T. I. Bogdanova

Activity of the Enzymes of Carbon Metabolism in Sulfobacillus sibiricus under Various Conditions of Cultivation

The thermoacidophilic iron-oxidizing chemolithotroph Sulfobacillus sibiricus N1T is characterized by steady growth and amplified cell yield when grown in vigorously aerated medium containing Fe2+, glucose, and yeast extract as energy sources. In this case, carbon dioxide, glucose, and yeast extract are used as carbon sources. Glucose is assimilated through the fructose-bisphosphate pathway and the pentose-phosphate pathway. The glyoxylate bypass does not function in S. sibiricus, and the tricarboxylic acid cycle is disrupted at the level of 2-oxoglutarate dehydrogenase. The presence of ribulose-bisphosphate carboxylase indicates that carbon dioxide fixation proceeds through the Calvin cycle. The activity of ribulose-bisphosphate carboxylase is highest in autotrophically grown cells. The cells also contain pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxytransphosphorylase.

Growth and Carbohydrate Metabolism of Sulfobacilli

The moderately thermophilic acidophilic bacteria Sulfobacillus thermosulfidooxidans, strain 1269, S. thermosulfidooxidanssubsp. “asporogenes,” strain 41, and the thermotolerant strain S. thermosulfidooxidanssubsp. “thermotolerans” K1 prefer mixotrophic growth conditions (the concomitant presence of ferrous iron, thiosulfate, and organic compounds in the medium). In heterotrophic and autotrophic growth conditions, these sulfobacilli can grow over only a few culture transfers. In cell-free extracts of these sulfobacilli, key enzymes of the Embden–Meyerhof–Parnas, pentose-phosphate, and Entner–Doudoroff pathways were found. The role of a particular pathway depended on the cultivation conditions. All of the enzymes assayed were most active under mixotrophic conditions in the presence of Fe2+and glucose, suggesting the operation of all of the three major pathways of carbohydrate metabolism under these conditions. However, the operation of the Entner–Doudoroff pathway in strain 41 was restricted under mixotrophic conditions. After the first culture transfer from mixotrophic to heterotrophic conditions, the utilization of glucose occurred only via the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways. After the first culture transfer from mixotrophic to autotrophic conditions, the activity of carbohydrate metabolism enzymes decreased in all of the strains studied; in strain K1, only the glycolytic pathway remained operative. The high activity of fructose-bisphosphate aldolase, remaining in strain 41 cells under these conditions, suggests the involvement of this enzyme in the reactions of the Calvin cycle or of gluconeogenesis.

Effect of Cultivation Conditions on the Growth and Activities of Sulfur Metabolism Enzymes and Carboxylases of Sulfobacillus thermosulfidooxidans subsp. asporogenes Strain 41

The moderately thermophilic acidophilic bacterium Sulfobacillus thermosulfidooxidans subsp. asporogenes strain 41 is capable of utilizing sulfides of gold–arsenic concentrate and elemental sulfur as a source of energy. Growth in the presence of S0 under auto- or mixotrophic conditions was less stable than in media containing iron monoxide. The enzymes involved in the oxidation of sulfur inorganic compounds—thiosulfate-oxidizing enzyme, tetrathionate hydrolase, rhodanase, adenylyl phosphosulfate reductase, sulfite oxidase, and sulfur oxygenase—were determined in the cells of the sulfobacilli grown in mineral medium containing 0.02% yeast extract and either sulfur or iron monoxide and thiosulfate. Cell-free extracts of the cultures grown in the medium with sulfur under auto- or mixotrophic conditions displayed activity of the key enzyme of the Calvin cycle—ribulose bisphosphate carboxylase—and several other enzymes involved in the heterotrophic fixation of carbon dioxide. Activities of carboxylases depended on the composition of the cultivation media.

Investigation of the Phylogenetic Position of Aerobic, Moderately Thermophilic Bacteria Oxidizing Fe2+, S0, and Sulfide Minerals and Affiliated to the Genus Sulfobacillus

At present, the genus Sulfobacillus includes three species of chemolithotrophic, acidophilic, moderately thermophilic or mesophilic bacteria. These are the type species of the genus S. thermosulfidooxidans (strains VKM B-1269 = DSM 9293 T and BC1) [1, 2], S. acidophilus (strains NAL T and ALV) [2], and S. disulfidooxidans (strain SD-11 T ) [3]. Based on their phenotypic properties, two additional strains, 41 and K1, were assigned as subspecies of S. thermosulfidooxidans , and named S. thermosulfidooxidans subsp. “ asporogenes ” [4] and S. thermosulfidooxidans subsp. “ thermotolerans ” [5], respectively. Several unidentified strains (C-MT1, YTH2, and YTH1) were also assigned to the genus Sulfobacillus based on 16S rDNA sequence data [6, 7]. Phylogenetic analysis demonstrated that Sulfobacillus clusters with species of the genus Alicyclobacillus [3, 8].

Sulfur-Metabolizing Enzymes in Thermoacidophilic Bacteria Sulfobacillus sibiricus

Sulfur oxygenase, sulfite oxidase, adenylyl sulfate reductase, rhodanase, sulfur : Fe(III) oxidoreductase, and sulfite : Fe(III) oxidoreductase were found in cells of aerobic thermoacidophilic bacteria Sulfobacillus sibiricus, strains N1 and SSO. Enzyme activity was revealed in the cells grown on medium with elemental sulfur or in the presence of various sulfide minerals and concentrates of sulfide ores. The activity of enzymes of sulfur metabolism depended little on the degree of aeration during bacterial growth.

Genotypic and phenotypic polymorphism of environmental strains of the moderately thermophilic bacterium Sulfobacillus sibiricus

Five cultures of moderately thermophilic spore-forming acidophilic chemolithotrophic bacteria were isolated from the zones of spontaneous heating of pyrrhotite-containing pyrite-arsenopyrite gold-arsenic sulfide ores in an operating open pit (strains B1, B2, B3, OFO, and SSO). Analysis of the chromosomal DNA structure revealed the differences between these cultures at the strain level (apart from B3 and SSO, which had identical restriction profiles). All the strains had a similar G+C DNA molar content (47.4–48.3%). The level of DNA reassociation was 85 to 95%. The similarity between the DNA of the type strain Sulfobacillus sibiricus N1 isolated from arsenopyrite ore concentrate and that of these strains (83–93%) indicates that they belong to the same species. The strains had similar values of pH and temperature optimal for growth on ferrous iron (1.6–2.0 and 45–55°C, respectively). They were mixotrophs; Fe(II), So, and sulfide minerals along with organic compounds were used as energy sources and electron donors. However, the kinetic parameters of growth and substrate oxidation varied from strain to strain. Genetic variety of the strains from diverse ecosystems and environments is possibly the result of the different rates of microevolution processes.

Non-species-specific effects of unacylated homoserine lactone and hexylresorcinol, low molecular weight autoregulators, on the growth and development of bacteria

We conducted a comparative study of the effects of α-amino-γ-butyrolactone, the common structural element of extracellular microbial regulators of the homoserine lactone (HSL) group, and of 4-n-hexylresorcinol, an autoregulator of the alkylhydroxybenzene (AHB) group, on the growth and development of grampositive and gram-negative bacteria. We revealed non-species-specific effects of HSL and AHB and characterized their concentration dependencies. The addition of 10−5−10−3 M HSL or 10−5−10−4 M AHB during the exponential growth phase of the cultures grown on balanced media resulted in cell division arrest and accelerated the transition to the stationary phase that culminated in endospore formation in Bacillus cereus, Alicyclobacillus tolerans, and Sulfobacillus thermosulfidooxidans. When bacilli grew under the cultivation conditions that resulted in a low-zero spore percentage, 10−4−10−3 M HSL cancelled the inhibition of spore formation. In the gram-negative bacteria Pseudomonas aurantiaca and Azotobacter vinelandii, AHB at concentrations of 10−4 to (1.5−2.5)×10−4 M induced the formation of dormant cells. Studies with the actinobacterium Streptomyces avermitilis revealed that the HSL effect varied depending on the age of the test cultures. The addition of 10−4 M HSL during the lag phase of a submerged streptomycete culture accelerated its transition to the stationary phase and induced the formation of endospores, the dormant cells that are regarded as alternatives to exospores (conidia). If HSL (3.64 and 4.55 mg per 1 cm2 disc) was locally added to a surface S. avermitilis culture, the growing mycelium formed rings that differed in their density, in the extent of the development of aerial mycelium, and in the presence/absence of exospores. Ring-shaped growth of streptomycete mycelia was also induced by 0.075–0.75 mg of AHB; however, unlike HSL, AHB repressed exospore formation. The data on non-species-specific effects of HSL and AHB suggest that they may perform regulatory functions at the microbial community level.

Regulation of metabolic pathways in sulfobacilli under different aeration regimes

A comparative study of the activities of the enzymes of carbon metabolism from the cells of moderately thermophilic chemolithotrophic bacteria Sulfobacillus sibiricus (strains N1 and SSO) and Sulfobacillus thermosulfidooxidans subsp. asporogenes (strain 41) was carried out grown in a high layer of medium without forced aeration and cells grown with intense aeration. Limited air access to the growing S. sibiricus N1 cells resulted in switching from the pentose phosphate pathway of glucose metabolism to the Entner-Doudoroff pathway while the Embden-Meyerhof-Parnas pathway persisted. Irrespective of the level of the aeration, in the cells of S. sibiricus SSO and S. thermosulfidooxidans subsp. asporogenes 41, degradation of the glucose occurred via the Entner-Doudoroff and pentose phosphate metabolic pathways, respectively, as well as via the Embden-Meyerhof-Parnas pathway. Prolonged growth of S. sibiricus, strains N1 and SSO, in a high layer of the medium without forced aeration led to the repression of synthesis of most of the tricarboxylic acid cycle (TCA cycle) enzymes, in particular dehydrogenases, as well as of some carboxylases including RuBisCO. The traits of carbon metabolism in various strains of Sulfobacillus under conditions of oxygen deficiency are discussed.

Phenotypic properties of Sulfobacillus thermotolerans: Comparative aspects

The phenotypic characteristics of the species Sulfobacillus thermotolerans Kr1T, as dependent on the cultivation conditions, are described in detail. High growth rates (0.22–0.30 h−1) and high oxidative activity were recorded under optimum mixotrophic conditions at 40 °C on medium with inorganic (Fe(II), S0, or pyrite-arsenopyrite concentrate) and organic (glucose and/or yeast extract) substrates. In cells grown under optimum conditions on medium with iron, hemes a, b, and, most probably, c were present, indicating the presence of the corresponding cytochromes. Peculiar extended structures in the form of cylindrical cords, never observed previously, were revealed; a mucous matrix, likely of polysaccharide nature, occurred around the cells. In the cells of sulfobacilli grown litho-, organo-, and mixotrophically at 40 °C, the enzymes of the three main pathways of carbon utilization and some enzymes of the TCA cycle were revealed. The enzyme activity was maximum under mixotrophic growth conditions. The growth rate in the regions of limiting temperatures (55 °C and 12–14 °C) decreased two-and tenfold, respectively; no activity of 6-phosphogluconate dehydrogenase, one of the key enzymes of the oxidative pentose phosphate pathway, could be revealed; and a decrease in the activity of almost all enzymes of glucose metabolism and of the TCA cycle was observed. The rate of 14CO2 fixation by cells under auto-, mixo-, and heterotrophic conditions constituted 31.8, 23.3, and 10.3 nmol/(h mg protein), respectively. The activities of RuBP carboxylase (it peaked during lithotrophic growth) and of carboxylases of heterotrophic carbon dioxide fixation were recorded. The physiological and biochemical peculiarities of the thermotolerant bacillus are compared versus moderately thermophilic sulfobacilli.

The dependence of intracellular ATP level on the nutrition mode of the acidophilic bacteria Sulfobacillus thermotolerans and Alicyclobacillus tolerans

The dynamics of the ATP pool in the aerobic spore-forming acidothermophilic mixotrophic bacteria Sulfobacillus thermotolerans Kr1T and Alicyclobacillus tolerans K1T were studied in the course of their chemolithoheterotrophic, chemoorganoheterotrophic, and chemolithoautotrophic growth. It was established that, during mixotrophic growth, the maximum ATP concentrations in the cells of S. thermotolerans Kr1 and A. tolerans K1 were 3.8 and 0.6 nmol/mg protein, respectively. The ATP concentrations in sulfobacilli and alicyclobacilli during organotrophic growth were 2.2 and 3.1 nmol/mg protein, respectively. In the cells of the obligately heterotrophic bacterium Alicyclobacillus cycloheptanicus 4006T, the maximum ATP concentration was several times higher and reached 12.3 nmol/mg protein. During lithotrophic growth, the maximum values of the ATP concentration in the cells of S. thermotolerans Kr1 and A. tolerans K1 were 0.3 and <0.1 nmol/mg protein, respectively; in the cells of the autotrophic bacterium Acidithiobacillus ferrooxidans TFBk, the ATP content was about 60–300 times higher (17.0 nmol/mg protein). It is concluded that low ATP content is among the possible causes of growth cessation of S. thermotolerans Kr1 and A. tolerans K1 under auto-and heterotrophic conditions after several culture transfers.