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Taxonomic relationships of Thiobacillus halophilus, T. aquaesulis, and other species of Thiobacillus, as determined using 16S rDNA sequencing.

Total base sequences of the 16S rRNA genes of Thiobacillus halophilus and Thiobacillus aquaesulis show that these bacteria fall into the gamma- and beta-subdivisions, respectively of the Proteobacteria. The closest relative of T. halophilus is Thiobacillus hydrothermalis (with 98.7% similarity), and the closest relative of T. aquaesulis is Thiobacillus thioparus (93.2% similarity). Physiological properties and mol% G+C content of their DNA serve to confirm that these four organisms are all distinct species. It is reiterated that the species currently assigned to the genus Thiobacillus are clearly so diverse that they need reclassification into several genera. The type species, T. thioparus, is unequivocally placed in the beta-subdivision of the Proteobacteria, thus requiring that the use of the genus name Thiobacillus be restricted to the chemolithoautotrophic species falling into that group. T. aquaesulis and T. thioparus may thus be regarded as true species of Thiobacillus. The relatively large number of obligately chemolithoautotrophic Thiobacillus species falling in the gamma-subdivision of the Proteobacteria need further study in order to assess the case for reclassification into one or more new or different genera.

Bacteriological Techniques↗

The mer operon of the acidophilic bacterium Thiobacillus T3.2 diverges from its Thiobacillus ferrooxidans counterpart.

The chromosomal mercury resistance (mer) region of the acidophilic bacterium Thiobacillus T3.2 was cloned, characterized, and compared to reported homologous sequences. The Thiobacillus T3.2 mer resistance system is organized as an operon that transcribes into a polycistronic mRNA encoding the Hg2+ ion transport MerT and MerP proteins and the mercuric reductase MerA. In contrast to the Thiobacillus ferrooxidans mer determinant, no merC gene was detected. Transcription of structural genes is regulated by the product of the regulatory merR gene. On the basis of sequence data and expression experiments in E. coli, both merTPA and merR transcription units could be located close to each other and in different strands, with their promoters (PTPA and PR, respectively) overlapping the putative MerR binding site in the intergenic operator/promoter (O/P) region. Amino acid sequences of mer gene products were compared to their homologs. Some sequence features, such as the number and position of cysteine residues, are unique for the Mer proteins of this bacterium. Similarities (-10 and -35 boxes are 19bp apart in both PR and PTPA promoters) and differences (inverted repeats in the Thiobacillus T3.2 MerR-binding site are 2bp shorter than in Thiobacillus ferrooxidans) exist between the O/P intergenic regions of both Thiobacilli. In vivo experiments showed inducible expression of mercury resistance in E. coli cells transformed with the entire Thiobacillus T3.2 mer genetic determinant (structural plus regulatory genes), and little or no expression in clones containing only the structural merT, merP, and merA genes.

Amino Acid Sequence↗

Confirmation that Thiobacillus halophilus and Thiobacillus hydrothermalis are distinct species within the gamma-subclass of the Proteobacteria.

Thiobacillus halophilus and Thiobacillus hydrothermalis share 98.7% similarity in 16S rRNA sequence, possess similar gross DNA composition (64.2 and 67.4 mol% G+C values, respectively), and have similar physiological properties. While this might have indicated that they were strains of a single species, DNA-DNA hybridization between the type strains of the two species showed only 59% hybridization, indicating the organisms to be different at the species level. Thiobacillus neapolitanus is the phylogenetically nearest neighbour of T. halophilus and T. hydrothermalis (91.6-92.1% similarity in 16S rRNA sequence) and is the only other Thiobacillus in the gamma-subclass of the Proteobacteria that can be regarded as exclusively related to these two species. The 16S rRNA gene sequences of these three species are so different from those of the other thiobacilli in the gamma-subclass that they justify recognition as a distinct phyletic group. Their comparative properties are summarized.

Cell Division↗

Confirmation of Thiobacillus denitrificans as a species of the genus Thiobacillus, in the beta-subclass of the Proteobacteria, with strain NCIMB 9548 as the type strain.

Thiobacillus denitrificans is physiologically similar to the type species of the genus Thiobacillus, Thiobacillus Thioparus, and both are located in the beta-subclass of the Proteobacteria. T. denitrificans is distinguished from all other Thiobacillus species by its ability to grow as a facultatively anaerobic chemolithotroph, coupling the oxidation of inorganic sulfur compounds to the reduction of nitrate, nitrite and other oxidized nitrogen compounds to dinitrogen. A definitive description of this species is provided and strain NCIMB 9548T is designated as the type strain of the species, thereby correcting an earlier error in the literature.

Base Composition↗

Thiobacillus acidophilus: a study of its presence in Thiobacillus ferrooxidans cultures.

A study has been undertaken to account for the presence of Thiobacillus acidophilus in iron-grown cultures of Thiobacillus ferrooxidans. Attempts to adapt T. acidophilus to ferrous iron were not successful but the facultative autotroph grew to a limited extent in the spent medium of T. ferrooxidans and was able to grow oligotrophically. Possible oligotrophic substrates were methanol, ethanol, and sulphide. Thiobacillus ferrooxidans may benefit from the presence of T. acidophilus because in mixed cultures some inhibiting organic compounds such as alcohols, organic acids, and amino acids were utilized by T. acidophilus. The number of T. acidophilus cells in heterogeneous cultures with T. ferrooxidans was of the same order of magnitude as the number of T. ferrooxidans cells as revealed by fluorescent-labelled antibodies.

Adaptation, Physiological↗

Preservation of Thiobacillus ferrooxidans and Thiobacillus thiooxidans with activity check.

Cultures of Thiobacillus ferrooxidans and Thiobacillus thiooxidans, used in biohydrometallurgical processes of economic importance, are very difficult to preserve by conventional methods. Hence, to preserve the cultures with their activity intact, various techniques were tried, after determining their respective activity in terms of Iron Oxidation Rate (IOR) and Sulfur Oxidation Rate (SOR). Among the methods tested, along with the recommended method of serial transfer in a liquid medium, were methods such as lyophilization, storage in a liquid nitrogen and mixing with sterile, inert carriers like lignite or chalcopyrite ores. After a period check-up at 4 months and 8 months storage, it was found that out of these methods, mixing with sterile ore followed by storage at 8 degrees C, kept both types of activities intact. The temperature of storage was observed to have a definite effect on activity, in that when the preserved cultures were stored at 8 degrees C, the activity was retained, whereas at 28-30 degrees C (RT) storage, the activity of all the cultures preserved by various techniques, dropped significantly.

Acidithiobacillus thiooxidans↗

New facultative Thiobacillus and a reevaluation of the heterotrophic potential of Thiobacillus novellus.

A new facultatively autotrophic Thiobacillus has been isolated in pure culture. The general physiological characteristics of the organism are described together with a redescription of Thiobacillus novellus. The new isolate differs from T. novellus in its ability to grow heterotrophically at faster rates and on a greater range of organic compounds. It can be transferred readily between autotrophic and heterotrophic conditions. It can grow anaerobically by nitrate respiration on a number of organic compounds, but not on thiosulfate. Some problems in the nomenclature and taxonomy of the thiobacilli are discussed with reference to the new isolate.

Cell-Free System↗

Enzymes of intermediary carbohydrate metabolism in the obligate autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus.

Levels of enzymes operative in the Embden-Meyerhof-Parnas (glycolytic) pathway, pentose phosphate cycle, citric acid cycle, and certain other phases of intermediary carbohydrate metabolism have been compared in Thiobacillus thioparus and T. neapolitanus. All enzymes of the glycolytic pathway except phosphofructokinase were demonstrated in both organisms. There were some striking quantitative differences between the two organisms with respect to the activities of the individual enzymes of the glycolytic pathway and the citric acid cycle. Qualitative differences were also found: the isocitrate dehydrogenase activity of T. thioparus is strictly nicotinamide adenine dinucleotide phosphate (NADP)-dependent, whereas that of T. neapolitanus is primarily nicotinamide adenine dinucleotide-dependent, activity with NADP being low; the glucose-6-phosphate dehydrogenase of T. thioparus is particulate, whereas that of T. neapolitanus is partly soluble and partly particulate; the 6-phosphogluconate dehydrogenase of T. thioparus is soluble, that of T. neapolitanus is partly soluble and partly particulate. All enzymes which function in the carbon reduction cycle were present at very high levels. In contrast, enzymes which operate exclusively in cycles other than the carbon reduction cycle were present at low levels. Of the enzymes not operative in the carbon reduction cycle that were examined, isocitric dehydrogenase had the highest specific activity. Both organisms possessed reduced nicotinamide adenine dinucleotide dehydrogenase activity. The qualitative and quantitative aspects of the data are discussed in relation to possible biochemical explanations of obligate autotrophy.

Carbohydrate Metabolism↗

Assimilation and metabolism of exogenous organic compounds by the strict autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus.

The assimilation and utilization of the individual carbon atoms of pyruvate and acetate by cells of Thiobacillus thioparus and T. neapolitanus, in the presence and absence of an energy source, were studied by use of radioactive substrates. Both organisms produced (14)CO(2) from (14)C-labeled pyruvate, but more came from carbon 1 than from carbons 2 or 3. The conversion of the carbons of acetate to CO(2) by both organisms was much less than that from any of the pyruvate carbons. When labeled pyruvate and acetate were incubated with these organisms, small amounts of radioactivity were found in the tricholoacetic acid-soluble material, nucleic acids, and lipids, and larger amounts were found in the protein fraction. The composition of the incubation medium affected the amount of utilization and incorporation of labeled substrates by both organisms. The presence of an exogenous energy source (Na(2)S(2)O(3)) suppressed incorporation of the labeled substrates into various cellular components by T. thioparus, but enhanced incorporation by T. neapolitanus. When (14)C-pyruvate was used as a substrate, as many as 12 radioactive compounds were found in the water-soluble fraction in the experiments with T. neapolitanus, whereas no more than three radioactive compounds were detected in this fraction in the experiments with T. thioparus. Of the total (14)C activity found in the water-soluble fractions, malic acid contained the highest percentage. These findings are discussed in light of the overall metabolism of these two sulfur-oxidizing obligate chemoautotrophs, as well as in relation to the biochemical basis of chemoautotrophy.

Acetates↗

Phylogenetic analysis of the genera Thiobacillus and Thiomicrospira by 5S rRNA sequences.

5S rRNA nucleotide sequences from Thiobacillus neapolitanus, Thiobacillus ferrooxidans, Thiobacillus thiooxidans, Thiobacillus intermedius, Thiobacillus perometabolis, Thiobacillus thioparus, Thiobacillus versutus, Thiobacillus novellus, Thiobacillus acidophilus, Thiomicrospira pelophila, Thiomicrospira sp. strain L-12, and Acidiphilium cryptum were determined. A phylogenetic tree, based upon comparison of these and other related 5S rRNA sequences, is presented. The results place the thiobacilli, Thiomicrospira spp., and Acidiphilium spp. in the "purple photosynthetic" bacterial grouping which also includes the enteric, vibrio, pseudomonad, and other familiar eubacterial groups in addition to the purple photosynthetic bacteria. The genus Thiobacillus is not an evolutionarily coherent grouping but rather spans the full breadth of the purple photosynthetic bacteria.

Base Sequence↗

Thiobacillus plumbophilus spec. nov., a novel galena and hydrogen oxidizer.

From an uranium mine three strains of rod-shaped, mesophilic, chemolithouautotrophic bacteria were isolated. They grow by oxidation of H2S, galena (PbS) and H2. Angelsite (PbSO4) is formed from galena. No ferrous iron is oxidized by the isolates. They grow between pH 4 and 6.5 at temperatures of about 9 to 41 degrees C (optimum around 27 degrees C). The G + C content of the DNA is around 66 mol %. Based on their ability to oxidize sulfur compounds, the new organisms belong to the genus Thiobacillus. No significant homology with Thiobacillus ferrooxidans and Thiobacillus cuprinus was detected by DNA-DNA hybridization. Therefore the new isolates represent a new species within the genus Thiobacillus. Based on the unusual growth on galena, we name the new species Thiobacillus plumbophilus (type strain Gro7; DSM 6690).

Base Composition↗

Cell yield and bioenergetics of Thiomicrospira denitrificans compared with Thiobacillus denitrificans.

From cell yields of Thiomicrospira denitrificans grown inthe chemostat at different growth rates under anaerobic conditions a value of 1.4 mM S2O3 = per g dry wt and per h could be calculated for maintenance energy requirements, and of 5.65 dry wt per mole S2O3 = for the true growth yield. Cell yields of Thiomicrospira denitrificans appeared to be almost half of those of Thiobacillus denitrificans. Though in Thiobacillus denitrificans at D = 0.03 h(-1) under anaerobic conditions a value was found of 11.60 g dry wt per mole of thiosulphate used for energetic purposes, a value of 5.72 g dry wt per mole of thiosulphate was found under comparable conditions in Thiomicrospira denitrificans. Under aerobic conditions at D = 0.03 h(-1) values of 18.54 g dry wt per mole of thiosulphate were found in Thiobacillus denitrificans whereas Thiomicrospira denitrificans yielded only 9.38 g dry wt per mole of thiosulphate. As in Thiobacillus denitrificans anaerobic cell yields on sulphide were comparable to those on thiosulphate. Calculations have been made which indicate that the biosynthetic efficiency of Thiomicrospira denitrificans is lower than that of Thiobacillus denitrificans. This can only partly be explained by the absence of adenosine-phosphosulphate (APS) reductase.

Adenosine Triphosphate↗