Kaneyoshi Yamamoto

Transcriptional response of Escherichia coli to external copper

Transcriptional response of Escherichia coli upon exposure to external copper was studied using DNA microarray and in vivo and in vitro transcription assays. Transcription of three hitherto‐identified copper‐responsive genes, copA (copper efflux transporter), cueO (multicopper oxidase) and cusC (tripartite copper pump component) became maximum at 5u2003min after addition of copper sulphate, and thereafter decreased to the preshift levels within 30u2003min. Microarray analysis at 5u2003min after addition of copper indicated that a total of at least 29 genes including these three known genes were markedly and specifically affected (28 upregulated and one downregulated). Transcription of the divergent operons, cusCFB and cusRS, was found to be activated by CusR, which bound to a CusR box between the cusC and cusR promoters. Except for this site, the CusR box was not identified in the entire E. coli genome. On the other hand, transcription of copA and cueO was found to be activated by another copper‐responsive factor CueR, which bound to a conserved inverted repeat sequence, CueR box. A total of 197 CueR boxes were identified on the E. coli genome, including the CueR box associated with the moa operon for molybdenum cofactor synthesis. At least 10 copper‐induced genes were found to be under the control of CpxAR two‐component system, indicating that copper is one of the signals for activation of the CpxAR system. In addition, transcription of yedWV, a putative two‐component system, was activated by copper in CusR‐dependent manner. Taken together we conclude that the copper‐responsive genes are organized into a hierarchy of the regulation network, forming at least four regulons, i.e. CueR, CusR, CpxR and YedW regulons. These copper‐responsive regulons appear to sense and respond to different concentrations of external copper.

The QseC Adrenergic Signaling Cascade in Enterohemorrhagic E. coli (EHEC)

The ability to respond to stress is at the core of an organisms survival. The hormones epinephrine and norepinephrine play a central role in stress responses in mammals, which require the synchronized interaction of the whole neuroendocrine system. Mammalian adrenergic receptors are G-coupled protein receptors (GPCRs); bacteria, however, sense these hormones through histidine sensor kinases (HKs). HKs autophosphorylate in response to signals and transfer this phosphate to response regulators (RRs). Two bacterial adrenergic receptors have been identified in EHEC, QseC and QseE, with QseE being downstream of QseC in this signaling cascade. Here we mapped the QseC signaling cascade in the deadly pathogen enterohemorrhagic E. coli (EHEC), which exploits this signaling system to promote disease. Through QseC, EHEC activates expression of metabolic, virulence and stress response genes, synchronizing the cell response to these stress hormones. Coordination of these responses is achieved by QseC phosphorylating three of the thirty-two EHEC RRs. The QseB RR, which is QseCs cognate RR, activates the flagella regulon which controls bacteria motility and chemotaxis. The QseF RR, which is also phosphorylated by the QseE adrenergic sensor, coordinates expression of virulence genes involved in formation of lesions in the intestinal epithelia by EHEC, and the bacterial SOS stress response. The third RR, KdpE, controls potassium uptake, osmolarity, and also the formation of lesions in the intestine. Adrenergic regulation of bacterial gene expression shares several parallels with mammalian adrenergic signaling having profound effects in the whole organism. Understanding adrenergic regulation of a bacterial cell is a powerful approach for studying the underlying mechanisms of stress and cellular survival.

Identification and Molecular Characterization of the Mg2+ Stimulon of Escherichia coli

Transcription profile microarray analysis in Escherichia coli was performed to identify the member genes of the Mg(2+) stimulon that respond to the availability of external Mg(2+) in a PhoP/PhoQ two-component system-dependent manner. The mRNA levels of W3110 in the presence of 30 mM MgCl(2), WP3022 (phoP defective), and WQ3007 (phoQ defective) were compared with those of W3110 in the absence of MgCl(2). The expression ratios of a total of 232 genes were <0.75 in all three strains (the supplemental data are shown at http://www.nara.kindai.ac.jp/nogei/seiken/array.html), suggesting that the PhoP/PhoQ system is involved directly or indirectly in the transcription of these genes. Of those, 26 contained the PhoP box-like sequences with the direct repeats of (T/G)GTTTA within 500 bp upstream of the initiation codon. Furthermore, S1 nuclease assays of 26 promoters were performed to verify six new Mg(2+) stimulon genes, hemL, nagA, rstAB, slyB, vboR, and yrbL, in addition to the phoPQ, mgrB, and mgtA genes reported previously. In gel shift and DNase I footprinting assays, all of these genes were found to be regulated directly by PhoP. Thus, we concluded that the phoPQ, mgrB, mgtA, hemL, nagA, rstAB, slyB, vboR, and yrbL genes make up the Mg(2+) stimulon in E. coli.

PdhR (Pyruvate Dehydrogenase Complex Regulator) Controls the Respiratory Electron Transport System in Escherichia coli

The pyruvate dehydrogenase (PDH) multienzyme complex plays a key role in the metabolic interconnection between glycolysis and the citric acid cycle. Transcription of the Escherichia coli genes for all three components of the PDH complex in the pdhR-aceEF-lpdA operon is repressed by the pyruvate-sensing PdhR, a GntR family transcription regulator, and derepressed by pyruvate. After a systematic search for the regulation targets of PdhR using genomic systematic evolution of ligands by exponential enrichment (SELEX), we have identified two novel targets, ndh, encoding NADH dehydrogenase II, and cyoABCDE, encoding the cytochrome bo-type oxidase, both together forming the pathway of respiratory electron transport downstream from the PDH cycle. PDH generates NADH, while Ndh and CyoABCDE together transport electrons from NADH to oxygen. Using gel shift and DNase I footprinting assays, the PdhR-binding site (PdhR box) was defined, which includes a palindromic consensus sequence, ATTGGTNNNACCAAT. The binding in vitro of PdhR to the PdhR box decreased in the presence of pyruvate. Promoter assays in vivo using a two-fluorescent-protein vector also indicated that the newly identified operons are repressed by PdhR and derepressed by the addition of pyruvate. Taken together, we propose that PdhR is a master regulator for controlling the formation of not only the PDH complex but also the respiratory electron transport system.

Characterization of Copper-Inducible Promoters Regulated by CpxA/CpxR in Escherichia coli

The copper stimulon in Escherichia coli consists of four regulons, the CueR-, CusS/CusR-, CpxA/CpxR-, and YedV/YedW regulons. E. coli mutants defective in cpxRA showed higher sensitivity to copper than the wild type. A total of 15 promoters were found to be induced in E. coli culture upon exposure to copper in a CpxA/CpxR-dependent manner. After gel-shift and DNase I foot-printing analyses, a conserved tandem repeat of pentanucleotide sequence, GTAAA(N)4–8GTAAA, with a conserved A of 4-bp upstream of each pentamer, was identified to be the CpxR-binding site. The difference in the orientation and location of the CpxR box is discussed with respect to the regulation mechanism among CpxR-regulon genes.

A Novel Two-Component Signaling System That Activates Transcription of an Enterohemorrhagic Escherichia coli Effector Involved in Remodeling of Host Actin

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for worldwide outbreaks of bloody diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. After colonizing the large intestine, EHEC forms attaching and effacing (AE) lesions on intestinal epithelial cells. These lesions cause destruction of the microvilli and elicit actin rearrangement to form pedestals that cup each bacterium individually. EHEC responds to a signal produced by the intestinal microbial flora, autoinducer-3 (AI-3), and the host hormones epinephrine and norepinephrine to activate transcription of the genes involved in AE lesion formation. These three signals, involved in interkingdom communication, are sensed by bacterial sensor kinases. Here we describe a novel two-component system, QseEF (quorum-sensing E. coli regulators E and F), which is part of the AI-3/epinephrine/norepinephrine signaling system. QseE is the sensor kinase and QseF the response regulator. The qseEF genes are cotranscribed, and transcription of qseEF is activated by epinephrine through the QseC sensor. A qseF mutant does not form AE lesions. QseF activates transcription of the gene encoding EspFu, an effector protein translocated to the host cell by the EHEC, which mimics a eukaryotic SH2/SH3 adapter protein to engender actin polymerization during pedestal formation. Expression of the espFu gene from a plasmid restored AE lesion formation to the qseF mutant, suggesting that lack of espFu expression in this mutant was responsible for the loss of pedestal formation. These findings suggest the QseEF is a two-component system involved in the regulation of AE lesion formation by EHEC.

Two different modes of transcription repression of the Escherichia coli acetate operon by IclR

IclR is a repressor for the Escherichia coli aceBAK operon, which encodes isocitrate lyase (aceB), malate synthase (aceA) and isocitrate dehydroge‐nase kinase/phosphorylase (aceK) in the glyoxylate bypass. IclR also represses the expression of iclR in an autogenous manner. DNase I footprinting and in vitro transcription assays indicated that IclR binds to an IclR box (−21 to +14), which overlaps the iclR promoter and thus competes with the RNA polymerase for DNA binding, leading to transcription repression. In the case of the aceBAK operon, IclR binds to IclR box II between −52 and −19 of the aceB promoter and interferes with binding of the RNA polymerase to this promoter. A secondary IclR binding site (IclR box I) was identified between −125 and −99 of the aceB promoter. IclR binds to this IclR box I even after formation of the aceB promoter open complex and, moreover, induces disassembly of the open complex, leading to repression of aceB transcription. In parallel, the location of the C‐terminal domain of the RNA polymerase α subunit (αCTD) on DNA is shifted close to the IclR box I, indicating that direct interaction between the αCTD and the IclR box I‐associated IclR caused the repression.

Genomic SELEX Search for Target Promoters under the Control of the PhoQP-RstBA Signal Relay Cascade

RstBA, a two-component regulatory system of Escherichia coli with an unidentified regulatory function, is under the control of a Mg(2+)-sensing PhoQP two-component system. In order to identify the network of transcription regulation downstream of RstBA, we isolated a set of RstA-binding sequences from the E. coli genome by using the genomic SELEX system. A gel mobility shift assay indicated the binding of RstA to two SELEX DNA fragments, one including the promoter region of asr (acid shock RNA) and another including the promoter for csgD (a regulator of the curli operon). Using a DNase I footprinting assay, we determined the RstA-binding sites (RstA boxes) with the consensus sequence TACATNTNGTTACA. Transcription of the asr gene was induced 10- to 60-fold either in low-pH (pH 4.5) LB medium or in low-phosphate minimal medium as detected by promoter assay. The acid-induced in vivo transcription of asr was reduced after the deletion of rstA. In vivo transcription of the asr promoter was observed only in the presence of RstA. In agreement with the PhoQP-RstBA network, the addition of Mg(2+) led to a severe reduction of the asr promoter activity, and the disruption of phoP also reduced the asr promoter activity, albeit to a lesser extent. These observations altogether indicate that RstA is an activator of asr transcription. In contrast, transcription of csgD was repressed by overexpression of RstA, indicating that RstA is a repressor for csgD. With these data taken together, we conclude that the expression of both asr and csgD is under the direct control of the PhoQP-RstBA signal relay cascade.

Transcriptional Response of Escherichia coli to External Zinc

Transcriptional response of Escherichia coli to extracellular zinc was studied using DNA microarray and S1 mapping assays. Addition of external zinc induced the expression of zinc exporter ZntA and inhibited the expression of zinc importer ZnuC. In the continuous presence of zinc, ZnuC repression took place at lower zinc concentrations than ZntA induction. The microarray assay indicated that the addition of excess external zinc induces the expression of many genes that are organized in the regulon for cysteine biosynthesis, implying that cysteine plays a role in transient trapping of free zinc for maintenance of zinc homeostasis. Besides the RpoE regulon, other genes were also induced by zinc, suggesting that periplasmic proteins denatured by zinc induce the genes for protein repair. The microarray data of the newly identified zinc-responsive promoters were confirmed by S1 mapping.

Signal transduction cascade between EvgA/EvgS and PhoP/PhoQ two-component systems of Escherichia coli.

Transcriptional analysis of a constitutively active mutant of the EvgA/EvgS two-component system of Escherichia coli resulted in enhanced expression of 13 PhoP/PhoQ-regulated genes, crcA, hemL, mgtA, ompT, phoP, phoQ, proP, rstA, rstB, slyB, ybjG, yrbL, and mgrB. This regulatory network between the two systems also occurred as a result of overproduction of the EvgA regulator; however, enhanced transcription of the phoPQ genes did not further activate expression of the PhoP/PhoQ-regulated genes. These results demonstrated signal transduction from the EvgA/EvgS system to the PhoP/PhoQ system in E. coli and also identified the genes that required the two systems for enhanced expression. This is one example of the intricate signal transduction networks that are posited to exist in E. coli.