Molecular Insights into the Copper-Sensitive Operon Repressor in Acidithiobacillus caldus.

The copper-sensitive operon repressor (CsoR) family, which is the main Cu(I)-sensing family, is widely distributed and regulates regulons involved in detoxification in response to extreme copper stress (a general range of ≥3 g/liter copper ions). Here, we identified CsoR in hyper-copper-resistant Acidithiobacillus caldus (CsoRAc), an organism used in the bioleaching process of copper ores. CsoRAc possesses highly conserved Cu(I) ligands and structures within the CsoR family members. Transcriptional analysis assays indicated that the promoter (PIII) of csoR was active but weakly responsive to copper in Escherichia coli. Copper titration assays gave a stoichiometry of 0.8 mol Cu(I) per apo-CsoRAc monomer in vitro combined with atomic absorption spectroscopy analysis. CuI-CsoRAc and apo-CsoRAc share essentially identical secondary structures and assembly states, as demonstrated by circular dichroism spectra and size exclusion chromatography profiles. The average dissociation constants (KD = 2.26 × 10-18 M and 0.53 × 10-15 M) and Cu(I) binding affinity of apo-CsoRAc were estimated by bathocuproine disulfonate (BCS) and bicinchoninic acid (BCA) competition assays, respectively. Site-directed mutations of conserved Cu(I) ligands in CsoRAc did not significantly alter the secondary structure or assembly state. Competition assays showed that mutants had the same order of magnitude of Cu(I) binding affinity as apo-CsoRAc. Moreover, apo-CsoRAc could bind to the DNA fragment P08430 in vitro, although with low affinity. Finally, a working model was developed to illustrate putative copper resistance mechanisms in A. caldus. IMPORTANCE Research on copper resistance among various species has attracted considerable interest. However, due to the lack of effective and reproducible genetic tools, few studies regarding copper resistance have been reported for A. caldus. Here, we characterized a major Cu(I)-sensing family protein, CsoRAc, which binds Cu(I) with an attomolar affinity higher than that of the Cu(I)-specific chelator bathocuproine disulfonate. In particular, CsoR family proteins were identified only in A. caldus, rather than A. ferrooxidans and A. thiooxidans, which are both used for bioleaching. Meanwhile, A. caldus harbored more copper resistance determinants and a relatively full-scale regulatory system involved in copper homeostasis. These observations suggested that A. caldus may play an essential role in the application of engineered strains with higher copper resistance in the near future.

Metabolic transcriptional analysis on copper tolerance in moderate thermophilic bioleaching microorganism Acidithiobacillus caldus.

Bioleaching, an alternative environmental smelting technology, typically uses high concentrations of heavy metal ions, especially in the subsequent phase, due to metal ion accumulation from the mineral. In this study, we analyzed the overall response of the bioleaching microorganism Acidithiobacillus caldus to copper stress through physiological and transcriptomic analyses. Scanning electron microscopy results showed higher extracellular polymeric substances secretion and cell aggregation under copper stress. Intracellular levels of glutamic acid, glycine and cysteine increased, favoring the synthesis of glutathione for maintenance of the oxidation-reduction state. GSH, during copper stress conditions, the activity of GSH-PX and CAT increased, resulting in reduced oxidative damage while maintaining stable intracellular pH. Higher unsaturated and cyclopropane fatty acid levels resulted in increased membrane fluidity and compactness and decreased ATP levels to support the energy requirements for stress resistance. Initially, H+-ATPase activity increased to provide energy for proton output and decreased later at higher copper ion stress. From transcriptome analysis, 140 genes were differentially expressed under low copper stress (1 g/L), while 250 genes exhibited altered transcriptional levels at higher copper stress (3 g/L). These differentially expressed genes were involved primarily in metabolic pathways such as energy metabolism, two-component systems, amino acid metabolism, and signal transduction. The Sox family cluster gene cluster involved in the conversion of thiosulfate to sulfate was upregulated in the sulfur metabolism pathway. In the oxidative phosphorylation pathway, genes participating in the synthesis of NADH oxidoreductase and cytochrome c oxidase, nuoL, cyoABD (cyoA, cyoB and cyoD) and cydAB (cydA and cydB), were downregulated. The TCS element ompR, closely associated with the osmotic pressure, exhibited active response, while Cu2+ efflux system gene cusRS was upregulated. In the amino acid metabolism, the glnA involved in nitrogen fixation was upregulated and promoted the synthesis of glutamine synthetase for reducing excessive oxidative stress. This study provides new insights into the mechanism underlying A. caldus response to heavy-metal ion stress under harsh bioleaching conditions.

Insights into the microbial community structure of anaerobic digestion of municipal solid waste landfill leachate for methane production by adaptive thermophilic granular sludge

BACKGROUND: The amount of municipal solid waste (MSW) gradually increased along with the rapid development of modern cities. A large amount of landfill leachate are generated with excessive chemical oxygen demand (COD), which create a great deal of pressure on the environment-friendly treatment process. Anaerobic digestion is an ideal technique to solve the above problem. RESULTS: A thermophilic granular sludge was successfully adapted for anaerobic digestion of MSW leachate (from an aging large-scale landfill) for methane production. The COD degradation efficiency improved by 81.8%, while the methane production rate reached 117.3 mL CH4/(g VS d), which was 2.34-fold more than the control condition. The bacterial and archaeal communities involved in the process were revealed by 16S rRNA gene high-throughput pyrosequencing. The richness of the bacterial community decreased in the process of thermophilic granular sludge, while the archaeal community structure presented a reverse phenomenon. The bacterial genus, Methanosaeta was the most abundant during the mesophilic process, while Methanobacterium, Methanoculleus, Methanosaeta and Methanosarcina were more evenly distributed. The more balanced community distribution between hydrogenotrophic and acetotrophic methanogens implied a closer interaction between the microbes, which further contributed to higher methane productivity. The detailed relationship between the key functional communities and anaerobic digestion performances were demonstrated via the multivariate canonical correspondence analysis. Conclusions: With the assistance of adaptive thermophilic granular sludge, microbial community structure was more evenly distributed, while both of COD degradation rate and methane production was improved during anaerobic digestion of MSW landfill leachate.