Biology of Frankia strains, actinomycete symbionts of actinorhizal plants.

The above statement, although meant to be tongue in cheek, contains an essential truism: all work with inhibitors is inherently suspect. This fact has been known by biochemists for some time. However, use of chemical inhibitors of enzymic systems and membranes continues to be a common approach taken toward unraveling the biochemistry and biophysics of plants, animals, and microorganisms. Various types of “broad-spectrum” biochemical inhibitors (e.g., poisons, respiratory inhibitors, and uncouplers) have been employed by ecologists for many years in order to demonstrate the active participation of microbes in chemical reactions occurring in natural samples (e.g., soils, sediments, and water). In recent years, considerable advances have been made in our understanding of the biochemistry of microorganisms of biogeochemical interest. Concurrent with these advances have been the discoveries of novel types of compounds that will block the metabolism of one particular group of microbes, but have little disruptive effect on other physiological types. Thus, the term “specific inhibitor” has been applied to these types of compounds when they are used to probe the functions of mixed populations of microorganisms. These substances provide powerful experimental tools for investigating the activity and function of certain types of microorganisms in natural samples.

Use of “Specific” Inhibitors in Biogeochemistry and Microbial Ecology

Sulfide-induced dissimilatory nitrate reduction to ammonia in anaerobic freshwater sediments

summary
More than 200 angiosperms, distributed in 25 genera, develop root nodule symbioses (actinorhizas) with soil bacteria of the actinomycetous genus Frankia. Although most soils studied contain infective Frankia, cultured strains are available only after isolation from root nodules. Frankia infects roots via root hairs in some hosts or via intercellular penetration in others. The nodule originates in the pericycle. The number of nodules in Alnus is determined by the plant in an autoregulated process that, in turn, is modulated by nutrients such as nitrogen and phosphate. Except in the genera Allocausarina and Casuarina, Frankia in nodules develops so-called vesicles where nitrogenase is localized. Sporulation of Frankia occurs in some symbioses. As a group, actinorhizal plants show a large range of anatomical and biochemical adaptations in order to balance the oxygen tension near nitrogenase. In symbioses with well aerated nodule tissue like Alnus, the vesicles have a multilayered envelope composed mainly of lipids, bacterio-hopanetetrol and their derivatives. This envelope is assumed to retard the diffusion of oxygen into the nitrogenase-containing vesicle. In symbioses like Casuarina, the infected plant cells themselves, rather than Frankia, appear to retard oxygen diffusion, and high concentrations of haemoglobin indicate an infected region with a low oxygen tension. At least in Alnus spp., ammonia resulting from N2 fixation is assimilated by glutamine synthetase in the plant. The carbon compound(s) used by Frankia in nodules is not yet known. Nitrogenase activity decreases in response to a number of environmental factors but recovers upon return to normal conditions. This dynamism in nitrogenase activity is often explained by loss and recovery of active nitrogenase and has been traced to loss and recovery of the nitrogenase proteins themselves. Recovery is partly due to growth of Frankia and to development of new vesicles in the Alnus nodules. In the field, varying conditions continuously affect the plants and the measured rate of N2 fixation is a result not only of the conditions prevailing at the moment but also of the conditions experienced over preceding days. N2 fixed by actinorhizal plants is substantial and actinorhizal plants have great potential in soil reclamation and in various types of forestry. Several species are also useful in horticulture.

Actinorhizal symbioses and their N2 fixation

Measurements of denitrification using the acetylene inhibition,15N isotope tracer, and N2 flux methods were carried out concurrently using sediment cores from Vilhelmsborg so, Denmark, in an attempt to clarify some of the limitations of each technique. Three experimental treatments of overlying water were used: control, nitrate enriched, and ammonia enriched water. The N2 flux and15N tracer experiments showed high rates of coupled nitrification/denitrification in the sediments. The acetylene inhibition method did not capture any coupled nitrification/denitrification. This could be explained by acetylene inhibition of nitrification. A combined15N tracer/acetylene inhibition experiment demonstrated that acetylene inhibition of N2O reduction was incomplete and the method, therefore, only measured approximately 50% of the denitrification due to nitrate from the overlying water. Similar rates of denitrification due to nitrate in the overlying water were measured by the N2 flux method and the acetylene inhibition method, after correcting for the 50% efficiency of acetylene inhibition. Rates of denitrification due to nitrate from the overlying water measured by the15N tracer method, however, were only approximately 35% or less of those measured by the acetylene inhibition or N2 flux methods.

Denitrification measurements in aquatic sediments: A comparison of three methods

The production and reduction of nitrous oxide (N2O) after the addition of N2O, nitrite (NO2−), or nitrate (NO3−) was studied in non-sterile soil, in sterilized soil inoculated with Pseudomonas aeruginosa, and in washed cell suspensions of this organism. Sodium sulfide (8 μmol S2− mL−1 or g−1) inhibited N2O reduction markedly in cell suspensions and also in soil, an effect which may cause sulfidic habitats to act as sources of N2O. Sodium thiosulfate (up to 64 μmol S2O32− g−1) showed no such effect. Acetylene (0.02 atm C2H2) completely inhibited the reduction of N2O by soil, but the combination of C2H2 with 8 μmol S2− g−1 permitted the complete reduction of 2 μmol added N2O g−1 within 3 days under the most favourable conditions. Under the same conditions, 8 μmol S2O32− g−1 permitted complete reduction of the N2O within 6 days. The rate of such reduction of N2O was decreased, but not inhibited completely, by raising the C2H2 concentration to 0.11 atm. The data have important implications for the effectivene...

Effects of sulfide and acetylene on nitrous oxide reduction by soil and by Pseudomonas aeruginosa

Nitrogen-fixing, field-obtained root nodules of the silky green alder were studied by transmission electron microscopy. The nodule endophyte exhibited a prokaryotic cytology and was present in two ...

Ultrastructure of the Alnus crispa var. mollis Fern. root nodule endophyte.

Hydrogen uptake by Azospirillum brasilense was studied in N2-fixing and NH4Cl-grown batch cultures. The Km for H2 of uptake hydrogenase (O2-dependent H3H uptake) in whole cells was 9 µM. In N2-flxi...

Effect of hydrogen and oxygen on uptake-hydrogenase activity in nitrogen-fixing and ammonium-grown Azospirillum brasilense

Early reports of Lampropedia suggested that it occurred in stagnant or muddy waters, rotting vegetation, and liquid manure. More recently, "window-pane sarcinae" have been observed in the rumen con...

Roger Knowles

Papers

Effects of sulfide and acetylene on nitrous oxide reduction by soil and by Pseudomonas aeruginosa

Ultrastructure of the Alnus crispa var. mollis Fern. root nodule endophyte.

Effect of hydrogen and oxygen on uptake-hydrogenase activity in nitrogen-fixing and ammonium-grown Azospirillum brasilense

The occurrence of lampropedia in the intestines of some reptiles and nematodes