Two Trk/Ktr/HKT-type potassium transporters, TrkG and TrkH, perform distinct functions in Escherichia coli K-12.

Escherichia coli K-12 possesses two versions of Trk/Ktr/HKT-type potassium ion (K+) transporters, TrkG and TrkH. The current paradigm is that TrkG and TrkH have largely identical characteristics, and little information is available regarding their functional differences. Here, we show using cation uptake experiments with K+ transporter knockout mutants that TrkG and TrkH have distinct ion transport activities and physiological roles. K+-transport by TrkG required Na+, whereas TrkH-mediated K+ uptake was not affected by Na+. An aspartic acid located five residues away from a critical glycine in the third pore-forming region might be involved in regulation of Na+-dependent activation of TrkG. In addition, we found that TrkG but not TrkH had Na+ uptake activity. Our analysis of K+ transport mutants revealed that TrkH supported cell growth more than TrkG; however, TrkG was able to complement loss of TrkH-mediated K+ uptake in E. coli. Furthermore, we determined that transcription of trkG in E. coli was downregulated but not completely silenced by the xenogeneic silencing factor H-NS (histone-like nucleoid structuring protein or heat-stable nucleoid-structuring protein). Taken together, the transport function of TrkG is clearly distinct from that of TrkH, and TrkG seems to have been accepted by E. coli during evolution as a K+ uptake system that coexists with TrkH.

Regulation of constant cell elongation and Sfm pili synthesis in Escherichia coli via two active forms of FimZ orphan response regulator.

Escherichia coli (E. coli) has multiple copies of the chaperone-usher (CU) pili operon in five fimbria groups: CU pili, curli, type IV pili, type III secretion pili, and type IV secretion pili. Commensal E. coli K-12 contains 12 CU pili operons. Among these operons, Sfm is expressed by the sfmACDHF operon. Transcriptome analyses, reporter assays, and chromatin immunoprecipitation PCR analyses reported that FimZ directly binds to and activates the sfmA promoter, transcribing sfmACDHF. In addition, FimZ regularly induces constant cell elongation in E. coli, which is required for F-type ATPase function. The bacterial two-hybrid system showed a specific interaction between FimZ and the α subunit of the cytoplasmic F1 domain of F-type ATPase. Studies performed using mutated FimZs have revealed two active forms, I and II. Active form I is required for constant cell elongation involving amino acid residues K106 and D109. Active form II additionally required D56, a putative phosphorylation site, to activate the sfmA promoter. The chromosomal fimZ was hardly expressed in parent strain but functioned in phoB and phoP double-gene knockout strains. These insights may help to understand bacterial invasion restricted host environments by the sfm γ-type pili.

The hdeD Gene Represses the Expression of Flagellum Biosynthesis via LrhA in Escherichia coli K-12.

Escherichia coli survives under acid stress conditions by the glutamic acid-dependent acid resistance (GAD) system, which enzymatically decreases intracellular protons. We found a linkage between GAD and flagellar systems in E. coli. The hdeD gene, one of the GAD cluster genes, encodes an uncharacterized membrane protein. A reporter assay showed that the hdeD promoter was induced in a GadE-dependent manner when grown in the M9 glycerol medium. Transcriptome analysis revealed that most of the transcripts were from genes involved in flagellum synthesis, and cell motility increased not only in the hdeD-deficient mutant but also in the gadE-deficient mutant. Defects in both the hdeD and gadE increased the intracellular level of FliA, an alternative sigma factor for flagellum synthesis, activated by the master regulator FlhDC. The promoter activity of the lrhA gene, which encodes repressor for the flhDC operon, was found to decrease in both the hdeD- and gadE-deficient mutants. Transmission electron microscopy showed that the number of flagellar filaments on the hdeD-, gadE-, and lrhA-deficient cells increased, and all three mutants showed higher motility than the parent strain. Thus, HdeD in the GAD system activates the lrhA promoter, resulting in a decrease in flagellar filaments in E. coli cells. We speculated that the synthesis of HdeD, stimulated in E. coli exposed to acid stress, could control the flagellum biosynthesis by sensing slight changes in pH at the cytoplasmic membrane. This could help in saving energy through termination of flagellum biosynthesis and improve bacterial survival efficiency within the animal digestive system. IMPORTANCE E. coli cells encounter various environments from the mouth down to the intestines within the host animals. The pH of gastric juice is lower than 2.0, and the bacterial must quickly respond and adapt to the following environmental changes before reaching the intestines. The quick response plays a role in cellular survival in the population, whereas adaptation may contribute to species survival. The GAD and flagellar systems are important for response to low pH in E. coli. Here, we identified the novel inner membrane regulator HdeD, encoding in the GAD cluster, to repress the synthesis of flagella. These insights provide a deeper understanding of how the bacteria enter the animal digestive system, survive, and form colonies in the intestines.

Cytotoxic Mechanism of Excess Polyamines Functions through Translational Repression of Specific Proteins Encoded by Polyamine Modulon.

Excessive accumulation of polyamines causes cytotoxicity, including inhibition of cell growth and a decrease in viability. We investigated the mechanism of cytotoxicity caused by spermidine accumulation under various conditions using an Escherichia coli strain deficient in spermidine acetyltransferase (SAT), a key catabolic enzyme in controlling polyamine levels. Due to the excessive accumulation of polyamines by the addition of exogenous spermidine to the growth medium, cell growth and viability were markedly decreased through translational repression of specific proteins [RMF (ribosome modulation factor) and Fis (rRNA transcription factor) etc.] encoded by members of polyamine modulon, which are essential for cell growth and viability. In particular, synthesis of proteins that have unusual locations of the Shine-Dalgarno (SD) sequence in their mRNAs was inhibited. In order to elucidate the molecular mechanism of cytotoxicity by the excessive accumulation of spermidine, the spermidine-dependent structural change of the bulged-out region in the mRNA at the initiation site of the rmf mRNA was examined using NMR analysis. It was suggested that the structure of the mRNA bulged-out region is affected by excess spermidine, so the SD sequence of the rmf mRNA cannot approach initiation codon AUG.

Epistatic Effect of Regulators to the Adaptive Growth of Escherichia coli.

Bacteria survive in the environment with three steps: a sensing environmental conditions, a responding to sensed signals, and an adaptation for proper survival in the environment. An adapting bacterial cell occurs cell division to increase the number of sister cells, termed adaptive growth. Two-component systems (TCSs), representing the main bacterial signal transduction systems, consist of a pair of one sensor kinase (SK) and one response regulator (RR), and RR genes are abundant in most bacterial genomes as part of the core genome. The OmpR gene family, a group of RR genes, is conserved in 95% of known bacterial genomes. The Escherichia coli genome has an estimated 34 RR genes in total, including 14 genes of OmpR family genes. To reveal the contribution of TCSs for fast growth as an adaptive growth strategy of E. coli, we isolated a set of gene knockout strains by using newly developed genome editing technology, the HoSeI (Homologous Sequence Integration) method, based on CRISPR-Cas9. The statistics of single cell observation show a knockout of an arbitrary pair of phoP, phoB, and ompR genes, stably expressed by positive feedback regulation, dramatically inhibit the optimum adaptive growth of E. coli. These insights suggest that the adaptive growth of bacteria is fulfilled by the optimum high intracellular level of regulators acquired during growth under environmental conditions.

Author Correction: Regulatory Role of PlaR (YiaJ) for Plant Utilization in Escherichia coli K-12.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Measurement of the Promoter Activity in Escherichia coli by Using a Luciferase Reporter.

The reporter system is widely used technique for measuring promoter activity in bacterial cells. Until now, a number of reporter system have been developed, but the bioluminescent reporter constructed from the bacterial luciferase genes is one of the useful systems for measuring in vivo dynamics of gene expression. The introduced bioluciferase lux reporter enables easy, fast, and sensitive measurement of the promoter activity without cell lysis because the substrates of bioluminescent reaction are synthesized inside the bacterial cell, thereby allowing low-cost experiments. This protocol describes a high throughput technique to measure the promoter activity in Escherichia coli K-12 using the lux reporter system.

Regulatory roles of pyruvate-sensing two-component system PyrSR (YpdAB) in Escherichia coli K-12.

When the rate of production of metabolites in bacteria exceeds the amounts needed for cell growth, excess metabolites are secreted into the extracellular environment. Upon entry into poor nutrient conditions, overflowed exometabolites are reused to continue cell growth and survival. At present, however, the genetic system for utilization of exometabolites is poorly understood even for the best-characterized model prokaryote Escherichia coli. A two-component system YpdAB of E. coli K-12 was predicted to participate in regulation of this process, and the yhjX gene encoding the MFS-family transporter with an as yet unidentified function was identified as a single regulatory target of YpdB. Using gSELEX screening in vitro, however, we have identified up to eight regulatory targets, including the yhjX gene. The predicted regulatory targets were all confirmed to be under the direct control of YpdB by gel shift assay in vitro and reporter assay in vivo. For induction of YpdAB function, the major exometabolite pyruvate in growing E. coli K-12 was identified as the inducer. We then propose to rename YpdAB as PyrSR (regulator of pyruvate reutilization). One unique feature of PyrSR is its cross-talk with another pyruvate-sensing BtsSR at the TCS stage 1 for fine-tuning of pyruvate reutilization.

Regulatory Role of PlaR (YiaJ) for Plant Utilization in Escherichia coli K-12.

Outside a warm-blooded animal host, the enterobacterium Escherichia coli K-12 is also able to grow and survive in stressful nature. The major organic substance in nature is plant, but the genetic system of E. coli how to utilize plant-derived materials as nutrients is poorly understood. Here we describe the set of regulatory targets for uncharacterized IclR-family transcription factor YiaJ on the E. coli genome, using gSELEX screening system. Among a total of 18 high-affinity binding targets of YiaJ, the major regulatory target was identified to be the yiaLMNOPQRS operon for utilization of ascorbate from fruits and galacturonate from plant pectin. The targets of YiaJ also include the genes involved in the utilization for other plant-derived materials as nutrients such as fructose, sorbitol, glycerol and fructoselysine. Detailed in vitro and in vivo analyses suggest that L-ascorbate and α-D-galacturonate are the effector ligands for regulation of YiaJ function. These findings altogether indicate that YiaJ plays a major regulatory role in expression of a set of the genes for the utilization of plant-derived materials as nutrients for survival. PlaR was also suggested to play protecting roles of E. coli under stressful environments in nature, including the formation of biofilm. We then propose renaming YiaJ to PlaR (regulator of plant utilization). The natural hosts of enterobacterium Escherichia coli are warm-blooded animals, but even outside hosts, E. coli can survive even under stressful environments. On earth, the most common organic materials to be used as nutrients by E. coli are plant-derived components, but up to the present time, the genetic system of E. coli for plant utilization is poorly understand. In the course of gSELEX screening of the regulatory targets for hitherto uncharacterized TFs, we identified in this study the involvement of the IclR-family YiaJ in the regulation of about 20 genes or operons, of which the majority are related to the catabolism of plant-derived materials such as ascorbate, galacturonate, sorbitol, fructose and fructoselysine. Therefore, we propose to rename YiaJ to PlaR (regulator of plant utilization).

Regulatory role of CsqR (YihW) in transcription of the genes for catabolism of the anionic sugar sulfoquinovose (SQ) in Escherichia coli K-12.

The binding sites of YihW, an uncharacterized DeoR-family transcription factor (TF) of Escherichia coli K-12, were identified using Genomic SELEX screening at two closely located sites, one inside the spacer between the bidirectional transcription units comprising the yihUTS operon and the yihV gene, and another one upstream of the yihW gene itself. Recently the YihUTS and YihV proteins were identified as catalysing the catabolism of sulfoquinovose (SQ), a hydrolysis product of sulfoquinovosyl diacylglycerol (SQDG) derived from plants and other photosynthetic organisms. Gel shift assay in vitro and reporter assay in vivo indicated that YihW functions as a repressor for all three transcription units. De-repression of the yih operons was found to be under the control of SQ as inducer, but not of lactose, glucose or galactose. Furthermore, a mode of its cooperative DNA binding was suggested for YihW by atomic force microscopy. Hence, as a regulator of the catabolism of SQ, we renamed YihW as CsqR.

Dimerization site 2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation.

The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing.

Altered Distribution of RNA Polymerase Lacking the Omega Subunit within the Prophages along the Escherichia coli K-12 Genome.

The RNA polymerase (RNAP) of Escherichia coli K-12 is a complex enzyme consisting of the core enzyme with the subunit structure α2ßß'ω and one of the σ subunits with promoter recognition properties. The smallest subunit, omega (the rpoZ gene product), participates in subunit assembly by supporting the folding of the largest subunit, ß', but its functional role remains unsolved except for its involvement in ppGpp binding and stringent response. As an initial approach for elucidation of its functional role, we performed in this study ChIP-chip (chromatin immunoprecipitation with microarray technology) analysis of wild-type and rpoZ-defective mutant strains. The altered distribution of RpoZ-defective RNAP was identified mostly within open reading frames, in particular, of the genes inside prophages. For the genes that exhibited increased or decreased distribution of RpoZ-defective RNAP, the level of transcripts increased or decreased, respectively, as detected by reverse transcription-quantitative PCR (qRT-PCR). In parallel, we analyzed, using genomic SELEX (systemic evolution of ligands by exponential enrichment), the distribution of constitutive promoters that are recognized by RNAP RpoD holoenzyme alone and of general silencer H-NS within prophages. Since all 10 prophages in E. coli K-12 carry only a small number of promoters, the altered occupancy of RpoZ-defective RNAP and of transcripts might represent transcription initiated from as-yet-unidentified host promoters. The genes that exhibited transcription enhanced by RpoZ-defective RNAP are located in the regions of low-level H-NS binding. By using phenotype microarray (PM) assay, alterations of some phenotypes were detected for the rpoZ-deleted mutant, indicating the involvement of RpoZ in regulation of some genes. Possible mechanisms of altered distribution of RNAP inside prophages are discussed. IMPORTANCE The 91-amino-acid-residue small-subunit omega (the rpoZ gene product) of Escherichia coli RNA polymerase plays a structural role in the formation of RNA polymerase (RNAP) as a chaperone in folding the largest subunit (ß', of 1,407 residues in length), but except for binding of the stringent signal ppGpp, little is known of its role in the control of RNAP function. After analysis of genomewide distribution of wild-type and RpoZ-defective RNAP by the ChIP-chip method, we found alteration of the RpoZ-defective RNAP inside open reading frames, in particular, of the genes within prophages. For a set of the genes that exhibited altered occupancy of the RpoZ-defective RNAP, transcription was found to be altered as observed by qRT-PCR assay. All the observations here described indicate the involvement of RpoZ in recognition of some of the prophage genes. This study advances understanding of not only the regulatory role of omega subunit in the functions of RNAP but also the regulatory interplay between prophages and the host E. coli for adjustment of cellular physiology to a variety of environments in nature.

Regulatory role of XynR (YagI) in catabolism of xylonate in Escherichia coli K-12.

The genome of Escherichia coli K-12 contains ten cryptic phages, altogether constituting about 3.6% of the genome in sequence. Among more than 200 predicted genes in these cryptic phages, 14 putative transcription factor (TF) genes exist, but their regulatory functions remain unidentified. As an initial attempt to make a breakthrough for understanding the regulatory roles of cryptic phage-encoded TFs, we tried to identify the regulatory function of CP4-6 cryptic prophage-encoded YagI with unknown function. After SELEX screening, YagI was found to bind mainly at a single site within the spacer of bidirectional transcription units, yagA (encoding another uncharacterized TF) and yagEF (encoding 2-keto-3-deoxy gluconate aldolase, and dehydratase, respectively) within this prophage region. YagEF enzymes are involved in the catabolism of xylose downstream from xylonate. We then designated YagI as XynR (regulator of xylonate catabolism), one of the rare single-target TFs. In agreement with this predicted regulatory function, the activity of XynR was suggested to be controlled by xylonate. Even though low-affinity binding sites of XynR were identified in the E. coli K-12 genome, they all were inside open reading frames, implying that the regulation network of XynR is still fixed within the CR4-6 prophage without significant influence over the host E. coli K-12.

Cross-regulation between two common ancestral response regulators, HprR and CusR, in Escherichia coli.

The uncharacterized two-component system YedVW of Escherichia coli is involved in stress response to hydrogen peroxide. To identify the H2O2-sensing role of YedV, a set of single Cys-to-Ala substitution mutants were constructed. One particular mutant with C165A substitution in the membrane domain rendered YedV inactive in H2O2-dependent transcription of its regulatory target hiuH. We then proposed to rename YedVW to HprSR (hydrogen peroxide response sensor/regulator). One unique characteristic of HprR is the overlapping of its recognition sequence with that of the Cu(II)-response two-component system regulator CusR. Towards understanding this unique regulation system, in this study we analysed the interplay between HprR and CusR with respect to transcription of hiuH, a regulatory target of HprR, and cusC, a target of CusR. Under low protein concentrations in vitro and in vivo, two regulators recognize and transcribe both hiuH and cusC promoters, albeit at different efficiency, apparently in a collaborative fashion. This is a new type of transcription regulation of the common target genes in response to different external signals. Upon increase in protein concentrations, however, HprR and CusR compete with each other in transcription of the common targets, thereby exhibiting a competitive interplay.

Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli.

Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order) and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3' proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX screening of promoters recognized by RNA polymerase (RNAP) holoenzyme containing RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon), within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons are expressed independent of rRNA synthesis under specific conditions where further synthesis of ribosomes is not needed.

Effect of Spermidine Analogues on Cell Growth of Escherichia coli Polyamine Requiring Mutant MA261.

The effects of spermidine analogues [norspermidine (NSPD, 33), spermidine (SPD, 34), homospermidine (HSPD, 44) and aminopropylcadaverine (APCAD, 35)] on cell growth were studied using Escherichia coli polyamine-requiring mutant MA261. Cell growth was compared at 32°C, 37°C, and 42°C. All four analogues were taken up mainly by the PotABCD spermidine-preferential uptake system. The degree of stimulation of cell growth at 32°C and 37°C was NSPD ≥ SPD ≥ HSPD > APCAD, and SPD ≥ HSPD ≥ NSPD > APCAD, respectively. However, at 42°C, it was HSPD ¼ SPD > NSPD > APCAD. One reason for this is HSPD was taken up effectively compared with other triamines. In addition, since natural polyamines (triamines and teteraamines) interact mainly with RNA, and the structure of RNA is more flexible at higher temperatures, HSPD probably stabilized RNA more tightly at 42°C. We have thus far found that 20 kinds of protein syntheses are stimulated by polyamines at the translational level. Among them, synthesis of OppA, RpoE and StpA was more strongly stimulated by HSPD at 42°C than at 37°C. Stabilization of the initiation region of oppA and rpoE mRNA was tighter by HSPD at 42°C than 37°C determined by circular dichroism (CD). The degree of polyamine stimulation of OppA, RpoE and StpA synthesis by NSPD, SPD and APCAD was smaller than that by HSPD at 42°C. Thus, the degree of stimulation of cell growth by spermidine analogues at the different temperatures is dependent on the stimulation of protein synthesis by some components of the polyamine modulon.

Transcription factor CecR (YbiH) regulates a set of genes affecting the sensitivity of Escherichia coli against cefoperazone and chloramphenicol.

Genomic SELEX (systematic evolution of ligands by exponential enrichment) screening was performed for identification of the binding site of YbiH, an as yet uncharacterized TetR-family transcription factor, on the Escherichia coli genome. YbiH was found to be a unique single-target regulator that binds in vitro within the intergenic spacer located between the divergently transcribed ybiH-ybhGFSR and rhlE operons. YbhG is an inner membrane protein and YbhFSR forms a membrane-associated ATP-binding cassette (ABC) transporter while RhlE is a ribosome-associated RNA helicase. Gel shift assay and DNase footprinting analyses indicated one clear binding site of YbiH, including a complete palindromic sequence of AATTAGTT-AACTAATT. An in vivo reporter assay indicated repression of the ybiH operon and activation of the rhlE operon by YbiH. After phenotype microarray screening, YbiH was indicated to confer resistance to chloramphenicol and cefazoline (a first-generation cephalosporin). A systematic survey of the participation of each of the predicted YbiH-regulated genes in the antibiotic sensitivity indicated involvement of the YbhFSR ABC-type transporter in the sensitivity to cefoperazone (a third-generation cephalosporin) and of the membrane protein YbhG in the control of sensitivity to chloramphenicol. Taken together with the growth test in the presence of these two antibiotics and in vitro transcription assay, it was concluded that the hitherto uncharacterized YbiH regulates transcription of both the bidirectional transcription units, the ybiH-ybhGFSR operon and the rhlE gene, which altogether are involved in the control of sensitivity to cefoperazone and chloramphenicol. We thus propose to rename YbiH as CecR (regulator of cefoperazone and chloramphenicol sensitivity).

Expanded roles of two-component response regulator OmpR in Escherichia coli: genomic SELEX search for novel regulation targets.

The two-component system (TCS) is a sophisticated bacterial signal transduction system for regulation of genome transcription in response to environmental conditions. The EnvZ-OmpR system is one of the well-characterized TCS of Escherichia coli, responding to changes in environmental osmolality. Regulation has largely focused on the differential expression of two porins, OmpF and OmpC, which transport small molecules across the outer membrane. Recently, it has become apparent that OmpR serves a more global regulatory role and regulates additional targets. To identify the entire set of regulatory targets of OmpR, we performed the genomic SELEX screening of OmpR-binding sites along the E. coli genome. As a result, more than 30 novel genes have been identified to be under the direct control of OmpR. One abundant group includes the genes encoding a variety of membrane-associated transporters that mediate uptake or efflux of small molecules, while another group encodes a set of transcription regulators, raising a concept that OmpR is poised to control a diverse set of responses by altering downstream transcriptional regulators.

Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases.

Hybrid sensor kinase, which contains a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, conveys signals to its cognate response regulator by means of a His-Asp-His-Asp phosphorelay. We examined the multistep phosphorelay of a recombinant EvgAS system in Escherichia coli and performed in vitro quantitative analyses of phosphorylation by using Phos-tag SDS-PAGE. Replacement of Asp in the receiver domain of EvgS by Ala markedly promoted phosphorylation at His in the HK domain compared with that in wild-type EvgS. Similar Ala-substituted mutants of other hybrid sensor kinases BarA and ArcB showed similar characteristics. In the presence of sufficient ATP, autophosphorylation of the HK domain in the mutant progressed efficiently with nearly pseudo-first-order kinetics until the phosphorylation ratio reached a plateau value of more than 95% within 60 min, and the value was maintained until 180 min. However, both wild-type EvgS and the Ala-substituted mutant of His in the HPt domain showed a phosphorylation ratio of less than 25%, which gradually decreased after 10 min. These results showed that the phosphorylation level is regulated negatively by the receiver domain. Furthermore, our in vivo assays confirmed the existence of a similar hyperphosphorylation reaction in the HK domain of the EvgS mutant in which the Asp residue was replaced with Ala, confirming the validity of the control mechanism proposed from profiling of phosphorylation in vitro [corrected].

Cross talk in promoter recognition between six NarL-family response regulators of Escherichia coli two-component system.

Bacterial two-component system (TCS) is composed of the sensor kinase (SK) and the response regulator (RR). After monitoring an environmental signal or condition, SK activates RR through phosphorylation, ultimately leading to the signal-dependent regulation of genome transcription. In Escherichia coli, a total of more than 30 SK-RR pairs exist, each forming a cognate signal transduction system. Cross talk of the signal transduction takes place at three stages: signal recognition by SK (stage 1); RR phosphorylation by SK (stage 2); and target recognition by RR (stage 3). Previously, we analyzed the stage 2 cross talk between the whole set of E. coli SK-RR pairs and found that the cross talk takes place for certain combinations. As an initial attempt to identify the stage 3 cross talk at the step of target promoter recognition by RR, we analyzed in this study the cross-recognition of target promoters by six NarL-family RRs, EvgA, NarL, NarP, RcsB, UhpA, and UvrY. Results of both in vivo and in vitro studies indicated that the stage 3 cross talk takes place for limited combinations, in particular, including a multifactor-regulated ydeP promoter.