Biomedical subjects

M R Betlach

Publications and source records attributed to M R Betlach.

4 recordsLinked to original sources

PubMed1982

Evolution of bacterial denitrification and denitrifier diversity.

Little is known about the role of nitrate in evolution of bacterial energy-generating mechanisms. Denitrifying bacteria are commonly regarded to have evolved from nitrate-respiring bacteria. Some researchers regard denitrification to be the precursor of aerobic respiration; others feel the opposite is true. Currently recognized denitrifying bacteria such as Hyphomicrobium, Paracoccus, Pseudomonas and Thiobacillus form a very diverse group. However, inadequate testing procedures and uncertain taxonomic identification of many isolates may have overstated the number of genera with species capable of denitrification. Nitrate reductases are structurally similar among denitrifying bacteria, but distinct from the enzymes in other nitrate-reducing organisms. Denitryfying bacteria have one of two types of nitrite reductase, either a copper-containing enzyme or an enzyme containing a cytochrome cd moiety. Both types are distinct from other nitrate reductases. Organisms capable of dissimilatory nitrate reduction are widely distributed among eubacterial groups defined by 16S ribosomal RNA phylogeny. Indeed, nitrate reduction is an almost universal property of actinomycetes and enteric organisms. However, denitrification is restricted to genera within the purple photosynthetic group. Denitrification within the genus Pseudomonas is distributed in accordance with DNA and RNA homology complexes. Denitrifiers seem to have evolved from a common ancestor within the purple photosynthetic bacterial group, but not from a nitrate-reducing organism such as those found today. Although denitrification seems to have arisen at the same time as aerobic respiration, the evolutionary relationship between the two cannot be determined at this time.

Bacteria↗

PubMed1981

Assimilatory nitrate uptake in Pseudomonas fluorescens studied using nitrogen-13.

The mechanism of nitrate uptake for assimilation in procaryotes is not known. We used the radioactive isotope, 13N as NO3-, to study this process in a prevalent soil bacterium, Pseudomonas fluorescens. Cultures grown on ammonium sulfate or ammonium nitrate failed to take up labeled nitrate, indicating ammonium repressed synthesis of the assimilatory enzymes. Cultures grown on nitrite or under ammonium limitation had measurable nitrate reductase activity, indicating that the assimilatory enzymes need not be induced by nitrate. In cultures with an active nitrate reductase, the form of 13N internally was ammonium and amino acids; the amino acid labeling pattern indicated that 13NO3- was assimilated via glutamine synthetase and glutamate synthase. Cultures grown on tungstate to inactivate the reductase concentrated NO3- at least sixfold. Chlorate had no effect on nitrate transport or assimilation, nor on reduction in cell-free extracts. Ammonium inhibited nitrate uptake in cells with and without active nitrate reductases, but had no effect on cell-free nitrate reduction, indicating the site of inhibition was nitrate transport into the cytoplasm. Nitrate assimilation in cells grown on nitrate and nitrate uptake into cells grown with tungstate on nitrite both followed Michaelis-Menten kinetics with similar Km values, 7 muM. Both azide and cyanide inhibited nitrate assimilation. Our findings suggest that Pseudomonas fluorescens can take up nitrate via active transport and that nitrate assimilation is both inhibited and repressed by ammonium.

Ammonium Sulfate↗

PubMed1981

Kinetic explanation for accumulation of nitrite, nitric oxide, and nitrous oxide during bacterial denitrification.

The kinetics of denitrification and the causes of nitrite and nitrous oxide accumulation were examined in resting cell suspensions of three denitrifiers. An Alcaligenes species and a Pseudomonas fluorescens isolate characteristically accumulated nitrite when reducing nitrate; a Flavobacterium isolate did not. Nitrate did not inhibit nitrite reduction in cultures grown with tungstate to prevent formation of an active nitrate reductase; rather, accumulation of nitrite seemed to depend on the relative rates of nitrate and nitrite reduction. Each isolate rapidly reduced nitrous oxide even when nitrate or nitrite had been included in the incubation mixture. Nitrate also did not inhibit nitrous oxide reduction in Alcaligenes odorans, an organism incapable of nitrate reduction. Thus, added nitrate or nitrite does not always cause nitrous oxide accumulation, as has often been reported for denitrifying soils. All strains produced small amounts of nitric oxide during denitrification in a pattern suggesting that nitric oxide was also under kinetic control similar to that of nitrite and nitrous oxide. Apparent K(m) values for nitrate and nitrite reduction were 15 muM or less for each isolate. The K(m) value for nitrous oxide reduction by Flavobacterium sp. was 0.5 muM. Numerical solutions to a mathematical model of denitrification based on Michaelis-Menten kinetics showed that differences in reduction rates of the nitrogenous compounds were sufficient to account for the observed patterns of nitrite, nitric oxide, and nitrous oxide accumulation. Addition of oxygen inhibited gas production from NO(3) by Alcaligenes sp. and P. fluorescens, but it did not reduce gas production by Flavobacterium sp. However, all three isolates produced higher ratios of nitrous oxide to dinitrogen as the oxygen tension increased. Inclusion of oxygen in the model as a nonspecific inhibitor of each step in denitrification resulted in decreased gas production but increased ratios of nitrous oxide to dinitrogen, as observed experimentally. The simplicity of this kinetic model of denitrification and its ability to unify disparate observations should make the model a useful guide in research on the physiology of denitrifier response to environmental effectors.

Journal Article↗

PubMed1977

Numerically dominant denitrifying bacteria from world soils.

Nineteen soils, three freshwater lake sediments, and oxidized poultry manure were examined to determine the dominant denitrifier populations. The samples, most shown or expected to support active denitrification, were from eight countries and included rice paddy, temperate agricultural, rain forest, organic, and waste-treated soils. Over 1,500 organisms that could grow anaerobically on nitrate agar were isolated. After purification, 146 denitrifiers were obtained, as verified by production of N(2) from NO(3) (-). These isolates were characterized by 52 properties appropriate for the Pseudomonas-Alcaligenes group. Numerical taxonomic procedures were used to group the isolates and compare them with nine known denitrifier species. The major group isolated was representative of Pseudonomas fluorescens biotype II. The second most prevalent group was representative of Alcaligenes. Other Pseudomonas species as well as members of the genus Flavobacterium, the latter previously not known to denitrify, also were identified. One-third of the isolates could not utilize glucose or other carbohydrates as sole carbon sources. Significantly, none of the numerically dominant denitrifiers we isolated resembled the most studied species: Pseudomonas denitrificans, Pseudomonas perfectomarinus, and Paracoccus denitrificans. Denitrification appears to be a property of a very diverse group of gram-negative, motile bacteria, as shown by the large number (22.6%) of ungrouped organisms. The diversity of denitrifiers from a given sample was usually high, with at least two groups present. Denitrifiers, nitrite accumulators, and organisms capable of anaerobic growth were present in the ratio of 0.20+/-0.23:0.81+/-0.23:1. There were few correlations between their numbers and the sample characteristics measured. However, the temperatures at which isolates could grow were significantly related to the temperatures of the environments from which they were isolated. Regression analysis revealed few relationships between physical parameters and bacterial types, save for the anaerobe numbers, in which 94% of the variance could be accounted for.

Anaerobiosis↗