Thiosulphate oxidation by Thiobacillus thioparus and Halothiobacillus neapolitanus strains isolated from the petrochemical industry

Sulphur Oxidizing Bacteria (SOB) is a group of microorganisms widely used for the biofiltration of Total Reduced Sulphur compounds (TRS). TRS are bad smelling compounds with neurotoxic activity which are produced by different industries (cellulose, petrochemical). Thiobacillus thioparus has the capability to oxidize organic TRS, and strains of this bacterium are commonly used for TRS biofiltration technology. In this study, two thiosulphate oxidizing strains were isolated from a petrochemical plant (ENAP BioBio, Chile). They were subjected to molecular analysis by real time PCR using specific primers for T. thioparus. rDNA16S were sequenced using universal primers and their corresponding thiosulphate activities were compared with the reference strain T. thioparus ATCC 10801 in batch standard conditions. Real time PCR and 16S rDNA sequencing showed that one of the isolated strains belonged to the Thiobacillus branch. This strain degrades thiosulphate with a similar activity profile to that shown by the ATCC 10801 strain, but with less growth, making it useful in biofiltration.

Differential gene expression in response to copper in Acidithiobacillus ferrooxidans analyzed by RNA arbitrarily primed polymerase chain reaction.

Acidithiobacillus ferrooxidans is a chemoautotrophic bacterium that plays an important role in metal bioleaching processes. Despite the high level of tolerance to heavy metals shown by A. ferrooxidans, the genetic basis of copper resistance in this species remains unknown. We investigated the gene expression in response to copper in A. ferrooxidans LR using RNA arbitrarily primed polymerase chain reaction (RAP-PCR). One hundred and four differentially expressed genes were identified using eight arbitrary primers. Differential gene expression was confirmed by DNA slot blot hybridization, and approximately 70% of the RAP-PCR products were positive. The RAP-PCR products that presented the highest levels of induction or repression were cloned, sequenced and the sequences were compared with those in databases using the BLAST search algorithm. Seventeen sequences were obtained. The RAP-PCR product with the highest induction ratio showed similarity with the A. ferrooxidans cytochrome c. A high similarity with the thiamin biosynthesis gene thiC from Caulobacter crescentus was observed for another RAP-PCR product induced by copper. An RAP-PCR product repressed by copper showed significant similarity with the carboxysome operon that includes the ribulose-1,5-bisphosphate carboxylase/oxygenase complex from A. ferrooxidans and another copper-repressed product was significantly similar to the XyIN outer membrane protein from Pseudomonas putida. Finally, RAP-PCR products of unknown similarities were also present.

Molecular cloning, sequencing, and expression of omp-40, the gene coding for the major outer membrane protein from the acidophilic bacterium Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. Some of the surface components of this microorganism are probably involved in adaptation to their acidic environment and in bacterium-mineral interactions. We have isolated and characterized omp40, the gene coding for the major outer membrane protein from T. ferrooxidans. The deduced amino acid sequence of the Omp40 protein has 382 amino acids and a calculated molecular weight of 40,095.7. Omp40 forms an oligomeric structure of about 120 kDa that dissociates into the monomer (40 kDa) by heating in the presence of sodium dodecyl sulfate. The degree of identity of Omp40 amino acid sequence to porins from enterobacteria was only 22%. Nevertheless, multiple alignments of this sequence with those from several OmpC porins showed several important features conserved in the T. ferrooxidans surface protein, such as the approximate locations of 16 transmembrane beta strands, eight loops, including a large external L3 loop, and eight turns which allowed us to propose a putative 16-stranded beta-barrel porin structure for the protein. These results together with the previously known capacity of Omp40 to form ion channels in planar lipid bilayers strongly support its role as a porin in this chemolithoautotrophic acidophilic microorganism. Some characteristics of the Omp40 protein, such as the presence of a putative L3 loop with an estimated isoelectric point of 7.21 allow us to speculate that this can be the result of an adaptation of the acidophilic T. ferrooxidans to prevent free movement of protons across its outer membrane.

IST1 insertional inactivation of the resB gene: implications for phenotypic switching in Thiobacillus ferrooxidans.

Thiobacillus ferroxidans ATCC 19859 undergoes rapid phenotypic switching between a wild-type state characterized by the ability to oxidize ferrous iron (FeII) and reduced sulfur compounds and a mutant state where it has lost the capacity to oxidize FeII but retains the ability to oxidize sulfur. The mutant has also gained the capacity to swarm. It is proposed that loss of FeII oxidation is due to the reversible transposition of the insertion sequence IST1 into resB encoding a putative cytochrome c-type biogenesis protein. Downstream from resB and co-transcribed with it is resC, encoding another putative cytochrome biogenesis protein. IST1 insertional inactivation of resB could result in the loss of activity of its target c-type cytochrome(s). This putative target cytochrome(s) is proposed to be essential for FeII oxidation but not for sulfur oxidation. Curiously, resB and resC pertain to the proposed system II cytochrome biogenesis pathway whereas gamma Proteobacteria, of which T. ferrooxidans is a member, normally use system I. This could represent an example of lateral gene transfer.

[Metal resistance systems in Pseudomonas]. / Sistemas de resistencia a metales en Pseudomonas.

Several chromosome- and plasmid-encoded metal resistance genetic systems have been studied in Pseudomonas and related bacteria. Some systems are known with molecular detail whereas others are still poorly understood. The former include resistance genes for cations derived from mercury, cadmium and copper and anions from arsenic and chromium. Except for mercury, where a redox transformation occurs, extrusion of the toxic ions from the bacterial cytoplasm appears to be the most common mechanism of resistance.

Isolation and nucleotide sequence of the Thiobacillus ferrooxidans genes for the small and large subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase.

The genes encoding for the large (rbcL) and small (rbcS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisCO) were cloned from the obligate autotroph Thiobacillus ferrooxidans, a bacterium involved in the bioleaching of minerals. Nucleotide sequence analysis of the cloned DNA showed that the two coding regions are separated by a 30-bp intergenic region, the smallest described for the RuBisCO genes. The rbcL and rbcS genes encode polypeptides of 473 and 118 amino acids, respectively. Comparison of the nucleotide and amino acid sequences with those of the genes for rbcL and rbcS found in other species demonstrated that the T. ferrooxidans genes have the closest degree of identity with those of Chromatium vinosum and of Alvinoconcha hessleri endosymbiont. Both T. ferrooxidans enzyme subunits contain all the conserved amino acids that are known to participate in the catalytic process or in holoenzyme assembly.

[Isolation of transcription promoters from Thiobacillus ferrooxidans and T. acidophilus and its introduction by conjugation to T. intermedius]. / Aislamiento de promotores de transcripción de Thiobacillus ferrooxidans y de T. acidophilus y su introducción por conjugación a T. intermedius.

The chemolithotrophic acidophilic bacteria, Thiobacillus ferrooxidans is considered as the most important microorganism in relation to copper and uranium bioleaching ability. Since T. ferrooxidans is a strict chemolithotrophic microorganism, its genetic manipulation is a very hard task. Until now, all efforts have been unsuccessful. Therefore, we decided to approach the problem in steps, trying initially to manipulate some T. ferrooxidans related strains. We chose Thiobacillus acidophilus that shares its habitat with T. ferrooxidans and Thiobacillus intermedius, for its heterotrophic nature that makes them easier to grow and suitable for heterologous conjugation. The main objective of this work was directed towards the isolation and characterization of transcription promoters from T. acidophilus and T. ferrooxidans genomic DNA. Four different promoters from T. acidophilus and four from T. ferrooxidans were isolated and sequenced. In order to test their functional capacity in bacterial systems different from E. coli, they were subcloned and transferred to other bacteria. One of these recombinant plasmids was successfully transferred to T. intermedius and from it to Ps. putida. The subcloned promoter was able to confer streptomycin resistance to Ps. putida.