Isolation of the tetrathionate hydrolase from Thiobacillus acidophilus.

An enzyme capable of hydrolysing tetrathionate was purified from cell-free extracts of Thiobacillus acidophilus. The purified enzyme converts tetrathionate into thiosulfate, sulfur and sulfate. In addition, pentathionate could also be converted by the same enzyme. Measurement of the enzyme activity during purification is based on the absorbance of the initial intermediates formed from tetrathionate in the ultraviolet region, which have not been identified. Enzyme activity could also be measured by the scattering of insoluble sulfur in the visible region. The purified enzyme has a pH optimum of 2.5 and a temperature optimum of 65 degrees C. Enzyme activity is strongly stimulated by the presence of sulfate ions. The purified enzyme is a dimer with two identical subunits of 48 kDa. The ultraviolet-visible absorption spectra and denaturation experiments indicate the presence of an organic cofactor.

Purification and partial characterization of a thermostable trithionate hydrolase from the acidophilic sulphur oxidizer Thiobacillus acidophilus.

Cell-free extracts of Thiobacillus acidophilus catalysed the quantitative conversion of trithionate (S3O6(2-) to thiosulphate and sulphate. A continuous assay for quantification of experimental results was based on the difference in absorbance between trithionate and thiosulphate at 220 nm. Trithionate hydrolase was purified to near homogeneity from cell-free extracts of T. acidophilus. The molecular masses of the native enzyme and the subunit were 99 kDa (gel filtration) and 34 kDa (SDS/PAGE). The purified enzyme has a pH optimum of 3.5-4.5 and a temperature optimum of 70 degrees C. Enzyme activity was stimulated by sulphate. The stimulation of the enzyme activity by sulphate was half maximal at a concentration of 0.23 M. The Km for trithionate is 70 microM at 30 degrees C and 270 microM at 70 degrees C. Enzyme activity was lost after 36 days at 0 degrees C, 27 days at 70 degrees C; but after 97 days at 30 degrees C, 40% of the initial activity was still present: The enzyme activity was inhibited by mercury chloride, N-ethylmaleimide, thiosulphate and tetrathionate. Tetrathionate S4O6(2-) was not hydrolysed by trithionate hydrolase.

Growth of Thiobacillus ferrooxidans on Formic Acid.

A variety of acidophilic microorganisms were shown to be capable of oxidizing formate. These included Thiobacillus ferrooxidans ATCC 21834, which, however, could not grow on formate in normal batch cultures. However, the organism could be grown on formate when the substrate supply was growth limiting, e.g., in formate-limited chemostat cultures. The cell densities achieved by the use of the latter cultivation method were higher than cell densities reported for growth of T. ferrooxidans on ferrous iron or reduced sulfur compounds. Inhibition of formate oxidation by cell suspensions, but not cell extracts, of formate-grown T. ferrooxidans occurred at formate concentrations above 100 muM. This observation explains the inability of the organism to grow on formate in batch cultures. Cells grown in formate-limited chemostat cultures retained the ability to oxidize ferrous iron at high rates. Ribulose 1,5-bisphosphate carboxylase activities in cell extracts indicated that T. ferrooxidans employs the Calvin cycle for carbon assimilation during growth on formate. Oxidation of formate by cell extracts was NAD(P) independent.

Kinetics and energetics of reduced sulfur oxidation by chemostat cultures of Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans was grown in chemostat cultures with thiosulfate and tetrathionate as the limiting substrates. The yields at steady state on both substrates at different dilution rates were calculated. In a few experiments the air supply was supplemented with 2% CO2 (v/v). This resulted in a slightly increased yield. Cells from the chemostat cultures were used to study the kinetics of thiosulfate, tetrathionate, sulfite and sulfide oxidation. With all substrates mentioned the Ks values were in the micromolar range. The values for thiosulfate and tetrathionate were 2 orders of magnitude lower that those published previously.

Ethane oxidation by methane-oxidizing bacteria.

The relationship between the rates of methane and ethane oxidation by washed suspensions of methane-oxidizing bacteria has been investigated. Considerable differences between bacterial strains were observed. Two closely related Methylomonas strains which differed in their oxidizing capacity were further investigated. The low ethane oxidation rate of one strain could be strongly stimulated by the addition of oxidizable co-substrates and the presence of ethane stimulated formate oxidation. The other strain had a much higher ethane oxidation rate and stimulation by co-substrates was negligible. Differences between the levels of dissimilative enzymes in cell-free extracts could not be detected. Attempts to produce extracts with methane mono-oxygenase activity failed. When cells were made permeable with chitosan the results suggested that strains with a low ethane oxidizing capacity obtain the required reductant for the moo-oxygenase from endogenous respiration. In strains with a high ethane oxidation rate, the reductant appears to be derived from oxidation of ethanol or acetaldehyde.

Some cultural and physiological aspects of methane-utilizing bacteria.

A number of different methane-utilizing bacteria are described and compared with isolates of other investigators. The strains can be divided into three groups based on pigmentation, cell morphology and internal membrane structures. The oxidation of hydrocarbons, alcohols, aldehydes, fatty acids, methyl ethers and sugar phosphates by these bacteria was studied. There was much similarity between strains within the same group. Differences between groups as regards oxidative properties could be detected, but these were mainly quantitative and could not be used as taxonomical criteria. In addition, the inhibition of methane oxidation by metabolites and enzyme inhibitors was investigated. Formaldehyde proved to be the most active of the organic compounds tested. Iodoacetic acid inhibited both methane and methanol oxidation at concentrations of 0.03 M or above. Of the inorganic compounds, KCN completely suppressed methane oxidation at 5 times 10(-4) M and to more than 90% at 5 times 10(-5) M.