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). INTRODUCTIONBacteria can switch from a single-cell planktonic growth mode to a multicellular community (biofilm) mode. During such a growth transition state, cell morphology, physiology and metabolism are markedly altered (PrigentCombaret et al., 2001;Schembri et al., 2003;Beloin et al., 2004;Ren et al., 2004). In single-cell growth mode, cell motility using flagella is critical for adaptation to environments. The master regulator FlhCD complex plays a key role in controlling transcription of a set of genes for flagella formation (Claret & Huges, 2000). When Escherichia coli cells switch their life mode from single planktonic cell growth to multicellular community (biofilm) mode, the genetic system for flagella formation is turned off and the genes involved in cell-cell adhesion are activated. The biofilm matrix is a complex architecture of cell aggregates that are attached on the surface of inorganic solid materials in nature or on eukaryotic tissues in host animals. After a comprehensive analysis of a set of E. coli mutants, each lacking one of 3985 non-essential genes, a total of 110 genes were indicated to be involved in biofilm formation (Niba et al., 2007).During biofilm development, curli fimbriae, the major biofilm component, play a key role in both initial adhesion to solid surfaces and subsequent cell-cell interactions (Vidal et al., 1998;Chapman et al., 2002;Prigent-Combaret et al., 2000). Curli fimbriae also mediate bacterial adhesion to host cells and invasion, and activation of both the proinflammatory response and the immune system (Bian et al., 2000;Zogaj et al., 2001). Accordingly, curli participate in virulence phenotypes (Hammar et al., 1995; Vidal et al., 1998;Bian et al., 2000; Gophna et al., 2001;Cookson et al., 2002). A set of polysacch...

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 systemdependent 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.

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.

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.The pyruvate dehydrogenase (PDH) complex of Escherichia coli contains three components, pyruvate dehydrogenase (E1p), dehydrolipoate acyltransferase (E2p), and dihydrolipoate dehydrogenase (E3), and catalyzes the NAD-linked oxidative decarboxylation of pyruvate and the concomitant formation of acetyl coenzyme A (acetyl-CoA) (25, 34), which then reacts with oxalacetate to produce citrate in the first reaction of the citric acid (tricarboxylic acid [TCA] Transcription of the pdh operon is controlled by two major promoters: the upstream promoter P pdh generates a pdhR-lpdA readthrough transcript, and the internal promoter P lpd generates independent lpdA transcript (26). The primary pdh promoter P pdh is negatively autoregulated by PdhR, the product of the first gene in this pdh operon (26). PdhR is a member of the Gnt family of transcription factors and shares sequence similarity in its N-terminal DNA-binding domain with other members (14). PdhR senses the intracellular pyruvate pool, and its activity is controlled by pyruvate. Since the PdhR-pyruvate complex is unable to bind target DNA, the pdhR-aceEF-lpdA operon is derepressed when pyruvate is available (26). The secondary promoter P lpd is under the control of ArcA, which is the global repressor of TCA cycle genes under anaerobic conditions (8). Besides the pdhR operon, the only regulation target of PdhR identified so far is the yfiD gene, encoding a putative formate acetyltransferase (36), that is induced at low pH or by pyruvate (3).Since the PDH multienzyme complex is a key enzyme for the metabolic interconnection between glycolysis and the TCA cycle,...

CsgD, the master regulator of biofilm formation, activates the synthesis of curli fimbriae and extracellular polysaccharides in Escherichia coli. To obtain insights into its regulatory role, we have identified a total of 20 novel regulation target genes on the E. coli genome by using chromatin immunoprecipitation (ChIP)-on-chip analysis with a high-density DNA microarray. By DNase I footprinting, the consensus CsgD-binding sequence predicted from a total of 18 target sites was found to include AAAAGNG(N 2 )AAAWW. After a promoter-lacZ fusion assay, the CsgD targets were classified into two groups: group I genes, such as fliE and yhbT, are repressed by CsgD, while group II genes, including yccT and adrA, are activated by CsgD. The fliE and fliEFGH operons for flagellum formation are directly repressed by CsgD, while CsgD activates the adrA gene, which encodes an enzyme for synthesis of cyclic di-GMP, a bacterial second messenger, which in turn inhibits flagellum production and rotation. Taking these findings together, we propose that the cell motility for planktonic growth is repressed by CsgD, thereby promoting the switch to biofilm formation.