Selenite-Reduction Mechanism And Biogenesis Of Selenium Nanoparticles And Cd Se Quantum Dots In A Bacterium

Selenium is an essential element to living organisms.There are at least 25 sepecies of selenopeoteins in human,which play multiple vital roles for health.However,long term of selenium deficiency or over-uptake will cause severe diseases.Soluable selenite[Se(Ⅳ)]is the most hazardous form among the selenium species and is the major form of selenium contamination.Some microorganisms could reduce Se(Ⅳ)into Se(0)-nanoparticles(SeNPs)or selenides[Se(-Ⅱ)].Such processes are beneficial for bacterial detoxification and bioremediation of environmental selemium contamination.Some microorganisms could synthesize CdSe quantum dots(QDs)when Se(Ⅳ)and Cd(Ⅱ)are co-exist.The SeNPs and CdSe-QDs have great values in medical and biosensor applications.There are some Se(Ⅳ)-reducing bacteria with undetermined taxonomic classification and the knowledge of molecular mechanisms of Se(Ⅳ)reduction and SeNPs/CdSe QDs formation is very limited.Thus,we performed polyphasic taxonomy analyses and identified Paenirhodobacter enshiensis DW2-9T as a type strain representing a novel genus and species.We also performed genomic analysis of strain DW2-9T and its close relatives.In the meantime,the mechanism of Se(Ⅳ)reduction and SeNPs/CdSe QDs formation in Pseudomonas stutzeri TS44 were systematically analyzed.The major results and findings of this dissertation are as follows:1.Establishment of a novel genus of selenite reducing bacteria and genome analysis.Strain DW2-9T was isolated from the soil of a non-selenium enriched region in Enshi,Hubei province.Based on the distinctive differences in 16S rRNA gene similarity,nonphotosynthetic characteristic,cellular fatty acid and polar lipids compositions as well as the cultivation conditions with its close relatives,it was proposed as a representative of a novel genus and novel species in the family Rhodobacteraceae,namely Paenirhodobacter enshiensis gen.nov.,sp.nov..The highest 16S rRNA gene sequence similarity were found to Rhodobacter capsulatus ATCC 11166T(97.1%),others were species in the genera Rhodobacter and Haematobacter with the 16S rRNA gene sequence similarities ranged from 95.7%to 96.4%.The 16S rRNA gene sequence similarities between strain DW2-9T and other members of the family Rhodobacteraceae were less than 95.5%.Strain DW2-9T neither formed internal photosynthetic membranes nor produced photosynthetic pigments,and it contained C18:1 ω7c,C16:0,C19:0 cyclo ω8c and summed feature 3(one or more of iso-C15:0 2-OH,C16:1 ω6c and C16:1ω7c)as the major cellular fatty acids(>5%),while the major polar lipids were phosphatidylethanolamine,phosphatidylglycerol and an unknown aminophospholipid,which differed from the most related species of the genera Rhodobacter and Haematobacter.In addition to the differences in phylogenetic position and polar lipid types,strain DW2-9T could be distinguished from species of the genus Haematobacter by the cultivation conditions.Moreover,strain DW2-9T could be distinguished from members of the genus Thioclava by its inability to oxidize sulfur or to grow chemoautotrophically,and from species of the genus Roseicitreum by the absence of internal photo synthetic membranes and photosynthetic pigments and by its non-halophilic character.Based on the results,we conclude that strain DW2-9T representing a bacterial novel genus and novel species in the family Rhodobacteraceae,for which the name Paenirhodobacter enshiensis gen.nov.,sp.nov.is proposed.Genome analysis showed a highly consistence between the genotype and phenotype of strain DW2-9T,which provides extra supports for its taxonomic identification.In addition,enzymes potentially involved in Se(Ⅳ)reduction was found in the genome of strain DW2-9T.It was also the first time to report genomic sequences of the genera Paenirhodobacter and Haematobacter in this study,which is convenient for other taxonomy works of unknown species.2.Selenite reduction,SeNPs and CdSe QDs formation in Pseudomonas stutzeri TS44Pseudomonas stutzeri TS44 showed higher resistant level to Se(Ⅳ)and higher Se(Ⅳ)reduction rate compared to strain DW2-9T,thus,P.stutzeri TS44 was chosen for this study.The formation of SeNPs and CdSe-QDs in P.stutzeri TS44 were systematically analysed using TEM,elemental mapping,XPS,FTIR,molecular biological and enzymological methods.Our results sugguested that strain TS44 could aerobically reduce Se(Ⅳ)into SeNPs and transforms Se(Ⅳ)/Cd(Ⅱ)mixture into CdSe-QDs.When the final concentration of Se(Ⅳ)was added as 2.5 mmol/L,the gorA mutated strain TS44-AgorA showed obvious decrease of Se(Ⅳ)reduction,although there was no significant different in the intracellular GSH content compared to the wild type strain.The complementary strain TS44-gshA-C thoroughly restored the phenotype.In contrast,the gshA mutanted strain TS44-gshA540 didn’t show seriously impaired Se(Ⅳ)reduction,although the intracellular GSH content was decreased.The in vivo results above showed that GorA was involved in Se(Ⅳ)reduction in a GSH-independent manner when Se(Ⅳ)concentration was in a relative high level.In vitro,GorA could catalyzes Se(Ⅳ)reduction with NADPH as the electron donor(Vmax of 3.947±0.1061 μmol/min/mg protein under pH 7.0 and 28℃).In addition,we found that GSH rather than GorA is necessary for in vivo CdSe-QD formation.In vivo,the gshA mutated strain TS44-gshA540 always showed weaker fluorescence of CdSe-QDs compared to the wild type strain.The complementary strain TS44-gshA-C partially restored the phenotype.Both the in vitro and in vivo evidences showed that GSH played an important role in CdSe-QDs formation in strain TS44.In addition,CdSe-QDs were successfully synthesized by a one-step method,which is convenient for large scale of CdSe QDs biofabrication.Taken together,a novel genus of selenitereducing bacteria was established by polyphasic taxonomy,and the whole genome sequencing and comparative genomic analysis were performed with two closely related strains of the genera Paenirhodobacter and Haematobacter.Inaddition,in vivo and in vitro evidences revealed that the glutathione reductase GorA was involved in SeNPs formation and GSH was essential for CdSe quantum dots synthesis in P.stutzeri TS44,respectively(Figure A).This work enriched the microorganism resources and provided new insights in elucidating the mechanism of Se(Ⅳ)-reducing and biosynthesis of SeNPs and CdSe quantum dots in a single bacterium.