105 research outputs found
Improved protocol for chromatofocusing on the ProteomeLab PF2D
No full textBeckman-Coulter has recently introduced the ProteomeLab PF2D for 2-D liquid separation of protein samples. The system features separation in the first dimension by chromatofocusing, followed by RP chromatography in the second dimension, allowing the analysis of complex proteomics samples. When used by the standard protocol, reproducibility and column life times are limited, making the use of the instrument very costly. We here present an improved protocol for chromatofocusing, which enhances column life by at least fivefold
Evaluation of chocolate as a source of dietary copper
No full textChocolate has frequently been proposed to be a valuable source of dietary copper, but data on the copper content of major contemporary chocolate brands are scarce. The copper content of 22 brands of chocolate, many of which are sold worldwide, is thus presented here. A reliable hot ashing procedure to determine the copper content of chocolate by inductively coupled plasma atomic emission spectroscopy is also described. It was found that the copper contents of the chocolates analyzed here varied in the range of 1.85 ± 0.10 to 16.50 ± 1.29 μg/g. There was a linear correlation of the copper content of chocolate to its cocoa content with a correlation coefficient R2 of 0.89, showing that the copper was largely contributed to the chocolate by the cocoa. The value of chocolate as a source of dietary copper is discussed
Copper chaperone cycling and degradation in the regulation of the cop operon of Enterococcus hirae
No full textplasmon resonance Extensive insight into copper homeostasis has recently emerged. The Gram-positive bacterium Entero-coccus hirae has been a paradigm for many aspects of the process. The cop operon of E. hirae consists of four genes that encode a repressor, CopY, a copper chaperone, CopZ, and two CPx-type copper ATPases, CopA and CopB. CopA and CopB accomplish copper uptake and export, respectively, and the expression of the cop operon is regulated by copper via the CopY repressor and the CopZ chaperone. The functions of the four Cop proteins have been extensively studied in vivo as well as in vitro and a detailed understanding of the regulation of the cop operon by copper has emerged
Copper homeostasis in Enterococcus hirae
No full textCopper is an essential component of life because of its convenient redox potential of 200–800 mV when bound to protein. Extensive insight into copper homeostasis has only emerged in the last decade and Enterococcus hirae has served as a paradigm for many aspects of the process. The cop operon of E. hirae regulates copper uptake, availability, and export. It consists of four genes that encode a repressor, CopY, a copper chaperone, CopZ, and two CPx-type copper ATPases, CopA and CopB. Most of these components have been conserved across the three evolutionary kingdoms. The four Cop proteins have been studied in vivo as well as in vitro and their function is understood in some detail
Efficient transformation of Lactococcus lactis IL1403 and generation of knock-out mutants by homologous recombination
No full textLactococcus lactis IL1403 is a Gram-positive bacterium of great biotechnological interest for food grade applications. Its use is however hampered by the difficulty to efficiently transform this strain. We here describe a detailed, optimized electrotransformation protocol which yields a transformation efficiency of 10(6) cfu/microg of DNA with the two E. coli Gram-positive shuttle vectors pC3 and pVA838. The utility of the protocol was demonstrated by the generation of single- and double-knock-out mutants by homologous recombination
Desulfovibrio DA2_CueO is a novel multicopper oxidase with cuprous, ferrous and phenol oxidase activity
No full textDesulfovibrio sp. A2 is a novel Gram-negative sulfate-reducing bacterium that was isolated from sediments of the Norilsk mining/smelting area in Russia. The organism possesses a monocistronic operon encoding a 71 kDa periplasmic multicopperoxidase, which we call DA2_CueO. Histidine-tagged DA2_CueO expressed from a plasmid in Escherichia coli and purified by Ni–NTA affinity chromatography oxidizes Cu+ and Fe2+, and exhibits phenol oxidase activity with 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), 2,3-dihydroxybenzoic acid and 2,6-dimethoxyphenol as substrates, using O2 as the oxidant. When expressed in an E. coli cueO knock-out strain, DA2_CueO exhibits phenol oxidase activity in vivo and enhances the copper tolerance of the strain. These findings indicate that the DA2_CueO gene of Desulfovibrio sp. A2 encodes a multicopperoxidase with a role in metal ion resistance. The enzyme displays some novel structural features, which are discussed
Copper induction of lactate oxidase of Lactococcus lactis: a novel metal stress response
No full textLactococcus lactis IL1403, a lactic acid bacterium widely used for food fermentation, is often exposed to stress conditions. One such condition is exposure to copper, such as in cheese making in copper vats. Copper is an essential micronutrient in prokaryotes and eukaryotes but can be toxic if in excess. Thus, copper homeostatic mechanisms, consisting chiefly of copper transporters and their regulators, have evolved in all organisms to control cytoplasmic copper levels. Using proteomics to identify novel proteins involved in the response of L. lactis IL1403 to copper, cells were exposed to 200 muM copper sulfate for 45 min, followed by resolution of the cytoplasmic fraction by two-dimensional gel electrophoresis. One protein strongly induced by copper was LctO, which was shown to be a NAD-independent lactate oxidase. It catalyzed the conversion of lactate to pyruvate in vivo and in vitro. Copper, cadmium, and silver induced LctO, as shown by real-time quantitative PCR. A copper-regulatory element was identified in the 5' region of the lctO gene and shown to interact with the CopR regulator, encoded by the unlinked copRZA operon. Induction of LctO by copper represents a novel copper stress response, and we suggest that it serves in the scavenging of molecular oxygen
The copper-responsive repressor CopR of Lactococcus lactis is a 'winged helix' protein
No full textCopR of Lactococcus lactis is a copper-responsive repressor involved in copper homoeostasis. It controls the expression of a total of 11 genes, the CopR regulon, in a copper-dependent manner. In the absence of copper, CopR binds to the promoters of the CopR regulon. Copper releases CopR from the promoters, allowing transcription of the downstream genes to proceed. CopR binds through its N-terminal domain to a 'cop box' of consensus TACANNTGTA, which is conserved in Firmicutes. We have solved the NMR solution structure of the N-terminal DNA-binding domain of CopR. The protein fold has a winged helix structure resembling that of the BlaI repressor which regulates antibiotic resistance in Bacillus licheniformis. CopR differs from other copper-responsive repressors, and the present structure represents a novel family of copper regulators, which we propose to call the CopY family
Killing of bacteria by copper, cadmium, and silver surfaces reveals relevant physicochemical parameters.
No full textThe killing of bacteria on metallic copper surfaces in minutes to hours is referred to as contact killing. Why copper possesses such strong antimicrobial activity has remained enigmatic. Based on the physicochemical properties of metals, it was recently predicted that cadmium should also be active in contact killing [Hans et al., Biointerphases 11, 018902 (2010)]. Here, the authors show that cadmium is indeed antimicrobial. It kills three logs of bacteria in 9 h, compared to copper which kills eight logs of bacteria. Metallic silver kills less than one log of bacteria in 9 h. These findings support the novel concept whereby oxide formation, metal ion dissolution, and a Pearson soft character are the key factors for a metal to be antibacterial. Based on these parameters, copper and cadmium are expected to be the two most antibacterial metals
- …
