Identification Of Sphingomonas Sp.Nov.and The Effect Of Formate Dehydrogenase To Antimony Resistance And Oxidation In Comamonas Sp.JL40

Microorganisms have been widely used in food,medicine,agriculture,industry and environmental protection because of their biological value.Microbial resources are rich in our country,but only few of them have been developed and utilized.In order to further develop microbial resources,the classification of microbial identification is performed.Besides,antimony(Sb)is one of the most toxic heavy metals in the environment,causing severe environmental pollution.Sb(?)oxidation to the less toxic Sb(?)has potential as an environmental remediation tool.Abiotic Sb(?)oxidation is relatively slow,however,microbe can increase the speed of antimony oxidation.Hence,the study of potential biological oxidants could be of significant value.There are two parts in this study.In the first part,the strain E62-3T isolated from the soil of Enshi Grand Canyon was used as the research material to identify its polyphasic taxonomy and determine its classification status;in the second part,the antimony resistance and oxidation strain Comamonas sp.JL40 was taken as the research material to look for genes related to antimony oxidation and resistance and explore the antimony metabolic mechanism of bacteria.In the first part,a Gram-negative,non-motile,rod-shaped,strictly aerobic bacterium designated strain E62-3T,was isolated from the soil sample collected from Grand Canyon of Enshi,Hubei province.Based on the 16S rRNA gene sequences,strain E62-3T was most closely related to Sphingosiniceolla vermicomposti YC7378T(96.0%),Sphingobium xanthum NL9T(95.8%),Sphingobium boeckii 469T(95.7%)and Sphingomonas laterariae LNB2T(95.5%).The highest similarity lower than 97.0%indicated that strain E62-3T was a potential new species,but the new strain did not belong to some genus within Sphingomonadaceae exactly only based on the 16S rRNA gene sequences similarity.Phylogenetic trees constructed with the maximum-likelihood,neighbor-joining and maximum-parsimony methods all showed that strain E62-3T was clustered with Sphingomonas laterariae LNB2T and other species of Sphingomonas,Polyphasic taxonomic study revealed that:i)the growth temperature range of strain E62-3T was 15-37 ?(optimum at 28 ?);ii)pH 6.0-9.0(optimum,pH 7.0)and the growth occurred with NaCl concentrations in the range 0-0.5%(w/v);iii)the predominant respiratory quinone was Q-10;iv)The predominant polar lipids were diphosphatidylglycerol,phosphatidylglycerol,phosphatidylethanolamine and phosphatidylcholine,also included unknown phospholipid and unknown glycolipid;v)the major fatty acids(>5%)were C18:?7c(36.4%),summed feature 3(24.5%),C16;0(14.7%)and C14;02-OH(8.9%);vi)The predominant polymine was homospermidine;vii)the DNA G+C content of strain E62-3T was 66.5 mol%.The results of phenotypic,genetic and chemical taxonomic of strain E62-3T indicated that the strain belonged to the genus Sphingomonas,for which the name Sphingomonas faucium sp.nov.is proposed.The type strain is E62-3T(= KCTC 42834T= CCTCC AB 20153001).The species of the genus Sphingomonas have good function in degrading pollutants of aromatic hydrocarbon compounds.This strain E62-3T need to be further studied in environmental remediation.In the second part,formate dehydrogenase gene fdhO was found related to Sb resistance by this research group with the method of transposon mutagenesis.On the basis of this study,mutant strain JL40-?fdhO and complementary strain JL40-?fdhO-C were constructed.The MIC,growth and Sb(?)oxidation experiments were conducted with the strains JL40,JL40-AfdhO and JL40-?fdhO-C.The results showed that the antimony resistance and oxidation rate of mutant strain have decreased,and that of complementary strain JL40-?fdhO-C obtained a certain degree of recovery.Heterologous expression assay in Escherichia coli showed that gene fdhO could increase Sb(?)oxidation rate compared with the empty carrier.Moreover,the result of the reporter gene fusion experiment indicated that the promoter of gene fdhO was induced by Sb(?).The glutathione and hydrogen peroxide assays revealed no difference between wild strain and the mutant strain under the same condition with or without Sb(?).Comprehensive analysis showed gene fdhO actually have the function of Sb(?)oxidation,but it was not specific,and the knock-out of gene fdhO do not impact the non-enzymatic factors of GSH and H2O2 to Sb(?).The research of antimony resistance and oxidation of gene fdhO provided reference for further explore of bacterial antimony resistant mechanism.