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.     

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.     

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.     

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.     

Spermidine triggering effect to the signal transduction through the AtoS-AtoC/Az two-component system in Escherichia coli

Recent analysis revealed that, in Escherichia coli the AtoS-AtoC/Az two-component system (TCS) and its target atoDAEB operon regulate the biosynthesis of short-chain poly-(R)-3-hydroxybutyrate (cPHB) biosynthesis, a biopolymer with many physiological roles, upon acetoacetate-mediated induction. We report here that spermidine further enhanced this effect, in E. coli that overproduces both components of the AtoS-AtoC/Az TCS, without altering their protein levels. However, bacteria that overproduce either AtoS or AtoC did not display this phenotype. The extrachromosomal introduction of AtoS-AtoC/Az in an E. coli DeltaatoSC strain restored cPHB biosynthesis to the level of the atoSC(+) cells, in the presence of the polyamine. Lack of enhanced cPHB production was observed in cells overproducing the TCS that did not have the atoDAEB operon. Spermidine attained the cPHB enhancement through the AtoC/Az response regulator phosphorylation, since atoC phosphorylation site mutants, which overproduce AtoS, accumulated less amounts of cPHB, compared to their wild-type counterparts. Exogenous addition of N(8)-acetyl-spermidine resulted in elevated amounts of cPHB but at lower levels than those attained upon spermidine addition. Furthermore, AtoS-AtoC/Az altered the intracellular distribution of cPHB according to the inducer recognized by the TCS. Overall, AtoS-AtoC/Az TCS was induced by spermidine to regulate both the biosynthesis and the intracellular distribution of cPHB in E. coli.     

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.     

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.     

Involvement of the AtoSCDAEB regulon in the high molecular weight poly-(R)-3-hydroxybutyrate biosynthesis in phaCAB(+)Escherichia coli

AtoSC two-component system plays a pivotal role in many regulatory indispensable Escherichia coli processes. AtoSCDAEB regulon, comprising the AtoSC system and the atoDAEB operon, regulates the short-chain fatty acids catabolism. We report here, that AtoSC up-regulates the high-molecular weight PHB biosynthesis, in recombinant phaCAB(+)E. coli, with the Cupriavidus necator phaCAB operon. PHB accumulation was maximized upon the acetoacetate-mediated induction of AtoSC, under glucose 1% w/v, resulting in a yield of 1.73 g/l with a biopolymer content of 64.5% w/w. The deletion of the atoSC locus, in the ΔatoSC strains, resulted in a 5 fold reduction of PHB accumulation, which was restored by the extrachromosomal introduction of the AtoSC system. The deletion of the atoDAEB operon triggered a significant decrease in PHB synthesis in ΔatoDAEB strains. However, the acetoacetate-induced AtoSC system in those strains increased PHB to 1.55 g/l, while AtoC expression increased PHB to 1.4 g/l upon acetoacetate. The complementation of the ΔatoDAEB phenotype was achieved by the extrachromosomal introduction of the atoSCDAEB regulon. The individual inhibition of β-oxidation and mainly fatty-acid biosynthesis pathways by acrylic acid or cerulenin respectively, reduced PHB biosynthesis. Under those conditions the introduction of the atoSC locus or the atoSCDAEB regulon was capable to up-regulate the biopolymer accumulation. The concurrent inhibition of both the fatty acids metabolic pathways eliminated PHB production. PHB up-regulation in phaCAB(+)E. coli, by AtoSC signaling through atoDAEB operon and its participation in the fatty acids metabolism interplay, provide additional perceptions of AtoSC critical involvement in E. coli regulatory processes towards the biotechnologically improved polyhydroxyalkanoates biosynthesis.     

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 (ΔatoSC) synthesized lower amounts of cPHB compared to wild-type cells. When the ΔatoSC 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.     

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 Η-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.     

Spermidine triggering effect to the signal transduction through the AtoS–AtoC/Az two-component system in Escherichia coli

Recent analysis revealed that, in Escherichia coli the AtoS–AtoC/Az two-component system (TCS) and its target atoDAEB operon regulate the biosynthesis of short-chain poly-(R)-3-hydroxybutyrate (cPHB) biosynthesis, a biopolymer with many physiological roles, upon acetoacetate-mediated induction. We report here that spermidine further enhanced this effect, in E. coli that overproduces both components of the AtoS–AtoC/Az TCS, without altering their protein levels. However, bacteria that overproduce either AtoS or AtoC did not display this phenotype. The extrachromosomal introduction of AtoS–AtoC/Az in an E. coli ΔatoSC strain restored cPHB biosynthesis to the level of the atoSC⁺ cells, in the presence of the polyamine. Lack of enhanced cPHB production was observed in cells overproducing the TCS that did not have the atoDAEB operon. Spermidine attained the cPHB enhancement through the AtoC/Az response regulator phosphorylation, since atoC phosphorylation site mutants, which overproduce AtoS, accumulated less amounts of cPHB, compared to their wild-type counterparts. Exogenous addition of N⁸-acetyl-spermidine resulted in elevated amounts of cPHB but at lower levels than those attained upon spermidine addition. Furthermore, AtoS–AtoC/Az altered the intracellular distribution of cPHB according to the inducer recognized by the TCS. Overall, AtoS–AtoC/Az TCS was induced by spermidine to regulate both the biosynthesis and the intracellular distribution of cPHB in E. coli.     

Role of fadR and atoC(Con) mutations in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombinant pha+ Escherichia coli.

Recombinant Escherichia coli fadR atoC(Con) mutants containing the polyhydroxyalkanoate (PHA) biosynthesis genes from Alcaligenes eutrophus are able to incorporate significant levels of 3-hydroxyvalerate (3HV) into the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]. We have used E. coli fadR (FadR is a negative regulator of fatty acid oxidation) and E. coli atoC(Con) (AtoC is a positive regulator of fatty acid uptake) mutants to demonstrate that either one of these mutations alone can facilitate copolymer synthesis but that 3HV levels in single mutant strains are much lower than in the fadR atoC(Con) strain. E. coli atoC(Con) mutants were used alone and in conjunction with atoA and atoD mutants to determine that the function of the atoC(Con) mutation is to increase the uptake of propionate and that this uptake is mediated, at least in part, by atoD+. Similarly, E. coli fadR mutants were used alone and in conjunction with fadA, fadB, and fadL mutants to show that the effect of the fadR mutation is dependent on fadB+ and fadA+ gene products. Strains that were mutant in the fadB or fadA locus were unable to complement a PHA biosynthesis pathway that was mutant at the phaA locus (thiolase), but a strain containing a fadR mutation and which was fadA+ fadB+ was able to complement the phaA mutation and incorporated 3HV into P(3HB-co-3HV) to a level of 29 mol%.