Computational Analysis of the Ligand-Binding Sites of the Molecular Chaperone OppA from Yersinia pseudotuberculosis.

The function of chaperones is to correct or degrade misfolded proteins inside the cell. Classic molecular chaperones such as GroEL and DnaK have not been found in the periplasm of Yersinia pseudotuberculosis. Some periplasmic substrate-binding proteins could be bifunctional, such as OppA. Using bioinformatic tools, we try to elucidate the nature of the interactions between OppA and ligands from four proteins with different oligomeric states. Using the crystal structure of the proteins Mal12 alpha-glucosidase from Saccharomyces cerevisiae S288C, LDH rabbit muscle lactate dehydrogenase, EcoRI endonuclease from Escherichia coli and THG Geotrichum candidum lipase, a hundred models were obtained in total, including five different ligands from each enzyme with five conformations of each ligand. The best values for Mal12 stem from ligands 4 and 5, with conformation 5 for both; for LDH, ligands 1 and 4, with conformations 2 and 4, respectively; for EcoRI, ligands 3 and 5, with conformation 1 for both; and for THG, ligands 2 and 3, with conformation 1 for both. The interactions were analyzed with LigProt, and the length of the hydrogen bridges has an average of 2.8 to 3.0 Å. The interaction within the OppA pocket is energetically favored due to the formation of hydrogen bonds both of OppA and of the selected enzymes. The Asp 419 residue is important in these junctions.

Role of cis-trans proline isomerization in the function of pathogenic enterobacterial Periplasmic Binding Proteins.

Periplasmic Binding Proteins (PBPs) trap nutrients for their internalization into bacteria by ABC transporters. Ligand binding triggers PBP closure by bringing its two domains together like a Venus flytrap. The atomic determinants that control PBP opening and closure for nutrient capture and release are not known, although it is proposed that opening and ligand release occur while in contact with the ABC transporter for concurrent substrate translocation. In this paper we evaluated the effect of the isomerization of a conserved proline, located near the binding site, on the propensity of PBPs to open and close. ArgT/LAO from Salmonella typhimurium and HisJ from Escherichia coli were studied through molecular mechanics at two different temperatures: 300 and 323 K. Eight microseconds were simulated per protein to analyze protein opening and closure in the absence of the ABC transporter. We show that when the studied proline is in trans, closed empty LAO and HisJ can open. In contrast, with the proline in cis, opening transitions were much less frequent and characterized by smaller changes. The proline in trans also renders the open trap prone to close over a ligand. Our data suggest that the isomerization of this conserved proline modulates the PBP mechanism: the proline in trans allows the exploration of conformational space to produce trap opening and closure, while in cis it restricts PBP movement and could limit ligand release until in productive contact with the ABC transporter. This is the first time that a proline isomerization has been related to the control of a large conformational change like the PBP flytrap mechanism.

Sulfate-Binding Protein (Sbp) from Xanthomonas citri: Structure and Functional Insights.

The uptake and transport of sulfate in bacteria is mediated by an ATP-binding cassette transporter (ABC transporter) encoded by sbpcysUWA genes, whose importance has been widely demonstrated due to their relevance in cysteine synthesis and bacterial growth. In Xanthomonas citri, the causative agent of canker disease, the expression of components from this ABC transporter and others related to uptake of organic sulfur sources has been shown during in vitro growth cultures. In this work, based on gene reporter and proteomics analyses, we showed the activation of the promoter that controls the sbpcysUWA operon in vitro and in vivo and the expression of sulfate-binding protein (Sbp), a periplasmic-binding protein, indicating that this protein plays an important function during growth and that the transport system is active during Citrus sinensis infection. To characterize Sbp, we solved its three-dimensional structure bound to sulfate at 1.14 Å resolution and performed biochemical and functional characterization. The results revealed that Sbp interacts with sulfate without structural changes, but the interaction induces a significant increasing of protein thermal stability. Altogether, the results presented in this study show the evidence of the functionality of the ABC transporter for sulfate in X. citri and its relevance during infection.

Transcriptional and post-transcriptional regulation of pst2 operon expression in Vibrio cholerae O1.

One of the most abundant proteins in V. cholerae O1 cells grown under inorganic phosphate (Pi) limitation is PstS, the periplasmic Pi-binding component of the high-affinity Pi transport system Pst2 (PstSCAB), encoded in pst2 operon (pstS-pstC2-pstA2-pstB2). Besides its role in Pi uptake, Pst2 has been also associated with V. cholerae virulence. However, the mechanisms regulating pst2 expression and the non-stoichiometric production of the Pst2 components under Pi-limitation are unknown. A computational-experimental approach was used to elucidate the regulatory mechanisms behind pst2 expression in V. cholerae O1. Bioinformatics analysis of pst2 operon nucleotide sequence revealed start codons for pstS and pstC genes distinct from those originally annotated, a regulatory region upstream pstS containing potential PhoB-binding sites and a pstS-pstC intergenic region longer than predicted. Analysis of nucleotide sequence between pstS-pstC revealed inverted repeats able to form stem-loop structures followed by a potential RNAse E-cleavage site. Another putative RNase E recognition site was identified within the pstA-pstB intergenic sequence. In silico predictions of pst2 operon expression regulation were subsequently tested using cells grown under Pi limitation by promoter-lacZ fusion, gel electrophoresis mobility shift assay and quantitative RT-PCR. The experimental and in silico results matched very well and led us to propose a pst2 promoter sequence upstream of pstS gene distinct from the previously annotated. Furthermore, V. cholerae O1 pst2 operon transcription is PhoB-dependent and generates a polycistronic mRNA molecule that is rapidly processed into minor transcripts of distinct stabilities. The most stable was the pstS-encoding mRNA, which correlates with PstS higher levels relative to other Pst2 components in Pi-starved cells. The relatively higher stability of pstS and pstB transcripts seems to rely on the secondary structures at their 3' untranslated regions that are known to block 3'-5' exonucleolytic attacks.

Modification of glucose import capacity in Escherichia coli: physiologic consequences and utility for improving DNA vaccine production.

BACKGROUND: The bacterium Escherichia coli can be grown employing various carbohydrates as sole carbon and energy source. Among them, glucose affords the highest growth rate. This sugar is nowadays widely employed as raw material in industrial fermentations. When E. coli grows in a medium containing non-limiting concentrations of glucose, a metabolic imbalance occurs whose main consequence is acetate secretion. The production of this toxic organic acid reduces strain productivity and viability. Solutions to this problem include reducing glucose concentration by substrate feeding strategies or the generation of mutant strains with impaired glucose import capacity. In this work, a collection of E. coli strains with inactive genes encoding proteins involved in glucose transport where generated to determine the effects of reduced glucose import capacity on growth rate, biomass yield, acetate and production of an experimental plasmid DNA vaccine (pHN). RESULTS: A group of 15 isogenic derivatives of E. coli W3110 were generated with single and multiple deletions of genes encoding glucose, mannose, beta-glucoside, maltose and N-acetylglucosamine components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), as well as the galactose symporter and the Mgl galactose/glucose ABC transporter. These strains were characterized by growing them in mineral salts medium supplemented with 2.5 g/L glucose. Maximum specific rates of glucose consumption (qs) spanning from 1.33 to 0.32 g/g h were displayed by the group of mutants and W3110, which resulted in specific growth rates ranging from 0.65-0.18 h(-1). Acetate accumulation was reduced or abolished in cultures with all mutant strains. W3110 and five selected mutant derivatives were transformed with pHN. A 3.2-fold increase in pHN yield on biomass was observed in cultures of a mutant strain with deletion of genes encoding the glucose and mannose PTS components, as well as Mgl. CONCLUSIONS: The group of E. coli mutants generated in this study displayed a reduction or elimination of overflow metabolism and a linear correlation between qs and the maximum specific growth rate as well as the acetate production rate. By comparing DNA vaccine production parameters among some of these mutants, it was possible to identify a near-optimal glucose import rate value for this particular application. The strains employed in this study should be a useful resource for studying the effects of different predefined qs values on production capacity for various biotechnological products.

The Escherichia coli BtuE protein functions as a resistance determinant against reactive oxygen species.

This work shows that the recently described Escherichia coli BtuE peroxidase protects the bacterium against oxidative stress that is generated by tellurite and by other reactive oxygen species elicitors (ROS). Cells lacking btuE (ΔbtuE) displayed higher sensitivity to K(2)TeO(3) and other oxidative stress-generating agents than did the isogenic, parental, wild-type strain. They also exhibited increased levels of cytoplasmic reactive oxygen species, oxidized proteins, thiobarbituric acid reactive substances, and lipoperoxides. E. coli ΔbtuE that was exposed to tellurite or H(2)O(2) did not show growth changes relative to wild type cells either in aerobic or anaerobic conditions. Nevertheless, the elimination of btuE from cells deficient in catalases/peroxidases (Hpx(-)) resulted in impaired growth and resistance to these toxicants only in aerobic conditions, suggesting that BtuE is involved in the defense against oxidative damage. Genetic complementation of E. coli ΔbtuE restored toxicant resistance to levels exhibited by the wild type strain. As expected, btuE overexpression resulted in decreased amounts of oxidative damage products as well as in lower transcriptional levels of the oxidative stress-induced genes ibpA, soxS and katG.

The Escherichia coli btuE gene, encodes a glutathione peroxidase that is induced under oxidative stress conditions.

Most aerobic organisms are exposed to oxidative stress. Looking for enzyme activities involved in the bacterial response to this kind of stress, we focused on the btuE-encoded Escherichia coli BtuE, an enzyme that shares homology with the glutathione peroxidase (GPX) family. This work deals with the purification and characterization of the btuE gene product. Purified BtuE decomposes in vitro hydrogen peroxide in a glutathione-dependent manner. BtuE also utilizes preferentially thioredoxin A to decompose hydrogen peroxide as well as cumene-, tert-butyl-, and linoleic acid hydroperoxides, confirming that its active site confers non-specific peroxidase activity. These data suggest that the enzyme may have one or more organic hydroperoxide as its physiological substrate. The btuE gene was induced when cells were exposed to oxidative stress elicitors that included potassium tellurite, menadione and hydrogen peroxide, among others, suggesting that BtuE could participate in the E. coli response to reactive oxygen species. To our knowledge, this is the first report describing a glutathione peroxidase in E. coli.

Stability of the pstS transcript of Escherichia coli.

The pst operon of Escherichia coli is composed of five genes that encode a high-affinity phosphate transport system. As a member of the PHO regulon, pst transcription is activated under phosphate shortage conditions. Under phosphate-replete conditions, the pst operon also functions as a negative regulator of the PHO genes. Transcription of pst is initiated at the promoter located upstream to the first gene, pstS. Immediately after its synthesis, the primary transcript of pst is cleaved into shorter mRNA molecules. The transcription unit corresponding to pstS is significantly more abundant than the transcripts of the other pst genes due to stabilisation of pstS mRNA by a repetitive extragenic palindrome (REP) structure downstream to the pstS locus. The presence of the REP sequence also results in an increased level of PstS proteins. However, the surplus level of PstS proteins produced in the presence of REP does not contribute to the repressive role of Pst in PHO expression.

Crystallographic structure and substrate-binding interactions of the molybdate-binding protein of the phytopathogen Xanthomonas axonopodis pv. citri.

In Xanthomonas axonopodis pv. citri (Xac or X. citri), the modA gene codes for a periplasmic protein (ModA) that is capable of binding molybdate and tungstate as part of the ABC-type transporter required for the uptake of micronutrients. In this study, we report the crystallographic structure of the Xac ModA protein with bound molybdate. The Xac ModA structure is similar to orthologs with known three-dimensional structures and consists of two nearly symmetrical domains separated by a hinge region where the oxyanion-binding site lies. Phylogenetic analysis of different ModA orthologs based on sequence alignments revealed three groups of molybdate-binding proteins: bacterial phytopathogens, enterobacteria and soil bacteria. Even though the ModA orthologs are segregated into different groups, the ligand-binding hydrogen bonds are mostly conserved, except for Archaeglobus fulgidus ModA. A detailed discussion of hydrophobic interactions in the active site is presented and two new residues, Ala38 and Ser151, are shown to be part of the ligand-binding pocket.

The molybdate-binding protein (ModA) of the plant pathogen Xanthomonas axonopodis pv. citri.

The modABC operon of phytopathogen Xanthomonas axonopodis pv. citri (X. citri) encodes a putative ABC transporter involved in the uptake of the molybdate and tungstate anions. Sequence analyses showed high similarity values of ModA orthologs found in X. campestris pv. campestris (X. campestris) and Escherichia coli. The X. citri modA gene was cloned in pET28a and the recombinant protein, expressed in the E. coli BL21 (DE3) strain, purified by immobilized metal affinity chromatography. The purified protein remained soluble and specifically bound molybdate and tungstate with K(d) 0.29+/-0.12 microM and 0.58+/-0.14 microM, respectively. Additionally binding of molybdate drastically enhanced the thermal stability of the recombinant ModA as compared to the apoprotein. This is the first characterization of a ModA ortholog expressed by a phytopathogen and represents an important tool for functional, biochemical and structural analyses of molybdate transport in Xanthomonas species.

A differential effect of sigmaS on the expression of the PHO regulon genes of Escherichia coli.

The RNA polymerase core associated with sigma(S) transcribes many genes related to stress or to the stationary phase. When cells enter a phase of phosphate starvation, the transcription of several genes and operons, collectively known as the PHO regulon, is strongly induced. The promoters of the PHO genes hitherto analysed are recognized by sigma(D)-associated RNA polymerase. A mutation in the gene that encodes sigma(S), rpoS, significantly increases the level of alkaline phosphatase activity and the overproduction of sigma(S) inhibits it. Other PHO genes such as phoE and ugpB are likewise affected by sigma(S). In contrast, pstS, which encodes a periplasmic phosphate-binding protein and is a negative regulator of PHO, is stimulated by sigma(S). The effect of sigma(S) on the PHO genes is at the transcriptional level. It is shown that a cytosine residue at position -13 is important for the positive effect of sigma(S) on pst. The interpretation of these observations is based on the competition between sigma(S) and sigma(D) for the binding to the core RNA polymerase.