The heptameric structure of the flagellar regulatory protein FlrC is indispensable for ATPase activity and disassembled by cyclic-di-GMP

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. While, 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.

A trigger phosphodiesterase modulates the global c-di-GMP pool, motility and biofilm formation inVibrio parahaemolyticus

“…LafK, the master regulator of lateral flagellar gene expression, is a σ 54 -dependent transcriptional regulator phylogenetically related to FlrA and FlrC (19, 27). Both FlrA and FlrC have been shown to be c-di-GMP effectors (45, 46), so it would be interesting to evaluate if the activity of their relative LafK is also influenced by c-di-GMP levels. The expression of cpsA , on the other hand, is known to be regulated by the c-di-GMP effectors CpsQ and ScrO.…”

The N‐terminal FleQ domain of the Vibrio cholerae flagellar master regulator FlrA plays pivotal structural roles in stabilizing its active state

“…The recombinant proteins with the N‐terminal 6×His‐tag were overexpressed in BL21(DE3) cells, as per the protocol described before [24]. The cells were induced with 1 m m isopropyl β‐D‐thiogalactopyranoside and grown at 37 °C for 3 h. The cells were harvested by centrifugation at 15805 g for 20 min at 4 °C, resuspended in buffer A [containing 50 mM Tris–HCl (pH 8.0), 300 m m NaCl, 5 m m MgCl 2 , and 10% (v/v) glycerol], and lysed after addition of phenylmethylsulfonyl fluoride.…”

“…Spectrophotometric measurement of the release of inorganic phosphate (Pi) by the malachite green assay, as described previously [24], provided us with ATPase activities of Vc FlrA‐Fl and the other constructs. Reaction mixtures containing 2.5 μ m protein in the buffer containing 50 m m Tris–HCl (pH 8.0), 300 m m NaCl, and 5 m m MgCl 2 were incubated with ATP ranging from 0.1 to 0.8 m m at 25 °C for 10 min in a reaction volume of 1 mL.…”

“…The recombinant proteins with the N-terminal 6×His-tag were overexpressed in BL21(DE3) cells, as per the protocol described before [24]. The cells were induced with 1 mM isopropyl β-D-thiogalactopyranoside and grown at 37 °C for 3 h. The cells were harvested by centrifugation at 15805 g for 20 min at 4 °C, resuspended in buffer A…”

The polar flagellar transcriptional regulatory network in Vibrio campbellii deviates from canonical Vibrio species

“…The results of our RNA-seq analysis indicate that some flagellar genes are decreased in expression in both the flrA and flrC backgrounds compared to wild-type. FlrA and FlrC are both bacterial enhancer binding proteins (bEBPs) that share 39.79% identity and serve as σ 54 dependent transcriptional regulators (44,51,52). Thus, we hypothesized that FlrA shares a coregulon with FlrC, in addition to their distinct regulons of genes.…”