Background: Acidithiobacillus ferrooxidans is a major participant in consortia of microorganisms used for the industrial recovery of copper (bioleaching or biomining). It is a chemolithoautrophic, γ-proteobacterium using energy from the oxidation of iron-and sulfur-containing minerals for

Given the challenges to life at low pH, an analysis of inorganic sulfur compound (ISC) oxidation was initiated in the chemolithoautotrophic extremophile Acidithiobacillus caldus. A. caldus is able to metabolize elemental sulfur and a broad range of ISCs. It has been implicated in the production of environmentally damaging acidic solutions as well as participating in industrial bioleaching operations where it forms part of microbial consortia used for the recovery of metal ions. Based upon the recently published A. caldus type strain genome sequence, a bioinformatic reconstruction of elemental sulfur and ISC metabolism predicted genes included: sulfide–quinone reductase (sqr), tetrathionate hydrolase (tth), two sox gene clusters potentially involved in thiosulfate oxidation (soxABXYZ), sulfur oxygenase reductase (sor), and various electron transport components. RNA transcript profiles by semi quantitative reverse transcription PCR suggested up-regulation of sox genes in the presence of tetrathionate. Extensive gel based proteomic comparisons of total soluble and membrane enriched protein fractions during growth on elemental sulfur and tetrathionate identified differential protein levels from the two Sox clusters as well as several chaperone and stress proteins up-regulated in the presence of elemental sulfur. Proteomics results also suggested the involvement of heterodisulfide reductase (HdrABC) in A. caldus ISC metabolism. A putative new function of Hdr in acidophiles is discussed. Additional proteomic analysis evaluated protein expression differences between cells grown attached to solid, elemental sulfur versus planktonic cells. This study has provided insights into sulfur metabolism of this acidophilic chemolithotroph and gene expression during attachment to solid elemental sulfur.

Acidithiobacillus caldus has been proposed to play a role in the oxidation of reduced inorganic sulfur compounds (RISCs) produced in industrial biomining of sulfidic minerals. Here, we describe the regulation of a new cluster containing the gene encoding tetrathionate hydrolase (tetH), a key enzyme in the RISC metabolism of this bacterium. The cluster contains five cotranscribed genes, ISac1, rsrR, rsrS, tetH, and doxD, coding for a transposase, a two-component response regulator (RsrR and RsrS), tetrathionate hydrolase, and DoxD, respectively. As shown by quantitative PCR, rsrR, tetH, and doxD are upregulated to different degrees in the presence of tetrathionate. Western blot analysis also indicates upregulation of TetH in the presence of tetrathionate, thiosulfate, and pyrite. The tetH cluster is predicted to have two promoters, both of which are functional in Escherichia coli and one of which was mapped by primer extension. A pyrrolo-quinoline quinone binding domain in TetH was predicted by bioinformatic analysis, and the presence of an o-quinone moiety was experimentally verified, suggesting a mechanism for tetrathionate oxidation.

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We have found a direct relationship between protein production in Pichia pastoris and the number of introduced synthetic genes of miniproinsulin (MPI), fused to the Saccharomyces cerevisiae pre-pro alpha factor used as secretion signal, and inserted between the alcohol oxidase 1 (AOX1) promoter and terminator sequences. Two consecutive approaches were followed to increase the number of integrated cassettes: the head-to-tail expression cassette multimerization procedure and re-transformation with a dominant selection marker. This increased expression from 19 to 250 mg l(-1) when about 11 copies have been integrated. Further, the correct position of one of the disulphide bridges of the purified molecule was verified by digestion with Glu-C endoprotease, followed by mass spectrometry of the isolated fragments.

Acidithiobacillus thiooxidans is a mesophilic, extremely acidophilic, chemolithoautotrophic gammaproteobacterium that derives energy from the oxidation of sulfur and inorganic sulfur compounds. Here we present the draft genome sequence of A. thiooxidans ATCC 19377, which has allowed the identification of genes for survival and colonization of extremely acidic environments.Acidithiobacillus thiooxidans is an extremely acidophilic (pH 1 to 3), chemolithoautotrophic, motile gammaproteobacterium that inhabits acid drainage environments and is used for mineral solubilization during biomining operations (9, 11). Together with other members of the genus Acidithiobacillus (10), this bacterium has the capacity to obtain energy from inorganic sulfur compounds (ISCs) and survive in extremely high concentrations of heavy metals. A. thiooxidans is one of four members of the genus Acidithiobacillus characterized to date (6,15,16,18) whose shared metabolic and functional capabilities allow them to survive in extremely acidic environments. Little is known about the genomic and functional properties of this microbe, and most information regarding this genus comes from experimental and genome-based analyses of Acidithiobacillus ferrooxidans and Acidithiobacillus caldus (7,8,15,12).The draft genome sequence of A. thiooxidans ATCC 19377 T was determined by a whole-genome shotgun strategy. Genomic libraries of 4 kb and 40 kb were constructed, sequenced, and assembled using the Phred/Phrap programs (4), generating a draft assembly based on 46,391 high-quality reads. Using Consed (5), assemblies with at least 2-fold coverage were edited and curated. Gene modeling, annotation, and curation were performed as previously described (15, 16).The A. thiooxidans ATCC 19377 draft genome has a total of 3,019,868 bp distributed in 164 contigs and a GC content of 53.1%; 3,235 protein-encoding genes were predicted. From this set, 2,130 candidate genes have a predicted function and 2,101 were assigned to 1,349 clusters of orthologous groups (14). Forty-three tRNAs, one complete 5S-16S-23S operon, and one partial 5S-16S-23S operon were identified. Complete sets of genes for nucleotide, amino acid, and central carbon metabolism were identified except for the absence of genes encoding 2-oxogluatarate dehydrogenase in the tricarboxylic acid (TCA) cycle, which is a characteristic genome signature for obligate autotrophs (17).The following genes for ISC metabolism were found: two gene clusters encoding the sulfur oxidation complex SOX (soxYZB-hyp-resB-soxAX-resC and soxYZA-hyp-soxB) previously found in Acidithiobacillus caldus and the neutrophilic sulfur oxidizer Thiobacillus denitrificans (1, 2); the sulfur quinone oxidoreductase gene (sqr); the tetrathionate hydrolase gene (tetH); and a thiosulfate quinone oxidoreductase gene (doxD). The analysis revealed a complete suite of genes for flagellar formation and chemotaxis, as has been identified in A. caldus but not in A. ferrooxidans (15,16). The data also indicate the presence of several open reading...

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Background Acidithiobacillus caldus is a sulfur oxidizing extreme acidophile and the only known mesothermophile within the Acidithiobacillales. As such, it is one of the preferred microbes for mineral bioprocessing at moderately high temperatures. In this study, we explore the genomic diversity of A. caldus strains using a combination of bioinformatic and experimental techniques, thus contributing first insights into the elucidation of the species pangenome.Principal FindingsComparative sequence analysis of A. caldus ATCC 51756 and SM-1 indicate that, despite sharing a conserved and highly syntenic genomic core, both strains have unique gene complements encompassing nearly 20% of their respective genomes. The differential gene complement of each strain is distributed between the chromosomal compartment, one megaplasmid and a variable number of smaller plasmids, and is directly associated to a diverse pool of mobile genetic elements (MGE). These include integrative conjugative and mobilizable elements, genomic islands and insertion sequences. Some of the accessory functions associated to these MGEs have been linked previously to the flexible gene pool in microorganisms inhabiting completely different econiches. Yet, others had not been unambiguously mapped to the flexible gene pool prior to this report and clearly reflect strain-specific adaption to local environmental conditions.SignificanceFor many years, and because of DNA instability at low pH and recurrent failure to genetically transform acidophilic bacteria, gene transfer in acidic environments was considered negligible. Findings presented herein imply that a more or less conserved pool of actively excising MGEs occurs in the A. caldus population and point to a greater frequency of gene exchange in this econiche than previously recognized. Also, the data suggest that these elements endow the species with capacities to withstand the diverse abiotic and biotic stresses of natural environments, in particular those associated with its extreme econiche.