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Under stressful conditions in nature, Escherichia coil 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. coil 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). DOI 10.1099/mic.0.039131-0

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. doi: 10.1128/JB.00663-09

Proteins whose synthesis is enhanced by polyamines at the level of translation were identified with a polyaminerequiring mutant cultured in the presence of 0.1% glucose and 0.02% glutamate at 42[degrees]C. Polyamines had a greater effect on cell growth at 42[degrees]C than at 37[degrees]C. At 42[degrees]C, the synthesis of RpoE ([sigma.sup.24]) and StpA, which are involved in the transcription of a number of heat shock response genes, was stimulated by polyamines at the level of translation. In the rpoE and stpA mRNAs, a Shine-Dalgarno (SD) sequence is located at 13 and 12 nucleotides, respectively, upstream of the initiation codon AUG. When the SD sequences were moved to the more common position 7 nucleotides upstream of the initiation codon AUG, the degree of polyamine stimulation was reduced, although the level of RpoE and StpA synthesis was markedly increased. The mechanism underlying polyamine stimulation of RpoE synthesis was then studied. Polyamine stimulation of RpoE synthesis was reduced by changing the bulged-out structure in the initiation site of rpoE mRNA, although the level of RpoE synthesis increased. A selective structural change of this bulged-out region induced by spermidine at 42[degrees]C was observed by circular dichroism. Polyamine stimulation of fMet-tRNA binding to ribosomes at 42[degrees]C also disappeared by changing the bulged-out structure in the initiation site of rpoE mRNA. The results suggest that polyamines enhance the synthesis of RpoE by changing the bulged-out structure in the initiation site of rpoE mRNA.

LeuO, a LysR family transcription factor, exists in a wide variety of bacteria of the family Enterobacteriaceae and is involved in the regulation of as yet unidentified genes affecting the stress response and pathogenesis expression. Using genomic screening by systematic evolution of ligands by exponential enrichment (SELEX) in vitro, a total of 106 DNA sequences were isolated from 12 different regions of the Escherichia coli genome. All of the SELEX fragments formed complexes in vitro with purified LeuO. After Northern blot analysis of the putative target genes located downstream of the respective LeuO-binding sequence, a total of nine genes were found to be activated by LeuO, while three genes were repressed by LeuO. The LeuO target gene collection included several multidrug resistance genes. A phenotype microarray assay was conducted to identify the gene(s) responsible for drug resistance and the drug species that are under the control of the LeuO target gene(s). The results described herein indicate that the yjcRQP operon, one of the LeuO targets, is involved in sensitivity control against sulfa drugs. We propose to rename the yjcRQP genes the sdsRQP genes (sulfa drug sensitivity determinant).

Purines are degraded via uric acid to yield allantoin, Under anaerobic conditions, allantoin is further degraded via carbamoylphosphate to N[H.sup.+.sub.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 AIIR, together with AIIS, plays a key role in switching control of two pathways, nitrogen assimilation and energy production. The repressor function of AIIR 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, AIIR 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.

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.

The folA gene was identified as a new member of the TyrR regulon by genomic SELEX. Binding of TyrR to two sites in folA activated its transcription. Mutations in the N-terminal or central domain of TyrR, the [alpha] subunit of RNA polymerase, or integration host factor all abolished activation of the folA promoter.

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.

RstBA, a two-component regulatory system of Escherichia coli with an unidentified regulatory function, is under the control of a [Mg.sup.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.sup.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.

Proteins whose synthesis is enhanced by polyamines at the level of translation were identified in a polyaminerequiring mutant cultured in the presence of 0.1% glucose and 0.02% glutamate instead of 0.4% glucose as an energy source. Under these conditions, enhancement of cell growth by polyamines was almost the same as that in the presence of 0.4% glucose. It was found that synthesis of RpoN, Cra, and H-NS was enhanced by polyamines at the level of translation at the early logarithmic phase of growth ([A.sub.540] of 0.15). The effects of polyamines on synthesis of RpoN, H-NS, and Cra were due to the existence of unusual Shine-Dalgarno sequences (RpoN and H-NS) and an inefficient GUG initiation codon (Cra) in their mRNAs. Thus, rpoN, cra and hns genes were identified as new members of the polyamine modulon. Because most of the polyamine modulon genes thus far identified encode transcription factors (RpoS [[[sigma].sup.38]], Cya, FecI [[[sigma].sup.18]], Fis, RpoN [[[sigma].sup.54]], Cra, and H-NS), DNA microarray analysis of mRNA expressed in cells was performed. At the early logarithmic phase of growth, a total of 97 species of mRNAs that were up-regulated by polyamines more than twofold were under the control of seven polyamine modulon genes mentioned above.

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.