Carbon metabolism inSulfobacillus thermosulfidooxidans subsp.asporogenes, strain 41

The activities of carbon metabolism enzymes were determined in cellular extracts of the moderately thermophilic, chemolithotrophic, acidophilic bacteriumSulfobacillus thermosulfidooxidans subsp.asporogenes, strain 41, grown either at an atmospheric content of CO₂ in the gas phase (autotrophically, heterotrophically, or mixotrophically) or autotrophically at a CO₂ content increased to 5–10%. Regardless of the growth conditions, all TCA cycle enzymes (except for 2-oxoglutarate dehydrogenase), one glyoxylate bypass enzyme (malate synthase), and some carboxylases (ribulose bisphosphate carboxylase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase) were detected in the cell-free extracts of strain 411. During autotrophic cultivation of strains 41 and 1269, the increase in the CO2 content of the supplied air to 5–10% resulted in the activation of growth and iron oxidation, a 20–30% increase in the cellular content of protein, enhanced activity of the key TCA enzymes (citrate synthase and aconitase), and, in strain 41, a decrease in the activity of carboxylases.     

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. The growth in the presence of S0 under auto- or mixotrophic conditions was less stable compared with the media containing iron monoxide. The enzymes involved in oxidation of sulfur inorganic compounds--thiosulfate-oxidizing enzyme, tetrathionate hydrolase, rhodonase, adenylyl sulfate reductase, sulfite oxidase, and sulfur oxygenase--were discovered in the cells of Sulfobacillus grown in the 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 heterotrophic fixation of carbonic acid. Activities of carboxylases depended on the composition of cultivation media.     

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.     

Pentose metabolism byBacteroides ruminicola subsp.brevis strain B14

The fermentation ofd-arabinose byBacteroides ruminicola strain B₁4 occurs in a manner similar to or identical with that shown previously forl-arabinose metabolism by the organism, a combination of hexose resynthesis and the Embden-Meyerhof sequence. The use ofd-arabinose by strain B₁4 was repressed by prior growth in medium containingd-glucose and induced by prior growth in the presence ofl-arabinose ord-xylose. The use ofd-ribose andd-xylose by strain B₁4 is different from that ford-arabinose. During growth in the presence of 1-14C-d-arabinose, labeled acetate, propionate, and succinate were formed, whereas during 1-14C-d-ribose growth only labeled acetate and propionate were obtained. Under the conditions used,d-xylose growth failed to allow formation of acetate, propionate, or succinate. Strain B₁4 incorporates label from 1- or 2-labeled glycine into acetate, propionate, and succinate by a mechanism involving the cleavage of glycine and equilibration of glycine carbons 1 and 2 with different metabolic pools.     

Draft Genome Sequence of the Sulfobacillus thermosulfidooxidans Cutipay Strain,an Indigenous Bacterium Isolated from a Naturally Extreme Mining Environment in Northern Chile

Sulfobacillus thermosulfidooxidans strain Cutipay is a mixotrophic, acidophilic, moderately thermophilic bacterium isolated from mining environments of the north of Chile, making it an interesting subject for studying the bioleaching of copper. We introduce the draft genome sequence and annotation of this strain, which provide insights into its mechanisms for heavy metal resistance.     

Sulfur metabolism enzymes in thermoacidophilus 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 cells grown on the medium with elemental sulfur or in the presence of various sulfide elements and concentrates of sulfide ores. The activity of sulfur-metabolizing enzymes depended little on the degree of aeration during bacterial growth.     

Citrate metabolism in 16 Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum strains

Citrate metabolism was studied in non-growing cells of Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum with respect to energetics, formation of degradation products and stoichiometry. The use of selective ionophores and uncoupler showed that citrate utilization was coupled to the proton motive force generated by ATP hydrolysis. Differences in citrate metabolism observed in 20 Leuconostoc strains were related to strains but not to the species or subspecies studied. Citrate metabolism was stimulated by glucose up to a concentration of 25 mmol 1^-1 and decreased at higher concentrations. The main degradation products resulting from the co-metabolism of citrate (10 mmol 1^-1) and glucose (2 mmol 1^-1) were acetate, lactate and pyruvate. Only four Leuconostoc strains produced low levels of acetoin and diacetyl. No strains produced ethanol or acetaldehyde. Citrate degradation ability was stable for at least 130 generations in 81% of the Leuconostoc strains.     

Thiodiglycol metabolism in Alcaligenes xylosoxydans subsp. denitrificans

The investigation of the degradation of thiodiglycol (the major product of mustard gas hydrolysis) by Alcaligenes xylosoxydans subsp. denitrificans strain TD2 showed that thiodiglycol is metabolized through the oxidation of its primary alcohol groups and the subsequent cleavage of C-S bonds in the intermediate products, thiodiglycolic and thioglycolic acids. The end products of these reactions are SO4(2-) ions and acetate, the latter being involved in the central metabolism of strain TD2. The oxidation of the sulfur atom gives rise to diglycolsulfoxide, which is recalcitrant to further microbial degradation. Based on the data obtained, a metabolic pathway of thiodiglycol transformation by A. xylosoxydans subsp. denitrificans strain TD2 is proposed.     

Carbon dioxide fixation and mixotrophic metabolism by strain DCB-1, a dehalogenating anaerobic bacterium.

Fixation by strain DCB-1 of CO2 carbon into cell material and organic acids occurred during growth on pyruvate both with and without thiosulfate. By using sodium [14C]bicarbonate and sodium [2-14C]pyruvate, the isotopic composition of products and cells was investigated. Up to 70% of cell carbon was derived from CO2. CO2 carbon was also incorporated into succinate, formate, and acetate. Both carbons of acetate underwent exchange reactions with CO2, although the carboxyl-group exchange was twice as fast. Because strain DCB-1 uses CO2 as its major but not sole carbon source while deriving energy from pyruvate metabolism, we describe its metabolism as mixotrophic. Other mixotrophic conditions also supported growth. Lactate or butyrate, which could not support growth in mineral medium, could replace pyruvate as the oxidizable substrate only when acetate was added to the medium.     

Mixotrophic metabolism in Burkholderia kururiensis subsp. thiooxydans subsp. nov., a facultative chemolithoautotrophic thiosulfate oxidizing bacterium isolated from rhizosphere soil and proposal for classification of the type strain of Burkholderia kururiensis as Burkholderia kururiensis subsp. kururiensis subsp. nov.

A thiosulfate-oxidizing facultative chemolithoautotrophic Burkholderia sp. strain ATSB13^T was previously isolated from rhizosphere soil of tobacco plant. Strain ATSB13^T was aerobic, Gram-staining-negative, rod shaped and motile by means of sub-terminal flagellum. Strain ATSB13^T exhibited mixotrophic growth in a medium containing thiosulfate plus acetate. A phylogenetic study based on 16S rRNA gene sequence analysis indicated that strain ATSB13^T was most closely related to Burkholderia kururiensis KP23^T (98.7%), Burkholderia tuberum STM678^T (96.5%) and Burkholderia phymatum STM815^T (96.4%). Chemotaxonomic data [G+C 64.0 mol%, major fatty acids, C_18:1 ω7c (28.22%), C_16:1 ω7c/15 iso 2OH (15.15%), and C_16:0 (14.91%) and Q-8 as predominant respiratory ubiquinone] supported the affiliation of the strain ATSB13^T within the genus Burkholderia. Though the strain ATSB13^T shared high 16S rRNA gene sequence similarity with the type strain of B. kururiensis but considerably distant from the latter in terms of several phenotypic and chemotaxonomic characteristics. DNA–DNA hybridization between strain ATSB13^T and B. kururiensis KP23^T was 100%, and hence, it is inferred that strain ATSB13^T is a member of B. kururiensis. On the basis of data obtained from this study, we propose that B. kururiensis be subdivided into B. kururiensis subsp. kururiensis subsp. nov. (type strain KP23^T = JCM 10599^T = DSM 13646^T) and B. kururiensis subsp. thiooxydans subsp. nov. (type strain ATSB13^T = KACC 12758^T).     

Complete genome analysis of Sulfobacillus acidophilus strain TPY, isolated from a hydrothermal vent in the Pacific Ocean

Sulfobacillus acidophilus strain TPY is a moderately thermoacidophilic bacterium originally isolated from a hydrothermal vent in the Pacific Ocean. Ferrous iron and sulfur oxidation in acidic environments in strain TPY have been confirmed. Here we report the genome sequence and annotation of the strain TPY, which is the first complete genome of Sulfobacillus acidophilus.     

Enzymes of pyrimidine deoxyribonucleotide metabolism in Mycoplasma mycoides subsp. mycoides.

Cell-free extracts of Mycoplasma mycoides subsp. mycoides were assayed for enzymes associated with the salvage synthesis of pyrimidine deoxyribonucleotides. They possessed kinases for deoxycytidine, (d)CMP, thymidine (deoxyuridine), dTMP, and nucleoside diphosphates; dCTPase and dUTPase; dCMP deaminase; thymidine (deoxyuridine) phosphorylase; and dUMP (dTMP) phosphatase. The existence of these enzymic activities together with ribonucleoside diphosphate reductase explains the capacity of cytidine to provide M. mycoides with deoxyribose for the synthesis of thymidine nucleotides from thymine.     

Carbon dioxide fixation and mixotrophic metabolism by strain DCB-1, a dehalogenating anaerobic bacterium

Fixation by strain DCB-1 of CO2 carbon into cell material and organic acids occurred during growth on pyruvate both with and without thiosulfate. By using sodium [14C]bicarbonate and sodium [2-14C]pyruvate, the isotopic composition of products and cells was investigated. Up to 70% of cell carbon was derived from CO2. CO2 carbon was also incorporated into succinate, formate, and acetate. Both carbons of acetate underwent exchange reactions with CO2, although the carboxyl-group exchange was twice as fast. Because strain DCB-1 uses CO2 as its major but not sole carbon source while deriving energy from pyruvate metabolism, we describe its metabolism as mixotrophic. Other mixotrophic conditions also supported growth. Lactate or butyrate, which could not support growth in mineral medium, could replace pyruvate as the oxidizable substrate only when acetate was added to the medium.     

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

The thermoacidophilic iron-oxidizing chemolithotroph Sulfobacillus sibiricus N1^T 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.     

Carbon dioxide fixation in the metabolism of propylene and propylene oxide by Xanthobacter strain Py2.

Evidence for a requirement for CO2 in the productive metabolism of aliphatic alkenes and epoxides by the propylene-oxidizing bacterium Xanthobacter strain Py2 is presented. In the absence of CO2, whole-cell suspensions of propylene-grown cells catalyzed the isomerization of propylene oxide (epoxypropane) to acetone. In the presence of CO2, no acetone was produced. Acetone was not metabolized by suspensions of propylene-grown cells, in either the absence or presence of CO2. The degradation of propylene and propylene oxide by propylene-grown cells supported the fixation of 14CO2 into cell material, and the time course of 14C fixation correlated with the time course of propylene and propylene oxide degradation. The degradation of glucose and propionaldehyde by propylene-grown or glucose-grown cells did not support significant 14CO2 fixation. With propylene oxide as the substrate, the concentration dependence of 14CO2 fixation exhibited saturation kinetics, and at saturation, 0.9 mol of CO2 was fixed per mol of propylene oxide consumed. Cultures grown with propylene in a nitrogen-deficient medium supplemented with NaH13CO3 specifically incorporated 13C label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-beta-hydroxybutyrate. No specific label incorporation was observed when cells were cultured with glucose or n-propanol as a carbon source. The depletion of CO2 from cultures grown with propylene, but not glucose or n-propanol, inhibited bacterial growth. We propose that propylene oxide metabolism in Xanthobacter strain Py2 proceeds by terminal carboxylation of an isomerization intermediate, which, in the absence of CO2, is released as acetone.     

Stability of the delta-endotoxin gene from Bacillus thuringiensis subsp. kurstaki in a recombinant strain of Clavibacter xyli subsp. cynodontis.

Deletion of chromosomally inserted gene sequences from Clavibacter xyli subsp. cynodontis, a xylem-inhabiting endophyte, was studied in vitro and in planta. We found that nonreplicating plasmid pCG610, which conferred resistance to kanamycin and tetracycline and contained segments of C. xyli subsp. cynodontis genomic DNA, integrated into a homologous sequence in the bacterial chromosome. In addition, pCG610 contains two copies of the gene encoding the CryIA(c) insecticidal protein of Bacillus thuringiensis subsp. kurstaki HD73. Using drug resistance phenotypes and specific DNA probes, we found that the loss of all three genes arose both in vitro under nonselective conditions and in planta. The resulting segregants are probably formed by recombination between the repeated DNA sequences flanking pCG610 that resulted from the integration event into the chromosome. Eventually, segregants predominated in the bacterial population. The loss of the integrated plasmid from C. xyli subsp. cynodontis revealed a possible approach for decreasing the environmental consequences of recombinant bacteria for agricultural use.     

Stability of the delta-endotoxin gene from Bacillus thuringiensis subsp. kurstaki in a recombinant strain of Clavibacter xyli subsp. cynodontis.

Deletion of chromosomally inserted gene sequences from Clavibacter xyli subsp. cynodontis, a xylem-inhabiting endophyte, was studied in vitro and in planta. We found that nonreplicating plasmid pCG610, which conferred resistance to kanamycin and tetracycline and contained segments of C. xyli subsp. cynodontis genomic DNA, integrated into a homologous sequence in the bacterial chromosome. In addition, pCG610 contains two copies of the gene encoding the CryIA(c) insecticidal protein of Bacillus thuringiensis subsp. kurstaki HD73. Using drug resistance phenotypes and specific DNA probes, we found that the loss of all three genes arose both in vitro under nonselective conditions and in planta. The resulting segregants are probably formed by recombination between the repeated DNA sequences flanking pCG610 that resulted from the integration event into the chromosome. Eventually, segregants predominated in the bacterial population. The loss of the integrated plasmid from C. xyli subsp. cynodontis revealed a possible approach for decreasing the environmental consequences of recombinant bacteria for agricultural use.     

Genome sequence of Mycoplasma capricolum subsp. capripneumoniae strain M1601

Mycoplasma capricolum subsp. capripneumoniae is the causative agent of contagious caprine pleuropneumonia, a devastating disease of goats listed by the World Organization for Animal Health. Here we report the first complete genome sequence of this organism (strain M1601, a clinically isolated strain from China).     

Evidence for plasmid-associated lactose metabolism inLactobacillus casei subsp.casei

Lac variants ofLactobacillus casei subsp.casei DR1002 (formerly 64H) have been produced using acriflavin, ethidium bromide, mitomycin C, or combinations of these agents. Two successive transfers in the presence of acriflavin and mitomycin C or ethidium bromide and mitomycin C resulted in nearly a 100% loss of lactose fermentation. Cesium chloride-ethidium bromide isopycnic gradient ultracentrifugal analysis of purified lysates demonstrated that the 23-mdal plasmid (pDR101) found inL. casei DR1002 was consistently absent in Lac⁻ clones. We concluded that, as in lactic streptococci, lactose metablism is a plasmid-mediated train inL. casei DR1002.