Regulation Of Selenate Reduction By The Transcriptional Regulator CysB In Comamonas Testosteroni S44

Selenium(Se)is widespread in nature,and it is one of the essential trace elements for life.Deficiency or excess intake of selenium cause human diseases.Microbes play an important role on selenium cycle,which are involved in all the biochemical reactions of selenium oxidation and reduction.Previous studies of selenate reduction were mainly focused on the anaerobic bacteria,the molecular mechanism of selenate reduction remains unknown in aerobic bacteria.The strictly aerobic bacterium Comamonas testosteroni S44 had the ability to reduce selenate to red-colored selenium nano-particles.Eleven mutants of selenate reduction were obtained by using the transposon mutagenesis technique.The Tn-inserted sites in seven mutants were the gene encoding transcriptional regulator CysB of the sulfur metabolism.The Tn-inserted sites in two mutants were the gene encoding cell division protein FtsH.The others were genes encoding MFS family transporter and large subunit ribosomal RNA,respectively.CysB is involved in regulating cysteine anabolism.In this case,cysB mutants did not reduce selenate.Accordingly,we hypothesize that selenate is transported into cells and reduced through one of the cysteine sythesis pathways,which is regulated by CysB.Firstly,the competitive test of sulfate and selenate was processed.The reduction of selenate was inhibited by sulfate or cysteine,and the growth of C.testosteroni S44 was inhibited when the concentration of selenate was higher than that of sulfate,suggesting that selenate was reduced through sulfate reduction pathway.Moreover,the test of double exchange knockout and complement of the gene cysB demonstrated that CysB was associated with selenate reduction.In contrast,the selenate reduction was not processed as the gene cbl encoding sulfonate utilization transcriptional regulator was deleted,indicating that both the tau operon(responsible for taurine utilization)and ssu operon(responsible for alkanesulfonate utilization)regulated by Cbl were not involved in the selenate reduction.These results suggested that the cysteine sythesis cys operon regulated by CysB could be the genes involved in selenate reduction.Mutants of single exchange of cysl or double exchange of cysN lost the ability of selenate reduction,indicating that cysIHDN were responsible for selenate reduction.In addition,all the results of qRT-PCR,lacZ reporter gene fusion,bacterial one-hybrid reporter system and EMSA confirmed that the cysIHDN operon was regulated by the CysB and induced by selenate.In conclusion,selenate was reduced via the sulfate reduction pathway and regulated by CysB in C.testosteroni S44.