Identification Of Selenite Reductase In Comamonas Testosteroni S44 And A Novel Species Of The Genus Sphingoaurantiacus

Selenium is an essential trace element for organism.In nature,selenium exists in four states,including-II,0,IV and VI.Microorganisms are involved in the mutual transformation of all these four states.Many bacteria have the ability to reduce selenite to red elemental selenium nanoparticles（Se NPs）that is less toxic.In addition,the biosynthesized Se NPs play an essential role on various fields including antioxidant and anticancer agents,biosensors and environmental remediation.However,the molecular mechanism of selenite reduction in the aerobic bacteria is little known.Therefore,it is very important to elucidate the molecular mechanism in aerobic bacteria.In this study,a strictly aerobic bacterium Comamonas testosteroni S44 reducing selenite into red Se NPs under aerobic conditions was studied.Through combining with the protein native-PAGE and selenite reduction test in vitro,several candidate oxidoreductase were found by mass spectrometry and genomic analysis.The ability of selenite reduction of the single-crossover mutantΔ1962 was decreased compared with the wild type strain S44.The bioinformatics analysis showed that the protein 1962belongs to the sulfite oxidase family oxidoreductase,which is located in the periplasm and contains molybdenum cofactor,and has specific electron transfer cytochrome protein that can form heterodimer with protein 1962.To investigate the function of protein 1962 in vivo,we constructed the double-crossover mutant strain S44-Δ1962and complementary strain S44-Δ1962-C,and determined the growth and selenite reduction curve of the wild strain S44,mutant strain S44-Δ1962 and complementary strain S44-Δ1962-C.The growth was similar whersas the ability of selenite reduction of the mutant strain S44-Δ1962 was 75%lower than that of the wild strain S44 during64 hr,and the ability of selenite reduction of the complementary strain was completely restored.Furthermore,we constructed heterologous overexpressing strain Escherichia coli W3110::1962,and determined the growth and selenite reduction curves of overexpressing strains and wild strain.The result indicated that the ability of selenite reduction of the heterologous overexpressing strain W3110::1962 was significantly enhanced compared with wild strain W3110.To identify the function of protein 1962 in vitro,the 1962 was purified by heterologous expression and its selenite reductase activity was determined.The result showed that protein 1962 had selenite reductase activity,and its affinity constant Km and maximum reaction rate V_maxare 180±5μmol and 61±2 nmol min^-1mg^-1,respectively.In order to identify whether the cytochrome protein ccp is the specific cytochrome protein of the 1962,we constructed the overexpressing strain S44::（1962+ccp）,and determined the growth and selenite reduction curves of overexpressing strains and wild strain.The result showed that the ability of selenite reduction of S44::（1962+ccp）was not significantly enhanced compared with wild strain S44.The co-transcription experiment revealed that gene 1962 and ccp was not co-transcriptional.In summary,through combining with in vivo and in vitro experiments,we first comfirmed that a molybdenum-containing oxidoreductase 1962 at the periplasm is involved in selenite reduction in Comamonas testosteroni S44.However,the specific cytochrome protein of the selenite reductase 1962 was not unknown yet.The research on selenite reductase would be helpful for the application of bio-remediation in Se-polluted environments and production of biosynthesized Se NPs.In this study,a strain YLT33^Tisolated from mountain cliff soil located in Enshi Grand Canyon was identified as a novel species of the genus Sphingoaurantiacus by using the method of polyphasic taxonomic methods,for which the name Sphingoaurantiacus capsulatus sp.nov.is proposed.The type strain is YLT33^T（=CCTCC AB 2015150^T=KCTC 42644^T）.This carotenoid-producing bacterium would provide valuable gene resource and strain resource for the construction of engineering strain to produce carotenoids.