Phosphorylation of the Pseudomonas aeruginosa response regulator AlgR is essential for type IV fimbria-mediated twitching motility

The response regulator AlgR is required for both alginate biosynthesis and type IV fimbria-mediated twitching motility in Pseudomonas aeruginosa. In this study, the roles of AlgR signal transduction and phosphorylation in twitching motility and biofilm formation were examined. The predicted phosphorylation site of AlgR (aspartate 54) and a second aspartate (aspartate 85) in the receiver domain of AlgR were mutated to asparagine, and mutant algR alleles were introduced into the chromosome of P. aeruginosa strains PAK and PAO1. Assays of these mutants demonstrated that aspartate 54 but not aspartate 85 of AlgR is required for twitching motility and biofilm initiation. However, strains expressing AlgR D85N were found to be hyperfimbriate, indicating that both aspartate 54 and aspartate 85 are involved in fimbrial biogenesis and function. algD mutants were observed to have wild-type twitching motility, indicating that AlgR control of twitching motility is not mediated via its role in the control of alginate biosynthesis. In vitro phosphorylation assays showed that AlgR D54N is not phosphorylated by the enteric histidine kinase CheA. These findings indicate that phosphorylation of AlgR most likely occurs at aspartate 54 and that aspartate 54 and aspartate 85 of AlgR are required for the control of the molecular events governing fimbrial biogenesis, twitching motility, and biofilm formation in P. aeruginosa.     

Mucoid Pseudomonas aeruginosa in cystic fibrosis: signal transduction and histone-like elements in the regulation of bacterial virulence

The profuse production of the exopolysaccharide alginate results in mucoidy, a critical virulence factor expressed by Pseudomonas aeruginosa during chronic respiratory tract infections in cystic fibrosis. Studies of the regulation of this pathogenic determinant have unravelled at least two levels of control, including bacterial signal transduction systems and histone-like elements. Although only in its initial phase, an understanding of the dual control of mucoidy may help to illuminate adaptive processes that depend on the combination of these regulatory factors. Integration of specific signals transduced by the two-component systems with inputs generated by the general state of bacterial nucleoids may govern the expression of certain virulence determinants and provide a framework facilitating selection of phenotypes successful under particular environmental conditions and selective pressures.     

Genetic analysis of the catalytic domain of the chemotaxis-associated histidine kinase CheA.

Escherichia coli cells express two forms of CheA, the histidine kinase associated with chemotaxis. The long form, CheA(L), plays a critical role in chemotactic signal transduction by phosphorylating two chemotaxis-associated response regulators, CheY and CheB. CheA(L) first autophosphorylates amino acid His-48 before its phosphoryl group is transferred to these response regulators. The short form, CheA(S), lacks the amino-terminal 97 amino acids of CheA(L) and therefore does not possess the site of phosphorylation. The centrally located transmitter domain of both forms of CheA contains four regions, called N, G1, F, and G2, highly conserved among histidine kinases of the family of two-component signal transduction systems. On the basis of sequence similarity to highly conserved regions of certain eukaryotic kinases, the G1 and G2 regions are purported to be involved in the binding and hydrolysis of ATP. We report here that alleles mutated in the G1, G2, or F region synthesize CheA variants that cannot autophosphorylate in vitro and which cannot support chemotaxis in vivo. We also show that in vitro, the nonphosphorylatable CheA(S) protein mediates transphosphorylation of a CheA(L) variant defective in both G1 and G2. In contrast, CheA(L) variants defective for either G1 or G2 mediate transphosphorylation of each other poorly, if at all. These results are consistent with a mechanism by which the G1 and G2 regions of one protomer of a CheA dimer form a unit that mediates transphosphorylation of the other protomer within that dimer.     

Regulation of RssB-dependent proteolysis in Escherichia coli: a role for acetyl phosphate in a response regulator-controlled process

σ^S (RpoS) is a highly unstable global regulatory protein in Escherichia coli , whose degradation is inhibited by various stress signals, such as carbon starvation, high osmolarity and heat shock. As a consequence, these stresses result in the induction of σ^S-regulated stress-protective proteins. The two-component-type response regulator, RssB, is essential for the rapid proteolysis of σ^S and is probably involved in the transduction of some of these stress signals. Acetyl phosphate can be used as a phosphodonor for the phosphorylation of various response regulators in vitro and, in the absence of the cognate sensor kinases, acetyl phosphate can also modulate the activities of several response regulators in vivo . Here, we demonstrate increased in vivo half-lives of σS and the RpoS742::LacZ hybrid protein (also a substrate for RssB-dependent proteolysis) in acetyl phosphate-free ( pta – ackA ) deletion mutants, even though no sensor kinase was eliminated. The in vivo data indicate that acetyl phosphate acts through the response regulator, RssB. In vitro , efficient phosphotransfer from radiolabelled acetyl phosphate to the Asp-58 residue of RssB (the expected site of phosphorylation in the RssB receiver domain) was observed. Via such phosphorylation, acetyl phosphate may thus modulate RssB activity even in an otherwise wild-type background. While acetyl phosphate is not essential for the transduction of specific environmental stress signals, it could play the role of a modulator of RssB-dependent proteolysis that responds to the metabolic status of the cells reflected in the highly variable cellular acetyl phosphate concentration.     

Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis

Six cytoplasmic che gene products are required for signal transduction in bacterial chemotaxis, but the nature of their biochemical interactions is not known. We show that in vitro the CheA protein becomes autophosphorylated in the presence of ATP. In addition, the phosphate group on CheA can be rapidly transferred to CheB, a protein involved in adaptation to stimuli, or to CheY, a protein involved in the excitation response. The phosphorylation of CheB and CheY is transient; they readily dephosphorylate. We have also found that CheZ, a protein that appears to antagonize CheY function in vivo, accelerates the hydrolysis of the phosphate on CheY. These results suggest that signal transduction in bacterial chemotaxis may involve the flow of phosphate through a cascade of phosphorylated protein intermediates.     

Kinetic characterization of CheY phosphorylation reactions: comparison of P-CheA and small-molecule phosphodonors

In the chemotaxis system of Escherichia coli, phosphorylation of the CheY protein plays an important role in regulating the swimming pattern of the cell. In vitro, CheY can be phosphorylated either by phosphotransfer from phospho-CheA or by acquiring a phosphoryl group from any of a variety of small, high-energy phosphodonor molecules such as acetyl phosphate. Previous work explored the rapid kinetics of CheY phosphorylation by CheA. Here we extend that work and examine the kinetics of CheY phosphorylation by several small-molecule phosphodonors, including acetyl phosphate, benzoyl phosphate, carbamoyl phosphate, 2-methoxybenzoyl phosphate, and phosphoramidate. Our results indicate that these phosphodonors bind to CheY with relatively low affinity (Ks values ranging from 10 to 600 mM) and that the rate constant (kphos) for phosphotransfer at saturating phosphodonor concentrations is relatively slow (values ranging from 0.05 to 0.5 s-1). By contrast, under identical conditions, phosphorylation of CheY by phospho-CheA occurs much more rapidly (kphos approximately 800 s-1) and reflects CheY binding to phospho-CheA considerably more tightly (Ks approximately 60 microM) than it does to the small-molecule phosphodonors. In comparing CheA-mediated phosphorylation of CheY to small-molecule-mediated phosphorylation of CheY, the large difference in kphos values suggests that phospho-CheA makes significant contributions to the catalysis of CheY phosphorylation. The effects of pH and ionic strength on CheY phosphorylation kinetics were also investigated. For CheA-->CheY phosphotransfer, increasing ionic strength resulted in increased Ks values while kphos was unaffected. For CheY phosphorylation by small-molecule phosphodonors, increasing ionic strength resulted in decreasing Ks values and increasing kphos values. The significance of these effects is discussed in relation to the catalytic mechanism of CheY phosphorylation by phospho-CheA and small-molecule phosphodonors.     

Kinetics of CheY phosphorylation by small molecule phosphodonors.

The chemotaxis response regulator CheY can acquire phosphoryl groups either from its associated autophosphorylating protein kinase, CheA, or from small phosphodonor molecules such as acetyl phosphate. We report a stopped-flow kinetic analysis of CheY phosphorylation by acetyl phosphate. The results show that CheY has a very low affinity for this phosphodonor (K(s)&z.Gt;0.1 M), consistent with the conclusion that, whereas CheY provides catalytic functions for the phosphotransfer reaction, the CheA kinase may act simply to increase the effective phosphodonor concentration at the CheY active site.     

Evidence against the physiological role of acetyl phosphate in the phosphorylation of the ArcA response regulator in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The Arc two-component signal transduction system of Escherichia coli comprises the ArcB sensor kinase and the ArcA response regulator. Under anoxic growth conditions, ArcB autophosphorylates and transphos-phorylates ArcA, which, in turn, represses or activates its target operons. ArcA has been shown to be able to autophosphorylate in vitro at the expense of acetyl-P. Here, the in vivo effect of acetyl phosphate on the redox signal transduction by the Arc system was assessed. Our results indicate that acetyl phosphate can modulate the expression of ArcA-P target genes only in the absence of ArcB. Therefore, the acetyl phosphate dependent ArcA phosphorylation route does not seem to play a significant role under physiological conditions.     

Divergent regulatory pathways control A and S motility in Myxococcus xanthus through FrzE, a CheA-CheY fusion protein

Myxococcus xanthus moves on solid surfaces by using two gliding motility systems, A motility for individual-cell movement and S motility for coordinated group movements. The frz genes encode chemotaxis homologues that control the cellular reversal frequency of both motility systems. One of the components of the core Frz signal transduction pathway, FrzE, is homologous to both CheA and CheY from the enteric bacteria and is therefore a novel CheA-CheY fusion protein. In this study, we investigated the role of this fusion protein, in particular, the CheY domain (FrzECheY). FrzECheY retains all of the highly conserved residues of the CheY superfamily of response regulators, including Asp709, analogous to phosphoaccepting Asp57 of Escherichia coli CheY. While in-frame deletion of the entire frzE gene caused both motility systems to show a hyporeversal phenotype, in-frame deletion of the FrzECheY domain resulted in divergent phenotypes for the two motility systems: hyperreversals of the A-motility system and hyporeversals of the S-motility system. To further investigate the role of FrzECheY in A and S motility, point mutations were constructed such that the putative phosphoaccepting residue, Asp709, was changed from D to A (and was therefore never subject to phosphorylation) or E (possibly mimicking constitutive phosphorylation). The D709A mutant showed hyperreversals for both motilities, while the D709E mutant showed hyperreversals for A motility and hyporeversal for S motility. These results show that the FrzECheY domain plays a critical signaling role in coordinating A and S motility. On the basis of the phenotypic analyses of the frzE mutants generated in this study, a model is proposed for the divergent signal transduction through FrzE in controlling and coordinating A and S motility in M. xanthus.     

Phosphorylation in halobacterial signal transduction.

Regulated phosphorylation of proteins has been shown to be a hallmark of signal transduction mechanisms in both Eubacteria and Eukarya. Here we demonstrate that phosphorylation and dephosphorylation are also the underlying mechanism of chemo- and phototactic signal transduction in Archaea, the third branch of the living world. Cloning and sequencing of the region upstream of the cheA gene, known to be required for chemo- and phototaxis in Halobacterium salinarium, has identified cheY and cheB analogs which appear to form part of an operon which also includes cheA and the following open reading frame of 585 nucleotides. The CheY and CheB proteins have 31.3 and 37.5% sequence identity compared with the known signal transduction proteins CheY and CheB from Escherichia coli, respectively. The biochemical activities of both CheA and CheY were investigated following their expression in E.coli, isolation and renaturation. Wild-type CheA could be phosphorylated in a time-dependent manner in the presence of [gamma-32P]ATP and Mg2+, whereas the mutant CheA(H44Q) remained unlabeled. Phosphorylated CheA was dephosphorylated rapidly by the addition of wild-type CheY. The mutant CheY(D53A) had no effect on phosphorylated CheA. The mechanism of chemo- and phototactic signal transduction in the Archaeon H.salinarium, therefore, is similar to the two-component signaling system known from chemotaxis in the eubacterium E.coli.     

Intermolecular complementation of the kinase activity of CheA

CheA is a dimeric autophosphorylating protein kinase that plays a critical role in the signal transduction network controlling chemotaxis In Escherichia coli. The autophosphorylation reaction was analysed using mutant proteins defective in kinase and regulatory functions. Proteins in which the site of autophosphorylation was mutated (CheA48HQ) or missing (CheA_s) were found to phosphorylate the kinase-defective mutant, CheA470GK. The kinetics of this reaction support the hypothesis that autophosphorylation is the result of trans-phosphorylation within a dimer. The carboxy-terminal portion of CheA was previously shown to be dispensable for autophosphorylation, but required for regulation in response to environmental signals transmitted through a transducer and CheW. Mixing of CheA48HQ or CheA470GK with a truncated protein lacking this regulatory domain demonstrated that regulated autophosphoryltion requires the presence of both carboxy-terminal portions in a CheA dimer. These results indicate that the dimeric form of CheA plays an integral role in signal transduction in bacterial chemotaxis.     

An intersection of the cAMP/PKA and two-component signal transduction systems in Dictyostelium.

Terminal differentiation of both stalk and spore cells in Dictyostelium can be triggered by activation of cAMP-dependent protein kinase (PKA). A screen for mutants where stalk and spore cells mature in isolation produced three genes which may act as negative regulators of PKA: rdeC (encoding the PKA regulatory subunit), regA and rdeA. The biochemical properties of RegA were studied in detail. One domain is a cAMP phosphodiesterase (Km approximately 5 microM); the other is homologous to response regulators (RRs) of two-component signal transduction systems. It can accept phosphate from acetyl phosphate in a reaction typical of RRs, with transfer dependent on Asp212, the predicted phosphoacceptor. RegA phosphodiesterase activity is stimulated up to 8-fold by the phosphodonor phosphoramidate, with stimulation again dependent on Asp212. This indicates that phosphorylation of the RR domain activates the phosphodiesterase domain. Overexpression of the RR domain in wild-type cells phenocopies a regA null. We interpret this dominant-negative effect as due to a diversion of the normal flow of phosphates from RegA, thus preventing its activation. Mutation of rdeA is known to produce elevated cAMP levels. We propose that cAMP breakdown is controlled by a phosphorelay system which activates RegA, and may include RdeA. Cell maturation should be triggered when this system is inhibited.     

Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis.

The alternative sigma factor AlgU (Pseudomonas aeruginosa sigma E) is required for full resistance of P. aeruginosa to oxidative stress and extreme temperatures. AlgU also controls conversion of P. aeruginosa to the mucoid, alginate-overproducing phenotype associated with lethal infections in cystic fibrosis patients. Mutations that cause conversion to mucoidy in cystic fibrosis isolates occur frequently in mucA, the second gene within the algU mucABCD gene cluster. Here we analyze the biochemical basis of conversion to mucoidy. MucA was shown to act as an anti-sigma factor by binding to AlgU and inhibiting its activity. MucB, another negative regulator of AlgU, was localized in the periplasm. MucB exerts its function from this compartment, since deletion of the leader peptide and the cytoplasmic location of MucB abrogated its ability to inhibit mucoidy. These data support a model in which a multicomponent system, encompassing an anti-delta factor and elements in the periplasmic compartment, modulates activity of AlgU. Since factors controlling AlgU are conserved in other gram-negative bacteria, the processes controlling conversion to mucoidy in P. aeruginosa may be applicable to the regulation of AlgU (sigma E) equivalents in other organisms.