Metallosphaera javensis sp. nov., a novel species of thermoacidophilic archaea, isolated from a volcanic area.

A novel thermoacidophilic archeaon, strain J1T (=DSM 112778T,=JCM 34702T), was isolated from a hot pool in a volcanic area of Java, Indonesia. Cells of the strain were irregular, motile cocci of 1.0-1.2 µm diameter. Aerobic, organoheterotrophic growth with casamino acids was observed at an optimum temperature of 70 °C in a range of 55-78 °C and at an optimum pH of 3 in a range of 1.5 to 5. Various organic compounds were utilized, including a greater variety of sugars than has been reported for growth of other species of the genus. Chemolithoautotrophic growth was observed with reduced sulphur compounds, including mineral sulphides. Ferric iron was reduced during anaerobic growth with elemental sulphur. Cellular lipids were calditoglycerocaldarchaeol and caldarchaeol with some derivates. The organism contained the respiratory quinone caldariellaquinone. On the basis of phylogenetic and chemotaxonomic comparison with its closest relatives, it was concluded that strain J1T represents a novel species, for which the name Metallosphaera javensis is proposed. Low DNA-DNA relatedness values (16S rRNA gene <98.4%, average nucleotide identity (ANI) <80.1%) distinguished J1T from other species of the genus Metallosphaera and the DNA G+C content of 47.3% is the highest among the known species of the genus.

Salt-tolerant Acidihalobacter and Acidithiobacillus species from Vulcano (Italy) and Milos (Greece).

Ferrous iron- and sulfur-oxidizing Acidihalobacter species and similar so far unclassified bacteria have been isolated from the islands of Vulcano (Italy) and Milos (Greece), specifically from where seawater was acidified at sulfide-rich geothermal sites. Acidithiobacillus species which tolerated concentrations of chloride that inhibit most Acidithiobacillus spp. were also isolated from sites on both islands: these were At. thiooxidans strains and an unclassified species, Acidithiobacillus sp. strain V1. The potential of salt-tolerant acidophiles for industrial application in promoting copper extraction from mineral sulfides where chloride is naturally present at concentrations which would inhibit most acidophiles, or where seawater rather than fresh water is available, appears to be limited by the sensitivity of ferrous-iron oxidizing Acidihalobacter spp. to copper. However, tolerance of copper and chloride shown by At. thiooxidans strain A7 suggests it could oxidize sulfur and benefit acid leaching if ferric iron or copper was provided as the primary oxidant of sulfide ores.

Acidithiobacillus ferrianus sp. nov.: an ancestral extremely acidophilic and facultatively anaerobic chemolithoautotroph.

Strain MG, isolated from an acidic pond sediment on the island of Milos (Greece), is proposed as a novel species of ferrous iron- and sulfur-oxidizing Acidithiobacillus. Currently, four of the eight validated species of this genus oxidize ferrous iron, and strain MG shares many key characteristics with these four, including the capacities for catalyzing the oxidative dissolution of pyrite and for anaerobic growth via ferric iron respiration. Strain MG also grows aerobically on hydrogen and anaerobically on hydrogen coupled to ferric iron reduction. While the 16S rRNA genes of the iron-oxidizing Acidi-thiobacillus species (and strain MG) are located in a distinct phylogenetic clade and are closely related (98-99% 16S rRNA gene identity), genomic relatedness indexes (ANI/dDDH) revealed strong genomic divergence between strain MG and all sequenced type strains of the taxon, and placed MG as the first cultured representative of an ancestral phylotype of iron oxidizing acidithiobacilli. Strain MG is proposed as a novel species, Acidithiobacillus ferrianus sp. nov. The type strain is MGT (= DSM 107098T = JCM 33084T). Similar strains have been found as isolates or indicated by cloned 16S rRNA genes from several mineral sulfide mine sites.

Cytochromes in anaerobic growth of Acidithiobacillus ferrooxidans.

The mineral sulfide-oxidising Acidithiobacillus ferrooxidans has been extensively studied over many years but some fundamental aspects of its metabolism remain uncertain, particularly with regard to its anaerobic oxidation of sulfur. This label-free, liquid chromatography-electron spray ionisation-mass spectrometry-based proteomic analysis estimated relative protein abundance during aerobic and anaerobic growth of At. ferrooxidans. One of its two bc1 complexes, that encoded by the petII operon, was strongly implicated in anaerobic ferric iron-coupled sulfur oxidation, probably in conjunction with two cytochromes. These two cytochromes are homologs of the Cyc2 and Cyc1 proteins that are involved in ferrous iron oxidation. The previously undetected cytochromes apparently associated with anaerobic growth in At. ferrooxidans appear to be absent in many other ferrous iron-oxidising acidophiles that can also reduce ferric iron, which suggests a diversity in the ferric-iron-coupled sulfur oxidation pathways. For aerobic growth of At. ferrooxidans, this analysis was consistent with the generally accepted mechanism for its oxidation of ferrous iron. Unexpectedly, proteins encoded by the petI operon were not abundant and generally not detected in the proteomic analyses of cells grown aerobically on sulfur, although there was some expression of genes of the petI and petII operons in these cells.

Reclassification of 'Thiobacillus prosperus' Huber and Stetter 1989 as Acidihalobacter prosperus gen. nov., sp. nov., a member of the family Ectothiorhodospiraceae.

Analysis of phylogenomic metrics of a recently released draft genome sequence of the halotolerant, acidophile 'Thiobacillus prosperus' DSM 5130 indicates that it is not a member of the genus Thiobacillus within the class Betaproteobacteria as originally proposed. Based on data from 16S rRNA gene phylogeny, and analyses of multiprotein phylogeny and average nucleotide identity (ANI), we show that it belongs to a new genus within the family Ectothiorhodospiraceae, for which we propose the name Acidihalobacter gen. nov. In accordance, it is proposed that 'Thiobacillus prosperus' DSM 5130 be named Acidihalobacter prosperus gen. nov., sp. nov. DSM 5130T ( = JCM 30709T) and that it becomes the type strain of the type species of this genus.

Autotrophic, sulfur-oxidizing actinobacteria in acidic environments.

Some novel actinobacteria from geothermal environments were shown to grow autotrophically with sulfur as an energy source. These bacteria have not been formally named and are referred to here as "Acidithiomicrobium" species, as the first of the acidophilic actinobacteria observed to grow on sulfur. They are related to Acidimicrobium ferrooxidans with which they share a capacity for ferrous iron oxidation. Ribulose bisphosphate carboxylase/oxygenase (RuBisCO) is active in CO(2) fixation by Acidimicrobium ferrooxidans, which appears to have acquired its RuBisCO-encoding genes from the proteobacterium Acidithiobacillus ferrooxidans or its ancestor. This lateral transfer of RuBisCO genes between a proteobacterium and an actinobacterium would add to those noted previously among proteobacteria, between proteobacteria and cyanobacteria and between proteobacteria and plastids. "Acidithiomicrobium" has RuBisCO-encoding genes which are most closely related to those of Acidimicrobium ferrooxidans and Acidithiobacillus ferrooxidans, and has additional RuBisCO genes of a different lineage. 16S rRNA gene sequences from "Acidithiomicrobium" species dominated clone banks of the genes extracted from mixed cultures of moderate thermophiles growing on copper sulfide and polymetallic sulfide ores in ore leaching columns.

Ferrous iron oxidation and rusticyanin in halotolerant, acidophilic 'Thiobacillus prosperus'.

The halotolerant acidophile 'Thiobacillus prosperus' was shown to require chloride for growth. With ferrous iron as substrate, growth occurred at a rate similar to that of the well-studied acidophile Acidithiobacillus ferrooxidans. Previously, the salt (NaCl) requirement of 'T. prosperus' was not clear and its growth on ferrous iron was described as poor. A subtractive hybridization of cDNAs from ferrous-iron-grown and sulfur-grown 'T. prosperus' strain V6 led to identification of a cluster of genes similar to the rus operon reported to encode ferrous iron oxidation in A. ferrooxidans. However, the 'T. prosperus' gene cluster did not contain a homologue of cyc1, which is thought to encode a key cytochrome c in the pathway of electron transport from ferrous iron in A. ferrooxidans. Rusticyanin, another key protein in ferrous iron oxidation by A. ferrooxidans, was present in 'T. prosperus' at similar concentrations in cells grown on either ferrous iron or sulfur.

Ribulose bisphosphate carboxylase activity and a Calvin cycle gene cluster in Sulfobacillus species.

The Calvin-Benson-Bassham (CBB) cycle has been extensively studied in proteobacteria, cyanobacteria, algae and plants, but hardly at all in Gram-positive bacteria. Some characteristics of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) and a cluster of potential CBB cycle genes in a Gram-positive bacterium are described in this study with two species of Sulfobacillus (Gram-positive, facultatively autotrophic, mineral sulfide-oxidizing acidophiles). In contrast to the Gram-negative, iron-oxidizing acidophile Acidithiobacillus ferrooxidans, Sulfobacillus thermosulfidooxidans grew poorly autotrophically unless the CO(2) concentration was enhanced over that in air. However, the RuBisCO of each organism showed similar affinities for CO(2) and for ribulose 1,5-bisphosphate, and similar apparent derepression of activity under CO(2) limitation. The red-type, form I RuBisCO of Sulfobacillus acidophilus was confirmed as closely related to that of the anoxygenic phototroph Oscillochloris trichoides. Eight genes potentially involved in the CBB cycle in S. acidophilus were clustered in the order cbbA, cbbP, cbbE, cbbL, cbbS, cbbX, cbbG and cbbT.

A novel Acidimicrobium species in continuous cultures of moderately thermophilic, mineral-sulfide-oxidizing acidophiles.

A novel species of Acidimicrobium appeared to be the predominant ferrous iron oxidizer in a mixed culture that effected the continuous, efficient extraction of nickel from a mineral concentrate at 49 degrees C, but it was not isolated in pure culture. It outcompeted Acidimicrobium ferrooxidans, which was expected to have a major role in iron oxidation in reactors gassed with air, and was outnumbered at 49 degrees C only by the sulfur-oxidizing Acidithiobacillus caldus. Sulfobacillus species were expected to compete with Acidimicrobium species when culture aeration was enriched with carbon dioxide, but they were a minor component of the populations with and without this enrichment. Sulfobacillus thermosulfidooxidans replaced the Acidimicrobium species and Acidithiobacillus caldus when the temperature was increased to 55 degrees C.

Ferrous iron- and sulfur-induced genes in Sulfolobus metallicus.

Genes of Sulfolobus metallicus that appeared to be upregulated in relation to growth on either ferrous iron or sulfur were identified using subtractive hybridization of cDNAs. The genes upregulated during growth on ferrous iron were found in a cluster, and most were predicted to encode membrane proteins. Quantitative reverse transcription-PCR of cDNA showed upregulation of most of these genes during growth on ferrous iron and pyrite compared to results during growth on sulfur. The highest expression levels observed included those for genes encoding proteins with similarities to cytochrome c oxidase subunits and a CbsA-like cytochrome. The genes identified here that may be involved in oxidation of ferrous iron by S. metallicus are termed fox genes. Of three available genomes of Sulfolobus species (S. tokodaii, S. acidocaldarius, and S. solfataricus), only that of S. tokodaii has a cluster of highly similar open reading frames, and only S. tokodaii of these three species was also able to oxidize ferrous iron. A gene encoding sulfur oxygenase-reductase was identified as the source of the dominant transcript in sulfur-grown cells of S. metallicus, with the predicted protein showing high identities to the previously described examples from S. tokodaii and species of Acidianus.

Characteristics of Sulfobacillus acidophilus sp. nov. and other moderately thermophilic mineral-sulphide-oxidizing bacteria.

Several isolates of Gram-positive, acidophilic, moderately thermophilic, ferrous-iron- and mineral-sulphide-oxidizing bacteria were examined to establish unequivocally the characteristics of Sulfobacillus-like bacteria. Two species were evident: Sulfobacillus thermosulfidooxidans with 48-50 mol% G+C and Sulfobacillus acidophilus sp. nov. with 55-57 mol% G+C. Both species grew autotrophically and mixotrophically on ferrous iron, on elemental sulphur in the presence of yeast extract, and heterotrophically on yeast extract. Autotrophic growth on sulphur was consistently obtained only with S. acidophilus.

Acidimicrobium ferrooxidans gen. nov., sp. nov.: mixed-culture ferrous iron oxidation with Sulfobacillus species.

A new species of ferrous-iron-oxidizing, moderately thermophilic, acidophilic bacteria, Acidimicrobium ferrooxidans, has been described. Two isolates of the species differed only in the tendency of one, previously known as strain TH3, to grow in filaments. The chromosomal DNA base composition is between 67 and 69 mol% G + C. The capacity of this species to fix CO2 from air was greater than that of iron-oxidizing thermoacidophiles of the genus Sulfobacillus, which required an enhanced CO2 concentration for optimum autotrophic growth. Under air, ferrous iron oxidation in mixed cultures of A. ferrooxidans with either Sulfobacillus thermosulfidooxidans or Sulfobacillus acidophilus was more extensive than in pure cultures of these three strains. The greater part of ferrous iron oxidation in mixed cultures probably resulted from activity of the Sulfobacillus species, which possess a greater tolerance of ferric iron, and which presumably grew mixotrophically utilizing organic compounds from A. ferrooxidans.