Structural and nucleotide-binding properties of YajQ and YnaF, two Escherichia coli proteins of unknown function.

Structural genomics is a new approach in functional assignment of proteins identified via whole-genome sequencing programs. Its rationale is that nonhomologous proteins performing similar or related biological functions might have similar tertiary structure. We used dye pseudoaffinity chromatography, two-dimensional gel electrophoresis, and mass spectrometry to identify two novel Escherichia coli nucleotide-binding proteins, YnaF and YajQ. YnaF exhibited significant sequence identity with MJ0577, an ATP-binding protein from a hyperthermophile (Methanococcus jannaschii), and with UspA, a protein from Haemophilus influenzae that belongs to the Universal Stress Protein family. YnaF conserves the ATP-binding site and the dimeric structure observed in the crystal of MJ0577. The protein YajQ, present in many bacterial genomes, is missing in eukaryotes. In the absence of significant similarities of YajQ to any solved structure, we determined its structural and ligand-binding properties by NMR and isothermal titration calorimetry. We demonstrate that YajQ is composed of two domains, each centered on a beta-sheet, that are connected by two helical segments. NMR studies, corroborated with local sequence conservation among YajQ homologs in various bacteria, indicate that one of the beta-sheets is mostly involved in biological activity.

Identification and immunochemical location of UMP kinase from Bacillus subtilis.

Phosphorylation of CMP and UMP is accomplished in Bacillus subtilis, as in Escherichia coli, by two different enzymes exhibiting characteristic structural and catalytic properties. UMP kinase from B. subtilis is an oligomer whose activity is strictly dependent on GTP. The B. subtilis enzyme is unstable in the absence of UTP, which acts as an allosteric inhibitor. Antibodies raised against recombinant B. subtilis UMP kinase recognized the protein both in soluble extract and in immunoelectron microscopy. UMP kinase from B. subtilis has a peripheral distribution which is related most probably to its role in the synthesis of membrane sugar components and its putative role in cell division.

Structure-function relationships of UMP kinases from pyrH mutants of Gram-negative bacteria.

Bacterial uridine monophosphate (UMP) kinases are essential enzymes encoded by pyrH genes, and conditional-lethal or other pyrH mutants were analysed with respect to structure-function relationships. A set of thermosensitive pyrH mutants from Escherichia coli was generated and studied, along with already described pyrH mutants from Salmonella enterica serovar Typhimurium. It is shown that Arg-11 and Gly-232 are key residues for thermodynamic stability of the enzyme, and that Asp-201 is important for both catalysis and allosteric regulation. A comparison of the amino acid sequence of UMP kinases from several prokaryotes showed that these were conserved residues. Discussion on the enzyme activity level in relation to bacterial viability is also presented.

Transcription regulation coupling of the divergent argG and metY promoters in Escherichia coli K-12.

The cAMP-catabolite activator protein (CAP) complex is a pleiotropic regulator that regulates a vast number of Escherichia coli genes, including those involved in carbon metabolism. We identified two new targets of this complex: argG, which encodes the arginosuccinate synthase involved in the arginine biosynthetic pathway, and metY, which encodes one of the two methionine tRNA initiators, tRNAf2Met. The cAMP-CAP complex activates argG transcription and inhibits metY transcription from the same DNA position. We also show that ArgR, the specific repressor of the arginine biosynthetic pathway, together with its arginine cofactor, acts on the regulation of metY mediated by CAP. The regulation of the two divergent promoters is thus simultaneously controlled not only by the cAMP-CAP complex, a global regulator, but also by a specific regulator of arginine metabolism, suggesting a previously unsuspected link between carbon metabolism and translation initiation.

Effect of mild acid pH on the functioning of bacterial membranes in Vibrio cholerae.

In this paper, we initiated the first two-dimensional electrophoresis map of Vibrio cholerae, the aetiological agent of cholera disease. In this pathogen the efficient adaptation to detrimental conditions plays an important role in its survival in both the aquatic reservoir and human intestine. By proteome analysis we investigated the effect of mild acid treatment on the physiology of V. cholerae. More than 50 proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and database searching. Amongst them, pH regulated proteins belong to various functional classes such as intermediary metabolism and bacterial envelope. Several proteins whose accumulation level was decreased in response to acidic pH are known to be involved in the organization and the functioning of membranes, including lipopolysaccharide. Consistent with this, we observed an increased susceptibility to hydrophobic drugs, a loss of motility and a reduction in the ability to form a biofilm in cells grown at pH 6. Our results suggest that V. cholerae is able to sense a moderate decrease in pH and to modify accordingly its structure and physiology.

Regulation of bacterial motility in response to low pH in Escherichia coli: the role of H-NS protein.

The effect of detrimental conditions on bacterial motility in Escherichia coli was investigated. Expression profiling of mutant E. coli strains by DNA arrays and analysis of phenotypic traits demonstrated that motility and low-pH resistance are coordinately regulated. Analysis of transcriptional fusions suggests that bacterial motility in response to an acidic environment is mediated via the control by H-NS of flhDC expression. Moreover, the results suggested that the presence of an extended mRNA 5' end and DNA topology are required in this process. Finally, the presence of a similar regulatory region in several Gram-negative bacteria implies that this mechanism is largely conserved.