Molecular modeling and functional analysis of the AtoS-AtoC two-component signal transduction system of Escherichia coli

The AtoS-AtoC two-component signal transduction system positively regulates the expression of the atoDAEB operon in Escherichia coli. Upon acetoacetate induction, AtoS sensor kinase autophosphorylates and subsequently phosphorylates, thereby activating, the response regulator AtoC. In a previous work we have shown that AtoC is phosphorylated at both aspartate 55 and histidine73. In this study, based on known three-dimensional structures of other two component regulatory systems, we modeled the 3D-structure of the receiver domain of AtoC in complex with the putative dimerization/autophosphorylation domain of the AtoS sensor kinase. The produced structural model indicated that aspartate 55, but not histidine 73, of AtoC is in close proximity to the conserved, putative phosphate-donor, histidine (H398) of AtoS suggesting that aspartate 55 may be directly involved in the AtoS-AtoC phosphate transfer. Subsequent biochemical studies with purified recombinant proteins showed that AtoC mutants with alterations of aspartate 55, but not histidine 73, were unable to participate in the AtoS-AtoC phosphate transfer in support of the modeling prediction. In addition, these AtoC mutants displayed reduced DNA-dependent ATPase activity, although their ability to bind their target DNA sequences in a sequence-specific manner was found to be unaltered.     

Functional characterization of the histidine kinase of the E. coli two-component signal transduction system AtoS-AtoC

The Escherichia coli AtoS-AtoC two-component signal transduction system regulates the expression of the atoDAEB operon genes, whose products are required for short-chain fatty acid catabolism. In this study purified his-tagged wild-type and mutant AtoS proteins were used to prove that these proteins are true sensor kinases. The phosphorylated residue was identified as the histidine-398, which was located in a conserved Eta-box since AtoS carrying a mutation at this site failed to phosphorylate. This inability to phosphorylate was not due to gross structural alterations of AtoS since the H398L mutant retained its capability to bind ATP. Furthermore, the H398L mutant AtoS was competent to catalyze the trans-phosphorylation of an AtoS G-box (G565A) mutant protein which otherwise failed to autophosphorylate due to its inability to bind ATP. The formation of homodimers between the various AtoS proteins was also shown by cross-linking experiments both in vitro and in vivo.     

Phosphorylation activity of the response regulator of the two-component signal transduction system AtoS-AtoC in E. coli

Antizyme, long known to be a non-competitive inhibitor of ornithine decarboxylase, is encoded by the atoC gene in Escherichia coli. The present study reveals another role for AtoC, that of a response regulator of the AtoS-AtoC two component system regulating the expression of the atoDAEB operon upon acetoacetate induction. This operon encodes enzymes involved in short-chain fatty acid catabolism in E. coli. Evidence is presented to show that AtoS is a sensor kinase that together with AtoC constitutes a two-component signal transduction system. AtoS is a membrane protein which can autophosphorylate and then transfer that phosphoryl group to AtoC. This process can also be reproduced in vitro. AtoC contains in its amino acid sequence a conserved aspartic acid (D55), which is the putative phosphorylation site, as well as an unexpected "H box" consensus sequence (SHETRTPV), common to histidine kinases, with the histidine contained therein (H73) being a second potential target for phosphorylation. Substitution of either D55 or H73 in His10-AtoC diminished but did not abrogate AtoC phosphorylation suggesting that either both residues can be phosphorylated independently or that the phosphate group can be transferred between them. However, the D55 mutation in comparison to H73 had a more pronounced effect in vivo, on the activation of atoDAEB promoter after acetoacetate induction, although it was the presence of both mutations that rendered AtoC totally unresponsive to induction. These data provide evidence that the gene products of atoS and atoC constitute a two-component signal transduction system, with some unusual properties, involved in the regulation of the atoDAEB operon.     

Involvement of the AtoS-AtoC signal transduction system in poly-(R)-3-hydroxybutyrate biosynthesis in Escherichia coli

The AtoS-AtoC signal transduction system in E. coli, which induces the atoDAEB operon for the growth of E. coli in short-chain fatty acids, can positively modulate the levels of poly-(R)-3-hydroxybutyrate (cPHB) biosynthesis, a biopolymer with many physiological roles in E. coli. Increased amounts of cPHB were synthesized in E. coli upon exposure of the cells to acetoacetate, the inducer of the AtoS-AtoC two-component system. While E. coli that overproduce both components of the signal transduction system synthesize higher quantities of cPHB (1.5-4.5 fold), those that overproduce either AtoS or AtoC alone do not display such a phenotype. Lack of enhanced cPHB production was also observed in cells overexpressing AtoS and phosphorylation-impaired AtoC mutants. The results were not affected by the nature of the carbon source used, i.e., glucose, acetate or acetoacetate. An E. coli strain with a deletion in the atoS-atoC locus (delta atoSC) synthesized lower amounts of cPHB compared to wild-type cells. When the delta atoSC strain was transformed with a plasmid carrying a 6.4-kb fragment encoding the AtoS-AtoC system, cPHB biosynthesis was restored to the level of the atoSC+ cells. Introduction of a multicopy plasmid carrying a functional atoDAEB operon, but not one with a promoterless operon, resulted in increased cPHB synthesis only in atoSC+ cells in the presence of acetoacetate. These results indicate that the presence of both a functional AtoS-AtoC two-component signal transduction system and a functional atoDAEB operon is critical for the enhanced cPHB biosynthesis in E. coli.     

Analysis of two-component signal transduction by mathematical modeling using the KdpD/KdpE system of Escherichia coli

A mathematical model for the KdpD/KdpE two-component system is presented and its dynamical behavior is analyzed. KdpD and KdpE regulate expression of the kdpFABC operon encoding the high affinity K+ uptake system KdpFABC of Escherichia coli. The model is validated in a two step procedure: (i) the elements of the signal transduction part are reconstructed in vitro. Experiments with the purified sensor kinase and response regulator in presence or absence of DNA fragments comprising the response regulator binding-site are performed. (ii) The mRNA and molecule number of KdpFABC are determined in vivo at various extracellular K+ concentrations. Based on the identified parameters for the in vitro system it is shown, that different time hierarchies appear which are used for model reduction. Then the model is transformed in such a way that a singular perturbation problem is formulated. The analysis of the in vivo system shows that the model can be separated into two parts (submodels which are called functional units) that are connected only in a unidirectional way. Hereby one submodel represents signal transduction while the second submodel describes the gene expression.     

EvgA of the two-component signal transduction system modulates production of the yhiUV multidrug transporter in Escherichia coli

Overexpression of the EvgA regulator of the two-component signal transduction system was previously found to modulate multidrug resistance of Escherichia coli by increasing efflux of drugs (K. Nishino and A. Yamaguchi, J. Bacteriol. 183:1455-1458, 2001). Here we present data showing that EvgA contributes to multidrug resistance through increased expression of the multidrug transporter yhiUV gene.     

Functional characterization in vitro of all two-component signal transduction systems from Escherichia coli

Bacteria possess a signal transduction system, referred to as a two-component system, for adaptation to external stimuli. Each two-component system consists of a sensor protein-histidine kinase (HK) and a response regulator (RR), together forming a signal transduction pathway via histidyl-aspartyl phospho-relay. A total of 30 sensor HKs, including as yet uncharacterized putative HKs (BaeS, BasS, CreC, CusS, HydH, RstB, YedV, and YfhK), and a total of 34 RRs, including putative RRs (BaeR, BasR, CreB, CusR, HydG, RstA, YedW, YfhA, YgeK, and YhjB), have been suggested to exist in Escherichia coli. We have purified the carboxyl-terminal catalytic domain of 27 sensor HKs and the full-length protein of all 34 RRs to apparent homogeneity. Self-phosphorylation in vitro was detected for 25 HKs. The rate of self-phosphorylation differed among HKs, whereas the level of phosphorylation was generally co-related with the phosphorylation rate. However, the phosphorylation level was low for ArcB, HydH, NarQ, and NtrB even though the reaction rate was fast, whereas the level was high for the slow phosphorylation species BasS, CheA, and CreC. By using the phosphorylated HKs, we examined trans-phosphorylation in vitro of RRs for all possible combinations. Trans-phosphorylation of presumed cognate RRs by HKs was detected, for the first time, for eight pairs, BaeS-BaeR, BasS-BasR, CreC-CreB, CusS-CusR, HydH-HydG, RstB-RstA, YedV-YedW, and YfhK-YfhA. All trans-phosphorylation took place within less than 1/2 min, but the stability of phosphorylated RRs differed, indicating the involvement of de-phosphorylation control. In addition to the trans-phosphorylation between the cognate pairs, we detected trans-phosphorylation between about 3% of non-cognate HK-RR pairs, raising the possibility that the cross-talk in signal transduction takes place between two-component systems.     

沙门菌PhoP-PhoQ双组分信号转导系统

PhoP-PhoQ是调控沙门菌毒力的重要双组分信号转导系统,由组氨酸蛋白激酶PhoQ和反应调节蛋白PhoP组成。PhoP-PhoQ可调节沙门菌对Mg2+及其他周质环境的适应性,并调控沙门菌感染中毒力基因的转录和表达。PhoP-PhoQ调控的毒力基因参与沙门菌对上皮细胞的侵袭、胞内生存、对抗菌肽的抵抗反应、脂质A的修饰、Ⅲ型分泌系统效应蛋白的分泌等环节。PhoP-PhoQ还可与其他双组分信号转导系统或调节子合作,调控沙门菌的毒力。因此,PhoP-PhoQ双组分信号转导系统在沙门菌的毒力调控中发挥重要作用。     

Regulation of acid resistance by connectors of two-component signal transduction systems in Escherichia coli

Two-component signal transduction systems (TCSs), utilized extensively by bacteria and archaea, are involved in the rapid adaptation of the organisms to fluctuating environments. A typical TCS transduces the signal by a phosphorelay between the sensor histidine kinase and its cognate response regulator. Recently, small-sized proteins that link TCSs have been reported and are called "connectors." Their physiological roles, however, have remained elusive. SafA (sensor associating factor A) (formerly B1500), a small (65-amino-acid [65-aa]) membrane protein, is among such connectors and links Escherichia coli TCSs EvgS/EvgA and PhoQ/PhoP. Since the activation of the EvgS/EvgA system induces acid resistance, we examined whether the SafA-activated PhoQ/PhoP system is also involved in the acid resistance induced by EvgS/EvgA. Using a constitutively active evgS1 mutant for the activation of EvgS/EvgA, we found that SafA, PhoQ, and PhoP all contributed to the acid resistance phenotype. Moreover, EvgS/EvgA activation resulted in the accumulation of cellular RpoS in the exponential-phase cells in a SafA-, PhoQ-, and PhoP-dependent manner. This RpoS accumulation was caused by another connector, IraM, expression of which was induced by the activation of the PhoQ/PhoP system, thus preventing RpoS degradation by trapping response regulator RssB. Acid resistance assays demonstrated that IraM also participated in the EvgS/EvgA-induced acid resistance. Therefore, we propose a model of a signal transduction cascade proceeding from EvgS/EvgA to PhoQ/PhoP and then to RssB (connected by SafA and IraM) and discuss its contribution to the acid resistance phenotype.     

Inhibition of the signal transduction through the AtoSC system by histidine kinase inhibitors in Escherichia coli

AtoSC two-component system participates in many indispensable processes of Escherichia coli. We report here that the AtoSC signal transduction is inhibited by established histidine kinase inhibitors. Closantel, RWJ-49815 and TNP-ATP belonging to different chemical classes of inhibitors, abrogated the in vitro AtoS kinase autophosphorylation. However, when AtoS was embedded in the membrane fractions, higher inhibitor concentrations were required for total inhibition. When AtoS interacted with AtoC forming complex, the intrinsic histidine kinase was protected by the response regulator, requiring increased inhibitors concentrations for partially AtoS autophosphorylation reduction. The inhibitors exerted an additional function on AtoSC, blocking the phosphotransfer from AtoS to AtoC, without however, affecting AtoC~P dephosphorylation. Their in vivo consequences through the AtoSC inhibition were elucidated on atoDAEB operon expression, which was inhibited only in AtoSC-expressing bacteria where AtoSC was induced by acetoacetate or spermidine. The inhibitor effects were extended on the AtoSC regulatory role on cPHB [complexed poly-(R)-3-hydroxybutyrate] biosynthesis. cPHB was decreased upon the blockers only in acetoacetate-induced AtoSC-expressing cells. Increased ATP amounts during bacterial growth reversed the inhibitory TNP-ATP-mediated effect on cPHB. The alteration of pivotal E. coli processes as an outcome of AtoSC inhibition, establish this system as a target of two-component systems inhibitors.     

DjlA negatively regulates the Rcs signal transduction system in Escherichia coli

The Rcs signal transduction system of Escherichia coli regulating capsular polysaccharide synthesis (cps) genes is activated by overexpression of the djlA gene encoding a cytoplasmic membrane-anchored DnaJ-like protein. However, by monitoring the expression of a cpsB'-lac fusion in pgsA- and mdoH-null mutants in which the Rcs system is activated, we found that the Rcs activity was further increased by deletion of djlA and decreased by low-level extrachromosomal expression of djlA. Furthermore, deletion of djlA in a wild-type strain led to small but significant increase of the basal-level activity of the Rcs system. These results demonstrate that DjlA functions as a negative regulator of the Rcs system unless abnormally overproduced.     

Cpx two-component signal transduction in Escherichia coli: excessive CpxR-P levels underlie CpxA* phenotypes

In Escherichia coli, the CpxA-CpxR two-component signal transduction system and the sigma(E) and sigma(32) response pathways jointly regulate gene expression in adaptation to adverse conditions. These include envelope protein distress, heat shock, oxidative stress, high pH, and entry into stationary phase. Certain mutant versions of the CpxA sensor protein (CpxA* proteins) exhibit an elevated ratio of kinase to phosphatase activity on CpxR, the cognate response regulator. As a result, CpxA* strains display numerous phenotypes, many of which cannot be easily related to currently known functions of the CpxA-CpxR pathway. It is unclear whether CpxA* phenotypes are caused solely by hyperphosphorylation of CpxR. We here report that all of the tested CpxA* phenotypes depend on elevated levels of CpxR-P and not on cross-signalling of CpxA* to noncognate response regulators.     

The Cpx two-component signal transduction pathway is activated in Escherichia coli mutant strains lacking phosphatidylethanolamine.

The CpxA-CpxR two-component signal transduction pathway of Escherichia coli was studied in a mutant (pss-93) lacking phosphatidylethanolamine (PE). Several properties of this mutant are comparable to phenotypes of cpxA point mutants, indicating that this two-component pathway is activated in PE-deficient cells. In contrast to point mutants, cpx operon null mutants have a wild-type phenotype. By use of this information, a cpx operon null allele was introduced into a pss-93 mutant. Certain altered properties of PE-deficient mutants, which were consistent with activation of the Cpx pathway, returned to the wild-type phenotype, namely, active accumulation of proline and thiomethyl-beta-D-galactopyranoside was partially restored to wild-type levels, increased resistance to amikacin returned to wild-type sensitivity, and high levels of degP expression returned to repressed wild-type levels. Elevated levels of acetyl phosphate and nlpE gene product can result in activation of the Cpx pathway. However, inactivation of the nlpE gene or mutations eliminating the ability to make acetyl phosphate did not alter the high level of degP expression in pss-93 mutants. We propose that the lack of PE results in an alteration in cell envelope structure or physical properties, leading to direct activation of the Cpx pathway.     

葡萄球菌呼吸相关双组分系统SrrAB研究进展

葡萄球菌呼吸相关双组分系统SrrAB能感应外界O2浓度,并将信号传至胞内,调控下游基因的转录,以应对外界环境的变化。有研究表明,金黄色葡萄球菌SrrAB在有氧条件下促进毒力因子的表达,抑制生物膜的形成;在厌氧条件下抑制毒力因子的表达,促进生物膜的形成。另外,在有氧及厌氧条件下,金黄色葡萄球菌SrrAB调控生长代谢的途径也不一致。表皮葡萄球菌中也存在类似的双组分系统SrrAB,且与金黄色葡萄球菌SrrAB具有较高同源性,但目前尚不清楚两者在生长代谢及毒力调控方面的异同。结合课题组研究工作,简要综述葡萄球菌SrrAB的调控机制,着重比较其在有氧及厌氧条件下的调控差异,这对临床诊治葡萄球菌引起的感染具有一定的借鉴意义。