Identification Of A Bacterium Resistant To Copper&Cadmium And Construction Of Genome BAC Library

A bacterial strain NTG-01 which was resistant to 4.5mmol/L Cu2+and 2.0mmol/L Cd2+ was isolated from the soil of a copper mine in DaYe city of Hubei province. The high resistance of the strain to cadmium and copper is important in studying the resistant mechanisms of bacteria to heavy metals. Experiments showed that the expression of cadmium-resistant gene was induced by the copper-resistant gene. No plasmid was found in the cell after plasmid extraction, in situ plasmid fast detection and pulsed field gel electrophoresis determination. It is assumed that the gene was targeted in the chromosome. This result was different from the previous studies in which the heavy metal resistant gene was reported to be located in plasmid. Morphological and physiological identification showed that NTG-01 was Gram-negative, short rod shape, with flagella around the bacteria, V-P test positive and Methyl red test negative. The bacterium can use glucose as carbon suource and produce gases. No H2S produced on TTS medium. Lysine decarboxylase and orthinine decarboxylase, catalase and gelatine test are positive. Arginine decarboxylase, dehydrolase, oxidase and urase test are negative. Sequencing and alignment of 16S rDNA showed that it shared a 99% homologous sequence with Enterobacter aerogenes. Based on the above results along with morphological features and physiological traits, the strain was identified as Enterobacter aerogenes. In this study, MIC values of the isolated strain to 9 heavy metals were determined and the MIC values follow the order of Ag (0.00625 mmol/L) > Pb (0.9 mmol/L) > Zn (1.6 mmol/L) = Co > Ni (2.0 mmol/L) = Cd > Cr (2.11 mmol/L) > Cu (4.5 mmol/L) > Mn (26.7 mmol/L). It was concluded from the MIC values' that strain NTG-01 is highly resistant to most heavy metals. The genomic library was constructed in this study. According to previous investigations the size of copper-resistant gene is generally 7 kb and the fragment of all the clones should be four times lager than its containing gene. The fraction size of our genomic library was around 40 kb. Seven hundred clones were harvested and the library covered 99% of the chromosome. Therefore, the copper and cadmium-resistant gene could be cloned from the library in theory.This study attempted to clone the cadmium and copper-resistant genes from the isolated bacterial strain and would provide a basis for the construction of engineered strain to remediate heavy metal-contaminated environments.