisting of beta-linked N-acetylglucosamine.
Vibrio cholera is awfully fond of the chitinous exoskeleton of copepods, from which it can acquire nitrogen and carbon, in a process regulated by histidine kinase (ChiS); this protein is suppressed by periplasmic chitin binding protein (CBP) when chitin is absent. But once CBP binds chitin, ChiS gets busy; it is believed to phosphorylate (thereby deactivating) an unidentified regulator that inhibits expression of chitin degradation, uptake and utilization genes. Additionally, chitin also induces competence in V. cholera via regulation by TfoX and HapR, genes which are activated by chitin and quorum sensing, respectively.
However, the molecular dance linking chitin sensing to competence induction via TfoX activation is at present a mystery. This study has found and described a novel chitin sensing regulator that bridges this knowledge gap.
The methods do not make light reading, so I will summarize; transposon mutagenesis to give V. cholera spectinomycin resistance, bacteria grown either on chitin or chitin with kanamycin (to select for transformants), DNA isolated from both groups, DNA sequenced, sequences compared to identify the genes responsible for transformation, deletion mutants of these genes were then made, grown with tetracycline resistance plasmids, then tested for natural transformation by growing them as before but with tetracycline.
The previously known TfoS gene was found to be involved in competence induction; this gene is predicted to be a transmembrane protein with a DNA-binding site. TfoS deletion mutants did not transform in transformation inducing conditions. The position of TfoS in the regulatory cascade of natural competence was then examined; inducing overexpression of TfoX saves a TfoS mutant, so TfoS probably precedes TfoX. When grown in competence inducing and non-inducing conditions, TfoS could up regulate expression of TfoX. Using methods that I could not understand, they somehow confirmed that TfoS controls TfoX via regulation of the gene TfoR.
Afterwards, they fused a TfoR promoter to E. coli and found that it increased TfoS expression 30 fold, confirming that TfoR directly regulates TfoS.
So without chitin, TfoS cannot bind DNA. With chitin, it activates the TfoR gene, which interacts with the gene for TfoX, which activates the genes needed for binding and uptake of DNA from the environment. These genes have been previously shown to highly conserved amongst Vibrio species and likely serve similar functions. Since ChiS mutants could not grow on chitin, but TfoS mutants can, then these may be distinct chitin sensors involved in separate regulatory cascades. However another study has implicated ChiS as being part of the same regulatory cascade, so it is possible that it facilitates interaction of TfoS with chitin, or TfoR activation. Future work is proposed to be aimed at determining the binding site of TfoS and other binding targets of this protein to look for any additional roles it may have. I wonder that these cascades do during the transformation V. cholera undergoes as it becomes pathogenic in the human gut. Since HapR is quorum controlled, then surely it would be active in the gut when V. cholerae cell densities are high, so there must be some overlap between the cascades controlling the binding to the gut wall and to chitin. The human gut seems to be an ideal place for acquiring new genes, given the bacterial diversity there, but it is lacking in chitin, so it would be odd if Vibrio cholerae just turned off its competency once in a host and did not have another competence inducing system for when it is in the human gut.

Dalia, A. B., Lazinski, D. W., & Camilli, A. (2014). Identification of a Membrane-Bound Transcriptional Regulator That Links Chitin and Natural Competence in Vibrio cholerae. mBio, 5(1), e01028-13.

1 comment:

  1. So, what are the implications of this study? They've found that TfoX and TfoS are both vitally important in the competence of Vibrio (which you defined as up-taking DNA from the environment). Is it assumed that competence adds to its adaptability in the marine environment?