Insertion sequence elements in Cupriavidus metallidurans CH34: distribution and role in adaptation

Cupriavidus metallidurans CH34 is a β-proteobacterium well equipped to cope with harsh environmental conditions such as heavy metal pollution. The strain carries two megaplasmids specialized in the response to heavy metals and a considerable number of genomic islands, transposons and insertion sequence (IS) elements. The latter were characterized in detail in this study, which revealed nine new IS elements totaling to 21 distinct IS elements from 10 different IS families and reaching a total of 57 intact IS copies in CH34. Analysis of all fully sequenced bacterial genomes revealed that relatives of these IS elements were mostly found in the Burkholderiaceae family (β-proteobacteria) to which C. metallidurans belongs. Three IS elements were 100% conserved in other bacteria suggesting recent interaction and horizontal transfer between these strains. In addition, a number of these IS elements were associated with genomic islands, gene inactivation or rearrangements that alter the autotrophic growth capacities of CH34. The latter rearrangements gave the first molecular evidence for the mutator phenotype that is characteristic for various C. metallidurans strains. Furthermore, differential expression of some IS elements (or adjacent genes in the same strand orientation) was found under heavy metal stress, an environmental stress to which C. metallidurans CH34 is well adapted. These observations indicate that these IS elements play an active role in C. metallidurans CH34 lifestyle, including its metabolic potential and adaptation under selective pressure.     

Purification and characterization of the acetone carboxylase of Cupriavidus metallidurans strain CH34

Acetone carboxylase (Acx) is a key enzyme involved in the biodegradation of acetone by bacteria. Except for the Helicobacteraceae family, genome analyses revealed that bacteria that possess an Acx, such as Cupriavidus metallidurans strain CH34, are associated with soil. The Acx of CH34 forms the heterohexameric complex α(2)β(2)γ(2) and can carboxylate only acetone and 2-butanone in an ATP-dependent reaction to acetoacetate and 3-keto-2-methylbutyrate, respectively.     

Contributions of five secondary metal uptake systems to metal homeostasis of Cupriavidus metallidurans CH34

Cupriavidus metallidurans is adapted to high concentrations of transition metal cations and is a model system for studying metal homeostasis in difficult environments. The elemental composition of C. metallidurans cells cultivated under various conditions was determined, revealing the ability of the bacterium to shield homeostasis of one essential metal from the toxic action of another. The contribution of metal uptake systems to this ability was studied. C. metallidurans contains three CorA members of the metal inorganic transport (MIT) protein family of putative magnesium uptake systems, ZupT of the ZRT/IRT protein, or ZIP, family, and PitA, which imports metal phosphate complexes. Expression of the genes for all these transporters was regulated by zinc availability, as shown by reporter gene fusions. While expression of zupT was upregulated under conditions of zinc starvation, expression of the other genes was downregulated at high zinc concentrations. Only corA(1) expression was influenced by magnesium starvation. Deletion mutants were constructed to characterize the contribution of each system to transition metal import. This identified ZupT as the main zinc uptake system under conditions of low zinc availability, CorA(1) as the main secondary magnesium uptake system, and CorA(2) and CorA(3) as backup systems for metal cation import. PitA may function as a cation-phosphate uptake system, the main supplier of divalent metal cations and phosphate in phosphate-rich environments. Thus, metal homeostasis in C. metallidurans is achieved by highly redundant metal uptake systems, which have only minimal cation selectivity and are in combination with efflux systems that "worry later" about surplus cations.     

CzcP is a novel efflux system contributing to transition metal resistance in Cupriavidus metallidurans CH34

Cupriavidus metallidurans CH34 possesses a multitude of metal efflux systems. Here, the function of the novel P_IB4-type ATPase CzcP is characterized, which belongs to the plasmid pMOL30-mediated cobalt-zinc-cadmium (Czc) resistance system. Contribution of CzcP to transition metal resistance in C. metallidurans was compared with that of three P_IB2-type ATPases (CadA, ZntA, PrbA) and to other efflux proteins by construction and characterization of multiple deletion mutants. These data also yielded additional evidence for an export of metal cations from the periplasm to the outside of the cell rather than from the cytoplasm to the outside. Moreover, metal-sensitive Escherichia coli strains were functionally substituted in trans with CzcP and the three P_IB2-type ATPases. Metal transport kinetics performed with inside-out vesicles identified the main substrates for these four exporters, the K_m values and apparent turn-over numbers. In combination with the mutant data, transport kinetics indicated that CzcP functions as 'resistance enhancer': this P_IB4-type ATPase exports transition metals Zn²⁺, Cd²⁺ and Co²⁺ much more rapidly than the three P_IB2-type proteins. However, a basic resistance level has to be provided by the P_IB2-type efflux pumps because CzcP may not be able to reach all different speciations of these metals in the cytoplasm.     

Spectroscopic characterization of the metal-binding sites in the periplasmic metal-sensor domain of CnrX from Cupriavidus metallidurans CH34

CnrX, the dimeric metal sensor of the three-protein transmembrane signal transduction complex CnrYXH of Cupriavidus metallidurans CH34, contains one metal-binding site per monomer. Both Ni and Co elicit a biological response and bind the protein in a 3N2O1S coordination sphere with a nearly identical octahedral geometry as shown by the X-ray structure of CnrXs, the soluble domain of CnrX. However, in solution CnrXs is titrated by 4 Co-equiv and exhibits an unexpected intense band at 384 nm that was detected neither by single-crystal spectroscopy nor under anaerobiosis. The data from a combination of spectroscopic techniques (spectrophotometry, electron paramagnetic resonance, X-ray absorption spectroscopy) showed that two sites correspond to those identified by crystallography. The two extra binding sites accommodate Co(II) in an octahedral geometry in the absence of oxygen and are occupied in air by a mixture of low-spin Co(II) as well as EPR-silent Co(III). These extra sites, located at the N-terminus of the protein, are believed to participate to the formation of peroxo-bridged dimers. Accordingly, we hypothesize that the intense band at 384 nm relies on the formation of a binuclear μ-peroxo Co(III) complex. These metal binding sites are not physiologically relevant since they are not detected in full-length NccX, the closest homologue of CnrX. X-ray absorption spectroscopy demonstrates that NccX stabilizes Co(II) in two-binding sites similar to those characterized by crystallography in its soluble counterpart. Nevertheless, the original spectroscopic properties of the extra Co-binding sites are of interest because they are susceptible to be detected in other Co-bound proteins.     

Molecular structure and metal-binding properties of the periplasmic CopK protein expressed in Cupriavidus metallidurans CH34 during copper challenge

The copK gene is localized on the pMOL30 plasmid of Cupriavidus metallidurans CH34 within the complex cop cluster of genes, for which 21 genes have been identified. The expression of the corresponding periplasmic CopK protein is strongly upregulated in the presence of copper, leading to a high periplasmic accumulation. The structure and metal-binding properties of CopK were investigated by NMR and mass spectrometry. The protein is dimeric in the apo state with a dissociation constant in the range of 10^- 5 M estimated from analytical ultracentrifugation. Mass spectrometry revealed that CopK has two high-affinity Cu(I)-binding sites per monomer with different Cu(I) affinities. Binding of Cu(II) was observed but appeared to be non-specific. The solution structure of apo-CopK revealed an all-β fold formed of two β-sheets in perpendicular orientation with an unstructured C-terminal tail. The dimer interface is formed by the surface of the C-terminal β-sheet. Binding of the first Cu(I)-ion induces a major structural modification involving dissociation of the dimeric apo-protein. Backbone chemical shifts determined for the 1Cu(I)-bound form confirm the conservation of the N-terminal β-sheet, while the last strand of the C-terminal sheet appears in slow conformational exchange. We hypothesize that the partial disruption of the C-terminal β-sheet is related to dimer dissociation. NH-exchange data acquired on the apo-protein are consistent with a lower thermodynamic stability of the C-terminal sheet. CopK contains seven methionine residues, five of which appear highly conserved. Chemical shift data suggest implication of two or three methionines (Met54, Met38, Met28) in the first Cu(I) site. Addition of a second Cu(I) ion further increases protein plasticity. Comparison of the structural and metal-binding properties of CopK with other periplasmic copper-binding proteins reveals two conserved features within these functionally related proteins: the all-β fold and the methionine-rich Cu(I)-binding site.     

Structural and metal binding characterization of the C-terminal metallochaperone domain of membrane fusion protein SilB from Cupriavidus metallidurans CH34

Detoxification of heavy metal ions in Proteobacteria is tightly controlled by various systems regulating their sequestration and transport. In Cupriavidus metallidurans CH34, a model organism for heavy metal resistance studies, the sil determinant is potentially involved in the efflux of silver and copper ions. Proteins SilA, SilB, and SilC form a resistance nodulation cell division (RND)-based transport system in which SilB is the periplasmic adaptor protein belonging to the membrane fusion protein (MFP) family. In addition to the four domains typical of known MFPs, SilB has a fifth additional C-terminal domain, called SilB(440-521), which is characterized here. Structure and backbone dynamics of SilB(440-521) have been investigated using nuclear magnetic resonance, and the residues of the metal site were identified from (15)N- and (13)C-edited HSQC spectra. The solution structure and additional metal binding experiments demonstrated that this C-terminal domain folds independently of the rest of the protein and has a conformation and a Ag(+) and Cu(+) binding specificity similar to those determined for CusF from Escherichia coli. The small protein CusF plays a role in metal trafficking in the periplasm. The similarity with CusF suggests a potential metallochaperone role for SilB(440-521) that is discussed in the context of simultaneous expression of different determinants involved in copper resistance in C. metallidurans CH34.     

Differential proteomic analysis using isotope‐coded protein‐labeling strategies: Comparison,improvements and application to simulated microgravity effect on Cupriavidus metallidurans CH34

Among differential proteomic methods based on stable isotopic labeling, isotope‐coded protein labeling (ICPL) is a recent non‐isobaric technique devised to label primary amines found in proteins. ICPL overcomes some of the disadvantages found in other chemical‐labeling techniques, such as iTRAQ or ICAT. However, previous analyses revealed that more than 30% of the proteins identified in regular ICPL generally remain unquantified. In this study, we describe a modified version of ICPL, named Post‐digest ICPL, that makes it possible to label and thus to quantify all the peptides in a sample (bottom–up approach). Optimization and validation of this Post‐digest ICPL approach were performed using a standard protein mixture and complex protein samples. Using this strategy, the number of proteins that were identified and quantified was greatly increased in comparison with regular ICPL and cICAT approaches. The pros and cons of this improvement are discussed. This complementary approach to traditional ICPL was applied to the analysis of modification of protein abundances in the model bacterium Cupriavidus metallidurans CH34 after cultivation under simulated microgravity. In this context, two different systems – a 2‐D clinorotation and 3‐D random positioning device – were used and the results were compared and discussed.     

The Ability of Basalt to Leach Nutrients and Support Growth of Cupriavidus metallidurans CH34 Depends on Basalt Composition and Element Release

The influence of different rock mineralogical compositions on element leaching and bio-weathering processes is poorly understood. Here, the role of basalt composition on nutrient leaching (calcium, iron, phosphorus or magnesium) and subsequent support of Cupriavidus metallidurans CH34 growth was evaluated. Seven different basalts were examined. In iron-limiting media, growth could be restored for all basalts tested, while only two basalts could restore growth in magnesium-limiting medium. Only for magnesium growth complementation was correlated with the composition of the basalts tested. Our results showed that basalt composition can affect leaching and availability of different elements, in turn, affecting bacterial growth.     

Molecular basis of the cooperative binding of Cu(I) and Cu(II) to the CopK protein from Cupriavidus metallidurans CH34

The bacterium Cupriavidus metallidurans CH34 is resistant to high environmental concentrations of many metal ions. Upon copper challenge, it upregulates the periplasmic protein CopK (8.3 kDa). The function of CopK in the copper resistance response is ill-defined, but CopK demonstrates an intriguing cooperativity: occupation of a high-affinity Cu(I) binding site generates a high-affinity Cu(II) binding site, and the high-affinity Cu(II) binding enhances Cu(I) binding. Native CopK and targeted variants were examined by chromatographic, spectroscopic, and X-ray crystallographic probes. Structures of two distinct forms of Cu(I)Cu(II)-CopK were defined, and structural changes associated with occupation of the Cu(II) site were demonstrated. In solution, monomeric Cu(I)Cu(II)-CopK features the previously elucidated Cu(I) site in Cu(I)-CopK, formed from four S(δ) atoms of Met28, -38, -44, and -54 (site 4S). Binding of Cu(I) to apo-CopK induces a conformational change that releases the C-terminal β-strand from the β-sandwich structure. In turn, this allows His70 and N-terminal residues to form a large loop that includes the Cu(II) binding site. In crystals, a polymeric form of Cu(I)Cu(II)-CopK displays a Cu(I) site defined by the S(δ) atoms of Met26, -38, and -54 (site 3S) and an exogenous ligand (modeled as H(2)O) and a Cu(II) site that bridges dimeric CopK molecules. The 3S Cu(I) binding mode observed in crystals was demonstrated in solution in protein variant M44L where site 4S is disabled. The intriguing copper binding chemistry of CopK provides molecular insight into Cu(I) transfer processes. The adaptable nature of the Cu(I) coordination sphere in methionine-rich clusters allows copper to be relayed between clusters during transport across membranes in molecular pumps such as CusA and Ctr1.     

Chemical Forms of Selenium in the Metal-Resistant Bacterium Ralstonia metallidurans CH34 Exposed to Selenite and Selenate

Ralstonia metallidurans CH34, a soil bacterium resistant to a variety of metals, is known to reduce selenite to intracellular granules of elemental selenium (Se⁰). We have studied the kinetics of selenite (Se^IV) and selenate (Se^VI) accumulation and used X-ray absorption spectroscopy to identify the accumulated form of selenate, as well as possible chemical intermediates during the transformation of these two oxyanions. When introduced during the lag phase, the presence of selenite increased the duration of this phase, as previously observed. Selenite introduction was followed by a period of slow uptake, during which the bacteria contained Se⁰ and alkyl selenide in equivalent proportions. This suggests that two reactions with similar kinetics take place: an assimilatory pathway leading to alkyl selenide and a slow detoxification pathway leading to Se⁰. Subsequently, selenite uptake strongly increased (up to 340 mg Se per g of proteins) and Se⁰ was the predominant transformation product, suggesting an activation of selenite transport and reduction systems after several hours of contact. Exposure to selenate did not induce an increase in the lag phase duration, and the bacteria accumulated approximately 25-fold less Se than when exposed to selenite. Se^IV was detected as a transient species in the first 12 h after selenate introduction, Se⁰ also occurred as a minor species, and the major accumulated form was alkyl selenide. Thus, in the present experimental conditions, selenate mostly follows an assimilatory pathway and the reduction pathway is not activated upon selenate exposure. These results show that R. metallidurans CH34 may be suitable for the remediation of selenite-, but not selenate-, contaminated environments.     

Chemical forms of selenium in the metal-resistant bacterium Ralstonia metallidurans CH34 exposed to selenite and selenate

Ralstonia metallidurans CH34, a soil bacterium resistant to a variety of metals, is known to reduce selenite to intracellular granules of elemental selenium (Se(0)). We have studied the kinetics of selenite (Se(IV)) and selenate (Se(VI)) accumulation and used X-ray absorption spectroscopy to identify the accumulated form of selenate, as well as possible chemical intermediates during the transformation of these two oxyanions. When introduced during the lag phase, the presence of selenite increased the duration of this phase, as previously observed. Selenite introduction was followed by a period of slow uptake, during which the bacteria contained Se(0) and alkyl selenide in equivalent proportions. This suggests that two reactions with similar kinetics take place: an assimilatory pathway leading to alkyl selenide and a slow detoxification pathway leading to Se(0). Subsequently, selenite uptake strongly increased (up to 340 mg Se per g of proteins) and Se(0) was the predominant transformation product, suggesting an activation of selenite transport and reduction systems after several hours of contact. Exposure to selenate did not induce an increase in the lag phase duration, and the bacteria accumulated approximately 25-fold less Se than when exposed to selenite. Se(IV) was detected as a transient species in the first 12 h after selenate introduction, Se(0) also occurred as a minor species, and the major accumulated form was alkyl selenide. Thus, in the present experimental conditions, selenate mostly follows an assimilatory pathway and the reduction pathway is not activated upon selenate exposure. These results show that R. metallidurans CH34 may be suitable for the remediation of selenite-, but not selenate-, contaminated environments.     

Characteristics of zinc transport by two bacterial cation diffusion facilitators from Ralstonia metallidurans CH34 and Escherichia coli

CzcD from Ralstonia metallidurans and ZitB from Escherichia coli are prototypes of bacterial members of the cation diffusion facilitator (CDF) protein family. Expression of the czcD gene in an E. coli mutant strain devoid of zitB and the gene for the zinc-transporting P-type ATPase zntA rendered this strain more zinc resistant and caused decreased accumulation of zinc. CzcD, purified as an amino-terminal streptavidin-tagged protein, bound Zn2+, Co2+, Cu2+, and Ni2+ but not Mg2+, Mn2+, or Cd2+, as shown by metal affinity chromatography. Histidine residues were involved in the binding of 2 to 3 mol of Zn2+ per mol of CzcD. ZitB transported 65Zn2+ in the presence of NADH into everted membrane vesicles with an apparent Km of 1.4 microM and a Vmax of 0.57 nmol of Zn2+ min(-1) mg of protein(-1). Conserved amino acyl residues that might be involved in binding and transport of zinc were mutated in CzcD and/or ZitB, and the influence on Zn2+ resistance was studied. Charged or polar amino acyl residues that were located within or adjacent to membrane-spanning regions of the proteins were essential for the full function of the proteins. Probably, these amino acyl residues constituted a pathway required for export of the heavy metal cations or for import of counter-flowing protons.     

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.     

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.