Incorporation of membrane and secretory proteins into COPII vesicles are facilitated either by the direct interaction of cargo proteins with COPII coat proteins, or by ER exit adaptor proteins which mediate the interaction of cargo proteins with COPII coat proteins. Svp26 is one of the ER exit adaptor proteins in the yeast Saccharomyces cerevisiae. The ER exit of several type II membrane proteins have been reported to be facilitated by Svp26. We demonstrate here that the efficient incorporation of Mnn4, a type II membrane protein required for mannosyl phosphate transfer to glycoprotein-linked oligosaccharides, into COPII vesicles is also dependent on the function of Svp26. We show that Mnn4 is localized to the Golgi. In addition to Mnn4, Mnn6 is known to be also required for the transfer of mannosyl phosphate to the glycans. We show, by indirect immunofluorescence, that Mnn6 localizes to the ER. As in the case with Svp26, deletion of the MNN6 gene results in the accumulation of Mnn4 in ER. In vitro COPII vesicle budding assays show that Svp26 and Mnn6 facilitate the incorporation of Mnn4 into COPII vesicles. In contrast to Svp26, which is itself efficiently captured into the COPII vesicles, Mnn6 was not incorporated into the COPII vesicles. Mnn4 and Mnn6 have the DXD motif which is often found in the many glycosyltransferases and functions to coordinate a divalent cation essential for the reaction. Alcian blue dye binding assay shows that substitution of the first D in this motif present in Mnn4 by A impairs the Mnn4 function. In contrast, amino acid substitutions in DXD motifs present in Mnn6 did not affect the function of Mnn6. These results suggest that Mnn4 may be directly involved in the mannosyl phosphate transfer reaction.
Bioremediation may affect the ecological system around bioremediation sites. However, little is known about how microbial community structures change over time after the initial injection of degraders. In this study, we have assessed the ecological impact of bioaugmentation using metagenomic and metatranscriptomic approaches to remove trichlorinated ethylene/cis-dichloroethylene (TCE/cDCE) by Rhodococcus jostii strain RHA1 as an aerobic chemical compound degrader. Metagenomic analysis showed that the number of organisms belonging to the genus Rhodococcus, including strain RHA1, increased from 0.1% to 76.6% of the total microbial community on day 0 at the injection site. Subsequently, the populations of strain RHA1 and other TCE/cDCE-degrading bacteria gradually decreased over time, whereas the populations of the anaerobic dechlorinators Geobacter and Dehalococcoides increased at later stages. Metatranscriptomic analysis revealed a high expression of aromatic compound-degrading genes (bphA1-A4) in strain RHA1 after RHA1 injection. From these results, we concluded that the key dechlorinators of TCE/cDCE were mainly aerobic bacteria, such as RHA1, until day 1, after which the key dechlorinators changed to anaerobic bacteria, such as Geobacter and Dehalococcocides, after day 6 at the injection well. Based on the α-diversity, the richness levels of the microbial community were increased after injection of strain RHA1, and the microbial community composition had not been restored to that of the original composition during the 19 days after treatment. These results provide insights into the assessment of the ecological impact and bioaugmentation process of RHA1 at bioremediation sites.
Many organisms produce endogenous hydrogen sulfide (H2S) as a by-product of protein, peptide, or L-cysteine degradation. Recent reports concerning mammalian cells have demonstrated that H2S acts as a signaling molecule playing important roles in various biological processes. In contrast to mammals, bacterial H2S signaling remains unclear. In this work, we demonstrate that Escherichia coli generates H2S through the assimilation of inorganic sulfur, without L-cysteine degradation. Comparison of phenotypes and genomes between laboratory E. coli K-12 strains revealed a major contribution of CRP (a protein that controls the expression of numerous genes involved in glycolysis) to H2S generation. We found that H2S was produced by cells growing in a synthetic minimal medium containing thiosulfate as a sole inorganic sulfur source, but not in a medium only containing sulfate. Furthermore, E. coli generated H2S in a CRP-dependent manner as a response to glucose starvation. These results indicate that CRP plays a key role in the generation of H2S coupled to thiosulfate assimilation, whose molecular mechanisms remains to be elucidated. Here, we propose a potential biological role of the H2S as a signaling mediator for a cross-talk between carbon and sulfur metabolism in E. coli.
In this study, a mutant xylanase of high thermostability was obtained by site-directed mutagenesis. The homologous 3D structure of xylanase was successfully modeled and the mutation sites were predicted using bioinformatics software. Two amino acids of XynZF-2 were respectively substituted by cysteines (T205C and A52C) and a disulfide bridge was introduced into the C-terminal of XynZF-2. The mutant gene xynZFTA was cloned into pPIC9K and expressed in P. pastoris. The optimum temperature of the variant XynZFTA was improved from 45°C to 60°C, and XynZFTA retained greater than 90.0% activity (XynZF-2 retained only 50.0% activity) after treatment at 50°C for 5 min. The optimum pH of mutant xylanase was similar to XynZF-2 (pH = 5.0). The pH stability span (5.0~7.0) of the mutant xylanase was increased to 3.0~9.0. Overall, the results implied that the introduction of a disulfide bridge in the C-terminal structure improved the thermostability and pH stability of XynZF-2.
Tellurium (Te) has been increasingly used as a semiconductor material in copious amounts, with a concomitant increase in its discharge from industrial effluents and mining wastewater into the environment. However, soluble Te, such as tellurate (VI) and tellurite (IV), is toxic to organisms. Thus, highly efficient technologies need to be developed for a double-benefit detoxification and recovery of soluble Te from industrial and mining wastewater. Since industrial wastewater contains high concentrations of salt, salt-tolerant microorganisms that metabolize rare metals such as Te have been the subject of focus for the effective detoxification and recovery of Te. In the present study, a total of 52 salt-tolerant tellurate-reducing microorganisms were isolated from marine environmental samples. Of these, 18 strains achieved greater than, or equal to, 50% removal of water-soluble Te from a medium containing 0.4 mM tellurate after 72 h incubation. The 18 isolated strains belonged to 13 species of the following 9 genera: Sulfitobacter, Ruegeria, Hoeflea, Alteromonas, Marinobacter, Pseudoalteromonas, Shewanella, Idiomarina, and Vibrio. No microorganism has been reported to reduce tellurate and tellurite from six of the aforementioned genera, namely, Sulfitobacter, Ruegeria, Alteromonas, Marinobacter, Idiomarina, and Vibrio. Especially, one of the isolates Sulfitobacter sp. strain TK39B, removed 82% (w/w) of soluble Te with a 4% NaCl tolerance. These results showed that salt-tolerant tellurate-reducing bacteria that can be used in the detoxification and recovery of Te are widely present in the marine environment.
The research purpose was the characterization of indigenous heavy metal-resistant plant growth-promoting bacteria (PGPB) from the farmlands located on the Le'an River basin contaminated by acid mine drainage and their effects on plant growth, nutrient uptake, antioxidant enzyme activities and metal accumulation. The plant growth-promoting (PGP) traits, including 1-aminocyclopropane-1-carboxylic acid deaminase, indoleacetic acid, siderophore, ammonia production and phosphate solubilization, as well as antibiotics, acid/alkali and salt resistance were determined. Ten isolates with relatively high PGP activities were identified to belong to the genera Burkholderia, Paraburkholderia, Cupriavidus, Pseudomonas and Ralstonia. The numerical classification based on bacterial resistant characteristics was mostly consistent with their phylogenetic positions. Burkholderia sp. strain S6-1 and Pseudomonas sp. strain S2-3 possessed both greater PGP activities and resistant characteristics in overall comparison. Compared with non-inoculated plants, strains S6-1 and S2-3 significantly increased the height, dry weight and N uptake of sorghum (Sorghum bicolor L.). The presence of S6-1 significantly increased Pb accumulation and enhanced the translocation of Zn from root to shoot in sorghum. Strain S2-3 helped sorghum to uptake Cu and Zn and improved the remediation effect of sorghum on Cu and Zn. Overall, indigenous PGPB did not show better advantages in improving plant growth than non-indigenous P. putida UW4. Nevertheless, indigenous PGPB can be used as better candidates in heavy metal phytoremediation to minimize the potential risks of introducing invasive microbial species into an agricultural ecosystem.
The Gram-positive bacterium Bacillus subtilis plays important roles in both industrial applications and basic research. However, transformation of competent B. subtilis cells is more difficult to achieve compared with that of Escherichia coli. It has been reported that the conjugative broad host range plasmid RK2 can be transferred to various organisms, including B. subtilis. Nevertheless, the protocol for conjugation from E. coli to B. subtilis has not been properly established. Thus, we optimized interspecies conjugation from E. coli to B. subtilis using the RK2 system. We constructed mobilizable shuttle and integrative vectors pEB1 and pEB2, respectively. pEB1 was used to evaluate the effect of mating media, time, temperature, and genetic background of the recipient and donor strains. We found that conjugation was not significantly affected by the conjugation time or genetic background of the recipient and donor strains. Conjugation on agar was more efficient than that in a liquid medium. A low temperature (16°C and lower) drastically decreased conjugation efficiency. When using the optimized protocol for homologous recombination after conjugation, we could not obtain double crossover mutants, as only single crossover mutants were observed in the initial selection. We then established a two-step homologous recombination method whereby positive colonies were cultivated further, which finally allowed efficient yield of double crossover recombinants. The optimized conjugation method described here allowed facility and efficient gene introduction into B. subtilis from E. coli