Hexavalent Chromium Reduction from Pollutant Samples by <Emphasis Type="Italic">Achromobacter xylosoxidans</Emphasis> SHB 204 and its Kinetics Study

Cr(VI) is most toxic heavy metal and second most widespread hazardous metal compound worldwide. Present work focused on Cr(VI) reduction from synthetic solutions and polluted samples by Achromobacter xylosoxidans SHB 204. It could tolerate Cr(VI) up to 1600 ppm and reduce 500 ppm with 4.5 chromium reductase enzyme units (U) having protein size 30 kDa. Changes in morphology of cells on interaction with Cr(VI) metal ion was also studied using SEM–EDX and FTIR. Microcosm studies in pollutant samples for Cr(VI) reduction and adsorption isotherm with biomass of bacterium was best fitted with Langmuir model along with kinetic studies. This study focuses on significance of Cr reduction from synthetic solutions and polluted samples by A. xylosoxidans SHB 204 and its potential for bioremediation.     

Evidence for the presence of a critical histidine residue at the active site in glyceraldehyde-3-phosphate dehydrogenase of Ehrlich ascites carcinoma cells

Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (GA3PD) (EC. 1.2.1.12) was completely inactivated by diethyl pyrocarbonate (DEPC), a fairly specific reagent for histidine residues in the pH range of 6.0-7.5. The rate of inactivation was dependent on pH and followed pseudo-first order reaction kinetics. The difference spectrum of the inactivated and native enzymes showed an increase in the absorption maximum at 242 nm, indicating the modification of histidine residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of one essential histidine residue to be responsible for loss of the catalytic activity of EAC cell GA3PD. DEPC inactivation was protected by substrates, D-glyceraldehyde-3-phosphate and NAD, indicating the presence of essential histidine residue at the substrate-binding region of the active site. Double inhibition studies also provide evidence for the presence of histidine residue at the active site.     

Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility

A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS₂) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using intein technology, the isotope-labeled PrS₂-tag is replaced with non-isotope labeled PrS₂-tag, silencing the NMR signals from PrS₂-tag in isotope-filtered ¹H-detected NMR experiments. This method was applied to the E. coli ribosome binding factor A (RbfA), which aggregates and precipitates in the absence of a solubilization tag unless the C-terminal 25-residue segment is deleted (RbfAΔ25). Using the PrS₂-tag, full-length well-behaved RbfA samples could be successfully prepared for NMR studies. PrS₂ (non-labeled)-tagged RbfA (isotope-labeled) was produced with the use of the intein approach. The well-resolved TROSY-HSQC spectrum of full-length PrS₂-tagged RbfA superimposes with the TROSY-HSQC spectrum of RbfAΔ25, indicating that PrS₂-tag does not affect the structure of the protein to which it is fused. Using a smaller PrS-tag, consisting of a single N-terminal domain of protein S, triple resonance experiments were performed, and most of the backbone ¹H, ¹⁵N and ¹³C resonance assignments for full-length E. coli RbfA were determined. Analysis of these chemical shift data with the Chemical Shift Index and heteronuclear ¹H–¹⁵N NOE measurements reveal the dynamic nature of the C-terminal segment of the full-length RbfA protein, which could not be inferred using the truncated RbfAΔ25 construct. CS-Rosetta calculations also demonstrate that the core structure of full-length RbfA is similar to that of the RbfAΔ25 construct.     

Regulation of Cu(I)/Ag(I) efflux genes in Escherichia coli by the sensor kinase CusS

Two-component systems are widely used by bacteria to mediate adaptive responses to a variety of environmental stimuli. The CusR/CusS two-component system in Escherichia coli induces expression of genes involved in metal efflux under conditions of elevated Cu(I) and Ag(I) concentrations. As seen in most prototypical two-component systems, signal recognition and transmission is expected to occur by ligand binding in the periplasmic sensor domain of the histidine kinase CusS. Although discussed in the extant literature, little experimental evidence is available to establish the role of CusS in metal homeostasis. In this study, we show that the cusS gene is required for Cu(I) and Ag(I) resistance in E.?coli and that CusS is linked to the expression of the cusCFBA genes. These results show a metal-dependent mechanism of CusS activation and suggest an absolute requirement for CusS in Cu(I)- and Ag(I)-dependent upregulation of cusCFBA expression in E.?coli.