| The microbial metabolism of arsenic is involved in energy and material source flow, which play key roles in biogeochemical cycles. Pteris vittata L. (brake fern) is an arsenic hyper-accumulator that has been successfully used in arsenic bioremediation. However, little is known about how microbial communities and functional genes respond to arsenic contamination, rhizosphere and biogeochemical heterogeneities throughout the vertical soil profile, while those factors may ultimately influence on the arsenic transformation and phytoremediation. Five soil samples with different arsenic contaminated levels in P. vittata rhizosphere and non-rhizosphere,5 depths from the surface down to 4 m were respectively collected from an arsenic phytoremediation experimental station and its adjacent untreated region, where located at Chenzhuo city, Hunan province, Central South of China (25°48’N and 113°02’E). The samples were investigated by Biolog and functional gene microarray (GeoChip 3.0) analyses to illustrate the linkage between microbial metabolic potential, community structure, key functional genes with arsenic contaminated levels, rhizosphere, soil geochemical features and vertical soil profile. Moreover, bacteria zinc resistance molecular basis has been discussed, while the homoistasis mechanism is largely unknown. Here, we studied zinc efflux systems and their transcriptional model based on whole genomic sequence in a high zinc resistant strain, Comamonas testosteroni S44. The main results were as follows:1. At P. vittata rhizosphere with arsenic contaminated samples, the uncontaminated soil harbored the greatest diversity of sole carbon utilization ability, and that arsenic-contamination decreased the metabolic diversity, while rhizosphere soils had stronger metabolic activities than the non-rhizosphere soils. GeoChip 3.0 analysis showed low proportions of gene overlap across the five soil samples (16.52%-45.75%). The uncontaminated soil had a higher heterogeneity and more unique genes (48.09%) compared to the arsenic-contaminated soils. Canonical correspondence analysis (CCA) revealed that arsenic is the main driver in reducing the soil functional gene diversity; however, organic matter and phosphorus also have significant effects on the soil microbial community structure. Arsenic resistance, sulfur reduction, phosphorus utilization and denitrification related genes structure were remarkably distinct between P. vittata rhizosphere and non-rhizosphere soils, which provides evidence for a strong linkage among the level of arsenic contamination, the rhizosphere and the functional gene distribution. The results implied that soil arsenic phytoremediation is the interaction among P. vittata, microbia and arsenic levels, and rhizobacteria play an important role during the processes by P. vittata.2. At the vertical soil profile, the microbial metabolic potential and diversity were decreased strongly with soil depths. Dissimilarity tests showed the community structure of surface samples were significantly different (p< 0.05) from the subsurface samples, while no significant difference were detected among the subsurface samples. However, significant changes of genes involved in arsenic resistance, carbon and nitrogen cycling were detected among the vertical soil profile. The C/N ratios showed significant correlations with arsC/B/A (arsenic resistance, p= 0.069), carbon cycling (p=0.084) and nifH (nitrogenase, p=0.024), while rbcL genes (ribulose-1,5-bisphosphate carboxylase) were signicantly correlated (p=0.071) with the T-C (total carbon) contents. The variation of microbial structures and activities were observed when using the rbcL and nifH genes as biomarkers. The combination of P, NO3- and C:N showed the highest correlation (p=0.062, r=0.779) with the whole microbial structures across the vertical soil profile. The results revealed that short-term arsenic infiltration created a certain impact on microbial structures and functional genes, while variation of available nutrient and spatial isolation were the key factors in maintaining microbial functions and controlling As transformation.3. A highly zinc resistant bacterium, Comamonas testosteroni S44, isolated from mining soil, which harbored multiple metal resistance, such as Cd2+ and Pb2+ Reporter gene assays showed the putative regulator ZntRl responded to Zn2+, Cd2+ and Pb2+. When the zntR1 gene was disrupted, the mutant (S44ΔzntR1) displayed decrease resistances to Zn2+, Cd2+ or Pb2+(MICs:10 mM vs 6.0 mM,2 mM vs 1.5 mM and 3.5 mM vs 2.0 mM, respectively), and accumulated more intracellular Zn2+ Whole genome sequences analysis revealed that the strain contained nine putative Zn2+ transporters (4 zntA and 5 czcA genes). Real time RT-PCR revealed the 4 zntA-like genes were all induced by Zn2+, while czcA-like genes, three were Zn2+ induced and two were down-regulated. The large number of genes encoding putative metal(loid)s resistance proteins and mobile genetic elements implied that transposition and duplication events may have occurred in C. testosteroni S44 to adapt to heavy metal(loid)s contaminated environment.The results of this dissertation have made great contributions to the exploitation of the linkage between microbial structure and key functional genes with arsenic contaminated levels, rhizosphere and spatial isolation, and required the key factors in shaping microbial communities, which will be essential for future protection of biodiversity and arsenic remediation. Based on gene knockout, reporter assay and whole genomic sequence comparison, our study provided full understanding on the molecular mechanism of high zinc resistance. |
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