Cloning And Functional Analysis Of Copper Resistance Genes Of Bradyrhizobium Liaoningense

Copper in contaminated soil may present some selection pressure to soil bacterium. For surviving in the copper-rich environments and establishing valid symbiosis in the very host, rhizobia have to adopt effective strategies to cope with such conditions. Investigation of copper reisistance mechannism of rhizobia has great biological and ecological significances. Bradyrhizobium liaoningense CCNWSX0360 is a copper resistant strain isolated from root nodules of Vigna unguiculata growing in farmland near the copper/zinc mine tailings, and it exhibited more resistance to 2.0 mM Cu2+ on the TY agar medium.To identify genes involved in Cu resistance in the strain, a transposon mutant library?17,247 Tn5-insertions? was constructed. Upon screening, six Cu sensitive mutants were obtained, named Bln-d, Bln-163, Bln-c, Bln-29, Bln-32 and Bln-54, respectively. Of the mutants, Bln-d, Bln-163 and Bln-c showed specific sensitivity to copper while Bln-32, Bln-29 and Bln-54 the varying of decreased tolerance toward other metals ions,but did not exclusively affect the resistance to Cu2+. Plasmid rescue strategy was used to to map the sequences contiguous with the inserted transposon, and the results showed that Tn5 inserted into the same gene encoding a putative heavy metal-transporting P-type ATPase, named cueA, in both strains Bln-d and Bln-163. For the other four mutants?Bln-c, Bln-29, Bln-32 and Bln-54?, the Tn5 was inserted in gene copA?encoding a multicopper oxidase?, gene ctpA?encoding a carboxyl terminal processing enzyme?, gene tolC?encoding a TolC family protein? and lptE?encoding a LPS transporter chaperone LptE?, respectively. This indicated that B. liaoningense CCNWSX0360 adopts three mechanisms at least to protect cell from copper toxicity: transmembrane transport?cueA and tolC?; oxidation or sequestration?copA?; and protection of the membrane barrier?lptE and ctpA?.CueA was classed to the Cu⁺/Ag⁺ transporting P1B-type ATPase acording to the metal binding motif and signature sequences present in transmembrane segments?TMS?. Unlike other P1B1-type ATPases possessing the typical N-terminal GXXCXXC motif, CueA contains two His-rich stretchs. Using homologous combination knock-out strategy two in-frame deletion mutant ?csoR and ?cueA were constructed, and subjected to copper tolerance assay. The results showed that the MTC of the mutant ?cueA to copper was 0.8 mM which is identical to the Tn5 insertions?Bln-d and Bln-163?. Complementation of this mutant restored comparable copper resistance to that of the wild-type. qRT-RCR and lacZ fusion analysis data showed that expression of cueA could be induced by Cu²⁺ and displayed a dose-dependent manner. Addition of the Cu⁺-specific chelating agent bathocuproine disulfonate?BCS? to the cultures completely or partially eliminated the induction and toxicity caused by Cu²⁺. Analysis of the copper tolerance and of transcription via RT-PCR combined with transposon gene disruptions revealed cueA is involved in and plays the major role in intracellular Cu detoxification via monovalent Cu+ efflux. The transcription of csoR and cueA in response to Cu²⁺ were examined by qRT-PCR in the ?csoR mutant and the ?cso R?csoR? complemented strain. The result showed that csoR mutation result in a high and constitutive expression of csoR and cueA; complementation of the mutant with csoR restored the Cu²⁺-dependent induction of cueA demonstrating that CsoR acts as a repressor to regulate itself and cueA via depression mechanism. Our study also showed that csoR mutation significantly increased the strain more resistance to Cu, Zn and Cd; expression of csoR and cueA could be induced by the three metal ions; expression of CueA in Zn²⁺/Cd²⁺sensitive Escherichia coli GG48 significantly enhanced Zn²⁺/Cd²⁺ resistance of the strain. All together, these data clearly showed that CueA involved in detoxification of monovalent Cu+ and divalent Zn²⁺/Cd²⁺. In addition, both the putative His-rich regions were obligatory for the function?copper resistance? of CueA but not absolutely essential, however, deletion of the two His-rich regions completely incapacitated the function for Zn/Cd resistance. The role of His-rich MBDs were predicted to be involved in buffering “free” Zn²⁺/Cd²⁺ ions by binding these metal ions. In greenhouse studies, cso R mutant displayed a significant decrease in the nodule occupancy efficiency compared to wild-type strain, demonstrating that copper resistance gene gives B. liaoningense CCNWSX0360 a competitive advantage in the establishment of symbiotic association with V. unguiculata when faced with copper stress.According to the result of mutation and tolerance assay results,all of the six genes of cop operon can specifically mediate copper resistance, and the copA and copB were the central of the cop system. In addition, 2212 and 2213 were required for the full copper resistance of B. liaoningense CCNWSX0360 while copC and cusF were not absolutely essential for the tolerance. We can predict that the proteins encoded by the six genes may form a complex to detoxify the copper toxicity. Moreover, the six genes displayed a similar expression profiles at increased copper concentrations. The expression level of each gene increased gradually with the increase of the Cu²⁺ under low concentrations, and the level reached a maximum at 0.625 mM. However, the expression level decreased under the high concentration of Cu²⁺?> 0.625 mM?. Combined co-transcription validation experiments with the result of 5' RACE experiment revealed that the six genes were transcribed as a polycistronic mRNA under the control of the 2213 promoter. The transcription start site of 2213 was mapped to the G nucleotide located 42 nucleotides upstream of the putative start codon. Analysis of promoter activity and lacZ fusion expression showed that the intergenic region sequences between the gene 2213 and 2212 might play a critical role in regulating the operon expression. The specific regulatory mechanism is still not clear. Combined with bioinformatics analysis we conclude that cop operon may be an ancient copper resistance operon.