Previous study on refolding of sulfur oxygenase reductase (SOR) inclusion bodies from recombinant Escherichia coli showed that iron was critical to the activity of the SOR from Acidianus ambivalens. In this study, enzymatic assays showed that 2,2′-Dipyridyl, Tiron and 8-hydroxyquinoline, which are specific for chelating ferrous or ferric ions, strongly inhibited the activity of SOR from A. tengchongensis, suggesting that iron atom is essential for SOR activity. Alignment of several functionally identified SORs and SOR-like sequences from genome database revealed a conserved, putative iron binding motif, H86-X3-H90-Xn-E114-Xn-E129 (numbering according to the Acidianus tengchongensis SOR sequence). Three mutants of SOR were generated by site-directed mutagenesis of H86, H90 and E129 into phenyla-lanine or alanine residue in this study. Circular dichroism spectrum determination indicated that there was no change of the secondary structures of mutant SORs, H86F, H90F and E129A, but all mutants were completely inactive. Through determination of iron contents we found that SOR mutants of H86F, H90F and E129A completely or partially lost iron, while mutants of C31S, C101S, and C104S (generated in a previous study) did not. This result indicated that H86, H90 and E129 but not C31, C101, and C104 were involved in binding to iron atom. Based on this and previous studies, it is proposed that the conserved motifs, C31-Xn-C101-X2-C104 and H86-X3-H90-X23-E114-X14-(E/D)129, are respectively for sulfur and molecular oxygen binding and activation. These two conserved motifs are essential elements for the SOR activity.
Halophilic archaea (haloarchaea) inhabit hypersaline environments,tolerating extreme salinity,low oxygen and nutrient availability,and in some cases,high pH (soda lakes) and irradiation (saltern ponds).Membrane-associated proteins of haloarchaea,such as surface layer (S-layer) proteins,transporters,retinal proteins,and internal organellar membrane proteins including intracellular gas vesicle proteins and those associated with polyhydroxyalkanoate (PHA) granules,contribute greatly to their environmental adaptations.This review focuses on these haloarchaeal cellular and organellar membrane-associated proteins,and provides insight into their physiological significance and biotechnological potential.
CAI LeiZHAO DaHeHOU JingWU JinHuaCAI ShuangFengDASSARMA PriyaXIANG Hua
Microbial oxidation and reduction of iron and sulfur are important parts of biogeochemical cycles in acidic environments such as geothermal solfataric regions. Species of Acidithiobacillus and Leptospirillum are the common ferrous-iron and sulfur oxidizers from such environments. This study focused on the Tengchong sofataric region, located in Yunnan Province, Southwest China. Based on cultivation, 9 strains that grow on ferrous-iron and sulfuric compounds were obtained. Analysis of 16S rRNA genes of the 9 strains indicated that they were affiliated to AcidithiobaciUus, Alicyclobacillus, Sulfobacillus, Leptospirillum and Acidiphilium. Physiological and phylogenetic studies indicated that two strains （TC-34 and TC-71） might represent two novel members of Alicyclobacillus. Strain TC-34 and TC-71 showed 94.8%-97.1% 16S rRNA gene identities to other species of Alicyclobacillus. Different from the previously described Alicyclobacillus species, strains TC-34 and TC-71 were mesophilic and their cellular fatty acids do not contain w-cyclic fatty acids. Strain TC-71 was obligately dependent on ferrous-iron for growth. It was concluded that the ferrous-iron oxidizers were diversified and Alicyclobacillus species were proposed to take part in biochemical geocycling of iron in the Tengchong solfataric region.
By functional complementation of Escherichia coli mutants defective in potassium (K+) uptake, two genes that are required for K+ uptake in halo-alkaliphilic Alkalimonas amylolytica strain N10 were cloned. These two genes, Aa-trkA (1337 bp) and Aa-trkH (1452 bp), were adjacent on the A. amylolytica N10 chromosome and transcribed in opposite directions. Complementation experiments revealed that Aa-TrkA and Aa-TrkH from A. amylolytica strain N10 restored the ability to grow at low K+ concentration in E. coli △trkA and △trkG △trkH strains, respectively. In addition, Aa-TrkAH supported the growth of an E. coli △sapD strain, indicating that the ATP-binding protein TrkE was dispensable for the Trk system of A. amylolytica strain N10. The net K+ uptake was detected at different pH levels and the critical NaCl concentration indicated that Aa-TrkAH is an alkaline-adaptable and partially halo-adaptable K+ transporter. Kinetics determined by heterogeneous K+ transport experiments with an E. coli △trkA strain revealed that Aa-TrkAH has an alkaline pH optimum close to 8.5 or higher. Site-directed mutagenesis of Aa-TrkH showed that Phe103 and Ser229 play certain key roles in K+ selection and transportation. The molecular chaperones groES-groEL and tig promoted Aa-TrkH and Aa-TrkA overexpression in vitro.
GUO YongHao1, XUE YanFen1, LIU Jun2, WANG QuanHui1 & MA YanHe1 1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
