Marina C. Theodorou

Extracellular Ca2+ transients affect poly-(R)-3-hydroxybutyrate regulation by the AtoS-AtoC system in Escherichia coli

Escherichia coli is exposed to wide extracellular concentrations of Ca2+, whereas the cytosolic levels of the ion are subject to stringent control and are implicated in many physiological functions. The present study shows that extracellular Ca2+ controls cPHB [complexed poly-(R)-3-hydroxybutyrate] biosynthesis through the AtoS-AtoC two-component system. Maximal cPHB accumulation was observed at higher [Ca2+]e (extracellular Ca2+ concentration) in AtoS-AtoC-expressing E. coli compared with their DeltaatoSC counterparts, in both cytosolic and membrane fractions. The reversal of EGTA-mediated down-regulation of cPHB biosynthesis by the addition of Ca2+ and Mg2+ was under the control of the AtoS-AtoC system. Moreover, the Ca2+-channel blocker verapamil reduced total and membrane-bound cPHB levels, the inhibitory effect being circumvented by Ca2+ addition only in atoSC+ bacteria. Histamine and compound 48/80 affected cPHB accumulation in a [Ca2+]e-dependent manner directed by the AtoS-AtoC system. In conclusion, these data provide evidence for the involvement of external Ca2+ on cPHB synthesis regulated by the AtoS-AtoC two-component system, thus linking Ca2+ with a signal transduction system, most probably through a transporter.

Effect of histamine on the signal transduction of the AtoS–AtoC two component system and involvement in poly-(R)-3-hydroxybutyrate biosynthesis in Escherichia coli

Summary.AtoS–AtoC two-component system acts directly on the atoDAEB operon transcription to regulate the biosynthesis of short-chain poly-(R)-3-hydroxybutyrate. This study sought to investigate the effect of histamine and compound 48/80 on the regulation of AtoS–AtoC two-component system in Escherichia coli K-12 MA255 (speC−, speB−) and the isogenic E. coli strains BW25113 (atoSC+) and BW28878 (ΔatoSC) transformed with plasmids carrying related genes. Histamine or compound 48/80 induced or tended to reduce atoC transcription, respectively, while neither compound showed any effect on atoDAEB operon transcription. Moreover, histamine down-regulated poly-(R)-3-hydroxybutyrate biosynthesis, whereas compound 48/80 up-regulated its biosynthesis, maximal induction being obtained in the presence of multiple copies of AtoS–AtoC. Interestingly, co-administration of histamine counteracted this inductive effect of compound 48/80. The reported data provide the first evidence for a differential modulator role of histamine and compound 48/80 on the AtoS–AtoC two-component system signaling in potentially pathogenic bacteria, leading to a new perspective on their symbiotic behavior.

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.

AtoSC two-component system is involved in cPHB biosynthesis through fatty acid metabolism in E. coli

BACKGROUND We have shown previously that AtoSC two-component system regulates the biosynthesis of E. coli cPHB [complexed poly-(R)-3-hydroxybutyrate]. METHODS The AtoSC involvement on fatty acids metabolism, towards cPHB synthesis, was studied using cPHB determination, gene expression, and fatty acid metabolic pathways inhibitors. RESULTS Deletion of the atoDAEB operon from the E. coli genome resulted in a consistent reduction of cPHB accumulation. When in ΔatoDAEB cells, the atoDAEB operon and the AtoSC system were introduced extrachromosomally, a significant enhancement of cPHB levels was observed. Moreover, the introduction of a plasmid with atoSC genes regulated positively cPHB biosynthesis. A lesser cPHB enhancement was triggered when plasmids carrying either atoS or atoC were introduced. The intracellular distribution of cPHB was regulated by AtoSC or AtoC according to the inducer (acetoacetate or spermidine). Blockage of β-oxidation by acrylic acid reduced cPHB levels, suggesting the involvement of this pathway in cPHB synthesis; however, the overproduction of AtoSC or its constituents separately resulted in cPHB enhancement. Inhibition of fatty acid biosynthesis by cerulenin resulted to a major cPHB reduction, indicating the contribution of this pathway in cPHB production. Inhibition of both β-oxidation and fatty acid biosynthesis reduced dramatically cPHB, suggesting the contribution of both pathways in cPHB biosynthesis. CONCLUSIONS Short fatty acid catabolism (atoDAEB operon) and fatty acids metabolic pathways participate in cPHB synthesis through the involvement of AtoSC system. GENERAL SIGNIFICANCE The involvement of the AtoSC system in the fatty acids metabolic pathways interplay towards cPHB biosynthesis provides additional perceptions of AtoSC role on E. coli regulatory biochemical processes.

Histamine in two component system-mediated bacterial signaling.

Histamine is a key mediator governing vital cellular processes in mammals beyond its decisive role in inflammation. Recent evidence implies additional actions in both eukaryotes and prokaryotes. Besides its function in host defense against bacterial infections, histamine elicits largely undefined actions in microorganisms that may contribute to bacteria-host interactions. Bacterial proliferation and adaptation are governed by sophisticated signal transduction networks, including the versatile two-component systems (TCSs) that comprise sensor histidine kinases and response regulators and rely on phosphotransfer mechanisms to exert their modulatory function. The AtoSC TCS regulates fundamental cellular processes such as short-chain fatty acid metabolism, poly-(R)-3-hydroxybutyrate (cPHB) biosynthesis and chemotaxis in Escherichia coli. The implication of exogenous histamine in the AtoSC-mediated cPHB biosynthesis and in E. coli chemotactic behavior is indicative of a putative function of histamine in bacterial physiology. The data raise questions on the significance of histamine actions in bacteria-host symbiosis, dysbiosis and pathogenicity as well as on the possible consequences upon therapeutic administration of histamine receptor-targeting agents and in particular ligands of the recently identified immunomodulatory H4 receptor.

Involvement of AtoSC two-component system in Escherichia coli flagellar regulon

The AtoSC two-component system in Escherichia coli is a key regulator of many physiological processes. We report here the contribution of AtoSC in E. coli motility and chemotaxis. AtoSC locus deletion in ΔatoSC cells renders cells not motile or responsive against any chemoattractant or repellent independently of the AtoSC inducer’s presence. AtoSC expression through plasmid complemented the ΔatoSC phenotype. Cells expressing either AtoS or AtoC demonstrated analogous motility and chemotactic phenotypes as ΔatoSC cells, independently of AtoSC inducer’s presence. Mutations of AtoC phosphate-acceptor sites diminished or abrogated E. coli chemotaxis. trAtoC, the AtoC constitutive active form which lacks its receiver domain, up-regulated E. coli motility. AtoSC enhanced the transcription of the flhDC and fliAZY operons and to a lesser extent of the flgBCDEFGHIJKL operon. The AtoSC-mediated regulation of motility and chemotactic response required also the expression of the CheAY system. The AtoSC inducers enhanced the AtoSC-mediated motility and chemotaxis. Acetoacetate or spermidine further promoted the responses of only AtoSC-expressing cells, while Ca2+ demonstrated its effects independently of AtoSC. Histamine regulated bacterial chemotaxis only in atoSC+ cells in a concentration-dependent manner while reversed the AtoSC-mediated effects when added at high concentrations. The trAtoC-controlled motility effects were enhanced by acetoacetate or spermidine, but not by histamine. These data reveal that AtoSC system regulates the motility and chemotaxis of E. coli, participating in the transcriptional induction of the main promoters of the chemotactic regulon and modifying the motility and chemotactic phenotypes in an induction-dependent mechanism.

Activation of the AtoSC two-component system in the absence of the AtoC N-terminal receiver domain in E. coli

The AtoSC two-component system in E. coli consists of the AtoS sensor kinase and the AtoC response regulator. It regulates positively the transcriptional activation of atoDAEB operon, encoding enzymes involved in short-chain fatty acid catabolism upon acetoacetate-mediated induction. AtoSC acting on atoDAEB operon, regulates the biosynthesis and the intracellular distribution of short-chain poly-(R)-3-hydroxybutyrate (cPHB). A phosphorylation-incompetent AtoC form was constructed lacking its N-terminal receiver domain, trAtoC, to study the effects of AtoC domains on cPHB biosynthesis and atoDAEB operon regulation. Both cPHB biosynthesis and atoDAEB gene expression were regulated positively by trAtoC in the absence of any inducer in E. coli of both atoSC+ and ΔatoSC genotypes. The presence of acetoacetate or spermidine further promoted these trAtoC actions. Competitive regulatory functions between the full length AtoC and trAtoC were observed referring to atoDAEB and cPHB targets as well as growth of trAtoC-overproducing atoSC+ cells on butyrate as the sole carbon source. trAtoC in contrast to the wild-type AtoC presented different modes of cPHB and atoDAEB regulation in the presence of compounds involved in fatty acid metabolism including CoA-SH, acetyl-CoA, sodium acetate or 3-hydroxybutyryl-CoA. These data provide evidence for a role of the AtoC N-terminal receiver domain in regulating the biological activities of AtoSC as well as additional mechanisms of interactions between the AtoSC constituents including their established inducers or new effectors towards the accomplishment of the AtoSC TCS signal transduction.

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.

Calcium channels blockers inhibit the signal transduction through the AtoSC system in Escherichia coli

Verapamil, diltiazem and nifedipine are Ca(2+)-channel blockers used in cardiovascular diseases. We report here that the Escherichia coli AtoSC signaling is inhibited by those blockers. AtoSC two-component system plays a pivotal role in sophisticated signaling networks in E. coli regulating processes implicated in bacterial homeostasis and pathogenicity. The Ca(2+)-channel blockers abrogated the in vitro full-length AtoS kinase autophosphorylation. However, they demonstrated no effect on the AtoS cytoplasmic form autophosphorylation. AtoC protected AtoS from verapamil or diltiazem but not from nifedipine, when the two constituents formed complex. The blockers did not affect the AtoS≈P to AtoC phosphotransfer. The blockers-mediated AtoSC inhibition was verified in vivo on the atoDAEB expression, which was inhibited only in AtoSC-expressing bacteria upon acetoacetate. The AtoS and AtoC protein or their genes transcription levels were unaffected by the blockers. Blockers demonstrated differential effects in the regulation of both the cytosolic- and most potently the membrane-bound-cPHB. Extracellular Ca(2+) counteracted the verapamil-mediated effect on cPHB only in atoSC(+) cells. Extracellular Ca(2+) reversed the diltiazem-mediated cPHB decreases in cells of both genetic backgrounds, yet a Ca(2+)-concentration dependent reversion was observed only in the AtoSC-regulated cPHB. Nifedipine caused a more pronounced cPHB down-regulation that was not reversed by extracellular Ca(2+). The AtoSC signaling inhibition by Ca(2+)-channel blockers used for human treatment, and their differential effects on cPHB-formed Ca(2+)-channels, signify their implications in bacterial-host interactions through the two-component signaling and could stimulate the design of Ca(2+)-channels blockers derivatives acting as inhibitors of two-component systems.

TOX Regulates Growth, DNA Repair, and Genomic Instability in T-cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes. Using a transgenic screen in zebrafish, thymocyte selection-associated high mobility group box protein (TOX) was uncovered as a collaborating oncogenic driver that accelerated T-ALL onset by expanding the initiating pool of transformed clones and elevating genomic instability. TOX is highly expressed in a majority of human T-ALL and is required for proliferation and continued xenograft growth in mice. Using a wide array of functional analyses, we uncovered that TOX binds directly to KU70/80 and suppresses recruitment of this complex to DNA breaks to inhibit nonhomologous end joining (NHEJ) repair. Impaired NHEJ is well known to cause genomic instability, including development of T-cell malignancies in KU70- and KU80-deficient mice. Collectively, our work has uncovered important roles for TOX in regulating NHEJ by elevating genomic instability during leukemia initiation and sustaining leukemic cell proliferation following transformation.Significance: TOX is an HMG box-containing protein that has important roles in T-ALL initiation and maintenance. TOX inhibits the recruitment of KU70/KU80 to DNA breaks, thereby inhibiting NHEJ repair. Thus, TOX is likely a dominant oncogenic driver in a large fraction of human T-ALL and enhances genomic instability. Cancer Discov; 7(11); 1336-53. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1201.