The Impact Of High Concentration Of Selenate On The Growth And Selenocompound Metabolism Of Herbaspirillum Sp.WT00C

Herbaspirillum sp.WT00C is a Gram-negative bacterium isolated from a landscape tea plant.This bacterium can promote the growth and development of tea plants and reduce selenate to form elemental selenium(Se⁰).Our laboratory has conducted in-depth studies on its physiological and biochemical characteristics,microbiological traits,taxonomic status,genomic properties as well as infection and colonization in tea plants.Previous studies have found that Herbaspirillum sp.WT00C can grow in high concentration selenate(50-500m M)and effectively reduce selenate to elemental selenium(Se⁰),but the detailed mechanism is still unclear.In order to understand how high concentration selenate affects the growth and the selenocompound metabolism of Herbaspirillum sp.WT00C,the effects of high concentration selenate on bacterial growth and selenocompound metabolism have been studied by using microbiology,proteomics analysis,semi-quantitative RT-PCR and other techniques.After hard work for 3 years,we achieved results as follow:1.Herbaspirillum sp.WT00C showed a 12-hour inhibition period and a growth recovery period including its exponential and stationary phases when it grew in 200 m M Na₂Se O₄.At30 hours,the bacterial cells reached saturation.The bacterial cells cultured in the LB medium of 200 m M Na₂Se O₄ for 28 hours were again grown in the medium containing 200 m M selenate,and the growth inhibition period was shortened to 6 hours.When the same cells were inoculated into LB medium without selenate,the growth curve was the same as that of the original bacterium.2.The morphology of bacterial cells was observed by scanning electron microscopy(SEM).It was found that the cells of Herbaspirillum sp.WT00C cultured in the LB medium containing 200 m M Na₂Se O₄ gradually appeared as a thick-stick shape.Its width was nearly twice of the original bacterium.The crude rod-shaped Herbaspirillum sp.WT00C was transferred to the LB medium without selenate and grown at 37?for 24 hours,and then transferred to fresh LB medium once every 24 hours.After the 5^th transfer,bacterial cells almost returned to the original size.Hence,high concentration of selenate not only affected the physiological metabolic process of Herbaspirillum sp.WT00C but also changed the morphological structure of bacterial cells.Bacterial morphological change is just a physiological response to high oxidative stress caused by selenate rather than genetic variation.3.Analysis of the content of selenate and selenite in the bacterial culture at different times found that the selenate and selenite concentrations in the medium did not significantly decrease to the MIC value within 20 hours of cultivating Herbaspirillum sp.WT00C in 200m M Na₂Se O₄.This result indicates that the growth recovery of Herbaspirillum sp.WT00C is not due to significant decrease of selenate or selenite concentration,instead the bacterium itself establishes an effective antioxidant mechanism.4.Comparative analysis of proteomics found that:(1)high concentration of selenate caused high oxidative stress in bacterial cells during the growth inhibition period of Herbaspirillum sp.WT00C.High oxidative stress resulted in the down-regulation of certain important proteins involving in life activities,such as DNA replication and repair,glucose and lipid metabolisms.Hence,the bacterial cells were unable to grow and divide normally,which led to a long inhibition period;(2)bacterial cells did not die under high oxidative stress and began to grow after 12 hours.Herbaspirillum sp.WT00C counterpoised the act of oxidative stress via up-regulation of redox enzymes to produce plenty of NADPH;(3)in the bacterial growth recovery period,some proteins(e.g.DNA polymerase III and RNA helicase)down-regulated in bacterial growth inhibition period gradually returned to normal levels,whereas the NADPH/NADH-producing enzymes,thioredoxin reductase(Trx R)and glutathione peroxidase(GPx)were simultaneously up-regulated to maintain the antioxidant activity of the bacterial cells;(4)high oxidative stress caused by high concentration of selenate in the medium changed bacterial morphology.Bacterial deformation was due to the incompleteness of bacterial cell wall,envelope and extracellular membrane,which was caused by high oxidative stress;(5)the growth inhibition period was shortened to 6 hours when those cells with thick rod shape were transferred to the medium containing 200 m M Na₂Se O₄.This is due to stronger antioxidation of those cells and partial repair of cell wall structure.Those cells produced more NADPH/NADH and continuous up-regulation of lipid A-disaccharide synthase[EC:2.4.1.182].5.The effect of selenate on the transcription of 39 genes involved in the metabolism of selenocompounds were investigated by semi-quantitative RT-PCR.The expression of 5 genes related to the glutathione(GSH)metabolic pathway was significantly differen,in which gs,gsr,gcs were down-regulated but gpx and gst were up-regulated.Two genes sel A and trx B related to selenoprotein synthesis were significantly up-regulated.This result suggests that high concentration selenate retards glutathione synthesis,but accelerates selenoprotein synthesis.Less glutathione may lead to less production of elemental selenium.6.Herbaspirillum sp.WT00C was cultured in medium containing different concentrations of selenate and then the selenium content of bacterial proteins was detected.The results showed that the selenium content of bacterial proteins appeared as a nonlinear increase under 0—50 m M Na₂Se O₄.This result not only verifies that Herbaspirillum sp.WT00C can effectively synthesize selenoproteins via the selenoprotein synthesis pathway and proves an evidence to support that selenate indeed accelerates selenoprotein synthesis.Our studies found that high concentration of selenate not only affected the physiological and biochemical metabolism process of Herbaspirillum sp.WT00C,but also affected the bacterial morphology.Meanwhile,high concentration of selenate also affected selenoprotein synthesis.Under high oxidative stress caused by high concentration of selenate,Herbaspirillum sp.WT00C was able to recover its growth through its own antioxidant mechanism,and simultaneously reduced selenate to form elemental selenium(Se⁰)or synthesize selenoproteins to lower selenate concentration.Our studies also verify that Herbaspirillum sp.WT00C indeed holds a capability of synthesizing selenoproteins.These results provide theoretical basis and technical support for the production of elemental selenium(Se⁰)and selenoproteins by Herbaspirillum sp.WT00C in the future.