Refining the binding of the Escherichia coli flagellar master regulator, FlhD4C2, on a base-specific level.

The Escherichia coli flagellar master regulator, FlhD(4)C(2), binds to the promoter regions of flagellar class II genes, yet, despite extensive analysis of the FlhD(4)C(2)-regulated promoter region, a detailed consensus sequence has not emerged. We used in vitro and in vivo experimental approaches to determine the nucleotides in the class II promoter, fliAp, required for the binding and function of FlhD(4)C(2). FlhD(4)C(2) protects 48 bp (positions -76 to -29 relative to the σ(70)-dependent transcriptional start site) in the fliA promoter. We divided the 48-bp footprint region into 5 sections to determine the requirement of each DNA segment for the binding and function of FlhD(4)C(2). Results from an in vitro binding competition assay between the wild-type FlhD(4)C(2)-protected fragment and DNA fragments possessing mutations in one section of the 48-bp protected region showed that only one-third of the 48 bp protected by FlhD(4)C(2) is required for FlhD(4)C(2) binding and fliA promoter activity. This in vitro binding result was also seen in vivo with fliA promoter-lacZ fusions carrying the same mutations. Only seven bases (A(12), A(15), T(34), A(36), T(37), A(44), and T(45)) are absolutely required for the promoter activity. Moreover, A(12), A(15), T(34), T(37), and T(45) within the 7 bases are highly specific to fliA promoter activity, and those bases form an asymmetric recognition site for FlhD(4)C(2). The implications of the asymmetry of the FlhD(4)C(2) binding site and its potential impact on FlhD(4)C(2) are discussed.

The CheZ binding interface of CheAS is located in alpha-helix E.

Specific CheA-short (CheA(S)) residues, L123 and L126, were identified as critical for CheZ binding. In the CheA(S) 'P1-CheZ nuclear magnetic resonance structure, these residues form an interaction surface on alpha-helix E in the 'P1 domain. Both L123 and L126 are buried in CheA-long (CheA(L)), providing an explanation for why CheA(L) fails to bind CheZ.

Structure of the Escherichia coli FlhDC complex, a prokaryotic heteromeric regulator of transcription.

The hetero-oligomeric complex of the FlhD and FlhC proteins (FlhDC) regulates transcription from several flagellar and non-flagellar operons in bacteria. The crystallographic structure of the Escherichia coli FlhDC complex has been solved to 3.0 A resolution, revealing a hexameric FlhD4FlhC2 assembly. In the complex, each FlhC protomer binds an FlhD2 dimer; the conformation of the dimer in the complex differs significantly from its conformation in the absence of FlhC. FlhC has a novel tertiary fold that includes a heretofore unrecognized zinc-binding site in which the ion is ligated by four cysteine residues. Gel shift experiments show that binding of the FlhDC complex to a cognate promoter bends the DNA by approximately 111 degrees . The structure of the FlhDC complex is compatible with models in which a fragment of operator DNA, at least 48 base-pairs in length, wraps around the complex and bends significantly when binding.

ASM Journals Eliminate Impact Factor Information from Journal Websites.

Many scientists attempt to publish their work in a journal with the highest possible journal impact factor (IF). Despite widespread condemnation of the use of journal IFs to assess the significance of published work, these numbers continue to be widely misused in publication, hiring, funding, and promotion decisions (1, 2).

ASM Journals Eliminate Impact Factor Information from Journal Websites.

Many scientists attempt to publish their work in a journal with the highest possible journal impact factor (IF). Despite widespread condemnation of the use of journal IFs to assess the significance of published work, these numbers continue to be widely misused in publication, hiring, funding, and promotion decisions (1, 2).

Structure of the constitutively active double mutant CheYD13K Y106W alone and in complex with a FliM peptide.

CheY is a member of the response regulator protein superfamily that controls the chemotactic swimming response of motile bacteria. The CheY double mutant D13K Y106W (CheY**) is resistant to phosphorylation, yet is a highly effective mimic of phosphorylated CheY in vivo and in vitro. The conformational attributes of this protein that enable it to signal in a phosphorylation-independent manner are unknown. We have solved the crystal structure of selenomethionine-substituted CheY** in the presence of its target, a peptide (FliM16) derived from the flagellar motor switch, FliM, to 1.5A resolution with an R-factor of 19.6%. The asymmetric unit contains four CheY** molecules, two with FliM16 bound, and two without. The two CheY** molecules in the asymmetric unit that are bound to FliM16 adopt a conformation similar to BeF3- -activated wild-type CheY, and also bind FliM16 in a nearly identical manner. The CheY** molecules that do not bind FliM16 are found in a conformation similar to unphosphorylated wild-type CheY, suggesting that the active phenotype of this mutant is enabled by a facile interconversion between the active and inactive conformations. Finally, we propose a ligand-binding model for CheY and CheY**, in which Ile95 changes conformation in a Tyr/Trp106-dependent manner to accommodate FliM.

The accessibility of cys-120 in CheA(S) is important for the binding of CheZ and enhancement of CheZ phosphatase activity.

The cheA gene of Escherichia coli encodes two proteins from in-frame tandem translation start sites. The long form of CheA (CheA(L)) is the histidine kinase responsible for phosphorylating the response regulator, CheY. The short form of CheA (CheA(S)) is identical in domain structure to CheA(L) except that it is missing the first 97 amino acids. Reduced CheA(S) bound to and enhanced the activity of the phosphatase of phospho-CheY, CheZ. Oxidized CheA(S) was unable to interact with CheZ. Oxidized CheA(S) formed covalent dimers, whereas CheA(L) did not. This property was believed to be the result of an intermolecular disulfide bond. The CheA proteins contain three cysteine residues, one of which likely lies within the CheZ binding region of CheA(S) and is exposed to solvent. We identified the CheZ binding domain of CheA(S) by testing the various fragments of CheA(S) that contain cysteine residues for CheZ binding activity in an ELISA-based CheA(S)-CheZ binding assay. Fragments of CheA(S) lacking the truncated P1 domain of CheA(S) ('P1) were unable to bind CheZ. We also found that a fusion of the first 42 amino acids of CheA(S) ('P1 domain) to GST bound CheZ and enhanced its activity. The interaction between the GST-CheA[98-139] fusion protein and CheZ was dependent on the accessibility of a cysteine residue (Cys-120) located in the 'P1 domain.

Increased motility of Escherichia coli by insertion sequence element integration into the regulatory region of the flhD operon.

The flhD operon is the master operon of the flagellar regulon and a global regulator of metabolism. The genome sequence of the Escherichia coli K-12 strain MG1655 contained an IS1 insertion sequence element in the regulatory region of the flhD promoter. Another stock of MG1655 was obtained from the E. coli Genetic Stock Center. This stock contained isolates which were poorly motile and had no IS1 element upstream of the flhD promoter. From these isolates, motile subpopulations were identified after extended incubation in motility agar. Purified motile derivatives contained an IS5 element insertion upstream of the flhD promoter, and swarm rates were sevenfold higher than that of the original isolate. For a motile derivative, levels of flhD transcript had increased 2.7-fold, leading to a 32-fold increase in fliA transcript and a 65-fold increase in flhB::luxCDABE expression from a promoter probe vector. A collection of commonly used lab strains was screened for IS element insertion and motility. Five strains (RP437, YK410, MC1000, W3110, and W2637) contained IS5 elements upstream of the flhD promoter at either of two locations. This correlated with high swarm rates. Four other strains (W1485, FB8, MM294, and RB791) did not contain IS elements in the flhD regulatory region and were poorly motile. Primer extension determined that the transcriptional start site of flhD was unaltered by the IS element insertions. We suggest that IS element insertion may activate transcription of the flhD operon by reducing transcriptional repression.

Gene array analysis of Yersinia enterocolitica FlhD and FlhC: regulation of enzymes affecting synthesis and degradation of carbamoylphosphate.

This paper focuses on global gene regulation by FlhD/FlhC in enteric bacteria. Even though Yersinia enterocolitica FlhD/FlhC can complement an Escherichia coli flhDC mutant for motility, it is not known if the Y. enterocolitica FlhD/FlhC complex has an effect on metabolism similar to E. coli. To study metabolic gene regulation, a partial Yersinia enterocolitica 8081c microarray was constructed and the expression patterns of wild-type cells were compared to an flhDC mutant strain at 25 and 37 degrees C. The overlap between the E. coli and Y. enterocolitica FlhD/FlhC regulated genes was 25 %. Genes that were regulated at least fivefold by FlhD/FlhC in Y. enterocolitica are genes encoding urocanate hydratase (hutU), imidazolone propionase (hutI), carbamoylphosphate synthetase (carAB) and aspartate carbamoyltransferase (pyrBI). These enzymes are part of a pathway that is involved in the degradation of L-histidine to L-glutamate and eventually leads into purine/pyrimidine biosynthesis via carbamoylphosphate and carbamoylaspartate. A number of other genes were regulated at a lower rate. In two additional experiments, the expression of wild-type cells grown at 4 or 25 degrees C was compared to the same strain grown at 37 degrees C. The expression of the flagella master operon flhD was not affected by temperature, whereas the flagella-specific sigma factor fliA was highly expressed at 25 degrees C and reduced at 4 and 37 degrees C. Several other flagella genes, all of which are under the control of FliA, exhibited a similar temperature profile. These data are consistent with the hypothesis that temperature regulation of flagella genes might be mediated by the flagella-specific sigma factor FliA and not the flagella master regulator FlhD/FlhC.

FlhD/FlhC is a regulator of anaerobic respiration and the Entner-Doudoroff pathway through induction of the methyl-accepting chemotaxis protein Aer.

The regulation by two transcriptional activators of flagellar expression (FlhD and FlhC) and the chemotaxis methyl-accepting protein Aer was studied with glass slide DNA microarrays. An flhD::Kan insertion and an aer deletion were independently introduced into two Escherichia coli K-12 strains, and the effects upon gene regulation were investigated. Altogether, the flhD::Kan insertion altered the expression of 29 operons of known function. Among them was Aer, which in turn regulated a subset of these operons, namely, the ones involved in anaerobic respiration and the Entner-Doudoroff pathway. In addition, FlhD/FlhC repressed enzymes involved in aerobic respiration and regulated many other metabolic enzymes and transporters in an Aer-independent manner. Expression of 12 genes of uncharacterized function was also affected. FlhD increased gltBD, gcvTHP, and ompT expression. The regulation of half of these genes was subsequently confirmed with reporter gene fusions, enzyme assays, and real-time PCR. Growth phenotypes of flhD and flhC mutants were determined with Phenotype MicroArrays and correlated with gene expression.