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. 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. 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. Vibrio cholerae, the facultative human pathogen that causes diarrheal disease cholera, is highly motile by means of a single, polar sheathed flagellum. V. cholerae enters inside the human host through ingestion of contaminated food or water, adheres to the apical surface of the intestinal epithelial cell, and expresses virulence factors (1Holmgren J. Svennerholm A.-M. Mechanisms of disease and immunity in cholera: a review.J. Infect. Dis. 1977; 136: S105-S11210.1093/infdis/136.Supplement.S105Crossref PubMed Google Scholar, 2Taylor R.K. Miller V.L. Furlong D.B. Mekalanos J.J. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin.Proc. Natl. Acad. Sci. U.S.A. 1987; 84 (2883655): 2833-283710.1073/pnas.84.9.2833Crossref PubMed Google Scholar). Motility and colonization of V. cholerae are prerequisites of producing the virulence factors and immune-resistant biofilms which, in turn, are governed by flagellar synthesis (3Yildiz F.H. Visick K.L. Vibrio biofilms: so much the same yet so different.Trends Microbiol. 2009; 17 (19231189): 109-11810.1016/j.tim.2008.12.004Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 4Syed K.A. Beyhan S. Correa N. Queen J. Liu J. Peng F. Satchell K.J.F. Yildiz F. Klose K.E. The Vibrio cholerae flagellar regulatory hierarchy controls expression of virulence factors.J. Bacteriol. 2009; 191 (19717600): 6555-657010.1128/JB.00949-09Crossref PubMed Scopus (112) Google Scholar). Expression of the proteins required to synthesize the functional flagellum of V. cholerae is regulated by a four-tiered transcriptional hierarchy (4Syed K.A. Beyhan S. Correa N. Queen J. Liu J. Peng F. Satchell K.J.F. Yildiz F. Klose K.E. The Vibrio cholerae flagellar regulatory hierarchy controls expression of virulence factors.J. Bacteriol. 2009; 191 (19717600): 6555-657010.1128/JB.00949-09Crossref PubMed Scopus (112) Google Scholar, 5Prouty M.G. Correa N.E. Klose K.E. The novel σ54- and σ28-dependent flagellar gene transcription hierarchy of Vibrio cholerae.Mol. Microbiol. 2001; 39 (11260476): 1595-160910.1046/j.1365-2958.2001.02348.xCrossref PubMed Scopus (158) Google Scholar, 6Klose K.E. Mekalanos J.J. Distinct roles of an alternative sigma factor during both free-swimming and colonizing phases of the Vibrio cholerae pathogenic cycle.Mol. Microbiol. 1998; PubMed Scopus Google Scholar, Klose K.E. Vibrio flagellar Infect. Microbiol. PubMed Scopus Google Scholar). The gene and bacterial enhancer-binding protein σ54-dependent transcription of the genes which FlrC and (4Syed K.A. Beyhan S. Correa N. Queen J. Liu J. Peng F. Satchell K.J.F. Yildiz F. Klose K.E. The Vibrio cholerae flagellar regulatory hierarchy controls expression of virulence factors.J. Bacteriol. 2009; 191 (19717600): 6555-657010.1128/JB.00949-09Crossref PubMed Scopus (112) Google Scholar, 5Prouty M.G. Correa N.E. Klose K.E. The novel σ54- and σ28-dependent flagellar gene transcription hierarchy of Vibrio cholerae.Mol. Microbiol. 2001; 39 (11260476): 1595-160910.1046/j.1365-2958.2001.02348.xCrossref PubMed Scopus (158) Google Scholar). of class-III flagellar which flagellar and the are regulated by FlrC (4Syed K.A. Beyhan S. Correa N. Queen J. Liu J. Peng F. Satchell K.J.F. Yildiz F. Klose K.E. The Vibrio cholerae flagellar regulatory hierarchy controls expression of virulence factors.J. Bacteriol. 2009; 191 (19717600): 6555-657010.1128/JB.00949-09Crossref PubMed Scopus (112) Google Scholar, N.E. Klose K.E. of binding by the Vibrio cholerae flagellar regulatory protein Bacteriol. PubMed Scopus Google Scholar). The factor is through the to σ28-dependent transcription of which and of the (4Syed K.A. Beyhan S. Correa N. Queen J. Liu J. Peng F. Satchell K.J.F. Yildiz F. Klose K.E. The Vibrio cholerae flagellar regulatory hierarchy controls expression of virulence factors.J. Bacteriol. 2009; 191 (19717600): 6555-657010.1128/JB.00949-09Crossref PubMed Scopus (112) Google Scholar, 6Klose K.E. Mekalanos J.J. Distinct roles of an alternative sigma factor during both free-swimming and colonizing phases of the Vibrio cholerae pathogenic cycle.Mol. Microbiol. 1998; PubMed Scopus Google Scholar). Motility and of V. cholerae are regulated by at the transcriptional the mechanisms by which motility in V. cholerae are a to the and of V. polar flagellum Yildiz F.H. The and of in Vibrio Microbiol. PubMed Scopus Google Scholar, S. A. Yildiz F.H. and of Vibrio cholerae to Bacteriol. PubMed Scopus Google Scholar, A. Vibrio cholerae motility by of transcription and Microbiol. PubMed Scopus Google Scholar, Yildiz F.H. and binding of and activity of the Vibrio cholerae transcriptional PubMed Scopus Google Scholar). that V. cholerae to an of by the transcription of the and genes and that of flagellar genes Yildiz F.H. and binding of and activity of the Vibrio cholerae transcriptional PubMed Scopus Google Scholar). This a of of with the bEBPs in and flagellar FlrC of V. cholerae is of central ATPase and in the domain of FlrC by N.E. Klose K.E. of binding by the Vibrio cholerae flagellar regulatory protein Bacteriol. PubMed Scopus Google Scholar). by Klose and K.E. Mekalanos J.J. Distinct roles of an alternative sigma factor during both free-swimming and colonizing phases of the Vibrio cholerae pathogenic cycle.Mol. Microbiol. 1998; PubMed Scopus Google that a V. cholerae a of is and also a colonization whereas a a of FlrC an K.E. Mekalanos J.J. Distinct roles of an alternative sigma factor during both free-swimming and colonizing phases of the Vibrio cholerae pathogenic cycle.Mol. Microbiol. 1998; PubMed Scopus Google Scholar, N.E. Klose K.E. of binding by the Vibrio cholerae flagellar regulatory protein Bacteriol. PubMed Scopus Google Scholar, N.E. Klose K.E. of the flagellar regulatory protein FlrC is necessary for Vibrio cholerae motility and Microbiol. PubMed Scopus Google Scholar). showed that both and of FlrC colonization a FlrC which that both and forms of FlrC are required for the and FlrC an or an colonization K.E. Mekalanos J.J. Distinct roles of an alternative sigma factor during both free-swimming and colonizing phases of the Vibrio cholerae pathogenic cycle.Mol. Microbiol. 1998; PubMed Scopus Google Scholar, N.E. Klose K.E. of binding by the Vibrio cholerae flagellar regulatory protein Bacteriol. PubMed Scopus Google Scholar, N.E. Klose K.E. of the flagellar regulatory protein FlrC is necessary for Vibrio cholerae motility and Microbiol. PubMed Scopus Google Scholar). domain FlrC is atypical in The σ54-dependent to the of the binding site and at the by mechanism at a in Microbiol. 2001; PubMed Scopus Google Scholar). FlrC binds of the and transcriptional of the and N.E. Klose K.E. of binding by the Vibrio cholerae flagellar regulatory protein Bacteriol. PubMed Scopus Google Scholar). The same in of for and genes the for and the without J. N. the flagellar gene in binds to or of the binding Bacteriol. PubMed Scopus Google Scholar). However, the of binding at the is yet to for or oligomerization of bEBPs through the bEBPs in of ATP and by the which roles in the proteins The of bacterial binding proteins of σ54-dependent PubMed Scopus Google Scholar, S. S. of to ATP in ATPase for bacterial Full Text Full Text PDF PubMed Scopus Google Scholar, S. ATPase and ATP PubMed Scopus Google Scholar, N. N. ATPase site is required for and activity of a transcriptional Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. ATPase site ATP binding in heptameric domain of flagellar regulatory protein Full Text Full Text PDF PubMed Scopus Google Scholar, N. of an a Full Text Full Text PDF PubMed Scopus Google Scholar, J. of the in the domain of transcription PubMed Scopus Google Scholar). in the of oligomerization is by from the to S. S. of to ATP in ATPase for bacterial Full Text Full Text PDF PubMed Scopus Google Scholar, N. of an a Full Text Full Text PDF PubMed Scopus Google Scholar, J. of the in the domain of transcription PubMed Scopus Google Scholar). We the of the domain of FlrC (FlrCC) in and S. J. ATPase site ATP binding in heptameric domain of flagellar regulatory protein Full Text Full Text PDF PubMed Scopus Google Scholar). both FlrCC in the heptameric the of FlrC forms heptamer without and S. J. ATPase site ATP binding in heptameric domain of flagellar regulatory protein Full Text Full Text PDF PubMed Scopus Google Scholar). The of the heptameric species of FlrCC also in by size exclusion chromatography and S. J. ATPase site ATP binding in heptameric domain of flagellar regulatory protein Full Text Full Text PDF PubMed Scopus Google Scholar). for to the at S. ATPase and ATP PubMed Scopus Google heptameric FlrCC with a much central to for the same without any S. J. ATPase site ATP binding in heptameric domain of flagellar regulatory protein Full Text Full Text PDF PubMed Scopus Google Scholar). the of and ATP binds in the with Walker A of the to the motif of sensor I to the of ATP S. S. of to ATP in ATPase for bacterial Full Text Full Text PDF PubMed Scopus Google Scholar). a novel and solely ATP binding and in the heptameric of ATP is by cis-acting Arg349 of sensor the 285REDXXYR291 motif of sensor I in FlrC and to the ATP-binding site in of motif in binding ATP S. J. ATPase site ATP binding in heptameric domain of flagellar regulatory protein Full Text Full Text PDF PubMed Scopus Google Scholar). This atypical “cis-mediated” of ATP binding in FlrC heptamerization and ATPase activity. of with FlrC of binding and ATP in if the heptameric of FlrC is required for binding the at or it is for ATPase activity cis-acting Arg349 of sensor and trans-acting Tyr290 and to 285REDXXYR291 of sensor I by and FlrCC-R291A showed ATPase activity with destabilization of the heptameric in the structures of FlrCC-R349A and the of the and functional that heptamerization with conformation of Walker A is for ATP binding and by FlrCC forms heptamer through concentration-dependent cooperativity to maximal ATPase activity. the bEBPs in flagellar synthesis of V. cholerae, of with the of the to expression of the flagellar genes A. Vibrio cholerae motility by of transcription and Microbiol. PubMed Scopus Google Scholar). However, the of in of FlrC yet study revealed for the that of ATPase activity of FlrCC by heptameric and phylogenetic of mechanisms in several Vibrio species and a set of monotrichous Gram-negative bacteria. FlrCC and the proteins and for ATP the S. J. ATPase site ATP binding in heptameric domain of flagellar regulatory protein Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. and analysis of the domain of FlrC from Vibrio PubMed Scopus Google Scholar, A for protein PubMed Scopus Google Scholar). ATPase assays FlrCC and ATP at for from to The of at upon with from by with a showed that the of by FlrCC a of ATP with for the it However, ATPase activity of low the of ATP inside the bacterial host to J. of a of in Bacteriol. PubMed Scopus Google Scholar, in from Full Text Full Text PDF PubMed Scopus Google Scholar). ATPase of FlrCC and upon ATP to and the of the in of by the proteins and by the with FlrCC of the in of ATPase activity trans-acting Tyr290 and of sensor I with ATP drastic in ATP upon of to that Tyr290 and of trans yet roles in the ATPase activity of The of FlrCC and by size exclusion chromatography of an of of the heptamer of FlrCC I a FlrCC-Y290A at a species and of protein in the of FlrCC and FlrCC-R291A FlrCC-Y290A species us to concentration to which the same a species of FlrCC-Y290A in through FlrCC-R291A and a of heptameric and species the heptameric for a of FlrCC-R349A a species that to an the concentration of FlrCC-R349A not that of of the species of FlrCC-R349A at that concentration during and of a FlrCC-R291A maintain heptameric similar and Crystal structures of FlrCC-R349A and FlrCC-Y290A to and similar and of the to that of FlrCC or similar the of the the of FlrCC FlrCC-Y290A and FlrCC-R349A showed species in the heptameric species in structures that proteins to heptamer at the of FlrCC-Y290A and in the and the structures of FlrCC-R349A and FlrCC-Y290A to of and and are in and of of in a of FlrCC-Y290A heptamer and FlrCC structures of and for FlrCC-R349A of and of the of the from to This that the heptameric of both similar to the functional protein. for the that by the most of the at in the of in ATPase activity the of Arg349 to the of from the of FlrCC-R349A factors for ATP in the heptameric of FlrCC-R349A Walker A motif in an conformation in Walker A of is the of the Walker A a to shift of Walker A in FlrCC to binding of ATP S. J. ATPase site ATP binding in heptameric domain of flagellar regulatory protein Full Text Full Text PDF PubMed Scopus Google Scholar). the which in Walker A in a to to the and of A to of Walker A that by However, the drastic shift of Walker in the of from and FlrCC in in the to a the of and of FlrCC-R349A the FlrCC revealed that conformation of Walker A in the encroached the ATP-binding site ATP-binding of FlrCC-Y290A occupied by ADP a and or during protein ADP of the that ADP by FlrCC-Y290A from bacterial host This is from the that and by the same not any from the heptameric of FlrCC S. J. ATPase site ATP binding in heptameric domain of flagellar regulatory protein Full Text Full Text PDF PubMed Scopus Google Scholar). ADP in the of the of ATP during protein A. S. of the transcriptional of the regulatory and ATPase 17 PubMed Scopus Google Scholar). not during protein ADP binding to FlrCC-Y290A the of the of trans-acting Tyr290 to of FlrCC-Y290A FlrCC showed that ADP binds in similar to binding The with and and The with of ADP through polar with the of Walker A and and However, which to of remained to the ADP binding and The of cis-acting Arg349 to from the ATP-binding site and it to with trans-acting conformation of Arg349 in low from the ATP-binding site to a with cis-acting the of the and low of that not any conformation to Arg349 during of Walker A of FlrCC-Y290A similar to that of FlrCC. of FlrCC-Y290A of in most of the with cis-acting it in of FlrCC the conformation of Walker of FlrCC concentration ATPase activity of FlrCC with of protein to through to upon with activity of from against protein concentration A in activity protein of which and in activity by the with concentration of protein to of the that an in protein concentration oligomerization through cooperativity that to ATPase activity. in activity is the concentration the is that FlrCC a heptameric in a activity us to any of ATP of the heptameric of FlrCC at low the protein showed low ATPase activity. of FlrCC concentration in the in or upon with of the during the concentration of FlrCC to However, shift in that ATP or the of FlrCC Furthermore, to protein concentration of the maximal and at FlrCC through ATPase that FlrCC of with protein concentration of and the with of during the upon of FlrCC with ATP to through a to in of against ATP of ATPase in the in of and of for FlrCC of whereas the and for FlrCC of in upon of the protein from to ATP binding upon of protein. in protein concentration in the heptameric to in ATPase activity. This by the that of and with an FlrCC concentration in at that of is an of of V. that binds the Walker A motif of ATPase the binding site is from the ATP-binding site of the from Natl. Acad. Sci. U.S.A. PubMed Google Scholar). Furthermore, analysis that binds Walker A of the ATPase of are regulated by the Full Text Full Text PDF PubMed Scopus Google Scholar). The us to that the Walker A motif of FlrCC. FlrCC and and is the of Walker A in which is also the and The are from Walker A. of with FlrCC and the of to upon FlrCC showed quenching upon of with an of the FlrCC-R349A with from the of quenching in of the not which low or binding and We the of the ATPase activity of FlrCC The not ATP and FlrCC with of in for ATP of and for of by a and against the concentration The showed that an in concentration ATPase activity of FlrCC. in ATPase activity to a of activity at of with a in the in activity to that with a concentration that in ATPase activity of FlrCC is in the of of FlrC a in flagellar synthesis and our with a previous that of flagellar synthesis of V. cholerae by a concentration of Yildiz F.H. The and of in Vibrio Microbiol. PubMed Scopus Google Scholar). by the of in ATPase activity with and concentration-dependent oligomerization of FlrCC it to the of ATPase activity at of the protein. the of and FlrCC with and of of ATPase activity revealed that whereas of in activity of FlrCC the of to and with FlrCC of and of in activity at protein of whereas of with protein. This to the that of of ATPase activity of FlrCC is at of the protein. of previous to the of and of the of heptameric with of in A shift of upon of with during the of concentration-dependent ATPase activity and destabilization of heptameric assembly of a protein of the concentration to which of in the previous shift of and and that destabilization of heptameric assembly is at This is in with the previous in activity at or protein concentration of shift of with which upon of and of and destabilization of heptameric assembly at low of which by and of the functional for of the protein in an in the of of the from Natl. Acad. Sci. U.S.A. PubMed Google Scholar). and us to that of FlrC by the heptameric assembly and is the protein is in low Vibrio species for FlrC similar regulatory from V. cholerae, monotrichous Vibrio species Vibrio Vibrio Vibrio Vibrio Vibrio Vibrio Vibrio Vibrio Vibrio Vibrio Vibrio Vibrio Vibrio Vibrio Vibrio Vibrio Vibrio Vibrio and Vibrio which a of for flagellar of functional and Walker sensor sensor and of in FlrCs of Vibrio species the in are highly in Vibrio species The of us to a similar mechanism of oligomerization of FlrC to ATPase in a and of with the in of Vibrio The of in V. or in are necessary for σ54-dependent of class-III flagellar genes M.G. Correa N.E. Klose K.E. The novel σ54- and σ28-dependent flagellar gene transcription hierarchy of Vibrio cholerae.Mol. Microbiol. 2001; 39 (11260476): 1595-160910.1046/j.1365-2958.2001.02348.xCrossref PubMed Scopus (158) Google Scholar, 6Klose K.E. Mekalanos J.J. Distinct roles of an alternative sigma factor during both free-swimming and colonizing phases of the Vibrio cholerae pathogenic cycle.Mol. Microbiol. 1998; PubMed Scopus Google Scholar, N.E. Klose K.E. of binding by the Vibrio cholerae flagellar regulatory protein Bacteriol. PubMed Scopus Google Scholar). for any of We monotrichous Gram-negative that flagellar regulatory proteins or for flagellar with FlrC and of showed that functional of the domain are Arg349 of sensor is of sensor I is also Tyr290 is by in a of packing with the to Arg349 upon to in at of sensor A with FlrC and FlrCs of and of V. cholerae the that controls transcription of class-III flagellar forms heptamer in and ATP binding S. J. ATPase site ATP binding in heptameric domain of flagellar regulatory protein Full Text Full Text PDF PubMed Scopus Google Scholar). atypical of FlrC a question whether heptamerization is solely required for with the or any ATPase activity study that with the of ATPase and that heptamerization of FlrCC through concentration-dependent cooperativity to ATPase activity. Excess the the activity by the assembly the FlrCC-R349A and FlrCC-Y290A and showed or destabilization of the heptameric in FlrCC-Y290A a FlrCC-R349A and FlrCC-R291A both heptameric and in in a concentration-dependent manner. of the upon is in especially in low FlrCC is to ATPase ATP are activity similar of the of propose that the heptameric of FlrCC is for ATPase activity and the protein activity in in the of cis-acting Arg349 of sensor of ATP S. J. ATPase site ATP binding in heptameric domain of flagellar regulatory protein Full Text Full Text PDF PubMed Scopus Google Scholar). study that Arg349 roles through packing with trans-acting the of Walker and of the of the Arg349 Walker A a drastic shift to of the ATP-binding in FlrCC-R349A and of the of packing cis-acting Arg349 and trans-acting FlrCC-R349A to especially in a low concentration Furthermore, in the of Walker A not to in to a to for ATP The heptamer of FlrCC-R349A through packing trans-acting Tyr290 with at an shift of Walker A which that the Arg349 the to Walker A in the trans-acting motif 285REDXXYR291 with in FlrCC S. J. ATPase site ATP binding in heptameric domain of flagellar regulatory protein Full Text Full Text PDF PubMed Scopus Google our study that Tyr290 of motif in the of the cis-acting Arg349 not to the ATP-binding a FlrCC-Y290A for the ATP and to the ADP at the ATP-binding site The the ATPase activity in the of to Arg349 is not to the ATP-binding in the of trans-acting Tyr290 or to conformation of Tyr290 the in the protein to in ATP which is from ATPase assays or of Tyr290 for oligomerization of FlrCC. the of the heptameric of ATP and contribution of the S. J. ATPase site ATP binding in heptameric domain of flagellar regulatory protein Full Text Full Text PDF PubMed Scopus Google which are the factors for ATP binding and are from the of the heptameric of our the mechanism of heptamerization of The of the activity of FlrCC a concentration-dependent oligomerization of FlrCC Furthermore, a in the upon of FlrCC from to that destabilization of the heptameric upon in ATP binding and of oligomerization protein concentration in S. S. of to ATP in ATPase for bacterial Full Text Full Text PDF PubMed Scopus Google Scholar). in that ATP to in The mechanism of oligomerization of FlrC, which is of for the that of the FlrCC concentration a of heptameric assembly in a ATPase activity of destabilization of heptameric assembly shift of in upon of the protein the in ATPase activity of FlrCC by and the is at low of the protein and This a question whether ATPase activity through or the is through destabilization of the heptameric the of of the site is not to the ATP-binding site both are Walker and the domain of in an alternative to ATPase of the from Natl. Acad. Sci. U.S.A. PubMed Google Scholar). A of the FlrCC that in the heptameric to at the ATP-binding which in showed binding of with FlrCC binding of to FlrCC-R349A with FlrCC to the of to the that to Walker A. conformation of FlrCC is for binding and the of destabilization of the heptameric especially at the low concentration of that FlrCC-Y290A a in ADP Walker A and it to that a concentration of required to by V. cholerae expression to transcription of flagellar genes during S. A. Yildiz F.H. and of Vibrio cholerae to Bacteriol. PubMed Scopus Google Scholar). of to with the of the expression of flagellar genes A. Vibrio cholerae motility by of transcription and Microbiol. PubMed Scopus Google Scholar). a of FlrC and the concentration of FlrC in the We propose that at an concentration to FlrC, in low in the Vibrio from an to of FlrC is heptameric without that the of of FlrC a regulatory factor for flagellar gene of FlrC by is the of in transcription of class-III flagellar whereas heptamerization of FlrC expression of class-III genes during flagellar of heptamer at low and in the of transcription of flagellar genes during and phylogenetic similar mechanisms in several Vibrio species and monotrichous Gram-negative that a in the of the bEBPs in flagellar synthesis of pathogenic bacteria. FlrCC-Y290A and FlrCC-R291A by FlrCC of and in and of the by a of and at of with a and the at the The with and at for The at for at and the in of and to the and the by The at for at The a chromatography that with The proteins with the through FlrCC-Y290A and FlrCC-R291A to and FlrCC-R349A with the same and to FlrCC also to a concentration of by concentration for The of the proteins with ATPase of FlrCC and the by the of by the in S. J. ATPase site ATP binding in heptameric domain of flagellar regulatory protein Full Text Full Text PDF PubMed Scopus Google Scholar). protein and and with ATP from to at for in a of that of of in of and of to the and at of the by and against of protein with without ATP to the if and the at from the by that protein upon of the the ATP without protein to the of the with the of ATPase activity of the same the protein concentration the in exclusion chromatography in a with an The for The with of and The against in the is the and is the and the in the analysis of FlrCC and the in of protein to the with and and at a of for FlrCC and for and for The and in with FlrCC and by with a and at an of for a a of at with protein of in the and at for the The are in of size by and against The in by the in of of FlrCC-R349A by with in and of FlrCC-Y290A with the at in the of in and in for at and to and for FlrCC-R349A and at of FlrCC without and for both showed heptameric assembly in the by and and of with N. J.J. a for PubMed Scopus Google Scholar). of in for PubMed Scopus Google Scholar). at the of both the of the of FlrCC-R349A and FlrCC-Y290A to the with and a of in at a of and the and with for both and at the in and the in in the of of and for protein with is the in is the is the for upon binding with and is the from and to the analysis the and The with the of monotrichous Gram-negative by with and PubMed Scopus Google that and factor of the structures of FlrCC-R349A and FlrCC-Y290A in the with the set and are the We of for and at of We also and for during at at We of for in to We are also to A. for during at We of for during We are also to for in and phylogenetic We are to for and with bacterial binding protein with size exclusion chromatography

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