Abstract
The bacterial enhancer-binding protein (bEBP) FlrC, controls motility and colonization of Vibrio cholerae by regulating the transcription of class-III flagellar genes in σ54-dependent manner. However, the mechanism by which FlrC regulates transcription is not fully elucidated. Although, most bEBPs require nucleotides to stimulate the oligomerization necessary for function, our previous study showed that the central domain of FlrC (FlrCC) forms heptamer in a nucleotide-independent manner. Furthermore, heptameric FlrCC binds ATP in "cis-mediated" style without any contribution from sensor I motif 285REDXXYR291 of the trans protomer. This atypical ATP binding raises the question of whether heptamerization of FlrC is solely required for transcription regulation, or if it is also critical for ATPase activity. ATPase assays and size exclusion chromatography of the trans-variants FlrCC-Y290A and FlrCC-R291A showed destabilization of heptameric assembly with concomitant abrogation of ATPase activity. Crystal structures showed that in the cis-variant FlrCC-R349A drastic shift of Walker A encroached ATP-binding site, whereas the site remained occupied by ADP in FlrCC-Y290A. We postulated that FlrCC heptamerizes through concentration-dependent cooperativity for maximal ATPase activity and upon heptamerization, packing of trans-acting Tyr290 against cis-acting Arg349 compels Arg349 to maintain proper conformation of Walker A. Finally, a Trp quenching study revealed binding of cyclic-di-GMP with FlrCC Excess cyclic-di-GMP repressed ATPase activity of FlrCC through destabilization of heptameric assembly, especially at low concentration of protein. Systematic phylogenetic analysis allowed us to propose similar regulatory mechanisms for FlrCs of several Vibrio species and a set of monotrichous Gram-negative bacteria.
Highlights
Vibrio cholerae, the facultative human pathogen that causes diarrheal disease cholera, is highly motile by means of a single, polar sheathed flagellum
This clearly indicates a distinctive mode of interactions of c-di-GMP with the bacterial enhancer-binding protein (bEBP) involved in exopolysaccaride production and flagellar synthesis
Apart from V. cholerae, we have identified 19 other monotrichous Vibrio species namely, Vibrio mimicus, Vibrio diazotrophicus, Vibrio alginolyticus, Vibrio campbelii, Vibrio harveyi, Vibrio nereis, Vibrio rotiferianus, Vibrio nigripulchritudo, Vibrio anguillarum, Vibrio caribbeanicus, Vibrio tubiashii, Vibrio fluvialis, Vibrio gazogenes, Vibrio mediterranei, Vibrio tapetis, Vibrio aerogenes, Vibrio azureus, Vibrio galatheae, and Vibrio xiamenesis, which possess a two-component system of FlrBC for flagellar synthesis
Summary
The facultative human pathogen that causes diarrheal disease cholera, is highly motile by means of a single, polar sheathed flagellum. We measured ATPase activities of FlrCC and variants upon elevating ATP concentrations to 0.3 and 0.5 mM as well (Fig. 1f).
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