Cadmium Toxicity Is Alleviated By AtLCD And AtDCD In Escherichia Coli

Hydrogen sulfide (H2S) is emerging as an important gasotransmitter in vivo parallel to nitric oxide (NO) and Carbon Monoxide (CO). H2S plays an important role in many physiological processes in mammals. Metabolism and functional importance of H2S have also been shown in plants. But the detection and the functions of the important enzymes that catalyze the generation of endogenous H2S in plants just only start to research, so it is necessary to further learning.AtLCD and AtDCD are important enzymes that catalyze the generation of endogenous H2S in Arabidopsis thaliana. To determine the effects of AtLCD and AtDCD proteins on cadmium (Cd) toxicity in Escherichia coli, we cloned AtLCD and AtDCD into pET28a expression vectors and transformed wild-type E. coli strain BL21(DE3) with these vectors. The AtLCD-and AtDCD-containing strains were named BL21(LCD) and BL21(DCD). The main results of experiments were as follows:1. AtLCD and AtDCD proteins were expressed in E. coli and showed cysteine desulfhydrase activity. The AtLCD protein band was46.4kDa, while the AtDCD protein band was41.7kDa by the software (http://www.expasy.ch/tools). As anticipated, there were two protein bands around45kDa by SDS-PAGE. In addition, In the induced E. coli strains BL21(LCD) and BL21(DCD) compared with wild type, a significantly higher H2S generation rates were observed. The experimental results indicated that the crude protein extract of BL21(LCD) decomposed L-cysteine into H2S, whose production rate was1.07times that of wild type, and that BL21(DCD) decomposed D-cysteine into H2S, whose production rate was7.2times that of wild type.2. Cd toxicity is alleviated by AtLCD and AtDCD in E. coli. Under220μM Cd stress, the survival rates of induced BL21(LCD) and BL21(DCD) were11.6and6.94times that of wild type respectively (Colony-forming units (CFU) with a diameter>1mm). Reduced contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2), decreased activities of superoxide dismutase (SOD) and catalase (CAT) under220μM Cd stress were noted in the strains BL21(LCD) and BL21(DCD). The above changes were substantially counteracted by the mixture of ammonia and pyruvic acid potassium (NC), which is used as a synthetic inhibitor of H2S.3. H2S, generating an ameliorating effect against Cd-induced oxidative stress, results in bacteria resistance to Cd toxicity. Addition of100μM NaHS, the exogenous H2S donor, restored CFU number and colony size significantly; at the same time, addition of NC eliminated the effect of NaHS on the bacterium.