Cloning And Functional Identification Of Copper-Resistance Genes Of Sinorhizobium Meliloti CCNWSX0020

It is well known that symbiotic nitrogen-fixing bacteria are commonly associated with the roots of leguminous plants. Rhizobia colonize the roots of legumes where they fix atmospheric N2, some of which can be utilized for plant growth. A root nodule bacterium, S.meliloti CCNWSX0020, resistant to 1.8 mM CuSO₄ was isolated from Medicago lupulina growing in mine tailings of Fengxian county, China. Six mutants sensitve to copper-resistance were obtained after screening about 14000 mutants by mutagenesis with Tn5-1064a, they were SXa-1, SXa-2, SXc-1, SXc-2, SXn and SXy. Zinc-resistant experiment showed that SXc-1, SXc-2, SXn and SXy were sensitive to zinc, while SXa-1 and SXa-2 were not, which suggested that mutant genes of SXc-1,SXc-2,SXn and SXy were probably involved in cation transport or Zn²⁺ involved in some metabolic pathways in these mutants. Meanwhile, SXc-1,SXc-2 and SXy showed sensitivity to Pb(NO₃)₂ while other mutants were slightly sensitive, which further indicated that the mutant genes of SXc-1,SXc-2 and SXy were probably related to divalent cations transport.Copper-sensitive phenotype of mutants were identified by the construction and screening of a transposon Tn5-1063a library and PCR. The results showed that single copy inserted into mutants, indicating that coppe-sensitive phenotype was the result of transposon insertion. Six copper-sensitive mutants were obtained and five different genes were indentified by sequencing. SXa-1 and SXa-2 were found with disruptions in the lpxXL gene, encoding LpxXL C28-acyltransferase; SXc-1 and SXc-2 were disrupted with cueR gene, which was found as regulator of copper- tolerance genes copA and cueO; SMc02281 of S. meliloti 1021 was inserted with SXn which encoded unknown membrane protein. It showed 74.63% similarity with the copper-tolerance protein in Agrobacterium sp. H13-3. Therefore, in this paper, SXn gene was lacated in copB and SXy was disrupted with fixI1 gene, belonging to E1-E2 ATPase family, involving in cellular aerobic respiration the electron transfer as a ion pump.The mutant genes were cloned into the vector, and through the tri-parental conjugation, each mutant gene was transferred into the corresponding mutants. The results showed that SXy-1 completely restored the growth at the concentration of 1.2 mM CuSO₄ and others could grow even at the concentration of 1.5 mM CuSO₄, furtherly suggesting that these mutant genes were related to copper-tolerance of S.meliloti CCNWSX0020.Nodule tests showed that lpxX and fixI1 mutant genes could remarably reduce the number of nodules of host plants. Comparing with the host plants inoculated wild S.meliloti CCNWSX0020, dry weight and copper accumulation in root as well as shoot of plants inoculated mutants obviously reduced. The findings suggested that mutants in this study affected the growth of host plants as well as absorption to copper, which indirectly indicating mutant genes were related to copper ion transport.