The rapid switch-off of nitrogenase activity in obligate methane-oxidizing bacteria

Nitrogenase activity in the obligate methaneoxidizing bacterium Methylococcus capsulatus (Bath) was added ammonia. This observation was extended to include other ammonia. This observation was extended to include other representative N₂-fixing species of methanotrophs. The ammonia switch-off of nitrogenase in M. capsulatus (Bath) was reversed on washing cells to remove excess ammonia, in the presence of chloramphenicol, suggesting that a form of covalent modification of nitrogenase may occur. Replacing the oxidizable substrate methanol with formaldehyde, formate, ethanol or hydrogen had no effect on nitrogenase switch-off. A number of potential nitrogen sources or intermediates of nitrogen metabolism such as glutamine, asparagine, glutamate and alanine when tested, did not effect switch-off. However, the rapid inhibition of nitrogenase activity of M. capsulatus (Bath) could be achieved by adding the uncoupler carbonylcyanide m-chlorophenylhydrazone or nitrite. The glutamine synthetase inhibitor methionine sulphoximine blocked the switch-off effect of ammonia, indicating that the metabolism of ammonia may be essential for switch-off to occur. Inhibitors of glutamate synthase did not alleviate the ammonia switch-off response. Methionine sulphoximine did not alleviate the rapid inhibition of nitrogenase by carbonylcyanide m-chlorophenylhydrazone indicating that the shortterm regulation of nitrogenase by uncouplers and ammonia proceed via different mechanisms.Abbreviations MSX methionine-DL-sulphoximine - DON 6-diazo-5-oxo-L-norleucine - GS glutamine synthetase - GOGAT glutamine 2-oxoglutarate aminotransferase (glutamate synthase) - CCCP carbonylcyanide m-chlorophenyl hydrazone     

Isolation and characterization of two vibrio-shaped methane-oxidizing bacteria

An enrichment method for, and the isolation of two related vibrio-shaped methane-oxidizing bacteria are described. Their morphological and physiological characteristics are given. As a name for the genus of the organisms Methy lovibrio is proposed.We wish to thank Miss W. E. de Boer and J. van der Toorn of this laboratory for making the photographs. One of the authors (P. J. Steennis) is indebted to the Royal Netherlands Fermentation Industries Ltd., Delft for a grant.     

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 mono-oxygenase from endogenous respiration. In strains with a high ethane oxidation rate, the reductant appears to be derived from oxidation of ethanol or acetaldehyde.     

Hydrogenase activity in nitrogen-fixing methane-oxidizing bacteria

Hydrogenase activity in cells of the nitrogen-fixing methane-oxidizing bacterium strain 41 of the Methylosinus type increased markedly when growth was dependent upon the fixation of gaseous nitrogen. A direct relationship may exist between hydrogenase and nitrogenase in this bacterium. Acetylene reduction was supported by the presence of hydrogen gas.     

Microbial transformations of selenite by methane-oxidizing bacteria

Methane-oxidizing bacteria are well known for their role in the global methane cycle and their potential for microbial transformation of wide range of hydrocarbon and chlorinated hydrocarbon pollution. Recently, it has also emerged that methane-oxidizing bacteria interact with inorganic pollutants in the environment. Here, we report what we believe to be the first study of the interaction of pure strains of methane-oxidizing bacteria with selenite. Results indicate that the commonly used laboratory model strains of methane-oxidizing bacteria, Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b, are both able to reduce the toxic selenite (SeO₃ ^2?) but not selenate (SeO₄ ^2?) to red spherical nanoparticulate elemental selenium (Se⁰), which was characterized via energy-dispersive X-ray spectroscopy (EDXS), X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS). The cultures also produced volatile selenium-containing species, which suggests that both strains may have an additional activity that can transform either Se⁰ or selenite into volatile methylated forms of selenium. Transmission electron microscopy (TEM) measurements and experiments with the cell fractions cytoplasm, cell wall and cell membrane show that the nanoparticles are formed mainly on the cell wall. Collectively, these results are promising for the use of methane-oxidizing bacteria for bioremediation or suggest possible uses in the production of selenium nanoparticles for biotechnology.

     

Anaerobic degradation of toluene by pure cultures of denitrifying bacteria

Several denitrifying Pseudomonas spp., isolated with various aromatic compounds, were tested for the ability to degrade toluene in the absence of molecular oxygen. Four out of seven strains were able to degrade toluene in the presence of N₂O. More than 50% of the ¹⁴C from ring-labelled toluene was released as CO₂, and up to 37% was assimilated into cell material. Furthermore it was demonstrated for two strains that they were able to grow on toluene as the sole carbon and energy source in the presence of N₂O. Suspensions of cells pre-grown on toluene degraded toluene, benzaldehyde or benzoate without a lag phase and without accumulation of intermediates. p-Cresol, p-hydroxybenzylalcohol, p-hydroxybenzaldehyde or p-hydroxybenzoate was degraded much slower or only after distinct lag times. In the presence of fluoroacetate [¹⁴C]toluene was transformed to [¹⁴C]benzoate, which suggests that anaerobic toluene degradation proceeds through oxidation of the methyl side chain to benzoate.     

Extraction of methane-oxidizing bacteria from soil particles

Abstract: We present a method for extraction of active methane (CH₄)-oxidizing bacteria from soil samples. The method is based on physical dispersion of bacteria from the soil particles followed by separation of bacteria and soil particles by floatation in the density media Nycodenz or Percoll. Separation on Nycodenz produced very pure bacterial suspensions while separation on Percoll produced rather impure suspensions. However, more than 60% of the methane-oxidizing activity was irreversibly inhibited in the procedure using Nycodenz compared to less than 10% irreversible inhibition when Percoll was employed. The bacterial suspensions extracted from soil can be used to study the physiology and ecology of soil bacteria that oxidize methane at atmospheric concentrations. Our data indicated that these bacteria are extremely difficult to dislodge from particles compared to the majority of bacteria in soil. Tentatively, we interpret the strong attachment to long residence time (i.e. slow turnover) of the methane-oxidizing bacteria. A slow turnover/growth rate would explain why soil disturbances, like cultivation, have a long lasting effect on the oxidation of atmospheric methane in soil.     

Ammonia-induced injury in pure cultures and natural populations of coliform bacteria

Ammonia-induced injury was investigated in pure cultures of Escherichia coli and Enterobacter aerogenes, and in natural coliform populations obtained from the oligotrophic Luxapallila and the eutrophic Sunflower Rivers in northern Mississippi. Pure cultures were affected by ammonia exposure as indicated by changes in the injury ratio (IR) of CFU on m-T7 agar/CFU on m-Endo agar. Ammonia concentrations between 0 and 20 (mg NH3-N/1) had little or no effect and concentrations between 40 and 80 caused the greatest injury. Natural coliform populations from the oligotrophic river were more prone to ammonia-induced injury than those from the eutrophic river. The results stress the need for the routine use of m-T7 media and the enumeration of injured cells when using the membrane filter procedure to ascertain domestic water quality.     

The taxonomy of obligate anaerobic bacteria

Studies on the chemotaxonomy of obligate anaerobic bacteria have been made. The combination of gas chromatography and mass spectrometry with computer-assisted analysis, permitting the multicomponent analysis of all products of bacterial metabolism and bacterial cell components, has been shown to be a research method, quite suitable for such studies. The chromatographic profiles of the end products of metabolism in anaerobic cultures of different age have been found to differ not in the set and number of peaks indicating various metabolites, but only in the concentration of metabolites, increasing in the process of prolonged incubation. The authors believe that the national microbiological "library" of the chromatographic profiles of anaerobic organisms should be created and the album of typing chromatographic profiles should be published; besides, data on new profiles should regularly appear in magazines.     

Screening for restriction endonucleases in methane-oxidizing bacteria.

51 methane-oxidizing bacteria strains such as Methylomonas methanica, M. rubra, Methylococcus capsulatus, M. thermophilus, M. luteus, M. ucrainicus, M. whittenburyi, Methylosinus trichosporium, M. sporium, Methylocystis parvus isolated from various ecological niches and geographical regions of the Ukraine and also the strains received from R. Whittenbury and Y. Heyer were screened for restriction endonucleases. Type II restriction endonucleases were detected in IMV B-3112 (= 12 b), IMV B-3027 (= 26), IMV B-3019 (= 9 c), IMV B-3017 (= 17 c), IMV B-3226 (= 26 v), IMV B-3033 (= Y), IMV B-3100 (= 100) and IMV B-3494 (= 1E494). The results obtained were indicative of relatively high frequency of restriction enzymes occurrence in methane-oxidizing bacteria. There were Kpn I (Asp 7181) restriction endonuclease isoschizomers in crude extracts of IMV B-3112, B-3017, B-3019, B-3027 isolated from fresh-water silt as well as in IMV B-3226 strain isolated from waste-water silt. Although these isolates had bee previously considered as untypical strains of M. ucrainicus, more detailed study of their properties allowed placing them with Methylovarius luteus (= Methylococcus luteus). IMV B-3494 strain was identified as Methylococcus capsulatus. Strain IMV B-3033 had earlier been allocated to Methylovarius whittenburyi (= Methylococcus whittenburyi). Specificity of restriction endonucleases of this strain was not tested. Therefore, for the first time restriction endonucleases were detected in methane-oxidizing bacteria. 8 strains (3 species) among 51 strains (13 species) were found to produce restriction endonucleases displaying three different types of specificity in the least. Producers of restriction endonucleases having Kpn I (Asp 7181) specificity were isolated from different water and silt samples of the Dnieper flood-land more than 20 years ago.     

Activity of methane-oxidizing bacteria in the adsorbed state]

Adsorption of pure cultures of methane oxidizing bacteria, Methylosinus trichosporium 20 and Methylococcus ucrainicus 21, on glass and coal was studied; the former strain was sorbed on both sorbents, the latter strain was sorbed on coal but not on glass. The rate of methane oxidation by the cells of adsorbed microorganisms was higher than in the case of free cells, and increased with a decrease in dimensions of the sorbent particles.     

Mobile DNA in obligate intracellular bacteria

The small genomes of obligate intracellular bacteria are often presumed to be impervious to mobile DNA and the fluid genetic processes that drive diversification in free-living bacteria. Categorized by reductive evolution and streamlining, the genomes of some obligate intracellular bacteria manifest striking degrees of stability and gene synteny. However, recent findings from complete genome sequences of obligate intracellular species and their mobile genetic associates favour the abandonment of these wholesale terms for a more complex and tantalizing picture.     

Degradation of trans-1,2-dichloroethene by mixed and pure cultures of methanotrophic bacteria

Summary Out of seven chlorinated aliphatic hydrocarbons tested, only trans-1,2-dichloroethene was relatively non-toxic for a mixed methanotrophic culture. The compound was degraded at a rate of 0.4 mol/mg protein·h^-1 and liberation of inorganic chloride was observed. Trans-2,3-dichlorooxirane was formed as an intermediate which was converted further only by chemical transformation with a half life of 31 h. From the consortium, a pure culture was isolated and found to be capable of degradation of trans-1,2-dichloroethene when grown in the presence of methane or methanol. The ability of cometabolic degradation of this compound was not specific for this isolate, since Methylomonas methanica NCIB11130 and Methylosinus trichosporium OB3b also showed degradation of trans-1,2-dichloroethene when grown with methane as sole carbon source.Abbreviations t12DCE trans-1,2-dichloroethene - t23DCO trans-2,3-dichlorooxirane - c23DCO cis-2,3-dichlorooxirane - ¹H-NMR proton nuclear magnetic resonance     

Rate of isolated hemicellulose degradation and utilization by pure cultures of rumen bacteria

Rate studies on the utilization or degradation (or both) of isolated hemicelluloses were conducted with six strains of rumen cellulolytic bacteria. Utilization was estimated by total pentose loss, and degradation values were based on solubilization of the hemicellulose in acidified 80% ethyl alcohol. With the various strains of ruminococci, degradation of flax and fescue grass hemicellulose was near the maximum within the first 12 hr of incubation. However, where applicable, the rates of utilization were considerably slower. Both degradation and utilization of corn hull hemicellulose occurred at much slower rates than observed with the other two substrates. With flax and fescue grass hemicellulose, the rates of degradation did not appear to be influenced by the organism's ability, or inability, to utilize the substrate as an energy source. The rates and extent of isolated hemicellulose degradation and utilization were compared between the cellulolytic ruminococci and three strains of bacteria isolated from the rumen with a xylan medium. Similar values were obtained with both types of bacteria. These observations would suggest that the cellulolytic ruminococci may be important in the overall fermentation of forage hemicelluloses in the rumen. The acidified 80% ethyl alcohol supernatant fluids, obtained from fermentations of isolated fescue grass hemicellulose by two strains of Ruminococcus flavefaciens, of which only one was able eventually to utilize the substrate, were investigated by thin-layer chromatography. Results indicated that soluble oligosaccharides were produced, which were observed to disappear gradually with time in fermentations with the utilizing strain and to accumulate in fermentations with the nonutilizing strain. Examination of the acidified 80% ethyl alcohol-insoluble residue hydrolysates, obtained from fermentations with the utilizing strain, revealed that the concentration of all the constituent sugars decreased uniformly.