Potential Role of Thiobacillus caldus in Arsenopyrite Bioleaching

We investigated the potential role of the three strains of Thiobacillus caldus (KU, BC13, and C-SH12) in arsenopyrite leaching in combination with a moderately thermophilic iron oxidizer, Sulfobacillus thermosulfidooxidans. Pure cultures of T. caldus and S. thermosulfidooxidans were used as well as defined mixed cultures. By measuring released iron, tetrathionate, and sulfur concentrations, we found that the presence of T. caldus KU and BC13 in the defined mixed culture lowered the concentration of sulfur, and levels of tetrathionate were comparable to or lower than those in the presence of S. thermosulfidooxidans. This suggests that T. caldus grows on the sulfur compounds that build up during leaching, increasing the arsenopyrite-leaching efficiency. This result was similar to leaching arsenopyrite with a pure culture of S. thermosulfidooxidans in the presence of yeast extract. Therefore, three possible roles of T. caldus in the leaching environment can be hypothesized: to remove the buildup of solid sulfur that can cause an inhibitory layer on the surface of the mineral, to aid heterotrophic and mixotrophic growth by the release of organic chemicals, and to solubilize solid sulfur by the production of surface-active agents. The results showed that T. caldus KU was the most efficient at leaching arsenopyrite under the conditions tested, followed by BC13, and finally C-SH12.     

Reduced sulfur compound oxidation by Thiobacillus caldus.

The oxidation of reduced inorganic sulfur compounds was studied by using resting cells of the moderate thermophile Thiobacillus caldus strain KU. The oxygen consumption rate and total oxygen consumed were determined for the reduced sulfur compounds thiosulfate, tetrathionate, sulfur, sulfide, and sulfite in the absence and in the presence of inhibitors and uncouplers. The uncouplers 2,4-dinitrophenol and carbonyl cyanide m-chlorophenyl-hydrazone had no affect on the oxidation of thiosulfate, suggesting that thiosulfate is metabolized periplasmically. In contrast, the uncouplers completely inhibited the oxidation of tetrathionate, sulfide, sulfur, and sulfite, indicating that these compounds are metabolized in the cytoplasm of T. caldus KU. N-Ethylmaleimide inhibited the oxidation of tetrathionate and thiosulfate at the stage of elemental sulfur, while 2-heptyl-4-hydroxyquinoline-N-oxide stopped the oxidation of thiosulfate, tetrathionate, and elemental sulfur at the stage of sulfite. The following intermediates in the oxidation of the sulfur compounds were found by using uncouplers and inhibitors: thiosulfate was oxidized to tetrathionate, elemental sulfur was formed during the oxidation of tetrathionate and sulfide, and sulfite was found as an intermediate of tetrathionate and sulfur metabolism. On the basis of these data we propose a model for the metabolism of the reduced inorganic sulfur compounds by T. caldus KU.     

Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA.

The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis.     

Presence of a family of plasmids (29 to 65 kilobases) with a 26-kilobase common region in different strains of the sulfur-oxidizing bacterium Acidithiobacillus caldus

Three large cryptic plasmids from different isolates of Acidithiobacillus caldus were rescued by using an in vitro transposition system that delivers a kanamycin-selectable marker and an Escherichia coli plasmid origin of replication. The largest of the plasmids, the 65-kb plasmid pTcM1, was isolated from a South African A. caldus strain, MNG. This plasmid was sequenced and compared to that of pTcF1 (39 kb, from strain "f," South Africa) and pC-SH12 (29 kb, from strain C-SH12, Australia). With the exception of a 2.7-kb insertion sequence, pC-SH12 appears to represent the DNA common to all three plasmids and includes a number of accessory genes plus the plasmid "backbone" containing the replication region. The two larger plasmids carry, in addition, a number of insertion sequences of the ISL3 family and a composite transposon related to the Tn21 subfamily containing a highly mosaic region within the borders of the inverted repeats. Genes coding for arsenic resistance, plasmid mobilization, plasmid stability, and a putative restriction-modification system occur within these mosaic regions.     

ATP generation during reduced inorganic sulfur compound oxidation by<Emphasis Type="Italic"> Acidithiobacillus caldus</Emphasis> is exclusively due to electron transport phosphorylation

The synthesis of adenosine 5-triphosphate (ATP) (increase in phosphorylation potential) during the oxidation of reduced inorganic sulfur compounds was studied in the moderately thermophilic acidophileAcidithiobacillus caldus (strain KU) (formerly Thiohacillus caldus). The phosphorylation potential increased during the oxidation of all reduced inorganic sulfur compounds tested compared with resting cells. The generation of ATP in whole cells was inhibited by the F0F1 ATPase inhibitor oligomycin, electron transport chain inhibitors, valinomycin and potassium ions. There was no increase in the phosphorylation potential, nor synthesis of ATP. in the absence of electron transport. An apparent lack of substrate-level phosphorylation was indicated by the lack of adenosine 5-phosphosulfate reductase in tetrathionate-grown At. caldus. Studies were also performed on the synthesis of ATP by membrane vesicles of At. caldus when presented with an artificial proton gradient. Complete inhibition of ATP synthesis in these vesicles occurred when they were loaded with N,N-dicyclohexylcarbodiimide (DCCD), but not when they were loaded with oligomycin, vanadate or electron transport chain inhibitors. The data presented here suggest that during the oxidation of reduced inorganic sulfur compounds by At. caldus, all ATP is synthesized by oxidative phosphorylation via a membrane-bound F0F1 ATPase driven by a proton gradient.     

Chromosomally encoded arsenical resistance of the moderately thermophilic acidophile Acidithiobacillus caldus

Arsenical resistance is important to bioleaching microorganisms because these organisms release arsenic from minerals such as arsenopyrite during bioleaching. The acidophile Acidithiobacillus caldus KU was found to be resistant to the arsenical ions arsenate, arsenite, and antimony via an inducible, chromosomally encoded resistance mechanism. Because no apparent alteration of the toxic ions was observed, Acidithiobacillus (At.) caldus was tested to determine if it was resistant as a result of decreased accumulation of toxic ions. Reduced accumulation of arsenate and arsenite by induced At. caldus cells supported this hypothesis. It was also found that, with the addition of an energy source, induced At. caldus could transport arsenate and arsenite out of the cell against a concentration gradient. The lack of efflux in the absence of an added energy source and in the presence of inhibitors suggested that efflux was energy dependent. Induced At. caldus also expressed arsenate reductase activity, indicating that At. caldus has an arsenical resistance mechanism that is analogous to previously described systems from other Bacteria. Southern hybridization analysis showed that At. caldus and other gram-negative acidophiles carry an Escherichia coli arsB homologue on the chromosome.     

Genome sequences of Alicycliphilus denitrificans strains BC and K601T

Alicycliphilus denitrificans strain BC and A. denitrificans strain K601(T) degrade cyclic hydrocarbons. These strains have been isolated from a mixture of wastewater treatment plant material and benzene-polluted soil and from a wastewater treatment plant, respectively, suggesting their role in bioremediation of soil and water. Although the strains are phylogenetically closely related, there are some clear physiological differences. The hydrocarbon cyclohexanol, for example, can be degraded by strain K601(T) but not by strain BC. Furthermore, both strains can use nitrate and oxygen as an electron acceptor, but only strain BC can use chlorate as electron acceptor. To better understand the nitrate and chlorate reduction mechanisms coupled to the oxidation of cyclic compounds, the genomes of A. denitrificans strains BC and K601(T) were sequenced. Here, we report the complete genome sequences of A. denitrificans strains BC and K601(T).     

The lipopolysaccharides of Neisseria gonorrhoeae colony types 1 and 4.

The lipopolysaccharides (LPS) of strains of Neisseria gonorrhoeae grown in type 1 (T1) and 4 (T4) colony forms have been isolated. LPS from T4 colony type cells on mild hydrolysis gave a lipid A and a core oligosaccharide composed of 2-amino-2-deoxy-D-glucose, D-glucose, D-galactose, L-glycero-D-manno-heptose and 3-deoxy-D-manno-octuosonic acid that appeared to be common to all the strains examined. LPS from T1 colony type cells on mild hydrolysis gave a lipid A and high molecular weight O polysacc,arides which showed considerable differences in glycose composition for each strain examined. In those strains examined, T4 cells appear to produce a common "R" type LPS whereas T1 cells produce an "S" type LPS with structurally different O polysaccharide structures which probably account for serologically differentiated strains of N. gonorrhoeae.     

Chemical and serological properties of lipopolysaccharides from Vibrio parahaemolyticus O-untypeable strains isolated from patients

Chemical and serological studies have been carried out on the O-antigenic lipopolysaccharides (LPS) of six strains, U-6443, W-90144, X-3972, AD-7999, 90A-6611 and KX-V212, of Vibrio parahaemolyticus isolated from patients. The O-serotypes of these strains have not been identified because they were not agglutinated by any diagnostic antisera against known O-serotype strains. A compositional sugar analysis of their LPS revealed that out of the six O-untypeable (OUT) strains, U-6443, W-90144 and AD-7999 strains belonged to chemotype II (chemotype of O2), 90A-6611 and KX-V212 strains to chemotype III (chemotype of O3, O5, O11 and O13) and X-3972 strain to chemotype IV (chemotype of O4). A structural analysis of LPS isolated from KX-V212 revealed that the inner core region of the LPS consisted of only one mole of 2-keto-3-deoxy-D-manno-octonic acid, which carried a phosphate group at position C4 and the outer core at position C5. In passive hemolysis tests performed by using LPS as the antigen to sensitize sheep red blood cells (SRBC), and diagnostic antisera (O1 to O11) or anti-whole-cell rabbit antisera raised against O12, O13 and the six OUT strains, strong cross-reactivity was observed among LPS derived from the strains belonging to chemotype II (U-6443, W-90144, AD-7999 and O2). Strong cross-reactivity was also observed between X-3972 (chemotype IV) and O4 LPS. In contrast, LPS from two of the strains belonging to chemotype III (90A-6611 and KX-V212) did not react with any of the antisera raised against known O-serotypes. Cross-absorption tests showed that the O-antigens of U-6443, W-90144 and AD-7999 were identical to that of O2, and the O-antigen of X-3972 to that of O4. On the other hand, after the absorption of antisera raised against 90A-6611 and KX-V212 with O2 cells, the hemolytic activities against SRBC sensitized with homologous LPS were still retained at a high titer, whereas the hemolytic activities against SRBC sensitized with LPS from other O-serotype strains were completely eliminated. A cross-absorption test revealed that the O-antigens of these two strains were identical to each other. Thus, it was demonstrated that the O-serotype of OUT strains 90A-6611 and KX-V212 was not involved in the known O-serotypes; rather it represented a novel serotype which has not hitherto been reported.     

The structure of the carbohydrate backbone of the rough type lipopolysaccharides from Proteus penneri strains 12, 13, 37 and 44

The following structure of the lipid A-core backbone of the rough type lipopolysaccharides (LPS) from Proteus penneri strains 12, 13, 37, and 44 was determined using NMR and mass spectroscopy and chemical analysis of the oligosaccharides obtained by mild-acid hydrolysis, alkaline O,N-deacylation, O-deacylation with hydrazine, and deamination of the LPSs:where K=H, R=PEtN, R(1)=alpha-Hep-(1-->2)-alpha-DDHep, and R(2)=alpha-GalN (strains 12 and 13) or beta-GlcNAc-(1-->4)-alpha-GlcN (strains 37 and 44). LPS from each strain contained several structural variants. LPS from strain 12 contained a variant with R(1)=alpha-DDHep, whereas LPS from strains 13, 37, and 44 contained structures with K=amide of beta-GalA with putrescine or spermidine. The phosphate group at O-1 of the alpha-GlcN residue in the lipid part was partially substituted with Ara4N.     

Surface antigens of Proteus mirabilis revealed by electroblotting from sodium dodecyl sulphate-polyacrylamide gels

Western Blotting of whole cell preparations of three strains of Proteus mirabilis after separation by electrophoresis on SDS-polyacrylamide gels revealed a complex pattern of antigens. Similar antigen profiles were obtained with isolated outer membranes indicating that the majority of cell surface antigens are located in the outer membrane. Major outer membrane proteins were strongly antigenic and cross-reactive. The highly immunogenic flagella were detected in whole cell preparations and visible in isolated outer membranes. Whereas the protein and flagellar antigens were cross-reactive, lipopolysaccharide (LPS) could only be detected as immunoreactive material using homologous antisera for each strain. The LPS appeared as two broad bands (high and low Mr, respectively) in immunoblots of whole cells, isolated outer membranes and purified LPS. However, isolated LPS could be resolved into multiple sharp bands when 4 M urea was included in the gel system. These discrete bands are assumed to represent differing O antigen chain lengths of the LPS as reported for other Gram-negative organisms.     

Neokomagataea gen. nov., with descriptions of Neokomagataea thailandica sp. nov. and Neokomagataea tanensis sp. nov., osmotolerant acetic acid bacteria of the α-Proteobacteria

Isolates AH11(T) and AH13(T) were isolated from flowers of lantana and candle bush respectively collected in Thailand. In phylogenetic trees based on 16S rRNA gene sequences, the two isolates formed an independent cluster, which was then connected to the type strain of Saccharibacter floricola. The calculated pair-wise 16S rRNA gene sequence similarities of isolate AH11(T) were 95.7-92.3% to the type strains of the type species of the 12 genera of acetic acid bacteria. The DNA base composition was from 51.2 to 56.8 mol % G+C, with a range of 5.6 mol %. When isolate AH11(T) was labeled, DNA-DNA similarities were 100, 12, 4, 5, and 4% respectively to isolates AH11(T) and AH13(T) and the type strains of Saccharibacter floricola, Gluconobacter oxydans, and Acetobacter aceti. The two isolates were non-motile and did not oxidize either acetate or lactate. No growth was found in the presence of 0.35% acetic acid w/v. The two isolates were not osmophilic but osmotolerant, produced 2,5-diketo-D-gluconate from D-glucose, and did not oxidize lactate, thus differing from strains of Saccharibacter floricola, which showed weak lactate oxidation. The two isolates contained unsaturated C(18:1)ω7c fatty acid as the major fatty acid, and were unique in the presence of a considerable amount of straight-chain C(18:1)2OH fatty acid. Q-10 was present as the major isoprenoid quinone. Neokomagataea gen. nov. was proposed with the two species, Neokomagataea thailandica sp. nov. for isolate AH11(T) (=BCC 25710(T)=NBRC 106555(T)), which has 56.8 mol % G+C, and Neokomagataea tanensis sp. nov. for isolate AH13(T) (=BCC 25711(T)=NBRC 106556(T)), which has 51.2 mol % G+C.     

猪Ⅱ型圆环病毒四川分离株鉴定及其ORF2基因序列分析

猪圆环病毒(Porcine circovirus,PCV),属于圆环病毒科(Circoviridae),圆环病毒属(Circovirus),血清型为PCV-1和PCV-2[1]。PCV-1首先由Tis-cher[2]于1974年从PK-15猪肾传代细胞系中分离获得,PCV-2首先由Clark[3]报道了是断奶仔猪多系统衰竭综合征(PMWS)的主要病原,随即相继报道了PCV-2与PDNSS(猪皮炎与肾炎综合症)、NP(增生性坏死性肿炎)、PRDC(猪呼吸道综合征)、繁殖障碍、先天性颤抖、肠炎等疾病亦有重要关联[4,5];它常与猪呼吸与繁殖综合征病毒(PRRSV)或猪细小病毒(PPV)并发感染或继发细菌感染[6]。我国自从2000年郎…     

Immunoblot analysis of trypomastigote excreted-secreted antigens as a tool for the characterization of Trypanosoma cruzi strains and isolates

An analysis of antibody recognition of Trypanosoma cruzi exoantigens by immunoblotting revealed a unique banding pattern that seems to be characteristic of each strain or isolate. Trypomastigote excreted-secreted antigens (TESA) present in supernatants of LLC-MK2 cells infected with 5 strains and 10 isolates of T. cruzi produced 13 different immunoblotting patterns. The same bands were observed when probed with acute-phase Chagas' disease serum or with serum from a rabbit immunized with the repetitive domain of T. cruzi transialidase recombinant protein (anti-shed acute-phase antigens). Three similar patterns were observed with TESA from 3 human isolates that probably belong to the same T. cruzi strain. When clone CL Brener, clone CL-14, and CL parental strain were analyzed, the same bands were observed, although they presented different biological behavior. These results suggest that immunoblotting analysis of TESA may be a useful tool for characterization of T. cruzi strains and isolates.     

O-chain expression in the rough Brucella melitensis strain B115: induction of O-polysaccharide-specific monoclonal antibodies and intracellular localization demonstrated by immunoelectron microscopy.

Spleen cells from mice infected with the rough Brucella melitensis strain B115 were fused with NSO myeloma cells. Hybridoma supernatants were screened in ELISA with cell walls (CW), sonicated cell extracts (CE) and rough lipopolysaccharide (R-LPS) of B. melitensis strain B115 and whole B. melitensis B115 cells. Surprisingly, 22 monoclonal antibodies (mAbs) reacting in ELISA with both CW and CE but not with R-LPS and bacterial cells were shown by immunoblot analysis and ELISA to react with smooth lipopolysaccharide (S-LPS). These mAbs also reacted in ELISA with O polysaccharides (OPS) from the smooth Brucella abortus strain 99 and the smooth B. melitensis strain 16M and thus recognize epitopes present on the O-chain. Proteinase K LPS preparations from B. melitensis B115 analysed by immunoblotting with one mAb (12G12) recognizing S-LPS of both A and M specificity displayed the typical S-LPS high-molecular-mass ladder pattern but no S-LPS was detected in the phenol/water/chloroform/light petroleum LPS preparation of the same strain. mAb 12G12, specific for S-LPS, and a mAb (A68/03F03/D05) specific for R-LPS were used to localize the O-chain and R-LPS expressed in B. melitensis strain B115 by immunoelectron microscopy. Immunogold labelling was observed at the surface of B. melitensis B115 cells with the anti-R-LPS mAb but not with the anti-S-LPS mAb. In ultrathin sections, immunogold labelling with the S-LPS specific mAb was observed in the cytoplasm and in the periphery of the cytoplasm, probably at the cytoplasmic membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and differentiation of species and strains of Arthrobacter and Microbacterium barkeri isolated from smear cheeses with Amplified Ribosmal DNA Restriction Analysis (ARDRA) and pulsed field gel electrophoresis (PFGE)

ARDRA (Amplified Ribosomal-DNA Restriction Analysis) was used to differentiate among species and genera of Arthrobacter and Microbacteria. Species-specific restriction patterns of PCR-products were obtained with NciI for Arthrobacter citreus (DSM 20133T), A. sulfureus (DSM 20167T), A. globiformis (DSM 20124T) and A. nicotianae strains (DSM 20123T, MGE 10D, CA13, CA14, isolate 95293, 95294, and 95299), A. rhombi CCUG 38813T, and CCUG 38812, and Microbacterium barkeri strains (DSM 30123T, MGE 10D, CA12 and CA15, isolate 95292, and isolate 95207). All yellow pigmented coryneforme bacteria isolated from the smear of surface ripened cheeses were identified as either A. nicotianae or M. barkeri strains. Using pulsed field gel electrophoresis (PFGE) strain specific restriction pattern for all Arthrobacter species and Microbacteria tested were obtained with restriction enzymes AscI and SpeI.