The RSP_2889 gene product of Rhodobacter sphaeroides is a CueR homologue controlling copper-responsive genes

Microbiology ◽  
2011 ◽  
Vol 157 (12) ◽  
pp. 3306-3313 ◽  
Author(s):  
Verena Peuser ◽  
Jens Glaeser ◽  
Gabriele Klug
Keyword(s):  
Gene Product ◽  
Rhodobacter Sphaeroides ◽  
Copper Ions ◽  
Copper Metabolism ◽  
Photosynthetic Bacterium ◽  
Copper Resistance ◽  
Copper Chaperone ◽  
Promoter Regions ◽  
Low Copy Number ◽  
Increased Sensitivity

Metal homeostasis is important in all living cells in order to provide sufficient amounts of metal ions for biological processes but to prevent toxic effects by excess amounts. Here we show that the gene product of RSP_2889 of the facultatively photosynthetic bacterium Rhodobacter sphaeroides is homologous to CueR, a regulator of copper metabolism in Escherichia coli and other bacteria. CueR binds to the promoter regions of genes for a copper-translocating ATPase and for a copper chaperone and is responsible for their high expression when cells are exposed to elevated levels of copper ions. While deletion of RSP_2889 has no significant effect on copper resistance, expression from a low-copy-number plasmid mediates increased sensitivity to copper.

scholarly journals Characterization of the CopR Regulon of Lactococcus lactis IL1403

2007 ◽  
Vol 190 (2) ◽  
pp. 536-545 ◽  
Author(s):  
David Magnani ◽  
Olivier Barré ◽  
Simon D. Gerber ◽  
Marc Solioz
Keyword(s):  
Lactococcus Lactis ◽  
Copper Metabolism ◽  
Copper Resistance ◽  
Copper Chaperone ◽  
Sequence Motifs ◽  
Lactate Oxidase ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Two Dimensional Gel Electrophoresis ◽  
A Genome

ABSTRACT To identify components of the copper homeostatic mechanism of Lactococcus lactis, we employed two-dimensional gel electrophoresis to detect changes in the proteome in response to copper. Three proteins upregulated by copper were identified: glyoxylase I (YaiA), a nitroreductase (YtjD), and lactate oxidase (LctO). The promoter regions of these genes feature cop boxes of consensus TACAnnTGTA, which are the binding site of CopY-type copper-responsive repressors. A genome-wide search for cop boxes revealed 28 such sequence motifs. They were tested by electrophoretic mobility shift assays for the interaction with purified CopR, the CopY-type repressor of L. lactis. Seven of the cop boxes interacted with CopR in a copper-sensitive manner. They were present in the promoter region of five genes, lctO, ytjD, copB, ydiD, and yahC; and two polycistronic operons, yahCD-yaiAB and copRZA. Induction of these genes by copper was confirmed by real-time quantitative PCR. The copRZA operon encodes the CopR repressor of the regulon; a copper chaperone, CopZ; and a putative copper ATPase, CopA. When expressed in Escherichia coli, the copRZA operon conferred copper resistance, suggesting that it functions in copper export from the cytoplasm. Other member genes of the CopR regulon may similarly be involved in copper metabolism.

scholarly journals Functional and Expression Analyses of the cop Operon, Required for Copper Resistance in Agrobacterium tumefaciens

2009 ◽  
Vol 191 (16) ◽  
pp. 5159-5168 ◽  
Author(s):  
Sirikan Nawapan ◽  
Nisanart Charoenlap ◽  
Anchalee Charoenwuttitam ◽  
Panatda Saenkham ◽  
Skorn Mongkolsuk ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Copper Ions ◽  
Protein A ◽  
Copper Resistance ◽  
Copper Chaperone ◽  
Phytopathogenic Bacterium ◽  
Ion Concentrations ◽  
Increased Sensitivity ◽  
Cop Operon

ABSTRACT The copper resistance determinant copARZ, which encodes a CPx-type copper ATPase efflux protein, a transcriptional regulator, and a putative intracellular copper chaperone, was functionally characterized for the phytopathogenic bacterium Agrobacterium tumefaciens. These genes are transcribed as an operon, and their expression is induced in response to increasing copper and silver ion concentrations in a copR-dependent fashion. Analysis of the copARZ promoter revealed a putative CopR binding box located within the spacer of the −35 and −10 promoter motifs. In vitro, purified CopR could specifically bind to the box. The inactivation of the copARZ operon or copZ reduces the level of resistance to copper but not to other metal ions. Also, the copARZ operon mutant shows increased sensitivity to the superoxide generators menadione and plumbagin. In addition, the loss of functional copZ does not affect the ability of copper ions to induce the copARZ promoter, indicating that CopZ is not involved in the copper-sensing mechanism of CopR. Altogether, the results demonstrate a crucial role for the copARZ operon as a component of the copper resistance machinery in A. tumefaciens.

Evidence for two recA genes mediating DNA repair in Bacillus megaterium

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 775-787 ◽  
Author(s):  
Hannes Nahrstedt ◽  
Christine Schröder ◽  
Friedhelm Meinhardt
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Mitomycin C ◽  
Bacillus Megaterium ◽  
Functional Complementation ◽  
Homologous Gene ◽  
Gene Products ◽  
Null Mutant ◽  
Promoter Regions ◽  
Increased Sensitivity

Isolation and subsequent knockout of a recA-homologous gene in Bacillus megaterium DSM 319 resulted in a mutant displaying increased sensitivity to mitomycin C. However, this mutant did not exhibit UV hypersensitivity, a finding which eventually led to identification of a second functional recA gene. Evidence for recA duplicates was also obtained for two other B. megaterium strains. In agreement with potential DinR boxes located within their promoter regions, expression of both genes (recA1 and recA2) was found to be damage-inducible. Transcription from the recA2 promoter was significantly higher than that of recA1. Since a recA2 knockout could not be achieved, functional complementation studies were performed in Escherichia coli. Heterologous expression in a RecA null mutant resulted in increased survival after UV irradiation and mitomycin C treatment, proving both recA gene products to be functional in DNA repair. Thus, there is evidence for an SOS-like pathway in B. megaterium that differs from that of Bacillus subtilis.

scholarly journals Metal-binding mechanism of Cox17, a copper chaperone for cytochrome c oxidase

2004 ◽  
Vol 382 (1) ◽  
pp. 307-314 ◽  
Author(s):  
Peep PALUMAA ◽  
Liina KANGUR ◽  
Anastassia VORONOVA ◽  
Rannar SILLARD
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Metal Binding ◽  
Copper Ions ◽  
Copper Chaperone ◽  
Copper Chaperones ◽  
Rate Limiting Step ◽  
Charge State Distributions ◽  
Rate Limiting ◽  
Partner Proteins

Cox17, a copper chaperone for cytochrome c oxidase, is an essential and highly conserved protein. The structure and mechanism of functioning of Cox17 are unknown, and even its metalbinding stoichiometry is elusive. In the present study, we demonstrate, using electrospray ionization–MS, that porcine Cox17 binds co-operatively four Cu+ ions. Cu4Cox17 is stable at pH values above 3 and fluorescence spectra indicate the presence of a solvent-shielded multinuclear Cu(I) cluster. Combining our results with earlier EXAFS results on yeast CuCox17, we suggest that Cu4Cox17 contains a Cu4S6-type cluster. At supramillimolar concentrations, dithiothreitol extracts metals from Cu4Cox17, and an apparent copper dissociation constant KCu=13 fM was calculated from these results. Charge-state distributions of different Cox17 forms suggest that binding of the first Cu+ ion to Cox17 causes a conformational change from an open to a compact state, which may be the rate-limiting step in the formation of Cu4Cox17. Cox17 binds non-co-operatively two Zn2+ ions, but does not bind Ag+ ions, which highlights its extremely high metal-binding specificity. We further demonstrate that porcine Cox17 can also exist in partly oxidized (two disulphide bridges) and fully oxidized (three disulphide bridges) forms. Partly oxidized Cox17 can bind one Cu+ or Zn2+ ion, whereas fully oxidized Cox17 does not bind metals. The metal-binding properties of Cox17 imply that, in contrast with other copper chaperones, Cox17 is designed for the simultaneous transfer of up to four copper ions to partner proteins. Metals can be released from Cox17 by non-oxidative as well as oxidative mechanisms.

scholarly journals Sandwich Hybridization Assay for Sensitive Detection of Dynamic Changes in mRNA Transcript Levels in Crude Escherichia coli Cell Extracts in Response to Copper Ions

2008 ◽  
Vol 74 (24) ◽  
pp. 7463-7470 ◽  
Author(s):  
Daniel Thieme ◽  
Peter Neubauer ◽  
Dietrich H. Nies ◽  
Gregor Grass
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Time Course ◽  
Copper Ions ◽  
Copper Resistance ◽  
Hybridization Assay ◽  
Target Mrna ◽  
Cell Extracts ◽  
Sandwich Hybridization ◽  
Sandwich Hybridization Assay

ABSTRACT Transcript quantification techniques usually rely on purified mRNAs. We report here a solution-based sandwich hybridization assay for the quantification of mRNAs from Escherichia coli without the need of prior RNA isolation. This assay makes use of four DNA oligonucleotide probes adjacently hybridizing to target RNA in clarified cell extracts. Two helper probes facilitate the hybridization of a detection and a capture probe. The latter is biotin labeled, allowing binding to streptavidin-coated paramagnetic beads and the separation of the RNA-DNA hybrid from cellular constituents. Added antidigoxigenin Fab fragments conjugated to alkaline phosphatase bind to the digoxigenin-labeled detection probe, completing the sandwich of the paramagnetic bead, mRNA, probes, and alkaline phosphatase. The target transcript can be quantified by assessing phosphatase activity on a substrate that is converted into a fluorescent product. The amount of target mRNA is calculated from the fluorescence output and from a calibration curve for a known concentration of in vitro-synthesized target mRNA. This technique was used in time course experiments to investigate the expression of three genes responsible for the copper resistance of E. coli. The induction of gene expression by copper cations was rapid, but under aerobic conditions, the levels of expression returned to low, prestress levels within minutes. In anaerobiosis, high-level expression continued for at least 1 h. When cultures were shifted from anaerobiosis to aerobiosis, expression levels were diminished within minutes to prestress levels. The improved technique presented here is relatively simple, has very high degrees of sensitivity and robustness, is less laborious than other RNA quantification methods, and is not negatively affected by genomic DNA. These characteristics make it a powerful complementary application to genetic reporter fusions and to reverse transcription-PCR.

scholarly journals Pathways Involved in Reductant Distribution during Photobiological H2Production by Rhodobacter sphaeroides

2011 ◽  
Vol 77 (20) ◽  
pp. 7425-7429 ◽  
Author(s):  
Wayne S. Kontur ◽  
Eva C. Ziegelhoffer ◽  
Melanie A. Spero ◽  
Saheed Imam ◽  
Daniel R. Noguera ◽  
...  
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Production Capacity ◽  
Photosynthetic Bacterium ◽  
Content Type ◽  
Hydrogenase Enzymes

ABSTRACTWe used global transcript analyses and mutant studies to investigate the pathways that impact H2production in the photosynthetic bacteriumRhodobacter sphaeroides. We found that H2production capacity is related to the levels of expression of the nitrogenase and hydrogenase enzymes and the enzymes of the Calvin-Benson-Bassham pathway.

scholarly journals The Brucella abortus xthA-1 Gene Product Participates in Base Excision Repair and Resistance to Oxidative Killing but Is Not Required for Wild-Type Virulence in the Mouse Model

2006 ◽  
Vol 188 (4) ◽  
pp. 1295-1300 ◽  
Author(s):  
Michael L. Hornback ◽  
R. Martin Roop
Keyword(s):  
Mouse Model ◽  
Oxidative Damage ◽  
Gene Product ◽  
Base Excision Repair ◽  
Brucella Abortus ◽  
Excision Repair ◽  
Wild Type ◽  
Exonuclease Iii ◽  
Increased Sensitivity ◽  
Base Excision

ABSTRACT Exonuclease III, encoded by the xthA gene, plays a central role in the base excision pathway of DNA repair in bacteria. Studies with Escherichia coli xthA mutants have also shown that exonuclease III participates in the repair of oxidative damage to DNA. An isogenic xthA-1 mutant (designated CAM220) derived from virulent Brucella abortus 2308 exhibited increased sensitivity to the alkylating agent methyl methanesulfonate (MMS) compared to the parent strain. In contrast, 2308 and the isogenic xthA-1 mutant displayed similar levels of resistance to the DNA cross-linker mitomycin C. These phenotypic properties are those that would be predicted for a strain defective in base excision repair. The B. abortus xthA-1 mutant also displayed reduced resistance to killing by H2O2 and the ONOO−-generating compound 3-morpholinosydnonimine (SIN-1) compared to strain 2308, indicating that the xthA-1 gene product participates in protecting B. abortus 2308 from oxidative damage. Introducing a plasmid-borne copy of the parental xthA-1 gene into CAM220 restored wild-type resistance of this mutant to MMS, H2O2, and SIN-1. Although the B. abortus xthA-1 mutant exhibited increased sensitivity to oxidative killing compared to the parental strain in laboratory assays, CAM220 and 2308 displayed equivalent spleen colonization profiles in BALB/c mice through 8 weeks postinfection and equivalent intracellular survival and replication profiles in cultured murine macrophages. Thus, although the xthA-1 gene product participates in base excision repair and resistance to oxidative killing in B. abortus 2308, XthA-1 is not required for wild-type virulence of this strain in the mouse model.

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