scholarly journals Thioredoxin Is Involved in U(VI) and Cr(VI) Reduction in Desulfovibrio desulfuricans G20

2009 ◽  
Vol 191 (15) ◽  
pp. 4924-4933 ◽  
Author(s):  
Xiangkai Li ◽  
Lee R. Krumholz
Keyword(s):  
Specific Inhibitor ◽  
Thioredoxin Reductase ◽  
Desulfovibrio Desulfuricans ◽  
Receptor Protein ◽  
Insertion Mutant ◽  
Gene Encoding ◽  
Transposon Insertion ◽  
Expression Studies ◽  
Genes Encoding ◽  
Camp Receptor

ABSTRACT A transposon insertion mutant has been identified in a Desulfovibrio desulfuricans G20 mutant library that does not grow in the presence of 2 mM U(VI) in lactate-sulfate medium. This mutant has also been shown to be deficient in the ability to grow with 100 μM Cr(VI) and 20 mM As(V). Experiments with washed cells showed that this mutant had lost the ability to reduce U(VI) or Cr(VI), providing an explanation for the lower tolerance. A gene encoding a cyclic AMP (cAMP) receptor protein (CRP) was identified as the site of the transposon insertion. The remainder of the mre operon (metal reduction) contains genes encoding a thioredoxin, thioredoxin reductase, and an additional oxidoreductase whose substrate has not been predicted. Expression studies showed that in the mutant, the entire operon is downregulated, suggesting that the CRP may be involved in regulating expression of the whole operon. Exposure of the cells to U(VI) resulted in upregulation of the entire operon. CdCl2, a specific inhibitor of thioredoxin activity, inhibits U(VI) reduction by washed cells and inhibits growth of cells in culture when U(VI) is present, confirming a role for thioredoxin in U(VI) reduction. The entire mre operon was cloned into Escherichia coli JM109 and the transformant developed increased U(VI) resistance and the ability to reduce U(VI) to U(IV). The oxidoreductase protein (MreG) from this operon was expressed and purified from E. coli. In the presence of thioredoxin, thioredoxin reductase, and NADPH, this protein was shown to reduce both U(VI) and Cr(VI), providing a mechanism for the cytoplasmic reduction of these metals.

scholarly journals CAU-1, a Subclass B3 Metallo-β-Lactamase of Low Substrate Affinity Encoded by an Ortholog Present in the Caulobacter crescentus Chromosome

2002 ◽  
Vol 46 (6) ◽  
pp. 1823-1830 ◽  
Author(s):  
Jean-Denis Docquier ◽  
Fabrizio Pantanella ◽  
Francesco Giuliani ◽  
Maria Cristina Thaller ◽  
Gianfranco Amicosante ◽  
...  
Keyword(s):  
Caulobacter Crescentus ◽  
Hypothetical Protein ◽  
Exchange Chromatography ◽  
Substrate Affinity ◽  
Gene Encoding ◽  
Native Form ◽  
Significant Similarity ◽  
Expression Studies ◽  
Genes Encoding ◽  
Isoelectric Ph

ABSTRACT The sequenced chromosome of Caulobacter crescentus CB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to metallo-β-lactamases of subclass B3. An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned in Escherichia coli from C. crescentus type strain DSM4727. Expression studies confirmed the metallo-β-lactamase activity of its product, CAU-1. The enzyme produced in E. coli was purified by two ion-exchange chromatography steps. CAU-1 contains a 29-kDa polypeptide with an alkaline isoelectric pH (>9), and unlike the L1 enzyme of Stenotrophomonas maltophilia, the native form is monomeric. Kinetic analysis revealed a preferential activity toward penicillins, carbapenems, and narrow-spectrum cephalosporins, while oxyimino cephalosporins were poorly or not hydrolyzed. Affinities for the various β-lactams were poor overall (Km values were always >100 μM and often >400 μM). The interaction with divalent ion chelators appeared to occur by a mechanism similar to that prevailing in other members of subclass B3. In C. crescentus, the CAU-1 enzyme is produced independently of β-lactam exposure and, interestingly, the bla CAU determinant is bracketed by three other genes, including two genes encoding enzymes involved in methionine biosynthesis and a gene encoding a putative transcriptional regulator, in an operon-like structure. The CAU-1 enzyme is the first example of a metallo-β-lactamase in a member of the α subdivision of the class Proteobacteria.

scholarly journals Genes Contributing to Staphylococcus aureus Fitness in Abscess- and Infection-Related Ecologies

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Michael D. Valentino ◽  
Lucy Foulston ◽  
Ama Sadaka ◽  
Veronica N. Kos ◽  
Regis A. Villet ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
New Drugs ◽  
Insertion Mutant ◽  
Mutant Library ◽  
Entire Genome ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Transposon Insertion ◽  
Genes Encoding ◽  
Novel Targets

ABSTRACTStaphylococcus aureusis a leading cause of both community- and hospital-acquired infections that are increasingly antibiotic resistant. The emergence ofS. aureusresistance to even last-line antibiotics heightens the need for the development of new drugs with novel targets. We generated a highly saturated transposon insertion mutant library in the genome ofS. aureusand used Tn-seq analysis to probe the entire genome, with unprecedented resolution and sensitivity, for genes of importance in infection. We further identified genes contributing to fitness in various infected compartments (blood and ocular fluids) and compared them to genes required for growth in rich medium. This resulted in the identification of 426 genes that were important forS. aureusfitness during growth in infection models, including 71 genes that could be considered essential for survival specifically during infection. These findings highlight novel as well as previously known genes encoding virulence traits and metabolic pathways important forS. aureusproliferation at sites of infection, which may represent new therapeutic targets.IMPORTANCEStaphylococcus aureuscontinues to be a leading cause of antibiotic-resistant community and nosocomial infection. With the bacterium’s acquisition of resistance to methicillin and, more recently, vancomycin, the need for the development of new drugs with novel targets is urgent. Applying a highly saturated Tn-seq mutant library to analyze fitness and growth requirements in a murine abscess and in various infection-relevant fluids, we identifiedS. aureustraits that enable it to survive and proliferate during infection. This identifies potential new targeting opportunities for the development of novel therapeutics.

scholarly journals Interplay between Cyclic AMP-Cyclic AMP Receptor Protein and Cyclic di-GMP Signaling in Vibrio cholerae Biofilm Formation

2008 ◽  
Vol 190 (20) ◽  
pp. 6646-6659 ◽  
Author(s):  
Jiunn C. N. Fong ◽  
Fitnat H. Yildiz
Keyword(s):  
Cyclic Amp ◽  
Vibrio Cholerae ◽  
Transcriptome Analysis ◽  
Biofilm Formation ◽  
Matrix Proteins ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Genes Encoding ◽  
Camp Receptor ◽  
Biofilm Matrix

ABSTRACT Vibrio cholerae is a facultative human pathogen. The ability of V. cholerae to form biofilms is crucial for its survival in aquatic habitats between epidemics and is advantageous for host-to-host transmission during epidemics. Formation of mature biofilms requires the production of extracellular matrix components, including Vibrio polysaccharide (VPS) and matrix proteins. Biofilm formation is positively controlled by the transcriptional regulators VpsR and VpsT and is negatively regulated by the quorum-sensing transcriptional regulator HapR, as well as the cyclic AMP (cAMP)-cAMP receptor protein (CRP) regulatory complex. Transcriptome analysis of cyaA (encoding adenylate cyclase) and crp (encoding cAMP receptor protein) deletion mutants revealed that cAMP-CRP negatively regulates transcription of both VPS biosynthesis genes and genes encoding biofilm matrix proteins. Further mutational and expression analysis revealed that cAMP-CRP negatively regulates transcription of vps genes indirectly through its action on vpsR transcription. However, negative regulation of the genes encoding biofilm matrix proteins by cAMP-CRP can also occur independent of VpsR. Transcriptome analysis also revealed that cAMP-CRP regulates the expression of a set of genes encoding diguanylate cyclases (DGCs) and phosphodiesterases. Mutational and phenotypic analysis of the differentially regulated DGCs revealed that a DGC, CdgA, is responsible for the increase in biofilm formation in the Δcrp mutant, showing the connection between of cyclic di-GMP and cAMP signaling in V. cholerae.

fur-independent regulation of the Pasteurella multocida hbpA gene encoding a haemin-binding protein

Microbiology ◽  
2003 ◽  
Vol 149 (8) ◽  
pp. 2273-2281 ◽  
Author(s):  
M. Elena Garrido ◽  
Montserrat Bosch ◽  
Ricardo Medina ◽  
Anna Bigas ◽  
Montserrat Llagostera ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Pasteurella Multocida ◽  
Outer Membrane Proteins ◽  
Protein Composition ◽  
Electrophoretic Analysis ◽  
Gene Encoding ◽  
Expression Studies ◽  
Genes Encoding ◽  
Fur Protein ◽  
Iron Manganese

Treatment of bacterial cultures with chelating agents such as 2,2′-dipyridyl (DPD) induces expression of iron-regulated genes. It is known that in the γ-Proteobacteria, the Fur protein is the major regulator of genes encoding haem- or haemoglobin-binding proteins. Electrophoretic analysis of outer-membrane proteins of the γ-proteobacterium Pasteurella multocida has revealed the induction of two proteins of 60 and 40 kDa in DPD-treated cultures in both wild-type and fur-defective strains. These two proteins have the same N-terminal amino acid sequence, which identifies this protein as the product of the PM0592 ORF. Analysis of the sequence of this ORF, which encodes a protein of 60 kDa, revealed the presence of a hexanucleotide (AAAAAA) at which a programmed translational frameshift can occur giving rise to a 40 kDa protein. Analyses conducted in Escherichia coli, using the complete PM0592 ORF and a derivative truncated at the hexanucleotide position, have shown that both polypeptides bind haemin. For this reason, the PM0592 ORF product has been designated HbpA (for haemin-binding protein). Expression studies using both RT-PCR and lacZ fusions, as well as electrophoretic profiles of outer-membrane protein composition, have demonstrated that the hbpA gene is negatively regulated by iron, manganese and haemin through a fur-independent pathway. Despite the fact that serum of mice infected with P. multocida contained antibodies that reacted with both the 60 and 40 kDa products of the hbpA gene, these proteins did not offer protection when used in immunization assays against this micro-organism.

scholarly journals N-Acetyl-d-Glucosamine Induces the Expression of Multidrug Exporter Genes, mdtEF, via Catabolite Activation in Escherichia coli

2006 ◽  
Vol 188 (16) ◽  
pp. 5851-5858 ◽  
Author(s):  
Hidetada Hirakawa ◽  
Yoshihiko Inazumi ◽  
Yasuko Senda ◽  
Asuka Kobayashi ◽  
Takahiro Hirata ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signaling Pathways ◽  
Protein Gene ◽  
The Other ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Gene Encoding ◽  
E Coli ◽  
Camp Receptor ◽  
Multiple Signaling

ABSTRACT The expression of MdtEF, a multidrug exporter in Escherichia coli, is positively controlled through multiple signaling pathways, but little is known about signals that induce MdtEF expression. In this study, we investigated compounds that induce the expression of the mdtEF genes and found that out of 20 drug exporter genes in E. coli, the expression of mdtEF is greatly induced by N-acetyl-d-glucosamine (GlcNAc). The induction of mdtEF by GlcNAc is not mediated by the evgSA, ydeO, gadX, and rpoS signaling pathways that have been known to regulate mdtEF expression. On the other hand, deletion of the nagE gene, encoding the phosphotransferase (PTS) system for GlcNAc, prevented induction by GlcNAc. The induction of mdtEF by GlcNAc was also greatly inhibited by the addition of cyclic AMP (cAMP) and completely abolished upon deletion of the cAMP receptor protein gene (crp). Other PTS sugars, glucose and d-glucosamine, also induced mdtEF gene expression. These results suggest that mdtEF expression is stimulated through catabolite control.

An HcpR homologue from Desulfovibrio desulfuricans and its possible role in nitrate reduction and nitrosative stress

2011 ◽  
Vol 39 (1) ◽  
pp. 224-229 ◽  
Author(s):  
Ian T. Cadby ◽  
Steve J.W. Busby ◽  
Jeffrey A. Cole
Keyword(s):  
Nitrate Reduction ◽  
Nitrosative Stress ◽  
Consensus Sequence ◽  
Desulfovibrio Desulfuricans ◽  
Receptor Protein ◽  
Major Function ◽  
E Coli ◽  
Sulfate Reducing ◽  
Camp Receptor ◽  
Desulfovibrio Species

The Escherichia coli CRP (cAMP receptor protein), is a global regulator of transcription that modulates gene expression by activation or repression at a range of promoters in E. coli. A major function is to regulate the selection of nutrients required for growth. The anaerobic sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC27774 is capable of utilizing sulfate, nitrite and nitrate as terminal electron acceptors. In the presence of both sulfate and nitrate, sulfate is reduced preferentially despite nitrate being the thermodynamically more favourable electron acceptor. Three inverted repeat sequences upstream of the D. desulfuricans ATCC27774 nap (nitrate reduction in the periplasm) operon have high levels of similarity to the consensus sequence for the E. coli CRP DNA-binding site. In other Desulfovibrio species a putative CRP homologue, HcpR [regulator of hcp (hybrid cluster protein) transcription], has a predicted regulon comprising genes involved in sulfate reduction and nitrosative stress. The presence of CRP consensus sites within the D. desulfuricans ATCC27774 nap promoter prompted a search for CRP homologues in the genomes of sulfate-reducing bacteria. This revealed the presence of a potential CRP homologue that we predict binds to CRP consensus sites such as those of the nap operon. Furthermore, we predict that much of the core HcpR regulon predicted in other Desulfovibrio species is conserved in D. desulfuricans.

scholarly journals Control of Transposon-mediated Activation of theglpFKOperon ofEscherichia coliby two DNA binding Proteins

2016 ◽  
Author(s):  
Zhongge Zhang ◽  
Milton H. Saier
Keyword(s):  
Insertion Sequence ◽  
Gene Activation ◽  
Regulatory Region ◽  
Receptor Protein ◽  
Upstream Region ◽  
Upstream Regulatory Region ◽  
Wild Type ◽  
Transposon Insertion ◽  
Glycerol Utilization ◽  
Camp Receptor

AbstractEscherichia colicells deleted for the cyclic AMP (cAMP) receptor protein (Crp) gene (Δcrp) cannot utilize glycerol because cAMP-Crp is a required positive activator of glycerol utilization operonglpFK. We have previously shown that a transposon, Insertion Sequence 5 (IS5), can reversibly insert into the upstream regulatory region of the operon so as to activateglpFKand enable glycerol utilization. GlpR, which repressesglpFKtranscription, binds to theglpFKupstream region near the site of IS5insertion, and prevents insertion. We here show that the cAMP-Crp complex, which also binds to theglpFKupstream regulatory region, also inhibits IS5hopping into the activating site. This finding allowed us to identify conditions under which wild type cells can acquireglpFK-activating IS5insertions. Maximal rates of IS5insertion into the activating site require the presence of glycerol as well as a non-metabolizable sugar analogue that lowers cytoplasmic cAMP concentrations. Under these conditions, IS5insertional mutants accumulate and outcompete the wild type cells. Because of the widespread distribution of glucose analogues in nature, this mechanism of gene activation could have evolved by natural selection.

scholarly journals Functional Interactions between the Carbon and Iron Utilization Regulators, Crp and Fur, in Escherichia coli

2005 ◽  
Vol 187 (3) ◽  
pp. 980-990 ◽  
Author(s):  
Zhongge Zhang ◽  
Guillermo Gosset ◽  
Ravi Barabote ◽  
Claudio S. Gonzalez ◽  
William A. Cuevas ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Transcription Factors ◽  
Fatty Acid Synthase ◽  
Glycogen Synthesis ◽  
Krebs Cycle ◽  
Receptor Protein ◽  
Published Data ◽  
Cyclopropane Fatty Acid ◽  
Gene Encoding ◽  
Genes Encoding

ABSTRACT In Escherichia coli, the ferric uptake regulator (Fur) controls expression of the iron regulon in response to iron availability while the cyclic AMP receptor protein (Crp) regulates expression of the carbon regulon in response to carbon availability. We here identify genes subject to significant changes in expression level in response to the loss of both Fur and Crp. Many iron transport genes and several carbon metabolic genes are subject to dual control, being repressed by the loss of Crp and activated by the loss of Fur. However, the sodB gene, encoding superoxide dismutase, and the aceBAK operon, encoding the glyoxalate shunt enzymes, show the opposite responses, being activated by the loss of Crp and repressed by the loss of Fur. Several other genes including the sdhA-D, sucA-D, and fumA genes, encoding key constituents of the Krebs cycle, proved to be repressed by the loss of both transcription factors. Finally, the loss of both Crp and Fur activated a heterogeneous group of genes under σS control encoding, for example, the cyclopropane fatty acid synthase, Cfa, the glycogen synthesis protein, GlgS, the 30S ribosomal protein, S22, and the mechanosensitive channel protein, YggB. Many genes appeared to be regulated by the two transcription factors in an apparently additive fashion, but apparent positive or negative cooperativity characterized several putative Crp/Fur interactions. Relevant published data were evaluated, putative Crp and Fur binding sites were identified, and representative results were confirmed by real-time PCR. Molecular explanations for some, but not all, of these effects are provided.

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