RESUMO
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.
Assuntos
Proteínas de Bactérias , Chaperonas Moleculares , Proteínas Periplásmicas de Ligação , Yersinia pseudotuberculosis , Animais , Coelhos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Transporte/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ligantes , Chaperonas Moleculares/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Ligação Proteica , Yersinia pseudotuberculosis/metabolismoRESUMO
All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable signal. Most existing biosensing technologies utilize proteins that passively bind to their analytes and therefore require wasteful washing steps, specialized reagents, and expensive instruments for detection. To overcome these limitations, protein engineering strategies have been applied to develop new classes of protein-based sensor/actuators, known as protein switches, responding to small molecules. Protein switches change their active state (output) in response to a binding event or physical signal (input) and therefore show a tremendous potential to work as a biosensor. Synthetic protein switches can be created by the fusion between two genes, one coding for a sensor protein (input domain) and the other coding for an actuator protein (output domain) by domain insertion. The binding of a signal molecule to the engineered protein will switch the protein function from an "off" to an "on" state (or vice versa) as desired. The molecular switch could, for example, sense the presence of a metabolite, pollutant, or a biomarker and trigger a cellular response. The potential sensing and response capabilities are enormous; however, the recognition repertoire of natural switches is limited. Thereby, bioengineers have been struggling to expand the toolkit of molecular switches recognition repertoire utilizing periplasmic binding proteins (PBPs) as protein-sensing components. PBPs are a superfamily of bacterial proteins that provide interesting features to engineer biosensors, for instance, immense ligand-binding diversity and high affinity, and undergo large conformational changes in response to ligand binding. The development of these protein switches has yielded insights into the design of protein-based biosensors, particularly in the area of allosteric domain fusions. Here, recent protein engineering approaches for expanding the versatility of protein switches are reviewed, with an emphasis on studies that used PBPs to generate novel switches through protein domain insertion.
Assuntos
Técnicas Biossensoriais/métodos , Periplasma/química , Proteínas Periplásmicas de Ligação/química , Engenharia de Proteínas/métodos , Domínios ProteicosRESUMO
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.
Assuntos
Enterobacteriaceae/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Prolina/metabolismo , Isomerismo , Prolina/químicaRESUMO
Trypanosoma cruzi, the etiologic agent of Chagas disease, is covered by a dense glycocalix mainly composed by glycoproteins called mucins which are also the acceptors of sialic acid in a reaction catalyzed by a trans-sialidase (TcTS). Sialylation of trypomastigote mucins protects the parasite from lysis by the anti α-Galp antibodies from serum. The TcTS is essential for the infection process since T. cruzi is unable to biosynthesize sialic acid. The enzyme specifically transfers it from a terminal ß-d-Galp unit in the host glycoconjugate to terminal ß-d-Galp units in the parasite mucins to construct the d-NeuNAc(α2â3)ß-d-Galp motif. On the other hand, although galactose is the most abundant sugar in mucins of both, the infective trypomastigotes and the insect stage epimastigotes, α-d-Galp is only present in the infective stage whereas ß-d-Galf is characteristic of the epimastigote stage of the less virulent strains. Neither α-d-Galp nor d-Galf is acceptor of sialic acid. In the mucins, some of the oligosaccharides are branched with terminal ß-d-Galp units to be able to accept sialic acid in the TcTS reaction. Based on previous reports showing that anti α-Galp antibodies only partially colocalize with sialic acid, we have undertaken the synthesis of the trisaccharide α-d-Galp(1â3)-[ß-d-Galp(1â6)]-d-Galp, the smallest structure containing both, the antigenic d-Galp(α1â3)-d-Galp unit and the sialic acid-acceptor ß-d-Galp unit. The trisaccharide was obtained as the 6-aminohexyl glycoside to facilitate further conjugation for biochemical studies. The synthetic approach involved the α-galactosylation at O-4 of a suitable precursor of the reducing end, followed by ß-galactosylation at O-6 of the same precursor and introduction of the 6-aminohexyl aglycone. The fully deprotected trisaccharide was successfully sialylated by TcTS using either 3'-sialyllactose or fetuin as donors. The product, 6-aminohexyl α-d-NeuNAc(2â3)-ß-d-Galp(1â6)-[α-d-Galp(1â3)]-ß-d-Galp, was purified and characterized.
Assuntos
Anticorpos/química , Glicoproteínas/metabolismo , Neuraminidase/metabolismo , Trissacarídeos/síntese química , Trypanosoma cruzi/metabolismo , Anticorpos/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Sequência de Carboidratos , Técnicas de Química Sintética , Proteínas de Transporte de Monossacarídeos/imunologia , Proteínas Periplásmicas de Ligação/imunologia , Trissacarídeos/metabolismoRESUMO
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.
Assuntos
Proteínas Periplásmicas de Ligação/metabolismo , Proteômica/métodos , Sulfatos/metabolismo , Xanthomonas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Citrus sinensis/microbiologia , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Doenças das Plantas/microbiologia , Ligação Proteica , Domínios Proteicos , Homologia de Sequência de Aminoácidos , Sulfatos/química , Xanthomonas/genética , Xanthomonas/fisiologiaRESUMO
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.
Assuntos
Regulação Bacteriana da Expressão Gênica , Proteínas Periplásmicas de Ligação/genética , Proteínas de Ligação a Fosfato/genética , Processamento Pós-Transcricional do RNA , Transcrição Gênica , Vibrio cholerae O1/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Transporte Biológico , Códon/química , Códon/metabolismo , Biologia Computacional , Endorribonucleases/genética , Endorribonucleases/metabolismo , Sequências Repetidas Invertidas , Óperon , Proteínas Periplásmicas de Ligação/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Fosfatos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Vibrio cholerae O1/metabolismo , Vibrio cholerae O1/patogenicidade , VirulênciaRESUMO
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.
Assuntos
Escherichia coli/metabolismo , Vacinas de DNA/biossíntese , Ácido Acético/metabolismo , Biomassa , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Cinética , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismoRESUMO
Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane. We have cloned and sequenced the genes coding for the components of the iron ABC-type acquisition system of G. diazotrophicus. Sequence analysis revealed three ORFs, (feuA, feuB, and feuC) organized as an operon and encoding polypeptides of 346 (38 kDa), 342 (34.2 kDa), and 240 (26 kDa) amino acids, respectively. The deduced translation products of the feu operon showed similarity with a periplasmic solute-binding protein (FeuA), permease (FeuB), and ATPase (FeuC) involved in Fe transport. The role of FeuB in the survival of G. diazotrophicus under iron depletion was evaluated by comparing the ability of wild-type and FeuB-Km(R) -mutant strains in a medium without iron supplementation and in a medium containing 2, 2'-dipyridyl (DP). Growth of the mutant was affected in the medium containing DP. The operon was expressed at higher levels in cells depleted for iron than in those that contained the metal. A decrease in nitrogenase activity was observed with the FeuB-Km(R) -mutant strain that with the wild-type under iron deficiency conditions, suggesting that the Feu operon play role in Fe nutrition of G. diazotrophicus.
Assuntos
Proteínas de Bactérias/genética , Gluconacetobacter/genética , Ferro/metabolismo , Óperon , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Transporte Biológico , Gluconacetobacter/metabolismo , Proteínas de Membrana Transportadoras/genética , Fixação de Nitrogênio , Proteínas Periplásmicas de Ligação/genética , Saccharum/microbiologiaRESUMO
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.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Estresse Oxidativo , Proteínas Periplásmicas de Ligação/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas Periplásmicas de Ligação/genética , Peroxidases/metabolismo , Transcrição GênicaRESUMO
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.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Glutationa Peroxidase/metabolismo , Estresse Oxidativo , Proteínas Periplásmicas de Ligação/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Glutationa/química , Glutationa/metabolismo , Glutationa Peroxidase/química , Glutationa Peroxidase/genética , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genéticaRESUMO
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.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/genética , Estabilidade de RNA , Transcrição Gênica , Sequência de Bases , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Óperon , Proteínas Periplásmicas de Ligação/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Regiões Promotoras Genéticas , RNA Bacteriano/química , RNA Bacteriano/genética , Sequências Repetitivas de Ácido NucleicoRESUMO
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.
Assuntos
Molibdênio/química , Molibdênio/metabolismo , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/metabolismo , Xanthomonas axonopodis/química , Xanthomonas axonopodis/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Ligantes , Dados de Sequência Molecular , Proteínas Periplásmicas de Ligação/genética , Filogenia , Doenças das Plantas/microbiologia , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de Proteína , Xanthomonas axonopodis/genética , Xanthomonas axonopodis/patogenicidadeRESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Molibdênio/metabolismo , Proteínas Periplásmicas de Ligação/isolamento & purificação , Xanthomonas/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Óperon , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Dobramento de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Termodinâmica , Compostos de Tungstênio/metabolismo , Xanthomonas/metabolismoRESUMO
Xanthomonas axonopodis pv. citri ModA protein is the ABC periplasmic binding component responsible for the capture of molybdate. The protein was crystallized with sodium molybdate using the hanging-drop vapour-diffusion method in the presence of PEG or sulfate. X-ray diffraction data were collected to a maximum resolution of 1.7 A using synchrotron radiation. The crystal belongs to the orthorhombic space group C222(1), with unit-cell parameters a = 68.15, b = 172.14, c = 112.04 A. The crystal structure was solved by molecular-replacement methods and structure refinement is in progress.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Molibdênio/metabolismo , Proteínas Periplásmicas de Ligação/química , Xanthomonas/química , Cristalização/métodos , Cristalografia por Raios X , Escherichia coli/metabolismo , Proteínas Recombinantes/químicaRESUMO
A homologue of the ferric uptake regulator gene, fur, was identified from a Azospirillum brasilense Sp7 genomic DNA clone. Experiments performed with transcriptional lacZ fusions demonstrated that the A. brasilense fur homologue regulated the expression of two fur regulated Escherichia coli genes: fiu (ferric iron uptake) and fhuF (ferric hydroxamate uptake). A differential regulation by the cognate Fur and the heterologous Fur homologue in response to the iron status of the growth medium was also observed.
Assuntos
Azospirillum brasilense/genética , Proteínas de Bactérias/metabolismo , Ferro/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Azospirillum brasilense/enzimologia , Proteínas da Membrana Bacteriana Externa , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Proteínas de Ligação ao Ferro , Dados de Sequência Molecular , Proteínas Periplásmicas de Ligação , Proteínas Repressoras/genética , Alinhamento de Sequência , Transformação BacterianaRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fosfatos/metabolismo , Regulon , Fator sigma/metabolismo , Transcrição Gênica , Fosfatase Alcalina/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Transporte/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas Periplásmicas de Ligação/metabolismo , Proteínas de Ligação a Fosfato , Porinas/metabolismo , Regiões Promotoras Genéticas , RNA Bacteriano/análise , RNA Bacteriano/isolamento & purificação , RNA Mensageiro/análise , RNA Mensageiro/isolamento & purificação , Fator sigma/genéticaRESUMO
RT-PCR is a powerful technique used in the amplification and detection of rare mRNAs. However, one of the most serious drawbacks of this method is the amplification of false-positive products due to DNA contamination in the RNA samples. This pitfall is particularly hard to overcome when RNA from prokaryotic origin is used. We present here a modification of the EXACT RT-PCR method that was successfully employed in the amplification of the low abundant full-length polycistronic pst operon mRNA of Escherichia coli. No DNase treatment of the RNA template is required, but unlike the original EXACT RT-PCR, a hybrid primer that is not composed of oligo(dT) was used. A nonhomologous sequence was incorporated at the reverse transcription step into the 5' end of the first-strand cDNA by means of the hybrid primer. For the PCR, a gene-specific primer and a second primer identical to the nonhomologous portion of the hybrid primer were used. To avoid amplification of genomic DNA, the hybrid-primer molecules that were not incorporated into the first-strand cDNA were removed by RNase H treatment followed by ultrafiltration.
Assuntos
Escherichia coli/genética , Óperon/genética , RNA Bacteriano/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Desoxirribonucleases/química , Desoxirribonucleases/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Proteínas de Ligação a Fosfato , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonuclease H/metabolismoRESUMO
Trypanosoma cruzi must invade mammalian host cells to replicate and complete its life cycle. Almost all nucleated mammalian cells can be invaded by the parasite following a receptor-ligand recognition as an early prerequisite. In this work, we describe a 67-kDa lectin-like glycoprotein that binds to desialylated human erythrocyte membranes in a galactose-dependent way. This protein is present on the parasite surface in both infective and non-infective stages of T. cruzi. More interestingly, we demonstrate by lectin-immuno-histochemistry assays that the 67kDa protein is involved in the recognition of host-cell receptors in mouse cardiac tissue and human cardiac aortic endothelium and mammary artery tissue. Moreover, antibodies against the 67kDa glycoprotein inhibit in vitro host-cell invasion by 63%. These data suggest that the 67kDa glycoprotein in vivo is needed for host-cell invasion by T. cruzi.
Assuntos
Proteínas de Ligação ao Cálcio , Membrana Eritrocítica/metabolismo , Proteínas de Helminto/isolamento & purificação , Proteínas de Transporte de Monossacarídeos/isolamento & purificação , Proteínas Periplásmicas de Ligação , Trypanosoma cruzi/fisiologia , Animais , Western Blotting , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/metabolismo , Endotélio Vascular/parasitologia , Membrana Eritrocítica/parasitologia , Imunofluorescência , Galactose/metabolismo , Coração/parasitologia , Proteínas de Helminto/imunologia , Proteínas de Helminto/fisiologia , Humanos , Soros Imunes/imunologia , Imuno-Histoquímica , Lectinas , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Transporte de Monossacarídeos/imunologia , Proteínas de Transporte de Monossacarídeos/fisiologia , Coelhos , Trypanosoma cruzi/químicaRESUMO
Our approach to the isolation of plant-inducible bacterial genes of Azospirillum brasilense, based on the analysis of protein patterns of bacteria grown in the presence and in the absence of plant root exudates, led to the identification of an acidic 40 kDa protein. Cloning and sequencing analysis of the corresponding coding DNA region revealed the presence of two open reading frames transcribed in the same orientation. The deduced ORF1 protein, which corresponds to the 40 kDa protein, is very similar to the periplasmic ChvE protein, identified in Agrobacterium tumefaciens and involved in enhanced virulence. The deduced ORF2 protein shows homology to members of the LysR family of transcriptional regulators. The function of the ChvE-like protein in A. brasilense was investigated further. The protein, designated as SbpA (sugar binding protein A), is involved in the uptake of D-galactose and functions in the chemotaxis of A. brasilense towards several sugars, including D-galactose, L-arabinose and D-fucose. Expression of the sbpA gene requires the presence of the same sugars in the growth medium and is enhanced further in combination with carbon starvation of A. brasilense cells.
Assuntos
Azospirillum brasilense/genética , Metabolismo dos Carboidratos , Proteínas de Membrana Transportadoras , Proteínas de Transporte de Monossacarídeos/genética , Proteínas Periplásmicas de Ligação , Agrobacterium tumefaciens/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Quimiotaxia/genética , Clonagem Molecular , Galactose/metabolismo , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Fases de Leitura Aberta , Raízes de Plantas/microbiologia , Ligação Proteica , Alinhamento de Sequência , Triticum/metabolismoRESUMO
Azospirillum brasilense is able to use glycine betaine as a powerful osmoprotectant; the uptake of this compound is strongly stimulated by salt stress, but significantly reduced by cold osmotic shock. Non-denaturing PAGE in the presence of [methyl-14C] glycine betaine and autoradiography demonstrated the presence of one glycine betaine-binding protein (GBBP) in periplasmic shock fluid obtained from high-osmolarity-grown cells. The binding activity was absent in periplasmic fractions from cells grown at low osmolarity. SDS-PAGE analysis showed that the osmotically inducible GBBP has an apparent molecular weight of 32,000. The isoelectric point was between 5.9 and 6.6, as determined by isoelectric focusing. This protein bound glycine betaine with high affinity (KD of 3 microM), but had no affinity for either other betaines (proline betaine, gamma-butyrobetaine, pipecolate betaine, trigonelline, homarine) or related compounds (choline, glycine betaine aldehyde, glycine and proline). Optimum binding activity occurred at pH 7.0 to 7.5, and was not altered whether or not the binding assays were done at low or high osmolarity. Immunoprecipitation and Western blotting showed that immunoadsorbed anti-GBBP antibody from E coli cross-reacted with the GBBP produced by A brasilense cells grown at high osmolarity.