CzcE from Cupriavidus metallidurans CH34 is a copper-binding protein

CzcE is encoded by the most distal gene of the czc determinant that allows Cupriavidus metallidurans CH34 to modulate its internal concentrations of cobalt, zinc and cadmium by regulation of the expression of the efflux pump CzcCBA. We have overproduced and purified CzcE. CzcE is a periplasm-located dimeric protein able to bind specifically 4 Cu-equivalent per dimer. Spectrophotometry and EPR are indicative of type II copper with typical d-d transitions. Re-oxidation of fully reduced CzcE led to the formation of an air stable semi-reduced form binding both 2 Cu(I) and 2 Cu(II) ions. The spectroscopic characteristics of the semi-reduced form are different of those of the oxidized one, suggesting a change in the environment of Cu(II).     

The response of <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> CH34 to spaceflight in the international space station

The survival and behavior of Cupriavidus metallidurans strain CH34 were tested in space. In three spaceflight experiments, during three separate visits to the ‘International Space Station’ (ISS), strain CH34 was grown for 10–12 days at ambient temperature on mineral agar medium. Space- and earth-grown cells were compared post-flight by flow cytometry and using 2D-gel protein analysis. Pre-, in- and post-flight incubation conditions and experiment design had a significant impact on the survival and growth of CH34 in space. In the CH34 cells returning from spaceflight, 16 proteins were identified which were present in higher concentration in cells developed in spaceflight conditions. These proteins were involved in a specific response of CH34 to carbon limitation and oxidative stress, and included an acetone carboxylase subunit, fructose biphosphate aldolase, a DNA protection during starvation protein, chaperone protein, universal stress protein, and alkyl hydroperoxide reductase. The reproducible observation of the over-expression of these same proteins in multiple flight experiments, indicated that the CH34 cells could experience a substrate limitation and oxidative stress in spaceflight where cells and substrates are exposed to lower levels of gravity and higher doses of ionizing radiation. Bacterium C. metallidurans CH34 was able to grow normally under spaceflight conditions with very minor to no effects on cell physiology, but nevertheless specifically altered the expression of a few proteins in response to the environmental changes.     

Biostructural analysis of the metal-sensor domain of CnrX from <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> CH34

In Cupriavidus metallidurans CH34, the proteins CnrX, CnrY, and CnrH regulate the expression of the cnrCBA operon that codes for a cation-efflux pump involved in cobalt and nickel resistance. The periplasmic part of CnrX can be defined as the metal sensor in the signal transduction complex composed of the membrane-bound anti-sigma factor CnrY and the extra-cytoplasmic function sigma factor CnrH. A soluble form of CnrX was overproduced and purified. This protein behaves as a dimer in solution as judged from gel filtration, sedimentation velocity experiments, and NMR. Native crystals diffracting to 2.3 A using synchrotron radiation were obtained using the hanging-drop vapor-diffusion method. They belong to the primitive monoclinic space group P2(1), with unit cell parameters a = 31.87, b = 74.80, c = 93.67 A, beta = 90.107 degrees. NMR data and secondary structure prediction suggest that this protein is essentially formed by helices.     

Precipitation of Silver-Thiosulfate Complex and Immobilization of Silver by <Emphasis Type="BoldItalic">Cupriavidus metallidurans</Emphasis> CH34

Cupriavidus metallidurans CH34 is a facultative chemolithotrophic bacterium that possesses two megaplasmids (pMOL28 and pMOL30) that confer resistance to eleven metals. The ability of Cupriavidus metallidurans CH34 to resist silver is described here. Electronic microscopy, energy-dispersive X-ray (EDX) and X-ray diffractometry (DRX) observations revealed that C. metallidurans CH34 strongly associated silver with the outer membrane, under chloride chemical form. Using derivate strains of C. metallidurans CH34, which carried only one or no megaplasmid, we show that this resistance seems to be carried by pMOL30.     

X-ray structure of the metal-sensor CnrX in both the apo- and copper-bound forms

Both the X-ray structures of the apo- and the copper-bound forms of the metal-sensor domain (residues 31-148) of CnrX from Cupriavidus metallidurans CH34 were obtained at 1.74 Å resolution from a selenomethionine derivative. This four-helix hooked-hairpin is the first structure of a metal-sensor in an ECF-type signaling pathway. The copper ion is bound in a type 2-like center with a 3N1O coordination in the equatorial plane and shows an unprecedented remote fifth axial ligand with Met93 contributing a weak S-Cu bond. The signal onset cannot be explained by conformational changes associated with CnrX metallation.     

In silico optimization and low structured kinetic model of poly[(R)-3-hydroxybutyrate] synthesis by Cupriavidus necator DSM 545 by fed-batch cultivation on glycerol

Glycerol was utilized by Cupriavidus necator DSM 545 for production of poly-3-hydroxybutyrate (PHB) in fed-batch fermentation. Maximal specific growth rates (0.12 and 0.3 h⁻¹) and maximal specific non-growth PHB production rate (0.16 g g⁻¹ h⁻¹) were determined from two experiments (inocula from exponential and stationary phase). Saturation constants for nitrogen (0.107 and 0.016 g L⁻¹), glycerol (0.05 g L⁻¹), non-growth related PHB synthesis (0.011 g L⁻¹) and nitrogen/PHB related inhibition constant (0.405 g L⁻¹), were estimated. Five relations for specific growth rate were tested using mathematical models. In silico performed optimization procedures (varied glycerol/nitrogen ratio and feeding) has resulted in a PHB content of 70.9%, shorter cultivation time (23 h) and better PHB yield (0.347 g g⁻¹). Initial concentration of biomass 16.8 g L⁻¹ and glycerol concentration in broth between 3 and 5 g L⁻¹ were decisive factors for increasing of productivity.     

Biophysical characterization of the MerP-like amino-terminal extension of the mercuric reductase from<Emphasis Type="Italic"> Ralstonia metallidurans</Emphasis> CH34

The purified native mercuric reductase (MerA) from Ralstonia metallidurans CH34 contains an N-terminal sequence of 68 amino acids predicted to be homologous to MerP, the periplasmic mercury-binding protein. This MerP-like protein has now been expressed independently. The protein was named MerAa by homology with Ccc2a, the first soluble domain of the copper-transporting ATPase from yeast. a has been characterized using a set of biophysical techniques. The binding of mercury was followed using circular dichroism spectroscopy and electrospray mass spectrometry. The two cysteine residues contained in the consensus sequence GMTCXXC are involved in the binding of one mercury atom, with an apparent affinity comparable to that of MerP for the same metal. The metal-binding site is confirmed by NMR chemical shift changes observed between apo- and metal-bound MerAa in solution. NMR shift and NOE data also indicate that only minor structural changes occur upon metal binding. Further NMR investigation of the fold of MerAa using long-range methyl–methyl NOE and backbone residual dipolar coupling data confirm the expected close structural homology with MerP. ¹⁵N relaxation data show that MerAa is a globally rigid molecule. An increased backbone mobility was observed for the loop region connecting the first -strand and the first -helix and comprising the metal-binding domain. Although significantly reduced, this loop region keeps some conformational flexibility upon metal binding. Altogether, our data suggest a role of MerAa in mercury trafficking.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-003-0495-yAbbreviations CCA -cyano-4-hydroxy-trans-cinnamic acid - CSI chemical shift index - HSQC ¹H-detected heteronuclear single-quantum coherence - MerAa the 68 amino acid N-terminal extension of the mercuric reductase - NOE nuclear Overhauser effect - RDCs residual dipolar couplings - TCEP-HCl tris(2-carboxyethyl)phosphine hydrochloride     

Site-directed mutagenesis studies of acetylglutamate synthase delineate the site for the arginine inhibitor

N-acetyl-l-glutamate synthase (NAGS), the first enzyme of bacterial/plant arginine biosynthesis and an essential activator of the urea cycle in animals, is, respectively, arginine-inhibited and activated. Site-directed mutagenesis of recombinant Pseudomonas aeruginosa NAGS (PaNAGS) delineates the arginine site in the PaNAGS acetylglutamate kinase-like domain, and, by extension, in human NAGS. Key residues for glutamate binding are identified in the acetyltransferase domain. However, the acetylglutamate kinase-like domain may modulate glutamate binding, since one mutation affecting this domain increases the K_m for glutamate. The effects on PaNAGS of two mutations found in human NAGS deficiency support the similarity of bacterial and human NAGSs despite their low sequence identity.     

Bef medaka mutant reveals the essential role of c-myb in both primitive and definitive hematopoiesis

Vertebrate hematopoiesis is characterized by two evolutionally conserved phases of development, i.e., primitive hematopoiesis, which is a transient phenomenon in the early embryo, and definitive hematopoiesis, which takes place in the later stages. Beni fuji (bef) was originally isolated as a medaka mutant that has an apparently reduced number of erythrocytes in its peripheral blood. Positional cloning revealed that the bef mutant has a nonsense mutation in the c-myb gene. Previous studies have shown that c-myb is essential for definitive hematopoiesis, and c-myb is now widely used as a marker gene for the onset of definitive hematopoiesis. To analyze the phenotypes of the bef mutant, we performed whole-mount in situ hybridization with gene markers of hematopoietic cells. The bef embryos showed decreased expression of α-globin and l-plastin, and a complete loss of mpo1 and rag1 expression, suggesting that the bef embryos had defects not only in erythrocytes but also in other myeloid cells, which indicates that their definitive hematopoiesis was aberrant. Interestingly, we observed a diminution in the number of primitive erythrocytes and a delay in the emergence of primitive macrophages in the bef embryos. These results suggest that c-myb also functions in the primitive hematopoiesis, potentially demonstrating a link between primitive and definitive hematopoiesis.     

Gene silencing reveals a crucial role for anti-lipopolysaccharide factors from Penaeus monodon in the protection against microbial infections

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides previously identified in various crustaceans. Out of five isoforms identified in Penaeus monodon, ALFPm3 is the best characterized, exhibits antibacterial and antifungal activities and can protect the shrimp from viral infections. Herein, the most recent identified ALFPm, called ALFPm6, is characterized for its potential role in the shrimp’s immunity. RNA interference-mediated gene silencing was used to study the function of ALFPm6 in comparison to ALFPm3. Knockdown of ALFPm3 gene led to rapid death with a cumulative shrimp mortality of 86% within 7 days, accompanied by a 12- and 50-fold higher bacterial count after 2 days in the haemolymph and hepatopancreas, respectively, compared to the control shrimp injected with GFP dsRNA. In contrast, gene silencing of ALFPm6 alone had no effect on the shrimp mortality, but led to a significant increase in the cumulative mortality and a faster mortality rate following Vibrio harveyi and white spot syndrome virus (WSSV) infections, respectively. These results support the roles of ALFPm6 and ALFPm3 in the protection of shrimp against microbial infections.     

The role of C. elegans Ena/VASP homolog UNC-34 in neuronal polarity and motility

Ena/VASP proteins mediate the effects of guidance cues on the actin cytoskeleton. The single C. elegans homolog of the Ena/VASP family of proteins, UNC-34, is required for the migrations of cells and growth cones. Here we show that unc-34 mutant alleles also interact genetically with Wnt mutants to reveal a role for unc-34 in the establishment of neuronal polarity along the C. elegans anterior-posterior axis. Our mutant analysis shows that eliminating UNC-34 function results in neuronal migration and polarity phenotypes that are enhanced at higher temperatures, revealing a heat-sensitive process that is normally masked by the presence of UNC-34. Finally, we show that the UNC-34 protein is expressed broadly and accumulates in axons and at the apical junctions of epithelial cells. While most mutants lacked detectable UNC-34, three unc-34 mutants that contained missense mutations in the EVH1 domain produced full-length UNC-34 that failed to localize to apical junctions and axons, supporting the role for the EVH1 domain in localizing Ena/VASP family members.     

Cloning, nucleotide sequence and primary biochemical characterization of esterase EstA from Ralstonia eutropha CH34

A genomic library of Ralstonia eutropha CH34 was screened in Escherichia coli S17-1 for esterase activity by using -naphthyl acetate and Fast Blue RR. A 1,711 bp DNA fragment was subcloned from an esterase-positive clone and sequenced. Esterase EstA was encoded by a 825-bp open reading frame and exhibited significant amino acid similarities with the enzymes involved in the meta-cleavage pathway. EstA is composed of 275 amino aicds with a predicted molecular mass of 30785 Da. The optimal pH for EstA was 7.0, and the enzyme retained more than 65% activity when incubated in buffers with pH 3.8–9.2 for 2 h. EstA was active at temperatures up to 80 °C and retained more than 77% activity after exposure to temperatures below 60 °C for 2 h.     

His92 and His110 selectively affect different heme centers of adrenal cytochrome b561

Adrenal cytochrome b₅₆₁ (cyt b₅₆₁), a transmembrane protein that shuttles reducing equivalents derived from ascorbate, has two heme centers with distinct spectroscopic signals and reactivity towards ascorbate. The His54/His122 and His88/His161 pairs furnish axial ligands for the hemes, but additional amino acid residues contributing to the heme centers have not been identified. A computational model of human cyt b₅₆₁ (Bashtovyy, D., Berczi, A., Asard, H., and Pali, T. (2003) Protoplasma 221, 31-40) predicts that His92 is near the His88/His161 heme and that His110 abuts the His54/His122 heme. We tested these predictions by analyzing the effects of mutations at His92 or His110 on the spectroscopic and functional properties. Wild type cytochrome and mutants with substitutions in other histidine residues or in Asn78 were used for comparison. The largest lineshape changes in the optical absorbance spectrum of the high-potential (b_H) peak were seen with mutation of His92; the largest changes in the low-potential (b_L) peak lineshape were observed with mutation of His110. In the EPR spectra, mutation of His92 shifted the position of the g = 3.1 signal (b_H) but not the g = 3.7 signal (b_L). In reductive titrations with ascorbate, mutations in His92 produced the largest increase in the midpoint for the b_H transition; mutations in His110 produced the largest decreases in ΔA₅₆₁ for the b_L transition. These results indicate that His92 can be considered part of the b_H heme center, and His110 part of the b_L heme center, in adrenal cyt b₅₆₁.     

Site-directed mutagenesis of a potential catalytic and formyl phosphate binding site and substrate inhibition of N10-formyltetrahydrofolate synthetase

Structural studies of N(10)-formyltetrahydrofolate synthetase (FTHFS) have indicated the involvement of Arg 97 in the binding of the formyl phosphate intermediate. Two site-directed mutants were constructed to test this hypothesis: R97S (Ser substitution) and R97E (Glu substitution). The k(cat) of R97S was approximately 60% that of the wild-type enzyme and had K(m) for ATP and formate twofold higher than those of wild type. R97E was completely inactive and had a K(m) for ATP nearly six times that of wild type. Substrate inhibition by tetrahydrofolate was shown to occur in wild-type and R97S enzymes using both steady-state and transient-state kinetic approaches. These results lend greater insight into the mechanistic function of FTHFS by confirming the interaction of both ATP and formate with Arg 97 and introducing the aspect of substrate inhibition by tetrahydrofolate with regard to substrate binding and dissociation.     

Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy

Cytochrome bd is a terminal quinol:O₂ oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b₅₅₈, b₅₉₅, and d. The role of heme b₅₉₅ remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b₅₉₅ causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b₅₉₅ and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with τ ∼ 12 μs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with τ ∼ 14 ns, 14 μs, and 280 μs. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-μs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ∼ 4% of the enzyme population. The final, 280-μs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b₅₉₅, and not that of heme b₅₅₈, controls the pathway(s) by which CO migrates between heme d and the medium.