Molecular Mechanism Of The Inhibitory Effect Of Phosphate On Uptake And Transformation Of Roxarsone By Enterobacter Sp.CZ-1

The arsenic-containing compound Roxarsone（ROX）has been widely used as a feed additive in livestock and poultry production,which has the functions of accelerating animal growth,brightening fur color,anti-coccidioidal and anti-bacterial.ROX introduced into the environment by livestock and poultry farming can be transformed into other forms by microorganisms.Previously,research on ROX biotransformation mainly focused on anaerobic microorganisms.Enterobacter sp.CZ-1,an aerobic bacterium,was isolated from an As-contaminated paddy soil and could transform ROX into five different As-containing metabolites under aerobic conditions.However,the mechanism of ROX uptake by strain CZ-1 is still unclear.In this study,we found that the uptake and transformation of ROX by strain CZ-1 were inhibited by inorganic phosphate（Pi）,a medium component.Using various molecular biological methods,such as gene knockout,prokaryotic transcriptome analysis and heterologous expression,we investigated the mechanism of the inhibitory effect of Pi on uptake and transformation of ROX by strain CZ-1.The main results of this thesis are as follows:1)By comparing the ROX transformation ability of strain CZ-1 in three different media ST10^-1,M9 and LB,it was found that strain CZ-1 transformed ROX only in ST10^-1.In the other two media（LB and M9）,The strain CZ-1 completely lost the transformation activity of ROX.The inhibitory effect induced by LB and M9 was a reversible process.Further research showed that the rich inorganic phosphate ions(HPO₄^2-and H₂PO₄^-)in M9were the key components to inhibit the transformation of ROX by strain CZ-1.Moreover,compared with ST10^-1,ROX intake was significantly reduced in LB and M9.Adding 50m M Pi to ST10^-1 medium could significantly inhibit the uptake of ROX by CZ-1 cells.After knocking out the Pi transport system coding genes pit,pst BACS and phn CDE in Enterobacter sp.CZ-1,it was found that there was no difference in the ROX uptake capacity between the mutants and wild-type strain.However,exogenous addition of 50 m M Pi still significantly inhibited the uptake of ROX by the three mutants.These results indicated that ROX uptake was not mediated by the three Pi transport systems.Interestingly,we found thatΔpit mutant andΔpst BACS mutant did not affect ROX transformation,whereasΔphn CDE mutant significantly inhibited ROX transformation.2)Through transcriptome analysis and relative quantitative PCR,we obtained a total of 20 genes whose expression levels were decreased by Pi by 3 times or more.They are:pst A,pst S,gud P,iat A,yeb E,ywk B,mae N,cus C,arg T,orf02426,phn D,phn E,rbs A,rbs C,rbs B,orf04219,ugp E,ugp A,ugp B and lld P.Among them,pst AS and phn DE are the components of the Pi transport system encoding genes pst BACS and phn CDE,respectively,and have been proved not to be involved in the uptake of ROX.We constructed mutants with deletion of these genes and measured the intracellular accumulation of ROX in these mutants.It was found that knocking out lld P significantly reduced the intracellular accumulation of ROX by strain CZ-1,while knocking out other 15 genes did not affect the uptake of ROX.According to the genome annotation,lld P encodes an L-lactate permease.The growth curve results showed that knocking out lld P had no significant effect on the growth of the strain.Compared with the wild-type strain,the ROX uptake capacity ofΔlld P mutant was significantly decreased,while there was no significant difference in As（III）uptake between the two strains.The result indicated that the protein coded by lld P participated in the uptake of ROX,but not As（III）.Besides,we found that knocking out lld P gene significantly reduced the ROX transformation ability of strain CZ-1 within a certain period of time（≤8 h）.Exogenous addition of L-and D-lactic acid had no significant effect on the uptake of ROX by strain CZ-1 andΔlld P mutants.In order to further explore the function of Lld P protein,we attempted to express Lld P protein in E.coli BL21（DE3）,but this was unsuccessful.Taken together,the results suggested that Lld P was involved in the uptake of ROX.Phosphate inhibited the expression of lld P gene,thereby inhibiting the uptake and transformation of ROX by the strain CZ-1.