Sulfobacillus thermotolerans: new insights into resistance and metabolic capacities of acidophilic chemolithotrophs.

The first complete genome of the biotechnologically important species Sulfobacillus thermotolerans has been sequenced. Its 3 317 203-bp chromosome contains an 83 269-bp plasmid-like region, which carries heavy metal resistance determinants and the rusticyanin gene. Plasmid-mediated metal resistance is unusual for acidophilic chemolithotrophs. Moreover, most of their plasmids are cryptic and do not contribute to the phenotype of the host cells. A polyphosphate-based mechanism of metal resistance, which has been previously unknown in the genus Sulfobacillus or other Gram-positive chemolithotrophs, potentially operates in two Sulfobacillus species. The methylcitrate cycle typical for pathogens and identified in the genus Sulfobacillus for the first time can fulfill the energy and/or protective function in S. thermotolerans Kr1 and two other Sulfobacillus species, which have incomplete glyoxylate cycles. It is notable that the TCA cycle, disrupted in all Sulfobacillus isolates under optimal growth conditions, proved to be complete in the cells enduring temperature stress. An efficient antioxidant defense system gives S. thermotolerans another competitive advantage in the microbial communities inhabiting acidic metal-rich environments. The genomic comparisons revealed 80 unique genes in the strain Kr1, including those involved in lactose/galactose catabolism. The results provide new insights into metabolism and resistance mechanisms in the Sulfobacillus genus and other acidophiles.

Ferrous iron oxidation in moderately thermophilic acidophile Sulfobacillus sibiricus N1(T).

The iron-oxidizing system of a moderately thermophilic, extremely acidophilic, gram-positive mixotroph, Sulfobacillus sibiricus N1(T), was studied by spectroscopic, high-performance liquid chromatography and inhibitory analyses. Hemes B, A, and O were detected in membranes of S. sibiricus N1(T). It is proposed that the electron transport chain from Fe²(+) to O2 is terminated by 2 physiological oxidases: aa3-type cytochrome, which dominates in the early-exponential phase of growth, and bo3-type cytochrome, whose role in iron oxidation becomes more prominent upon growth of the culture. Both oxidases were sensitive to cyanide and azide. Cytochrome aa3 was more sensitive to cyanide and azide, with K(i) values of 4.1 and 2.5 µmol·L⁻¹, respectively, compared with K(i) values for cytochrome bo3, which were 9.5 µmol·L⁻¹ for cyanide and 7.0 µmol·L⁻¹ for azide. This is the first evidence for the participation of a bo3-type oxidase in ferrous iron oxidation. The respiratory chain of the mixotroph contains, in addition to the 2 terminal oxidases, a membrane-bound cytochrome b573.

Sulfobacillus thermotolerans sp. nov., a thermotolerant, chemolithotrophic bacterium.

A thermotolerant, Gram-positive, aerobic, endospore-forming, acidophilic bacterium (strain Kr1T) was isolated from the pulp of a gold-containing sulfide concentrate processed at 40 degrees C in a gold-recovery plant (Siberia). Cells of strain Kr1(T) were straight to slightly curved rods, 0.8-1.2 microm in diameter and 1.5-4.5 microm in length. Strain Kr1T formed spherical and oval, refractile, subterminally located endospores. The temperature range for growth was 20-60 degrees C, with an optimum at 40 degrees C. The pH range for growth on medium containing ferrous iron was 1.2-2.4, with an optimum at pH 2.0; the pH range for growth on medium containing S0 was 2.0-5.0, with an optimum at pH 2.5. Strain Kr1T was mixotrophic, oxidizing ferrous iron, S0, tetrathionate or sulfide minerals as energy sources in the presence of 0.02 % yeast extract or other organic substrates. The G+C content of the DNA of strain Kr1T was 48.2+/-0.5 mol%. Strain Kr1T showed a low level of DNA-DNA reassociation with the known Sulfobacillus species (11-44 %). 16S rRNA gene sequence analysis revealed that Kr1T formed a separate phylogenetic group with a high degree of similarity between the nucleotide sequences (98.3-99.6 %) and 100 % bootstrap support within the phylogenetic Sulfobacillus cluster. On the basis of its physiological properties and the results of phylogenetic analyses, strain Kr1T can be affiliated to a novel species of the genus Sulfobacillus, for which the name Sulfobacillus thermotolerans sp. nov. is proposed. The type strain is Kr1T (=VKM B-2339T=DSM 17362T).

Ultrastructural organization of Alicyclobacillus tolerans strain K1T cells.

Electron microscopy examinations of thin sections and freeze-fracture replicas revealed the specific ultrastructural features of Alicyclobacillus tolerans strain K1(T). In particular, the cell wall displayed an ultrastructure typical of gram-positive bacteria and consisted of a thin murein layer (50-60 A in thickness); cells exhibited a surface S-layer constituted by large hexagonally packed (p6-symmetry) rod-shaped subunits of 150-160 A in diameter and 200 A in height. In the cytoplasmic membrane, there were intramembrane vesicular structures that sometimes appeared as large leaflets in the central part. The cytoplasm contained numerous vesicular inclusions covered with a monolayered wall, dissimilar to bilamellar lipid membranes. Endospore coats displayed an intricate structure and consisted of three thick layers; the outer layer had an unusual fine structure; the exosporium was also found.

Reclassification of 'Sulfobacillus thermosulfidooxidans subsp. thermotolerans' strain K1 as Alicyclobacillus tolerans sp. nov. and Sulfobacillus disulfidooxidans Dufresne et al. 1996 as Alicyclobacillus disulfidooxidans comb. nov., and emended description of the genus Alicyclobacillus.

Comparative analysis of 16S rRNA gene sequences, DNA-DNA hybridization data and phenotypic properties revealed that 'Sulfobacillus thermosulfidooxidans subsp. thermotolerans' strain K1 is not a member of the genus Sulfobacillus. Phylogenetically, strain K1 is closely related to unclassified strains of the genus Alicyclobacillus: the 16S rRNA gene sequence of strain K1 is similar to that of Alicyclobacillus sp. AGC-2 (99.6 %), Alicyclobacillus sp. 5C (98.9 %) and Alicyclobacillus sp. CLG (98.6 %) and bacterium GSM (99.1 %). The 16S rRNA gene sequence similarity values for strain K1 and species of the genus Alicyclobacillus with validly published names were in the range 92.1-94.6 %, and for S. thermosulfidooxidans VKM B-1269(T) the value was 87.7 %. Sulfobacillus disulfidooxidans SD-11(T) was also phylogenetically related to strain K1 (92.6 % sequence similarity) and thus belonged to the genus Alicyclobacillus. Chemotaxonomic data, such as the major cell-membrane lipid components of strains K1 and SD-11(T) (omega-alicyclic fatty acids) and the major isoprenoid quinone (menaquinone MK-7) of strain K1, supported the affiliation of strains K1 and SD-11(T) to the genus Alicyclobacillus. Physiological and molecular biological tests allowed genotypic and phenotypic differentiation of strains K1 and SD-11(T) from the nine Alicyclobacillus species with validly published names. The G+C content of the DNA of strain K1 was 48.7+/-0.6 mol%; that of strain SD-11(T) was 53+/-1 mol%. DNA-DNA reassociation studies showed low relatedness (22 %) between strains K1 and SD-11(T), and even lower relatedness (3-5 %) between these strains and Alicyclobacillus acidocaldarius subsp. acidocaldarius ATCC 27009(T), DSM 446(T). DNA reassociation of strains K1 and SD-11(T) with Alicyclobacillus cycloheptanicus DSM 4006(T) gave values of 15 and 21, respectively. Based on the phenotypic and phylogenetic characteristics of strains K1 and SD-11(T), Alicyclobacillus tolerans sp. nov. (type strain, K1(T)=VKM B-2304(T)=DSM 16297(T)) and Alicyclobacillus disulfidooxidans comb. nov. (type strain, SD-11(T)=ATCC 51911(T)=DSM 12064(T)) are proposed.