| With the development of economy, cadmium pollution is increasingly serious and greatly threats the eco-system and human health. Microorganism provides an efficient way in cadmium pollution control and remediation. Cadmium resistance of microorganism is one of the key factors which affect the application of microbe in cadmium pollution treatment. Thus, the research on microbe mechanism of cadmium resistance will provide theoretical support for the application of cadmium remediation by microbe.We collected cadmium-polluted soil samples and cadmium hyperaccumulator Solamum nigrum from a lead-zinc mine in Nanjing, Jiangsu province and isolated34,20and18bacterial strains from soil, root and stem, respectively. Maximal tolerant concentration (MTC) to cadmium and antibiotic resistance of strains were determined, and18strains were identified based on16S rDNA sequence analysis. Based on the cadmium and antibiotic resistance of the strains, strain KQ46was chosen as the tested strain for analyzing cadmium-resistant related genes of bacterium. According to the results of morphological characterization and physiological-biochemical analysis, strain KQ46was primarily identified as Cupriavidus campinensis.A transposon insertion mutant library of strain KQ46was constructed by using mini-mariner insertion mutagenesis. Ten mutants sensitive to cadmium were isolated and MTC to cadmium of these mutants significantly decreased from4mM (wild type) to0.8-1.0mM. Then we used SEFA PCR to amplify the flanking sequences of the transposon insertion sites in these Cd sensitive mutants. Sequence analysis revealed that transposon inserted into different sites in these ten mutants and homologous alignment showed that the gene might be disrupted in each mutant:six genes related to growth and metabolism were aroG, ppd, alkK, ppc, cytochrome c533gene and Acyl-CoA transferase gene, respectively; hmp maybe contributed to the stress resistance; hmyF encoded an auxiliary protein of the heavy metal cation-transporting efflux system HmyCBA; one gene encoded signal transduction histidine kinase;fcuA encoded ferrichrome receptor protein, which is involved in ferrichrome-cation transportation.By analyzing the function of mutagenized genes, we supposed that fcuA could regulate the concentration of Cd2+in Cupriavidus campinensis KQ46. We used sau3Al to digest the genomic DNA of MT21and cloned the fcuA gene by subcloning and sequencing. It was72%homologous to fcuA gene in C. metallidurans CH34and GenBank accession number was KC776916. To investigate how fcuA gene works, we constructed strain DRfcuA by recovering fcuA in MT21. Determination of Cd2+concentration in medium and cell during KQ46growing and FTIR analysis revealed that intracellular and extracellular concentration of Cd2+kept constant during growth. After growing12h and24h the intracellular concentration of Cd2+were0.548±0.016mg/g and0.552±0.008mg/g, respectively, and increased to1.038±0.046mg/g when cell entered into decline phase. No change in peak locations of infrared spectrum was detected under treatment with0.5mM Cd2+. For these results, we purposed that cadmium resistance of KQ46was efflux-mediated. The same studies on MT21and DRfcuA showed that though the MTC of these two strains were the same, cadmium caused more toxicity to MT21than DRfcuA. The absorption of Cd2+in DRfcuA was just50%to that in MT21, but still more than that in KQ46. FTIR profile showed that a peak at1735cm-1changed and this indicated that some functional groups on cell surface might bind with Cd2+. Taken as a whole, we reasoned that fcuA from KQ46regulate the concentration of Cd2+in coordination with some other genes, but the concrete mechanism needs for further study. |
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