Genetic manipulation of cysH/cysJ in Citrobacter sp. XT1-2-2 enhanced cadmium immobilization by regulating metabolic...

The Citrobacter sp. XT1-2-2 strain has emerged as a promising candidate for cadmium immobilization; however, the genetic basis underlying its sulfur-mediated bioremediation mechanisms remains inadequately understood. To address this gap, we concentrated on two pivotal genes, cysH and cysJ, within the sulfate assimilation pathway. We constructed Citrobacter sp. XT1-2-2-::APS and Citrobacter sp. XT1-2-2-::SiR strains with overexpression of cysH or cysJ for functional characterization. Transmission electron microscopy demonstrated a significant enhancement in the biosynthesis of CdS nanoparticles in both overexpression strains. This increase was attributed to the elevated production of hydrogen sulfide. Complementary physicochemical analyses, including Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and powder X-ray diffraction, further indicated that overexpression of cysH or cysJ modified functional groups on the surface and enhanced the efficiency of sulfur metabolism. Microcosm experiments demonstrated that the contents of Cd²⁺ in the roots, culms, leaves, and grains inoculated with overexpression strains were significantly lower than those observed in the wild-type strain. These findings establish a critical role for cysH/cysJ-mediated metabolic pathway regulation in cadmium immobilization. They provide a theoretical foundation for the exploration of novel bacterial-assisted techniques, marking a breakthrough in environmental biotechnology from fundamental research to the engineering application of genetically engineered bacteria.IMPORTANCEThe presence of cadmium in paddy soil poses a significant concern, primarily due to its potential threat to food safety and public health within the soil-plant system and the broader food chain. The genes cysH and cysJ were overexpressed under the regulation of the erythromycin promoter within the sulfate assimilation pathway in the Citrobacter sp. XT1-2-2 strain. The resulting overexpression strains (XT1-2-2-::APS and XT1-2-2-::SiR) exhibited enhanced biosynthesis of CdS nanoparticles, attributed to increased hydrogen sulfide production. Compared to the wild-type strain, cadmium concentrations in the grains of XT1-2-2-::APS and XT1-2-2-::SiR were reduced by 36.49% and 62.56%, respectively. Furthermore, the residual cadmium content in the soil was elevated by 36.36% (P < 0.01) and 27.27% (P < 0.01), respectively. These results provided a theoretical foundation for the exploration of novel bacterial-assisted techniques aimed at cadmium remediation, marking a breakthrough in environmental biotechnology from fundamental research to the engineering application of genetically engineered bacteria.