Ayako Kori

Regulation of the Escherichia coli csgD promoter: interplay between five transcription factors.

Under stressful conditions in nature, Escherichia coli forms biofilms for long-term survival. Curli fimbriae are an essential architecture for cell-cell contacts within biofilms. Structural components and assembly factors of curli are encoded by two operons, csgBA and csgDEFG. The csgD gene product controls transcription of both operons. Reflecting the response of csgD expression to external stresses, a number of transcription factors participate in the regulation of the csgD promoter. Analysis of the csgD mRNA obtained from E. coli mutants in different transcription factors indicated that CpxR and H-NS act as repressors while OmpR, RstA and IHF act as activators. An acid-stress response regulator, RstA, activates csgD only under acidic conditions. These five factors bind within a narrow region of about 200 bp upstream of the csgD promoter. After pair-wise promoter-binding assays, the increase in csgD transcription in the stationary phase was suggested to be due, at least in part, to the increase in IHF level cancelling the silencing effect of H-NS. In addition, we propose a novel regulation model of this complex csgD promoter through cooperation between the two positive factors (OmpR-IHF and RstA-IHF) and also between the two negative factors (CpxR-H-NS).

RutR is the uracil/thymine-sensing master regulator of a set of genes for synthesis and degradation of pyrimidines

Using the genomic SELEX, a total of six Escherichia coli DNA fragments have been identified, which formed complexes with transcription factor RutR. The RutR regulon was found to include a large number of genes encoding components for not only degradation of pyrimidines but also transport of glutamate, synthesis of glutamine, synthesis of pyrimidine nucleotides and arginine, and degradation of purines. DNase I footprinting indicated that RutR recognizes a palindromic sequence of TTGACCAnnTGGTCAA. The RutR box in P1 promoter of carAB encoding carbamoyl phosphate synthetase, a key enzyme of pyrimidine synthesis, overlaps with the PepA (CarP) repressor binding site, implying competition between RutR and PepA. Adding either uracil or thymine abolished RutR binding in vitro to the carAB P1 promoter. Accordingly, in the rutR‐deletion mutant or in the presence of uracil, the activation in vivo of carAB P1 promoter was markedly reduced. Northern blot analysis of the RutR target genes indicated that RutR represses the Gad system genes involved in glutamate‐dependent acid resistance and allantoin degradation. Altogether we propose that RutR is the pyrimidine sensor and the master regulator for a large set of the genes involved in the synthesis and degradation of pyrimidines.

Intracellular Concentrations of 65 Species of Transcription Factors with Known Regulatory Functions in Escherichia coli

The expression pattern of the Escherichia coli genome is controlled in part by regulating the utilization of a limited number of RNA polymerases among a total of its approximately 4,600 genes. The distribution pattern of RNA polymerase changes from modulation of two types of protein-protein interactions: the interaction of core RNA polymerase with seven species of the sigma subunit for differential promoter recognition and the interaction of RNA polymerase holoenzyme with about 300 different species of transcription factors (TFs) with regulatory functions. We have been involved in the systematic search for the target promoters recognized by each sigma factor and each TF using the newly developed Genomic SELEX system. In parallel, we developed the promoter-specific (PS)-TF screening system for identification of the whole set of TFs involved in regulation of each promoter. Understanding the regulation of genome transcription also requires knowing the intracellular concentrations of the sigma subunits and TFs under various growth conditions. This report describes the intracellular levels of 65 species of TF with known function in E. coli K-12 W3110 at various phases of cell growth and at various temperatures. The list of intracellular concentrations of the sigma factors and TFs provides a community resource for understanding the transcription regulation of E. coli under various stressful conditions in nature.

The Uncharacterized Transcription Factor YdhM Is the Regulator of the nemA Gene, Encoding N-Ethylmaleimide Reductase

N-ethylmaleimide (NEM) has been used as a specific reagent of Cys modification in proteins and thus is toxic for cell growth. On the Escherichia coli genome, the nemA gene coding for NEM reductase is located downstream of the gene encoding an as-yet-uncharacterized transcription factor, YdhM. Disruption of the ydhM gene results in reduction of nemA expression even in the induced state, indicating that the two genes form a single operon. After in vitro genomic SELEX screening, one of the target recognition sequences for YdhM was identified within the promoter region for this ydhM-nemA operon. Both YdhM binding in vitro to the ydhM promoter region and transcription repression in vivo of the ydhM-nemA operon by YdhM were markedly reduced by the addition of NEM. Taken together, we propose that YdhM is the repressor for the nemA gene, thus hereafter designated NemR. The repressor function of NemR was inactivated by the addition of not only NEM but also other Cys modification reagents, implying that Cys modification of NemR renders it inactive. This is an addition to the mode of controlling activity of transcription factors by alkylation with chemical agents.

Participation of Regulator AscG of the β-Glucoside Utilization Operon in Regulation of the Propionate Catabolism Operon

The asc operon of Escherichia coli is one of the cryptic genetic systems for beta-D-galactoside utilization as a carbon source. The ascFB genes for beta-D-galactoside transport and catabolism are repressed by the AscG regulator. After genomic SELEX screening, AscG was found to recognize and bind the consensus palindromic sequence TGAAACC-GGTTTCA. AscG binding was detected at two sites upstream of the ascFB promoter and at three sites upstream of the prpBC operon for propionate catabolism. In an ascG-disrupted mutant, transcription of ascFB was enhanced, in agreement with the repressor model of AscG. This repression was indicated to be due to interference of binding of cyclic AMP-CRP to the CRP box, which overlaps with the AscG-binding site 1, as well as binding of RNA polymerase to the promoter. Under conditions of steady-state E. coli growth in a rich medium, the intracellular level of AscG stayed constant at a level supposedly leading to tight repression of the ascFB operon. The level of prpR, encoding the activator of prpBCDE, was also increased in the absence of AscG, indicating the involvement of AscG in repression of prpR. Taken together, these data suggest a metabolic link through interplay between the asc and prp operons.

The transcription regulator AllR senses both allantoin and glyoxylate and controls a set of genes for degradation and reutilization of purines.

Purines are degraded via uric acid to yield allantoin. Under anaerobic conditions, allantoin is further degraded via carbamoylphosphate to NH(4)+ to provide a nitrogen source and, under aerobic conditions, to 3-phosphoglycerate via glyoxylate for energy production. In this study, we found that a DNA-binding transcription factor AllR, together with AllS, plays a key role in switching control of two pathways, nitrogen assimilation and energy production. The repressor function of AllR is activated in the presence of allantoin, the common substrate for both pathways, leading to repression of the genes for energy production. On the other hand, when glyoxylate is accumulated, AllR is inactivated for derepression of the pathway for energy production. RutR, the master regulator for pyrimidines and arginine, is also involved in this pathway-switching control.

Screening of promoter-specific transcription factors: multiple regulators for the sdiA gene involved in cell division control and quorum sensing.

Prokaryotic DNA-binding transcription factors (TFs) bind in close vicinity of the promoter and regulate transcription through interplay with the DNA-dependent RNA polymerase. Promoters associated with the genes involved in stress response have recently been found to be under the control of multiple regulators, each monitoring one specific environmental condition or factor. In order to identify TFs involved in regulation of one specific promoter, we have developed a PS-TF (promoter-specific TF) screening system, in which the binding of purified TFs to a test promoter was analysed by gel-shift assay. This PS-TF screening system was applied for detection of TFs involved in regulation of the promoter for the Escherichia coli sdiA gene encoding the master regulator of cell division and quorum sensing. After screening of a total of 191 purified TFs (two-thirds of the predicted E. coli TFs), at least 15 TFs have been identified to bind to the sdiA promoter, including five two-component system (TCS) regulators, ArcA, CpxR, OmpR, RcsB and TorR. In this study, we focus on these five TFs for detailed analysis of their regulatory roles in vivo. Under normal growth conditions in LB medium, all these TFs repressed the sdiA promoter and the repression levels correlated with their intracellular levels. Taken together, we propose that these TCS regulators repress transcription in vivo of the sdiA gene, ultimately leading to suppression of cell division.

Single live‐bacterial cell assay of promoter activity and regulation

For measurement of the promoter activity and regulation within single bacterial cells, we constructed various types of the cell chip. After gelation of portions of E. coli culture using the cell chip, we have succeeded, for the first time, the real-time single-bacterial cell assay of E. coli gcl promoter strength.

Involvement of the ribose operon repressor RbsR in regulation of purine nucleotide synthesis in Escherichia coli

Escherichia coli is able to utilize d-ribose as its sole carbon source. The genes for the transport and initial-step metabolism of d-ribose form a single rbsDACBK operon. RbsABC forms the ABC-type high-affinity d-ribose transporter, while RbsD and RbsK are involved in the conversion of d-ribose into d-ribose 5-phosphate. In the absence of inducer d-ribose, the ribose operon is repressed by a LacI-type transcription factor RbsR, which is encoded by a gene located downstream of this ribose operon. At present, the rbs operon is believed to be the only target of regulation by RbsR. After Genomic SELEX screening, however, we have identified that RbsR binds not only to the rbs promoter but also to the promoters of a set of genes involved in purine nucleotide metabolism. Northern blotting analysis indicated that RbsR represses the purHD operon for de novo synthesis of purine nucleotide but activates the add and udk genes involved in the salvage pathway of purine nucleotide synthesis. Taken together, we propose that RbsR is a global regulator for switch control between the de novo synthesis of purine nucleotides and its salvage pathway.

Regulation of the E. coli csgD gene encoding the master regulator of biofilm formation: Interplay between multiple transcription factors

Under stressful conditions in nature, Escherichia coli forms biofilms for long-term survival. Curli fimbriae are an essential architecture for cell-cell contacts within biofilms. Structural components and assembly factors of curli are encoded by two divergently arranged operons, csgBA and csgDEFG, which are regulated by the csgD gene product. Thus, CsgD is a master regulator of bacterial biofilm formation. Reflecting the response of biofilm formation to various external factors and conditions, the csgD promoter is under the control of multiple transcription factors including CRP, CpxR, Crl, H-NS, IHF, MlrA, OmpR, RcsB and RstA. We have conducted a systematic analysis of the regulation mechanism of the csgD promoter by these multiple transcription factors. After the DNA binding assays in vitro of various combinations of these factors with various segments of the csgD promoter as DNA probes, we realized not only competition between positive and negative regulators but also cooperative interplay within both positive and negative factor groups. These findings raise a regulation model, in which the csgD promoter is controlled by the relative levels of functional molecules of these transcription factors.