Akinori Kato

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.

Closing the loop: The PmrA/PmrB two-component system negatively controls expression of its posttranscriptional activator PmrD

A fundamental question in biology is how an organism integrates multiple signals to mediate an appropriate cellular response. The PmrA/PmrB two-component system of Salmonella enterica can be activated independently by Fe3+, which is sensed by the PmrB protein, and in low Mg2+, which is sensed by the PhoQ protein. The low-Mg2+ activation requires pmrD, a PhoP/PhoQ-activated gene that activates the response regulator PmrA at a posttranscriptional level. We now report that pmrD expression is negatively regulated by the PmrA/PmrB system. Conditions that activate the PmrA protein independently of pmrD, such as exposure to Fe3+, resulted in lower levels of pmrD transcription. The PmrA protein footprinted the pmrD promoter upstream of the PhoP-binding site but did not interfere with binding of the PhoP protein. Mutation of the PmrA-binding site in the pmrD promoter abolished PmrA-mediated repression. Negative regulation of the PhoP/PhoQ-activated pmrD gene by the PmrA/PmrB system closes a regulatory circuit designed to maintain proper cellular levels of activated PmrA protein and constitutes a singular example of a multicomponent feedback loop.

The PhoQ/PhoP Regulatory Network of Salmonella enterica

The PhoQ/PhoP two-component regulatory system is a major regulator of virulence in the enteric pathogen Salmonella enterica serovar Typhimurium. It also controls the adaptation to low Mg2+ environments by governing the expression and/or activity of Mg2+ transporters and of enzymes modifying the Mg2+-binding sites on the bacterial cell surface. The regulator PhoP modifies expression of approximately 3% of the Salmonella genes in response to the periplasmic Mg2+ concentration detected by the PhoQ protein. Genes that are directly controlled by the PhoP protein often differ in their promoter structures, resulting in distinct expression levels and kinetics in response to the low Mg2+ inducing signal. PhoP regulates a large number of genes indirectly: via other transcription factors and two-component systems that form a panoply of regulatory architectures including transcriptional cascades, feedforward loops and the use of connector proteins that modify the activity of response regulators. These architectures confer distinct expression properties that may be important contributors to Salmonellas lifestyle.

B1500, a small membrane protein, connects the two-component systems EvgS/EvgA and PhoQ/PhoP in Escherichia coli

Two-component signal-transduction systems (TCSs) of bacteria are considered to form an intricate signal network to cope with various environmental stresses. One example of such a network in Escherichia coli is the signal transduction cascade from the EvgS/EvgA system to the PhoQ/PhoP system, where activation of the EvgS/EvgA system promotes expression of PhoP-activated genes. As a factor connecting this signal transduction cascade, we have identified a small inner membrane protein (65 aa), B1500. Expression of the b1500 gene is directly regulated by the EvgS/EvgA system, and b1500 expression from a heterologous promoter simultaneously activated the expression of mgtA and other PhoP regulon genes. This activation was PhoQ/PhoP-dependent and EvgS/EvgA-independent. Furthermore, deletion of b1500 from an EvgS-activated strain suppressed mgtA expression. B1500 is localized in the inner membrane, and bacterial two-hybrid data showed that B1500 formed a complex with the sensor PhoQ. These results indicate that the small membrane protein, B1500, connected the signal transduction between EvgS/EvgA and PhoQ/PhoP systems by directly interacting with PhoQ, thus activating the PhoQ/PhoP system.

Transcription of emrKY is Regulated by the EvgA-EvgS Two-Component System in Escherichia coli K-12

Spontaneous mutations have been isolated in Escherichia coli that result in the constitutive expression of an emrKY promoter. These mutations were found to be single-nucleotide substitutions within the linker region of the sensor protein EvgS, which is part of a two-component regulatory system along with EvgA. In the linker mutants (evgS1 and evgS4), emrKY expression became constitutive and MIC against sodium deoxycholate was 20 mg/ml, eight-fold higher than in the wild type. Furthermore, the start site of transcription from the promoter of emrKY was identified; EvgA was shown to bind at the -52 to -84 region by the footprinting experiment.

A connector of two-component regulatory systems promotes signal amplification and persistence of expression.

Organisms rely on a variety of regulatory architectures to control gene transcription. Whereas the functional characteristics of particular architectures are well understood, the properties of newly discovered regulatory designs cannot be easily predicted. One emerging design depends on small proteins that connect two-component regulatory systems, which constitute the dominant form of bacterial signal transduction. These connectors enable one system to respond to the signal perceived by a different system. To understand the functional properties of such connector-mediated architectures, we investigated the pathway controlled by the PhoP-dependent connector protein PmrD of Salmonella enterica and contrasted it to the circuit in which genes are regulated directly by the transcription factor PhoP. The PmrD-mediated pathway displayed both signal amplification and persistence of expression when compared with the direct pathway. Mathematical modeling of the two pathways allowed us to identify critical factors responsible for signal amplification.

Reciprocal Control between a Bacterium's Regulatory System and the Modification Status of Its Lipopolysaccharide

Gram-negative bacteria often modify their lipopolysaccharide (LPS), thereby increasing resistance to antimicrobial agents and avoidance of the host immune system. However, it is unclear how bacteria adjust the levels and activities of LPS-modifying enzymes in response to the modification status of their LPS. We now address this question by investigating the major regulator of LPS modifications in Salmonella enterica. We report that the PmrA/PmrB system controls expression of a membrane peptide that inhibits the activity of LpxT, an enzyme responsible for increasing the LPS negative charge. LpxTs inhibition and the PmrA-dependent incorporation of positively charged L-4-aminoarabinose into the LPS decrease Fe(3+) binding to the bacterial cell. Because Fe(3+) is an activating ligand for the sensor PmrB, transcription of PmrA-dependent LPS-modifying genes is reduced. This mechanism enables bacteria to sense their cell surface by its effect on the availability of an inducing signal for the system regulating cell-surface modifications.

A connecter-like factor, CacA, links RssB/RpoS and the CpxR/CpxA two-component system in Salmonella

BackgroundBacteria integrate numerous environmental stimuli when generating cellular responses. Increasing numbers of examples describe how one two-component system (TCS) responds to signals detected by the sensor of another TCS. However, the molecular mechanisms underlying this phenomenon remain poorly defined.ResultsHere, we report a connector-like factor that affects the activity of the CpxR/CpxA two-component system in Salmonella enterica serovar Typhimurium. We isolated a clone that induced the expression of a cpxP-lac gene fusion from a high-copy-number plasmid pool of random Salmonella genomic fragments. A 63-amino acid protein, CacA, was responsible for the CpxA/CpxR-dependent activation of the cpxP gene. The CpxR-activated genes cpxP and spy exhibited approximately 30% and 50% reductions in transcription, respectively, in a clean cacA deletion mutant strain in comparison to wild-type. From 33 response regulator (RR) deletion mutants, we identified that the RssB regulator represses cacA transcription. Substitution mutations in a conserved -10 region harboring the RNA polymerase recognition sequence, which is well conserved with a known RpoS -10 region consensus sequence, rendered the cacA promoter RpoS-independent. The CacA-mediated induction of cpxP transcription was affected in a trxA deletion mutant, which encodes thioredoxin 1, suggesting a role for cysteine thiol-disulfide exchange(s) in CacA-dependent Cpx activation.ConclusionsWe identified CacA as an activator of the CpxR/CpxA system in the plasmid clone. We propose that CacA may integrate the regulatory status of RssB/RpoS into the CpxR/CpxA system. Future investigations are necessary to thoroughly elucidate how CacA activates the CpxR/CpxA system.

Characterization of H-box region mutants of WalK inert to the action of waldiomycin in Bacillus subtilis

The WalK/WalR two-component system is essential for cell wall metabolism and thus for cell growth in Bacillus subtilis. Waldiomycin was previously isolated as an antibiotic that targeted WalK, the cognate histidine kinase (HK) of the response regulator, WalR, in B. subtilis. To gain further insights into the action of waldiomycin on WalK and narrow down its site of action, mutations were introduced in the H-box region, a well-conserved motif of the bacterial HKs of WalK. The half-maximal inhibitory concentrations (IC50s) of waldiomycin against purified WalK protein with triple substitutions in the H-box region, R377M/R378M/S385A and R377M/R378M/R389M, were 26.4 and 55.1 times higher than that of the wild-type protein, respectively, indicating that these residues of WalK are crucial for the inhibitory effect of waldiomycin on its kinase activity. Surprisingly, this antibiotic severely affected cell growth in a minimum inhibitory concentration (MIC) assay, but not transcription of WalR-regulated genes or cell morphology in B. subtilis strains that harbored the H-box triple substitutions on the bacterial chromosome. We hypothesized that waldiomycin targets other HKs as well, which may, in turn, sensitize B. subtilis cells with the H-box triple mutant alleles of the walK gene to waldiomycin. Waldiomycin inhibited other HKs such as PhoR and ResE, and, to a lesser extent, CitS, whose H-box region is less conserved. These results suggest that waldiomycin perturbs multiple cellular processes in B. subtilis by targeting the H-box region of WalK and other HKs.

Identification of the Three Genes Involved in Controlling Production of a Phytotoxin Tropolone in Burkholderia plantarii

UNLABELLED Tropolone, a phytotoxin produced by Burkholderia plantarii, causes rice seedling blight. To identify genes involved in tropolone synthesis, we systematically constructed mutations in the genes encoding 55 histidine kinases and 72 response regulators. From the resulting defective strains, we isolated three mutants, KE1, KE2, and KE3, in which tropolone production was repressed. The deleted genes of these mutants were named troR1, troK, and troR2, respectively. The mutant strains did not cause rice seedling blight, and complementation experiments indicated that TroR1, TroK, and TroR2 were involved in the synthesis of tropolone in B. plantarii However, tropolone synthesis was repressed in the TroR1 D52A, TroK H253A, and TroR2 D46A site-directed mutants. These results suggest that the putative sensor kinase (TroK) and two response regulators (TroR1 and TroR2) control the production of tropolone in B. plantarii IMPORTANCE A two-component system is normally composed of a sensor histidine kinase (HK) and a cognate response regulator (RR) pair. In this study, HK (TroK) and two RRs (TroR1 and TroR2) were found to be involved in controlling tropolone production in B. plantarii These three genes may be part of a bacterial signal transduction network. Such networks are thought to exist in other bacteria to regulate phytotoxin production, as well as environmental adaptation and signal transduction.