A transposon for green fluorescent protein transcriptional fusions: application for bacterial transport experiments

The movement of bacteria through groundwater is a poorly understood process. Factors such as soil porosity and mineralogy, heterogeneity of soil particle size, and response of the bacteria to their environment contribute to the pattern of bacterial flow. The identification of transported bacteria is often a limiting factor in both laboratory and field transport experiments. Two bacterial strains were modified for use in bacterial transport experiments: a strain of Escherichia coli harboring the pGFP plasmid and a strain of Pseudomonas putida modified with a Tn5 derivative, Tn5GFP1. The Tn5GFP1 transposon incorporates the gene (gfp) encoding green fluorescent protein (GFP) and can be used to mutagenize Gram^- bacteria. Fluorescent colonies were suspended in phosphate-buffered saline (PBS) at a concentration of approx. 10⁹ bacteria/ml. A 10-cm glass column packed with quartz sand (diameter range 177–250 μm) was equilibrated with PBS prior to the forced flow introduction of the bacteria. Collected fractions were analyzed and the bacteria quantitated using a fluorescence spectrometer. Results demonstrate that the bacteria can be accurately tracked using their fluorescence, and that the intensity of the signal can be used to determine a C/Co ratio for the transported bacteria. The data show a rapid breakthrough of the bacteria followed by a characteristic curve pattern. A lower limit of detection of 10⁵ cells was estimated based on these experiments. The Tn5GFP1 transposon should become a valuable tool for labeling bacteria.     

Interaction of Escherichia coli and Soil Particles in Runoff

A laboratory-scale model system was developed to investigate the transport mechanisms involved in the horizontal movement of bacteria in overland flow across saturated soils. A suspension of Escherichia coli and bromide tracer was added to the model system, and the bromide concentration and number of attached and unattached E. coli cells in the overland flow were measured over time. Analysis of the breakthrough curves indicated that the E. coli and bromide were transported together, presumably by the same mechanism. This implied that the E. coli was transported by advection with the flowing water. Overland-flow transport of E. coli could be significantly reduced if the cells were preattached to large soil particles (>45 μm). However, when unattached cells were inoculated into the system, the E. coli appeared to attach predominantly to small particles (<2 μm) and hence remained unattenuated during transport. These results imply that in runoff generated by saturation-excess conditions, bacteria are rapidly transported across the surface and have little opportunity to interact with the soil matrix.     

Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Continuously Percolated Soil Columns

Although the absence of nitrate formation in grassland soils rich in organic matter has often been reported, low numbers of nitrifying bacteria are still found in these soils. To obtain more insight into these observations, we studied the competition for limiting amounts of ammonium between the chemolithotrophic ammonium-oxidizing species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis in the presence of Nitrobacter winogradskyi with soil columns containing calcareous sandy soil. The soil columns were percolated continuously at a dilution rate of 0.007 h^-1, based on liquid volumes, with medium containing 5 mM ammonium and different amounts of glucose ranging from 0 to 12 mM.A. globiformis was the most competitive organism for limiting amounts of ammonium. The numbers of N. europaea and N. winogradskyi cells were lower at higher glucose concentrations, and the potential ammonium-oxidizing activities in the uppermost 3 cm of the soil columns were nonexistent when at least 10 mM glucose was present in the reservoir, although 10⁷ nitrifying cells per g of dry soil were still present. This result demonstrated that there was no correlation between the numbers of nitrifying bacteria and their activities. The numbers and activities of N. winogradskyi cells decreased less than those of N. europaea cells in all layers of the soil columns, probably because of heterotrophic growth of the nitrite-oxidizing bacteria on organic substrates excreted by the heterotrophic bacteria or because of nitrate reduction at reduced oxygen concentrations by the nitrite-oxidizing bacteria. Our conclusion was that the nitrifying bacteria were less competitive than the heterotrophic bacteria for ammonium in soil columns but that they survived as viable inactive cells. Inactive nitrifying bacteria may also be found in the rhizosphere of grassland plants, which is rich in organic carbon. They are possibly reactivated during periods of net mineralization.     

Cesium Accumulation and Growth Characteristics of Rhodococcus erythropolis CS98 and Rhodococcus sp. Strain CS402

Growth and cesium accumulation characteristics of two cesium-accumulating bacteria isolated from soils were investigated. Rhodococcus erythropolis CS98 and Rhodococcus sp. strain CS402 accumulated high levels of cesium (approximately 690 and 380 μmol/g [dry weight] of cells or 92 and 52 mg/g [dry weight] of cells, respectively) after 24 h of incubation in the presence of 0.5 mM cesium. The optimum pH for cesium uptake by both Rhodococcus strains was 8.5. Rubidium and cesium assumed part of the role of potassium in the growth of both Rhodococcus strains. Potassium and rubidium inhibited cesium accumulation by these Rhodococcus strains. It is likely that both Rhodococcus strains accumulated cesium through a potassium transport system.     

Protozoa Drive the Dynamics of Culturable Biocontrol Bacterial Communities

Some soil bacteria protect plants against soil-borne diseases by producing toxic secondary metabolites. Such beneficial biocontrol bacteria can be used in agricultural systems as alternative to agrochemicals. The broad spectrum toxins responsible for plant protection also inhibit predation by protozoa and nematodes, the main consumers of bacteria in soil. Therefore, predation pressure may favour biocontrol bacteria and contribute to plant health. We analyzed the effect of Acanthamoeba castellanii on semi-natural soil bacterial communities in a microcosm experiment. We determined the frequency of culturable bacteria carrying genes responsible for the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN) and hydrogen cyanide (HCN) in presence and absence of A. castellanii. We then measured if amoebae affected soil suppressiveness in a bioassay with sugar beet seedlings confronted to the fungal pathogen Rhizoctonia solani. Amoebae increased the frequency of both DAPG and HCN positive bacteria in later plant growth phases (2 and 3 weeks), as well as the average number of biocontrol genes per bacterium. The abundance of DAPG positive bacteria correlated with disease suppression, suggesting that their promotion by amoebae may enhance soil health. However, the net effect of amoebae on soil suppressiveness was neutral to slightly negative, possibly because amoebae slow down the establishment of biocontrol bacteria on the recently emerged seedlings used in the assay. The results indicate that microfaunal predators foster biocontrol bacterial communities. Understanding interactions between biocontrol bacteria and their predators may thus help developing environmentally friendly management practices of agricultural systems.     

Effects of pantolactone and butyrolaectone on the pleiotropic phenotypes of lon mutants and on thermal induction of the SOS phenomena in a tif mutant of Escherichia coli K12

Hydrophobic and charge-charge interactions of Salmonella typhimirium and Serratia marcescens were determined and related to their content of fimbriae and lipopolysaccharide (LPS). The cell surface structures were characterized with hydrophobic interaction chromatography (HIC), electrostatic interaction chromatography (ESIC) and particle electrophoresis measurements. The degree of interaction at the air-water interface was tested using a monolayered lipid film applied to an aqueous surface. The cell surface hydrophobicity of S. typhimurium in the presence of fimbriae was less in smooth than in rought bacteria. Examination of a series of rough mutants of S. typhimurium indicates that reduction of the O-side chain and core oligosaccharides was correlated with increased cell hydrophobicity. The enrichment factors at the air-water interface were significantly higher for fimbriated than for non-fimbriated S. typhimurium cells. Fimbriated S. marcescens cells were less hydrophobic and adhered to a lesser degree at the air-water surface than non-fimbriated counterparts. Electrophoretic measurements and adsorption to ion exchangers gives different information about the surface charge of bacteria. The latter technique gives the interaction between localized charged surfaces.Abbreviations HIC hydrophobic interaction chromatography - ESIC electrostatic interaction chromatography - LPS lipopolysaccharide - PBS phosphate buffered saline solution     

Microbial hitchhiking: how Streptomyces spores are transported by motile soil bacteria

Streptomycetes are sessile bacteria that produce metabolites that impact the behavior of microbial communities. Emerging studies have demonstrated that Streptomyces spores are distributed through various mechanisms, but it remains unclear how spores are transported to their preferred microenvironments, such as plant roots. Here, we show that Streptomyces spores are capable of utilizing the motility machinery of other soil bacteria. Motility assays and microscopy studies reveal that Streptomyces spores are transported to plant tissues by interacting directly with the flagella of both gram-positive and gram-negative bacteria. Genetics experiments demonstrate that this form of motility is facilitated by structural proteins on the spore coat. These results demonstrate that nonmotile bacteria are capable of utilizing the motility machinery of other microbes to complete necessary stages of their lifecycle.Subject terms: Bacteria, Cellular microbiology     

Chs6p-dependent Anterograde Transport of Chs3p from the Chitosome to the Plasma Membrane in Saccharomyces cerevisiae

Chitin synthase III (CSIII), an enzyme required to form a chitin ring in the nascent division septum of Saccharomyces cerevisiae, may be transported to the cell surface in a regulated manner. Chs3p, the catalytic subunit of CSIII, requires the product of CHS6 to be transported to or activated at the cell surface. We find that chs6Δ strains have morphological abnormalities similar to those of chs3 mutants. Subcellular fractionation and indirect immunofluorescence indicate that Chs3p distribution is altered in chs6 mutant cells. Order-of-function experiments using end4–1 (endocytosis-defective) and chs6 mutants indicate that Chs6p is required for anterograde transport of Chs3p from an internal endosome-like membrane compartment, the chitosome, to the plasma membrane. As a result, chs6 strains accumulate Chs3p in chitosomes. Chs1p, a distinct chitin synthase that acts during or after cell separation, is transported normally in chs6 mutants, suggesting that Chs1p and Chs3p are independently packaged during protein transport through the late secretory pathway.     

Surface Properties of Bifidobacterial Strains of Human Origin

The adherence of Bifidobacterium strains isolated from infant feces and commercial fermented dairy products to enterocyte-like cells was correlated with the autoagglutination and hemagglutination properties of these organisms. These results allowed us to define two groups: (i) cell-adherent bacteria showing hemagglutination and autoagglutination and (ii) non-cell-adherent, nonhemagglutinating, nonautoagglutinating bacteria. Glass adherence was shown to be nonspecific and was discarded as a criterion for selection of adherent cells. Hydrophobicity appeared to be necessary for adhesion to enterocyte-like cells and autoagglutination. Adhesive strains were highly hydrophobic, and the degree of adherence was slightly dependent on the surface potential. Cells autoagglutinated more when the electrostatic negative charges on the cell surface were shielded by a decrease in the pH from 7 to 2. However, in some strains negative charges at the cell surface were adjuvant to adhesion, thus suggesting that specific chemical interactions occurred. The present results provide a method for preliminary selection of bacteria potentially adherent to epithelial cells by means of autoagglutination.     

Direct Measurements of the Outer Membrane Stage of Ferric Enterobactin Transport: POSTUPTAKE BINDING*

When Gram-negative bacteria acquire iron, the metal crosses both the outer membrane (OM) and the inner membrane, but existing radioisotopic uptake assays only measure its passage through the latter bilayer, as the accumulation of the radionuclide in the cytoplasm. We devised a methodology that exclusively observes OM transport and used it to study the uptake of ferric enterobactin (FeEnt) by Escherichia coli FepA. This technique, called postuptake binding, revealed previously unknown aspects of TonB-dependent transport reactions. The experiments showed, for the first time, that despite the discrepancy in cell envelope concentrations of FepA and TonB (∼35:1), all FepA proteins were active and equivalent in FeEnt uptake, with a maximum turnover number of ∼5/min. FepA-mediated transport of FeEnt progressed through three distinct phases with successively decreasing rates, and from its temperature dependence, the activation energy of the OM stage was 33–35 kcal/mol. The accumulation of FeEnt in the periplasm required the binding protein and inner membrane permease components of its overall transport system; postuptake binding assays on strains devoid of FepB, FepD, or FepG did not show uptake of FeEnt through the OM. However, fluorescence labeling data implied that FepA was active in the ΔfepB strain, suggesting that FeEnt entered the periplasm but then leaked out. Further experiments confirmed this futile cycle; cells without FepB transported FeEnt across the OM, but it immediately escaped through TolC.     

Physico-Chemical Characteristics of Lipoplexes Influence Cell Uptake Mechanisms and Transfection Efficacy

Background

Formulation of DNA/cationic lipid complexes (lipoplexes) designed for nucleic acid delivery mostly results in positively charged particles which are thought to enter cells by endocytosis. We recently developed a lipoplex formulation called Neutraplex that allows preparation of both cationic and anionic stable complexes with similar lipid content and ultrastructure.

Methodology/Principal Findings

To assess whether the global net charge could influence cell uptake and activity of the transported oligonucleotides (ON), we prepared lipoplexes with positive and negative charges and compared: (i) their physicochemical properties by zeta potential analysis and dynamic light scattering, (ii) their cell uptake by fluorescence microscopy and flow cytometry, and (iii) the biological activity of the transported ON using a splicing correction assay. We show that positively or negatively charged lipoplexes enter cells cells using both temperature-dependent and -independent uptake mechanisms. Specifically, positively charged lipoplexes predominantly use a temperature-dependent transport when cells are incubated OptiMEM medium. Anionic lipoplexes favour an energy-independent transport and show higher ON activity than cationic lipoplexes in presence of serum. However, lipoplexes with high positive global net charge and OptiMEM medium give the highest uptake and ON activity levels.

Conclusions

These findings suggest that, in addition to endocytosis, lipoplexes may enter cell via a temperature-independent mechanism, which could be mediated by lipid mixing. Such characteristics might arise from the specific lipoplex ultrastructure and should be taken into consideration when developing lipoplexes designed for in vivo or ex vivo nucleic acid transfer.     

Physiological Characterization of Dicarboxylate-Induced Pleomorphic Forms of Bradyrhizobium japonicum

When Bradyrhizobium japonicum I-110 was transferred into medium containing 40 mM succinate or 40 mM fumarate, over 90% of the bacteria acquired a swollen, pleomorphic form similar to that of bacteroids. The induction of pleomorphism was dependent on the carbon substrate and concentration but was independent of the hydrogen ion and sodium ion concentration. Cell extracts of rod-shaped and pleomorphic cells contained enzymes required for sugar catabolism and gluconeogenesis. Variations in these enzyme profiles were correlated with the carbon source used and not with the conversion to the bacteroid-like morphology. Rod-shaped cells cultured on glucose or 10 mM succinate transported glucose and succinate; however, the pleomorphic cells behaved similarly to symbiotic bacteroids in that they lacked the ability to transport glucose and transported succinate at lower rates than did rod-shaped cells.     

Relationship between the number of bacteria added to soil or seeds and their abundance and distribution in the rhizosphere of alfalfa

A study was conducted of the relationship between the density of several bacterial strains introduced into soil or onto seeds and their abundance in the rhizosphere of alfalfa. The abundance of six species in the rhizosphere was directly correlated with the density of bacteria initially added to soil. The density of six species in the rhizosphere of 15-day-old plants also was directly correlated with the density of each strain in nonrhizosphere soil. Tests of seven species added to soil at four inoculum densities showed that bacteria that survived well in the soil attained the highest densities in the rhizosphere and those that survived poorly in the soil were present at the lowest densities in the rhizosphere. Sixteen of 19 bacterial strains added to alfalfa seeds at 10⁷ or 10⁸ cells per g colonized the rhizosphere of 15-day-old plants, but nearly all of the cells were localized in the upper third of the rhizosphere. A study of 12 bacterial strains that failed to colonize the lower part of the rhizosphere if inoculated onto seeds showed that the bacteria colonized the entire rhizosphere of 15-day-old alfalfa plants if initially inoculated throughout the soil. The data suggest that the density of individual bacterial strains in the rhizosphere is dependent on their density in the soil and that seed inoculation only has an effect on the population in the proximal portion of the alfalfa root system.     

Active Transport and Accumulation of Iodide by Newly Isolated Marine Bacteria

Iodide (I⁻)-accumulating bacteria were isolated from marine sediment by an autoradiographic method with radioactive ¹²⁵I⁻. When they were grown in a liquid medium containing 0.1 μM iodide, 79 to 89% of the iodide was removed from the medium, and a corresponding amount of iodide was detected in the cells. Phylogenetic analysis based on 16S rRNA gene sequences indicated that iodide-accumulating bacteria were closely related to Flexibacter aggregans NBRC15975 and Arenibacter troitsensis, members of the family Flavobacteriaceae. When one of the strains, strain C-21, was cultured with 0.1 μM iodide, the maximum iodide content and the maximum concentration factor for iodide were 220 ± 3.6 (mean ± standard deviation) pmol of iodide per mg of dry cells and 5.5 × 10³, respectively. In the presence of much higher concentrations of iodide (1 μM to 1 mM), increased iodide content but decreased concentration factor for iodide were observed. An iodide transport assay was carried out to monitor the uptake and accumulation of iodide in washed cell suspensions of iodide-accumulating bacteria. The uptake of iodide was observed only in the presence of glucose and showed substrate saturation kinetics, with an apparent affinity constant for transport and a maximum velocity of 0.073 μM and 0.55 pmol min⁻¹ mg of dry cells⁻¹, respectively. The other dominant species of iodine in terrestrial and marine environments, iodate (IO₃⁻), was not transported.     

Citrate transport in Salmonella typhimurium.

Citrate was rapidly metabolized in wild-type cells of Salmonella typhimurium but actively accumulated in both aconitase mutants and fluorocitrate-poisoned cells. In aconitase mutants citrate was transported by a single high affinity system (K_m 23 μm, V_max 27.2 nmol min^?1 mg^?1), characterized by a single pH optimum of 7.0 and a Q₁₀ of 3.0, and was stimulated by Na⁺. cis-Aconitate, tricarballylate, trans-aconitate, and dl-fluorocitrate were weak competitive inhibitors of citrate transport whereas various other tricarboxylic acid cycle intermediates and carboxylates were ineffective. Spontaneous citrate transport mutants were unable to oxidize citrate, cis-aconitate, or tricarballylate. Such mutants were specific for citrate and transported dicarboxylates normally whereas dicarboxylate transport mutants transported and oxidized citrate normally. In whole cells of an aconitase mutant citrate transport was strongly dependent on an energy source. d(?)-Lactate dehydrogenase mutants were singularly defective in energization by d(?)-lactate. Membrane vesicles of wild-type cells were capable of energized transport by d(?)-lactate or ascorbate-phenyl-methyl sulfonate. Citrate transport in whole cells was primarily energized aerobically, and ATPase deficient mutants were still able to transport citrate in whole cells.     

Occurrence of Choline and Glycine Betaine Uptake and Metabolism in the Family Rhizobiaceae and Their Roles in Osmoprotection

The role of glycine betaine and choline in osmoprotection of various Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, and Bradyrhizobium reference strains which display a large variation in salt tolerance was investigated. When externally provided, both compounds enhanced the growth of Rhizobium tropici, Sinorhizobium meliloti, Sinorhizobium fredii, Rhizobium galegae, Agrobacterium tumefaciens, Mesorhizobium loti, and Mesorhizobium huakuii, demonstrating their utilization as osmoprotectants. However, both compounds were inefficient for the most salt-sensitive strains, such as Rhizobium leguminosarum (all biovars), Agrobacterium rhizogenes, Rhizobium etli, and Bradyrhizobium japonicum. Except for B. japonicum, all strains exhibit transport activity for glycine betaine and choline. When the medium osmolarity was raised, choline uptake activity was inhibited, whereas glycine betaine uptake was either increased in R. leguminosarum and S. meliloti or, more surprisingly, reduced in R. tropici, S. fredii, and M. loti. The transport of glycine betaine was increased by growing the cells in the presence of the substrate. With the exception of B. japonicum, all strains were able to use glycine betaine and choline as sole carbon and nitrogen sources. This catabolic function, reported for only a few soil bacteria, could increase competitiveness of rhizobial species in the rhizosphere. Choline dehydrogenase and betaine-aldehyde dehydrogenase activities were present in the cells of all strains with the exception of M. huakuii and B. japonicum. The main physiological role of glycine betaine in the family Rhizobiaceae seems to be as an energy source, while its contribution to osmoprotection is restricted to certain strains.     

Adhesion of Biodegradative Anaerobic Bacteria to Solid Surfaces

In order to exploit the ability of anaerobic bacteria to degrade certain contaminants for bioremediation of polluted subsurface environments, we need to understand the mechanisms by which such bacteria partition between aqueous and solid phases, as well as the environmental conditions that influence partitioning. We studied four strictly anaerobic bacteria, Desulfomonile tiedjei, Syntrophomonas wolfei, Syntrophobacter wolinii, and Desulfovibrio sp. strain G11, which theoretically together can constitute a tetrachloroethylene- and trichloroethylene-dechlorinating consortium. Adhesion of these organisms was evaluated by microscopic determination of the numbers of cells that attached to glass coverslips exposed to cell suspensions under anaerobic conditions. We studied the effects of the growth phase of the organisms on adhesion, as well as the influence of electrostatic and hydrophobic properties of the substratum. Results indicate that S. wolfei adheres in considerably higher numbers to glass surfaces than the other three organisms. Starvation greatly decreases adhesion of S. wolfei and Desulfovibrio sp. strain G11 but seems to have less of an effect on the adhesion of the other bacteria. The presence of Fe³⁺ on the substratum, which would be electropositive, significantly increased the adhesion of S. wolfei, whereas the presence of silicon hydrophobic groups decreased the numbers of attached cells of all species. Measurements of transport of cells through hydrophobic-interaction and electrostatic-interaction columns indicated that all four species had negatively charged cell surfaces and that D. tiedjei and Desulfovibrio sp. strain G11 possessed some hydrophobic cell surface properties. These findings are an early step toward understanding the dynamic attachment of anaerobic bacteria in anoxic environments.     

Carbohydrate Uptake and Utilization byClostridium beijerinckiiNCIMB 8052

The clostridia are a diverse group of obligately anaerobic bacteria with potential for the fermentative production of fuels, solvents and other chemicals. Several species exhibit a broad substrate range, but there have been few studies of the mechanisms involved in regulation of uptake and metabolism of fermentable carbohydrates.Clostridium beijerinckii(formerlyClostridium acetobutylicum) NCIMB 8052 exhibited transport activity for hexoses and hexitols. Glucose-grown cells transported glucose and fructose, but not galactose, glucitol (sorbitol) or mannitol, transport of which was induced by growth on the respective substrates. Phosphorylation of glucose, fructose, glucitol and mannitol by cell extracts was supported by phosphoenolpyruvate, indicating the involvement of a phosphotransferase system in uptake of these substrates. Fructose phosphorylation was also demonstrated by isolated membranes in the presence of fructose 1-phosphate, thus identifying this derivative as the product of the fructose phosphotransferase system. The presence of phosphotransferase activities in extracts prepared from cells grown on different carbon sources correlated with transport activities in whole cells, and the pattern of transport activities reflected the substrate preference of cells growing in the presence of glucose and another carbon source. Thus, glucose and fructose were co-metabolised, while utilization of glucitol was prevented by glucose, even in cells which were previously induced for glucitol metabolism. Of the substrates examined, only galactose appeared to be transported by a non-phosphotransferase mechanism, since a significant rate of phosphorylation of this sugar was supported by ATP rather than phosphoenolpyruvate.     

Response of bacteria to earthworm surface excreta

Response of bacteria to the surface excreta of the Aporrectodea caliginosa earthworm was studied. The excreta were obtained by a 1 h incubation of the earthworms in petri dishes with subsequent collection of the slime. Both inhibition and stimulation of growth were revealed, as well as suppression of the respiratory activity of some bacterial species treated with A. caliginosa surface excreta. The organisms studied included various taxa of soil bacteria (19 strains), bacteria isolated from A. caliginosa intestine and excrements (82 strain), and 48 Bacillus thuringiensis strains. For the cultures of soil bacteria, the respiratory activity was determined using the formazan color reaction due to the activity of the respiratory cycle enzymes. Earthworm excreta caused a consistent 30–50% decrease of dehydrogenase activity in 13 out of the 19 cultures. Determination of the growth rates (derived from OD₆₂₀ of cell suspensions) after 10 h of incubation revealed growth stimulation in 48 out of the 82 strains isolated from intestines and excrement. Other strains exhibited no reaction to the excreta. For 29 out of 45 B. thuringiensis strains, growth stimulation was observed, while growth of two strains was suppressed; other strains exhibited no reaction to the excreta. No relation was found between bacterial reaction to the excreta and their taxonomic position. These results correlate with the research, demonstrating antibacterial and antifungal activity of the extracts from the earthworm body and digestive tract. Thus, earthworms, apart from their medium-forming function, affect the formation of soil microbial communities by direct stimulation or suppression of specific microbial populations.     

Microbial decomposition of coumarin in soil

Microbial decomposition of coumarin was studied in samples of chernozem soil by manometric measurement of oxygen consumption, paper chromatography of aromatic metabolic intermediates in soil extract and measurement of their UV spectra, and by the technique of simultaneous adaptation. Coumarin is decomposed in soil viao-coumaric and melilotic acids and at least one other compound of aromatic character. The metabolic pathway including salicylic acid and catechol was not proved. A total of 39 strains of coumarin-decomposing bacteria were isolated from the soil, out of which 25 belong to the genusPseudomonas, 7 to the genusCellulomonas and 7 to the genusAchromobacter. A comparison of the counts of bacteria utilizing coumarin as a sole carbon source in garden soil, in two chernozem soil samples and in acidic brown soil showed that their occurrence bears no relation to the so-called total number of bacteria (grown on agar medium with yeast and soil extracts and with tryptone) or to the content of carbon and nitrogen in the soil, or to its acidity.