Research Of Copper Resistance Related Gene In S. Meliloti CCNWSX0020

Rhizobia are important agricultural microorganisms which can fix nitrogen by symbiosewith legumes. If long-term growth in the heavy metal polluted soil, Rhizobium cells may beform some mechanism resistant to heavy metal ions. Sinorhizobium. meliloti CCNWSX0020,isolated from root nodules of Medicago lupulina growing in Fengxian gold mine tailings inthe northwest of China, was resistant up to1.4mM Cu²⁺in tryptone-yeast （TY） extractmedium. By culturing the bacteria in the containing0.5mM Cu²⁺and Cu²⁺-free TY medium,the difference gene expression were analyzed by using cDNA-AFLP technique and obtained3differential transcript Derived Fragments. The differential fragments were sequenced andanalyzed with BLAST, the result showed that the TDF1corresponding gene encodes a proteinwith97%similarity to copper ion efflux P-type ATPase of S. meliloti1021; The TDF2corresponding gene encodes a protein with42%identity to RNase H of CandidatusAccumulibacter phosphatis clade IIA str. UW-1; The TDF3is a part of omp gene, omp contains1446nucleotides which encodes a putative protein with75%similarity to outer membraneprotein of Rhizobium leguminosarum bv. viciae3841.omp gene was amplified from S. meliloti CCNWSX0020and built omp gene knockoutvector.0.8mM Cu²⁺ could significantly inhibit the growth of the homologous recombinationmutant. The omp gene was cloned into pBBR1MCS-5and transferred into mutant. The resultshowed that omp gene can restore about80%copper resistance. Two genes SM0020₁8782and SM0020₁8787encode two unknown functions putative proteins in the upstream of Ompgene, there are three other genes SM0020₁8797, SM0020₁8802and SM0020₁8807encodecopper ion multicopper oxidase, the putative binding protein and a putative copper chaperonein the downstream of the omp gene, these6genes constitute an operon which plays animportant role in copper resistance.The metabolites of CCNWSX0020and omp mutant growing in TY medium with andwithout0.5mM copper ions were determined by GC-MS. The data were analyzed using PCA,PLS and TICL, the results showed that intracellular metabolites of cells cultured in mediumwith and without copper have obvious difference. The metabolites of wild type and mutanthave certain difference induced by copper, the concentration of alanine, glycine, leucine andvaline increased in mutant which can accelerate protein synthesis and repair damage proteincaused by copper. Malic acid, fructose, glycerol-3-phosphate content increased in wild-typecells which can accumulate antioxidant NADPH by adjusting the TCA. At the same time, two kinds of bacteria can produce IAA, SOD and CAT in the medium with copper. But comparedwith wild-type, the CAT, SOD activity of mutant reaches the maximum under lowconcentration copper, it showed mutant was more sensitive to copper, so copper lead freeradicals may cause inactivation of some enzyme and decreased the IAA yield in mutant.The draft genome sequence of Sinorhizobium meliloti CCNWSX0020comprises7,001,588bases. The genome of S. meliloti CCNWSX0020has a G+C content of59.9%.There are a total of7,086genes, including6rRNA genes,47tRNA genes, and7,033putativeprotein-coding sequences. Additionally, Sinorhizobium meliloti CCNWSX0020carried51predicted protein-coding genes involved in heavy metal resistance. Multiple genes relate toantioxidant SOD, CAT, GSH metabolism have been found in CCNWSX0020genome; Thereare some other genes encode proteins relate to synthesis of ACC deaminase, trehalose, IAA,this kind of metabolites produced by CCNWSX0020can promote plant growth and increasethe biomass to absorb more copper ions, thus reduce the copper concentration in the soil andenhance tolerance of bacteria to copper. The expression of six genes encoding P-type ATPasewere detected by RT-PCR in the MerR-like mutant, the results show that the SM0020₀5727and SM0020₀5862genes were regulated by MerR-like transcription factor. However, theP-type ATPases encode by these genes are not contain N-terminal CXXC motif. In addition,the gene product of SM0020₀5862probably is a special P-type ATPase since it contains nota CPC motif but TCP which is highly unusual.