Cd²⁺ Tolerance Mechanisms Of Serratia Marcescens KMR-3

Cadmium pollution has brought a great threat to environmental safety and human health.Biosorption removal technology,especially by using microbial as adsorbent,has been thought as a highly efficient,low-cost,and eco-friendly technique.However,the mechanisms involved in cadmium ions tolerance in bacteria is still unclear,which has been a serious problem in development of this technology in bio-remediation.To understand the toxicity of the heavy metals and the detoxification mechanisms in microorganisms will provide important theoretical and practical knowledge for the biological restoration of wastewater and soil in heavy metal pollution environments.To illustrate mechanisms involved in bacterial resistance against Cd²⁺,Serratia marcescen KMR-3 was used in this research,and Cd²⁺was used as heavy ion.The effects of different concentrations of Cd²⁺on the biomass,morphology,and metabolic activity of KMR-3,as well as the series of response mechanisms and cellular damages treated with Cd²⁺was investigated in this research.KMR-3 showed a high tolerance capacity under the Cd²⁺binding characteristics of KMR-3 were analyzed by SEM（Scanning electron microscopy）,XPS（X-ray photoelectron spectroscopy）and FTIR（Fourier transform infrared spectroscopy）.A significant difference in cell adhesion,membrane thickening,and shrinkage on the surface of the KMR-3 cells were identified.In addition,the amount of biofilm increased up to 1.5 to 3 times when KMR-3 was treated with concentration of Cd²⁺≥200 mg/m L,which suggested benefit for the adsorption of Cd²⁺,which help KMR-3 absorb and fix Cd²⁺outside of cell.The adsorption efficiency of Cd²⁺was increased by 19.5%when prodigiosin was produced in KMR-3.It was suggested that prodigiosin can form a network at outside of membrane to prevent Cd²⁺from entering cytoplasm,which better improve the ability of resistance against Cd²⁺in KMR-3.The Cd²⁺oxidative damage and cellular response mechanisms of antioxidant system in KMR-3 was investigated.The role of antioxidant enzymes and small molecules involved in cellular stress protection and the regulatory mechanisms were investigated thoroughly.It was found that increase in the level of active oxygen in KMR-3 cells will promote the production of H₂O₂ and peroxidation product of MDA（malondialdehyde）under Cd²⁺stress.The dose-effect curve showed a desired dose-effect relationship between Cd²⁺concentration and oxidative damage,which confirmed that the enrichment of intracellular Cd²⁺is one of reasons leading to damage of KMR-3 cells.The growth and metabolism were inhibited by enrichment of Cd²⁺,which disrupt the dynamic balance of the generation and clearance of active oxygen in KMR-3.On the other hands,the excess of active oxygen which produced by Cd²⁺induced the production of small molecule antioxidant and enzymes（SOD,CAT and GSH-Px）when treated with Cd²⁺,which eliminate excess intracellular active oxygen in KMR-3.The results showed that the content of total polyphenols and flavonoids were positively correlated with the concentration of Cd²⁺,which certified that antioxidant system play an important role in keeping the balance of oxidation and reduction status in the KMR-3 cells under Cd²⁺stress.In the end,we found that the transcription levels of genes related to the cell membrane biosynthesis pathway,the prodigiosin synthesis pathway,the P-type ATPase efflux pathway,and the Znu protein chelation pathway showed changes under Cd²⁺stress based on the transcriptome analysis of KMR-3.The RT-q PCR results indicated that the expression level of the protein chelating system genes of znu（znu A,znu B and znu C）and P-type ATPase efflux system regulating genes（znt A,znt B and znt R）increased significantly when treated with Cd²⁺.The results imply that those two systems played important roles in KMR-3 against Cd²⁺.To test this hypothesis,two knockout mutant strains were constructed in which the whole znu system（znu A,znu B and znu C）and znt A of P-type system were deleted and substituted with chlorampenicol resistant gene respectively.The results indicated that knockdown of the znt A decreased the resistance to Cd²⁺from 700 mg/L to 250 mg/L and the removal of Cd²⁺by 5.8%±0.5%,and knockdown of the znu decreased the resistance to Cd²⁺from 700 mg/L to300 mg/L and the removal of Cd²⁺by 7.19%±0.5%respectively.By those method,it has been demonstrated that the protein chelating system genes and P-type ATPase efflux system played significant roles involved in tolerance and removal of Cd²⁺+in KMR-3.The mechanisms involved in tolerance of Cd²⁺in KMR-3 were illustrated in this paper.It will not just provide a theoretical reference for the economic and effective application of Serratia marcescens KMR-3 in the bioremediation of heavy metal polluted water and soil,but also will help to improve the efficiency and capacity in treatment of heavy metals in the environment with microorganisms.