A sulfurtransferase gene (PcSft) with a coding region of 546 bp was cloned from the filamentous white-rot fungus Phanerochaere chrysosporium. The 181-amino acid protein contains a highly conserved “Rhodanese-like” domain and an ATP-binding site, with a molecular weight of 20.68 kDa. Semi-quantitative RT-PCR showed that the selective expression of PcSft was involved in secondary metabolism. The recombinant PcSFT protein was expressed in E. coli BL21 (DE3) and purified by Ni2+-chelating and size-exclusion chromatography. Its ATPase and sulfurtransferase (SFT) activities were indentified and characterized. PcSFT exhibited optimal SFT activity at pH 8 and 30 °C as well as stability at 20 °C and pH 8. The enzyme’s stability under different temperature and pH P. indicates a potential usefulness for the detoxification of cyanide in the environment. 相似文献
Mine tailings contain high concentrations of metal contaminants and only little nutrients, making the tailings barren for decades after the mining has been terminated. Effective phytoremediation of mine tailings calls for deep-rooted, metal accumulating, and soil fertility increasing plants with tolerance against harsh environmental conditions. We assessed the potential of the biofuel leguminous tree Pongamia pinnata inoculated with plant growth promoting rhizobia to remediate iron–vanadium–titanium oxide (V–Ti magnetite) mine tailing soil by pot experiment and in situ remediation test. A metal tolerant rhizobia strain PZHK1 was isolated from the tailing soil and identified as Bradyrhizobium liaoningense by phylogenetic analysis. Inoculation with PZHK1 increased the growth of P. pinnata both in V–Ti magnetite mine tailings and in Ni-contaminated soil. Furthermore, inoculation increased the metal accumulation capacity and superoxide dismutase activity of P. pinnata. The concentrations of Ni accumulated by inoculated plants were higher than the hyperaccumulator threshold. Inoculated P. pinnata accumulated high concentration of Fe, far exceeding the upper limit (1000 mg kg−1) of Fe in plant tissue. In summary, P. pinnata–B. liaoningense PZHK1 symbiosis showed potential to be applied as an effective phytoremediation technology for mine tailings and to produce biofuel feedstock on the marginal land.
New antibiotic targets are urgently needed to tackle the multidrug resistant and latent Mycobacterium tuberculosis, the causative agent of the most formidable infectious disease tuberculosis. Sulfur metabolism is essential for the survival and virulence of many pathogens including M. tuberculosis. The absence of most genes involved in microbial sulfur metabolism in human beings suggests abundant novel potential antibiotic targets in pathogen sulfur metabolism. In this article, a comparative genomic landscape of Mycobacterium sulfur metabolism, such as the uptake, activation, and reduction of sulfate and allied enzymes, the biosynthesis pathway of some sulfated metabolites, and the enzymes involved in these pathways were presented. Novel clues for antibiotic targets are put forward. 相似文献
Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg2+, which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics. 相似文献
Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis, a process which depends on an array of virulence factors to colonize and replicate within the host. The M. tuberculosis iron regulated open reading frame (ORF) rv3402c, encoding a conserved hypothetical protein, was shown to be up-regulated upon infection in both human and mice macrophages. To explore the function of this ORF, we heterologously expressed the rv3402c gene in the non-pathogenic fast-growing Mycobacterium smegmatis strain, and demonstrated that Rv3402c, a cell envelope-associated protein, was able to enhance the intracellular survival of recombinant M. smegmatis. Enhanced growth was not found to be the result of an increased resistance to intracellular stresses, as growth of the Rv3402c expressing strain was unaffected by iron depletion, H2O2 exposure, or acidic conditions. Colonization of macrophages by M. smegmatis expressing Rv3402c was associated with substantial cell death and significantly greater amount of TNF-α and IL-1β compared with controls. Rv3402c-induced TNF-α and IL-1β production was found to be mediated by NF-κB, ERK and p38 pathway in macrophages. In summary, our study suggests that Rv3402c delivered in a live M. smegmatis vehicle can modify the cytokines profile of macrophage, promote host cell death and enhance the persistence of mycobacterium within host cells. 相似文献
We induced mutants, stripe1-2 (st1-2) and stripe1-3 (st1-3), from rice (Oryza sativa L.) Indica 9311 using Ethyl methanesulfonate (EMS). Both st1-2 and st1-3 mutants encoded the small subunit of ribonucleotide reductase 1 (RNRS1), differed in the location of the mutated base, and displayed white-stripe from the L2 stage through maturity. The mutants were sensitive to temperature, and their chlorophyll content increased with the increase in temperature; however, they did not revert to normal green leaf phenotype under field conditions. The mutant st1-2 showed loosely arranged thylakoid lamellar structure as compared with wild-type (WT) plants. Contrastingly, st1-3 displayed normal thylakoid lamellar structure, good agronomic traits, and higher yield than st1-2 but lower yield than WT. Three-dimensional structure prediction for RNRS1 indicated that the mutation in Val-171 residue in st1-2 influenced the connection of RNRS1 to iron, causing abnormal development of chloroplasts. Real-time PCR analysis showed that the expression levels associated with chlorophyll biosynthetic pathway and photosynthesis were affected in st1-2 and st1-3 at different temperatures and different developmental stages. 相似文献
The cloning, expression and purification of the glutathione (sulfur) import system ATP-binding protein (gsiA) was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP) reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3) was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3) provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving. 相似文献