Alteration of a Nonconserved Active Site Residue in the Chemotaxis Response Regulator CheY Affects Phosphorylation and Interaction with CheZ

Silversmith, Ruth E.; Smith, Jenny G.; Guanga, Gerald P.; Les, Jessica T.; Bourret, Robert B.

doi:10.1074/jbc.m011418200

Cited by 36 publications

(73 citation statements)

References 46 publications

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“…According to the structure predicted by SWISS-MODEL (Guex & Peitsch, 1997;Schwede et al, 2003), a replacement of Asn877 by Ser increases its contact with the site of phosphorylation, Asp875, through hydrogen bonding. Different substitutions at the corresponding positions in VirG (N54D) (Han et al, 1992;Jin et al, 1993;Pazour et al, 1992;Scheeren-Groot et al, 1994), CheB (E58K) (Stewart, 1993) and CheY (N59K, N49R) (Silversmith et al, 2001) also have an activating effect, thereby outlining the importance of this non-conserved residue in the activation of response regulator domains.…”

Section: Discussionmentioning

confidence: 99%

Virulence attenuation in Salmonella enterica rcsC mutants with constitutive activation of the Rcs system

et al. 2005

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Mutations in rcsC that result in constitutive colanic acid capsule synthesis were obtained in Salmonella enterica serovar Typhimurium. Most rcsC alleles were dominant; however, recessive rcsC alleles were also found, in agreement with the postulated double role (positive and negative) of RcsC on the activation of the RcsB/C phosphorelay system. Salmonella rcsC mutants with constitutive activation of the Rcs system are severely attenuated for virulence in BALB/c mice and their degree of attenuation correlates with the level of Rcs activation. Partial relief of attenuation by a gmm mutation indicates that capsule overproduction is one of the factors leading to avirulence in constitutively activated rcsC mutants. INTRODUCTIONThe Rcs phosphorelay system was initially characterized in Escherichia coli as a two-component positive regulator of colanic acid capsule synthesis encoded by rcsC and rcsB (Brill et al., 1988;Gottesman et al., 1985;Stout & Gottesman, 1990). The sensor protein RcsC, a hybrid histidine kinase, and the transcriptional activator RcsB are the main components of the system, which also includes a second transcriptional activator, RcsA (Stout et al., 1991) and an intermediate phosphotransmitter, YojN (Takeda et al., 2001). The Rcs system is also a negative regulator of the flagellar master operon flhDC (Francez-Charlot et al., 2003), whose products are necessary for the expression of genes involved in flagellum synthesis, motility and chemotaxis.The first environmental signal shown to strongly and rapidly induce colanic acid synthesis was osmotic upshift (Sledjeski & Gottesman, 1996). This effect was mediated by the Rcs system. More recently it has been shown that the Rcs phosphorelay can be activated when wild-type cells are grown at 20 uC in the presence of glucose and a high concentration of Zn 2+ (Hagiwara et al., 2003). Furthermore, several genetic conditions that result in alterations in envelope composition or integrity are known to activate the Rcs system. For example, a mutation in the mdoH gene involved in the biosynthesis of membrane-derived oligosaccharides (Ebel et al., 1997) Here, we describe the isolation of Salmonella rcsC mutants with constitutive activation of the Rcs system, and the characterization of amino acid substitutions that lead to different degrees of activation of the system. Virulence trials with constitutively activated rcsC mutants provide a direct demonstration of the link between activation of the Rcs system and avirulence in mice. We also show that virulence attenuation associated with activation of the RcsB/C system is partially due to colanic acid overproduction. METHODSBacterial strains, bacteriophages and strain construction.S. enterica serovar Typhimurium strains used in this study are described in Table 1. Unless otherwise indicated, the strains derive from the mouse-virulent strain 14028. Transductional crosses using phage P22 HT 105/1 int201 (Schmieger, 1972) were used for strain construction operations. The transduction protocol was described by Maloy (199...

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Section: Discussionmentioning

confidence: 99%

Virulence attenuation in Salmonella enterica rcsC mutants with constitutive activation of the Rcs system

et al. 2005

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“…Asn59 is not conserved among CheY proteins from different bacterial species but is located on the ␤3␣3 loop at the active site, with its side chain pointing into the channel where the phosphodonor enters. The arginine substitution at this position resulted in extreme resistance to CheZ-mediated dephosphorylation despite binding CheZ with the same affinity as activated wild-type CheY (24). Substitution with an alanine residue at position 59 did not affect CheZ sensitivity, indicating that the CheZ resistance was due to introduction of the arginine and not the removal of the asparagine.…”

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confidence: 96%

CheZ-Mediated Dephosphorylation of the Escherichia coli Chemotaxis Response Regulator CheY: Role for CheY Glutamate 89

Silversmith¹,

Guanga²,

Betts

et al. 2003

J Bacteriol

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In Escherichia coli chemotaxis, cells swim towards attractants and away from repellants by regulating the frequency of switching between smooth swims (counterclockwise flagellar rotation) and reorienting tumbles (clockwise flagellar rotation), so that the duration of smooth swims is lengthened when swimming in a favorable direction. The series of molecular events that mediate this flow of information have been characterized in considerable detail (6,8), and bacterial chemotaxis has long served as a prototype for the study of two-component regulatory systems, a large family of signal transduction systems found in bacteria, fungi, and plants (26).In chemotaxis, the sensor kinase CheA associates with the cytoplasmic portion of transmembrane chemoreceptors and autophosphorylates a histidyl residue (His48) at a rate which is regulated by the occupancy of the extracellular binding sites of the receptors with attractant or repellant molecules. CheA then transfers the phosphoryl group to an aspartyl residue (Asp57) of the freely diffusible response regulator protein CheY. Phosphorylated CheY (CheY-P) binds to the flagellar switch, inducing clockwise flagellar rotation. Removal of the phosphoryl group from CheY occurs via catalysis by the accessory protein CheZ.

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“…All active-site residues (19,41), including the phosphoacceptor site Asp57 and Asn59, which is nonconserved but functionally important in E. coli (35), are identical in the E. coli and A. brasilense CheY homologs (Table 3).…”

Section: Discussionmentioning

confidence: 99%

Different Evolutionary Constraints on Chemotaxis Proteins CheW and CheY Revealed by Heterologous Expression Studies and Protein Sequence Analysis

Alexandre

Zhulin

2003

J Bacteriol

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CheW and CheY are single-domain proteins from a signal transduction pathway that transmits information from transmembrane receptors to flagellar motors in bacterial chemotaxis. In various bacterial and archaeal species, the cheW and cheY genes are usually encoded within homologous chemotaxis operons. We examined evolutionary changes in these two proteins from distantly related proteobacterial species, Escherichia coli and Azospirillum brasilense. We analyzed the functions of divergent CheW and CheY proteins from A. brasilense by heterologous expression in E. coli wild-type and mutant strains. Both proteins were able to specifically inhibit chemotaxis of a wild-type E. coli strain; however, only CheW from A. brasilense was able to restore signal transduction in a corresponding mutant of E. coli. Detailed protein sequence analysis of CheW and CheY homologs from the two species revealed substantial differences in the types of amino acid substitutions in the two proteins. Multiple, but conservative, substitutions were found in CheW homologs. No severe mismatches were found between the CheW homologs in positions that are known to be structurally or functionally important. Substitutions in CheY homologs were found to be less conservative and occurred in positions that are critical for interactions with other components of the signal transduction pathway. Our findings suggest that proteins from the same cellular pathway encoded by genes from the same operon have different evolutionary constraints on their structures that reflect differences in their functions.

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Alteration of a Nonconserved Active Site Residue in the Chemotaxis Response Regulator CheY Affects Phosphorylation and Interaction with CheZ

Cited by 36 publications

References 46 publications

Virulence attenuation in Salmonella enterica rcsC mutants with constitutive activation of the Rcs system

Virulence attenuation in Salmonella enterica rcsC mutants with constitutive activation of the Rcs system

CheZ-Mediated Dephosphorylation of the Escherichia coli Chemotaxis Response Regulator CheY: Role for CheY Glutamate 89

Different Evolutionary Constraints on Chemotaxis Proteins CheW and CheY Revealed by Heterologous Expression Studies and Protein Sequence Analysis

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