Methanol Metabolism in Pseudomonad C

Cell suspensions of pseudomonad C, a bacterium capable of growth on methanol as sole carbon source, were able to oxidize methanol, formaldehyde, and formate, although the rates of oxidation for the latter two compounds were much slower. The latter compounds also could not serve as sole carbon sources. Through the use of labeled compounds, it was shown that in the presence of methanol, formaldehyde, formate, and bicarbonate were incorporated into trichloroacetic acid-precipitable material. Hexose phosphate synthetase activity was found, indicating the assimilation of methanol via an allulose pathway. No hydroxypyruvate reductase activity was found, nor was any complex membrane structure observed. Such a combination of characteristics has been observed in an obligate methylotroph (Pseudomonas W1), but pseudomonad C can utilize a variety of non-methyl substrates.     

Influence of Temperature on the Iron Metabolism of a Fluorescent Pseudomonad

The iron requirement for maximal cell yields of a fluorescent pseudomonad increases as the temperature of incubation is increased. On a succinate salts medium, maximal cell yields are attained at iron concentrations of 0.10 mug/ml of added iron at 20 C and at 3.0 mug/ml of added iron at 28 C. This bacterium does not grow in the basal medium at 31 C even in the presence of 0.01 to 10 mug/ml of added iron. The inability to grow at the higher temperature is due to the loss, by this organism, of its ability to biosynthesize hydroxamate iron transport compounds at temperatures of 28 C and above, since supplementation with such compounds produced by this organism at lower temperatures promoted growth at 31 C. The biosynthesis of these compounds at lower temperatures contributes to the efficient utilization of iron by the bacterium.     

Degradation of I-Naphthol by a Soil Pseudomonad

A soil Pseudomonas sp. grew with 1-naphthol as sole organic carbon source and produced a 3,4-dihydro-dihydroxy-1(2H)-naphthalenone as the main early intermediary metabolite. Washed 1-naphthol-grown organisms oxidized naphthalene, 1- or 2-naphthol, salicylic acid and, to some extent, 2,3-dihydroxybenzoic acid.     

Oxidation of L-glucose by a Pseudomonad.

A new enzyme, D-threo-aldolse dehydrogenase (2S,3R-aldose dehydrogenase), found in Pseudomonas caryophylli, was capable of oxidizing L-glucose L-xylose, D-arabinose, and L-fucose in the presence of NAD+. The enzyme was synthesized constitutively and purified about 120-fold from D-glucose-grown cells. The Km values for L-glucose, L-xylose, D-arabinose, and L-fucose were 1.5 . 10(-2), 4.5 . 10(-3), 2.8 . 10(-3), and 2.1 . 10(-3), respectively. D-glucose and other aldoses inhibited the enzyme reaction; this inhibition was competitive with L-glucose as substrate and D-glucose as inhibitor. The optimum pH for the enzyme reaction was 10; the molecular weight of the enzyme was determined by gel filtration to be 7 . 10(4).     

Degradation of Triphenyltin by a Fluorescent Pseudomonad

Triphenyltin (TPT)-degrading bacteria were screened by a simple technique using a post-column high-performance liquid chromatography using 3,3′,4′,7-tetrahydroxyflavone as a post-column reagent for determination of TPT and its metabolite, diphenyltin (DPT). An isolated strain, strain CNR15, was identified as Pseudomonas chlororaphis on the basis of its morphological and biochemical features. The incubation of strain CNR15 in a medium containing glycerol, succinate, and 130 μM TPT resulted in the rapid degradation of TPT and the accumulation of approximately 40 μM DPT as the only metabolite after 48 h. The culture supernatants of strain CNR15, grown with or without TPT, exhibited a TPT degradation activity, whereas the resting cells were not capable of degrading TPT. TPT was stoichiometrically degraded to DPT by the solid-phase extract of the culture supernatant, and benzene was detected as another degradation product. We found that the TPT degradation was catalyzed by low-molecular-mass substances (approximately 1,000 Da) in the extract, termed the TPT-degrading factor. The other fluorescent pseudomonads, P. chlororaphis ATCC 9446, Pseudomonas fluorescens ATCC 13525, and Pseudomonas aeruginosa ATCC 15692, also showed TPT degradation activity similar to strain CNR15 in the solid-phase extracts of their culture supernatants. These results suggest that the extracellular low-molecular-mass substance that is universally produced by the fluorescent pseudomonad could function as a potent catalyst to cometabolite TPT in the environment.     

Desulfuration of Dialkyl Thiophosphoric Acids by a Pseudomonad

A strain of Pseudomonas acidovorans used the organophosphorus pesticide breakdown products, ionic O,O-diethyl phosphorothioate and ionic O,O-diethyl phosphorodithioate, as sulfur sources. The growth yields from the thiophosphates and sulfate were 3.6 to 4.1 kg of protein per mol of sulfur. Elemental sulfur and sulfide also served as sulfur sources but gave lower growth yields.     

Active Transport of Choline by a Marine Pseudomonad

A marine pseudomonad, BAL-31, accumulates the phospholipid nitrogen base, choline, although no detectable amount of choline is incorporated into polar lipids. Metabolic inhibitors such as cyanide and azide block the uptake process as does starving for oxygen by using nitrogen gas. Only very close structural analogues show any inhibition of transport, indicating that the uptake process has great structural specificity. The export of choline out of the cells is also an energy-dependent process and is markedly reduced during oxygen depletion. The constitutive level of choline transport is increased by approximately a factor of three after a brief induction period. Two other gram-negative bacteria also accumulate choline, whereas a gram-positive bacterium, Bacillus subtilis, and a yeast, Saccharomyces cerevisiae, fail to show any detectable accumulation.     

Planktonic-Cell Yield of a Pseudomonad Biofilm

Biofilm cells differ phenotypically from their free-floating counterparts. Differential growth rates in biofilms are often referred to, particularly in response to limited diffusion of oxygen and nutrients. We observed growth rates of attached Pseudomonas sp. strain CT07 cells that were notably higher than the maximum specific growth rate measured in batch culture. Despite dilution rates in continuous flow cells that exceeded the maximum planktonic specific growth rate by 58 times, sampling of the effluent revealed >10⁹ cells ml⁻¹, suggesting that biofilms function as a source of planktonic cells through high cell yield and detachment. Further investigation demonstrated considerable planktonic cell yield from biofilms as young as 6 h, indicating that detachment is not limited to established biofilms. These biofilm-detached cells were more sensitive to a commercial biocide than associated biofilm- and chemostat-cultivated populations, implying that detached biofilm cells exhibit a character that is distinct from that of attached and planktonic cell populations.     

Decomposition of β-Naphthol by a Soil Pseudomonad

Summary: A pseudomonad resembling Pseudomonas fluorescens , which grows with β-naphthol as sole source of carbon, was isolated from soil. It did not grow on either naphthalene, α-naphthol, 1,2- or 2,3 dihydroxynaphthalene. Phenol, benzoic acid, o-, p - and (to a small extent) m -hydroxybenzoic acids supported growth of the organism. A maroon coloured substance was produced from β-naphthol in cultures and by washed organisms. β-Naphthol oxidation depended on an induced enzyme system. β-Naphthol-grown organisms oxidized β-naphthol and 2,3- and 2,6-dihydroxynaphthalene immediately and several mono- and di-hydroxybenzoic acids, including salicylic acid, only after a lag. 2,3-Dihydroxynaphthalene may be a metabolite of β-naphthol.     

Regulatory Influences on the Production of Gamma-Aminobutyric Acid by a Marine Pseudomonad

A pseudomonad capable of producing γ-aminobutyric acid (GABA) was isolated from seawater via an enrichment in which glutamate was the sole carbon and nitrogen source. The organism grew optimally at pH 7.3 and at 25°C. Putrescine, alanine, and glucose-nitrate also served as effective growth substrates. The isolate grew poorly on GABA. Cell suspensions of the organism in 0.02 M phosphate buffer (pH 7.6) containing NaCl (19.4 g liter^-1) and MgCl₂. 6H₂O(3 g liter^-1) produced GABA from succinic semialdehyde in combination with glutamate or alanine but not from any substrate alone. Little or no GABA was produced with putrescine or glucose-nitrate as substrates. GABA production in the amino acid cosubstrate systems was transitory with optimum levels occurring in the suspension fluid after 3 h of incubation (0.3 and 0.03 mM for glutamate and alanine cosubstrates, respectively). However, yields of GABA in the cell suspension fluid were low, and quantities near that predicted from stoichiometry could be obtained only by extracting cell suspensions with methanol. GABA release in the suspension fluid was increased with higher pH or by decreasing NaCl. Substitution of the salt by the equivalent Tris-HCl or KCl likewise resulted in increased GABA release. When nigericin (10 μg ml^-1) was added to cell suspensions in which NaCl was not decreased, GABA release increased in a way similar to that observed in suspensions with decreased NaCl. The ionophore also decreased GABA uptake by cell suspensions of GABA-grown cells, and the effect was duplicated by lowering NaCl in cell suspensions. The results indicate a role for an Na⁺-dependent transport system in GABA release.     

Chromate Reduction by a Pseudomonad Isolated from a Site Contaminated with Chromated Copper Arsenate

A pseudomonad (CRB5) isolated from a decommissioned wood preservation site reduced toxic chromate [Cr(VI)] to an insoluble Cr(III) precipitate under aerobic and anaerobic conditions. CRB5 tolerated up to 520 mg of Cr(VI) liter⁻¹ and reduced chromate in the presence of copper and arsenate. Under anaerobic conditions it also reduced Co(III) and U(VI), partially internalizing each metal. Metal precipitates were also found on the surface of the outer membrane and (sometimes) on a capsule. The results showed that chromate reduction by CRB5 was mediated by a soluble enzyme that was largely contained in the cytoplasm but also found outside of the cells. The crude reductase activity in the soluble fraction showed a K_m of 23 mg liter⁻¹ (437 μM) and a V_max of 0.98 mg of Cr h⁻¹ mg of protein⁻¹ (317 nmol min⁻¹ mg of protein⁻¹). Minor membrane-associated Cr(VI) reduction under anaerobiosis may account for anaerobic reduction of chromate under nongrowth conditions with an organic electron donor present. Chromate reduction under both aerobic and anaerobic conditions may be a detoxification strategy for the bacterium which could be exploited to bioremediate chromate-contaminated or other toxic heavy metal-contaminated environments.     

Modulation of Affinity of a Marine Pseudomonad for Toluene and Benzene by Hydrocarbon Exposure

Trace (microgram liter⁻¹) quantities of either toluene or benzene injected into an amino-acid-limited continuous culture of Pseudomonas sp. strain T2 were utilized immediately with affinities of 2.6 and 6.8 liters g of cells⁻¹ h⁻¹, respectively, and yielded large amounts of organic products, carbon dioxide, and cells. The immediate utilization of hydrocarbons by hydrocarbon-deprived organisms helps to establish the nutritional value of nonpolar substrates in the environment. The observation of small Michaelis constants for toluene transport led to tests of metabolic competition between hydrocarbons; however, competitive inhibition of toluene metabolism was not found for benzene, naphthalene, xylene, dodecane, or amino acids. Benzene and terpenes were inhibitory at milligram liter⁻¹ concentrations. Toluene was metabolized by a strongly inducible system when compared with benzene. The capacity of toluene to effect larger affinity values increased with exposure time and concentration. The kinetics of induction suggested saturation phenomena, resulting in an induction constant, K_ind, of 96 μg of toluene liter⁻¹. Maximal induction of amino-acid-grown cells required about 80 h, with the affinity reaching 317 liters g of cells⁻¹ h⁻¹.     

Terminal oxydation pattern of a soil Pseudomonad (PL-strain)

Summary The organism grown on ¹-p-menthene was found to grow without any lag on methyl isopropyl ketone, isobutyrate, succinate, malate, lactate and acetate. Isobutyrate or acetate grown cells grew on ¹-p-menthene after a lag and showed comparatively little growth on -isopropyl pimelic acid.¹-p-menthene grown cells oxidized readily isobutyrate, acetate, succinate, malate, -ketoglutarate and methacrylate. Methylmalonate, methyl isopropyl ketone and -isopropyl pimelic acid were rather oxidized at slow rates. Isobutyrate grown cells, on the other hand, showed from very good to very fair oxidation rates with succinate, isobutyrate, acetate, malate, methacrylate, -ketoglutarate. Methylmalonate was oxidized much better and methyl isopropyl ketone was oxidized slowly.¹-p-menthene and isobutyrate grown cells were used under resting conditions with different substrates in the presence of arsenite. Analysis of the reaction products indicated the accumulation of a keto acid. Qualitative analysis of the keto acid formed by TLC showed pyruvate as the major ketocarboxylic acid with one or two other minor components. The major component had been isolated and identified as pyruvic acid. Similar results had been obtained by working with crude cell-free enzyme preparations.Based on these results two possible mechanisms of degradation of isobutyrate have been suggested. A plausible pathway has been outlined for the terminal oxidation pattern in the Pseudomonad (PL-strain).Abbreviations NAD Nicotinamide adenine dinucleotide - FAD Flavine adenine dinucleotide - -KGA -keto-glutaric acid - CFE cell-free extract - CoA coenzyme A in its reduced state Communication number 1426 from the National Chemical Laboratory.     

Studies on a 2,3-diaminopropionate: ammonia-lyase from a Pseudomonad.

2,3-Diaminopropionate:ammonia-lyase, an induced enzyme in a Pseudomonas isolate, has been purified 40-fold and found to be homogeneous by disc gel electrophoresis and by ultracentrifugation. Some of its properties have been studied. The optimum pH and temperature for activity are 8 and 40 degrees C, respectively. The enzyme shows a high degree of substrate specificity, acting only on 2,3-diaminopropionate; the D-isomer is only one-eighth as effective as the L-form. L-Homoserine and DL-cystathionine are not substrates, and 3-cyanolalanine does not inhibit its activity. It is a pyridoxal phosphate enzyme which requires free enzyme sulphhydryls for activity. The Km values for L-2,3-diaminopropionate and pyridoxal phosphate are 1mM and 25 muM, respectively. The molecular weight of the enzyme is about 80 000 as determined by gel filtration. On treatment with 0.5M urea or guanidine by hydrochloride, the enzyme dissociates into inactive subunits with an approximate molecular weight of 45 000. One mole of the active enzyme binds one mole of pyridoxal phosphate. The bacterial enzyme seems to be quite different in many of its properties from the rat liver enzyme which also exhibits the substrate specificity of cystathionine gamma-lyase.     

Ecological and physiological analyses of Pseudomonad species within a phenol remediation system

A diverse collection of 700 bacteria obtained from an operational phenolic remediating industrial treatment plant was made to select potential strains as microbial biosensors. Pseudomonads were the most abundant group, of which 48 selected from the liquor or suspended solids were assessed for their physiological response to phenolic pollutant loading and niche specialisation. By FAME-MIS identification the Pseudomonads were clustered into six major species groups. Those isolates able to utilise phenol as a sole carbon source predominantly belonged to a non-clonal Pseudomonas pseudoalcaligenes cluster determined by REP-PCR genotyping. Rapid microtitre based respiration assays were developed to contrast activity in response to increasing concentrations of phenol. A considerable range in response for both phenol degrader and non-degrader strains was observed. This natural phenotypic and physiological heterogeneity could facilitate the selection of isolates for the development of a suite of ecologically relevant, custom designed sensors with predictable toxicity susceptibilities to monitor process efficacy.     

Fluorescence Spectroscopy as a Promising Tool for a Polyphasic Approach to Pseudomonad Taxonomy

Fluorescence spectroscopy is an emerging tool for the analysis of biomolecules from complex matrices. We explored the potentialities of the method for the pseudomonad taxonomic purpose at the genus and species level. Emission spectra of three intrinsic fluorophores (namely, NADH, tryptophan, and the complex of aromatic amino acids and nucleic acid) were collected from whole bacterial cells. Their comparisons were performed through principal component analysis and factorial discriminant analysis. Reference strains from the Xanthomonas, Stenotrophomonas, Burkholderia, and Pseudomonas genera were well separated, with sensitivity and selectivity higher than 90%. At the species level, P. lundensis, P. taetrolens, P. fragi, P. chlororaphis, and P. stutzeri were also well separated, in a distant group, from P. putida, P. pseudoalcaligenes, and P. fluorescens. These results are in agreement with the generally admitted rRNA and DNA bacterial homology grouping but they also provide additional information about strain relatedness. In the case of environmental isolates, the method allows good discrimination, even for strains for which ambiguity still remained after PCR and API 20NE identification. Rapid, easy to perform, and low cost, fluorescence spectroscopy provides substantial information on cell components. Statistical analysis of collected data allows in-depth comparison of strains. Our results strongly support the view that fluorescence spectroscopy fingerprinting can be used as a powerful tool in a polyphasic approach to pseudomonad taxonomy.