Molecular Mechanism of Resistance of heavy Metal Resistant Bacteria and Their Application of to Heavy Metal-Containing Waste Water.

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l. MOLECULAR CLONING OF HEAVY METAL-RESISTANCE GENE.(1) P. Putida GAM-1 harboring pGUIOO can grow at 7mM CdCl_2. The Cd(II)-resistance was encoded in pGUlOO. By shot-gun cloning, a Cd(II)-resistant Ecoli C600 transformant was obtained, and it harbored a plasmid (pCDR1, 5.0 kb) which was constructed by BamHI treatment. But, BamHI could cut only one site in pCDR1. Southern analysis was done by using BamHI-EcoRI DNA fragment of pCDR1. One of BamHI-digested pGU1OO fragments was hybridized with BamHI-EcoRI fragment. DNA sequence of the BamHI-EcoRI fragment was highly homologous for that of IS1. (2) P. ambigua G-1 harboring 7 plasmids can grow at 1OOmM K_2CrO_4. Ecoli C600 was transformed to be Cr(VI)-resistant with the plasmids. Three Cr(VI)-resistant Ecoli C600 transformants were obtained, and they harbored 7.4 kb plasmid in common.2. MECHANISM OF HEAVY METAL-RESISTANCE.(1) Cd(II)-resistance of P. putida GAM-1 was induced by 1mM CdCl_2 , but that of E. coli C600(pGUIOO) was constitutive. T … More he Cd(II) content of E. coli C600 was 3-fold, as compared with that of E. coli C600(pGUIOO). This reduced acccumulation of Cd(II) by this transformant was due to an energy-dependent Cd(II) efflux system. (2) Cr(VI) reductase of P. ambigua G-1 was highly purified with several columnchromatographies. The molecular weight of the enzyme was estimated to be 65, 000 and that of subunit was 25, 000, indicating that the reductase consists two identical subunits. The enzyme required NAD(P) H as electron donor. The optimal temperature and pH were 60ﾟC and 8.1-8.6, respectively. Stoichiometry of Cr(VI) reduction and ESR spectra revealed that the Cr(VI) reductase reducod Cr(VI) to Cr(VI) to Cr(III) through Cr(V) as an intermed. iate.3. MICROBIAL TREATMENT OF HEAVY METAL-CONTAINING WASTE WATER.(1) P. Putida GAM-1 and Cd(II)-resistant E. coli C600 transformants were able to grow in the presence of 3.5mM CdCl_2. As P. putida GAM-1 can not grow at below pH 5, heavy metal-containing waste water in low pH requires the neutralization of the waste water. (2) P. ambigua G-1 was capable of growing in L-broth containing 150 ppm Cr(VI) and reduced actively Cr(VI) to Cr(III). Less