Uptake modes of octadecane by Pseudomonas sp. DG17 and synthesis of biosurfactant

Aims: In order to gain more insight into the uptake modes of octadecane by bacteria. Methods and Results: A strain that could utilize octadecane well was isolated from crude oil contaminated soil, and named as Pseudomonas sp. DG17 by 16S rDNA analysis. Culture growth result showed that Pseudomonas sp. DG17 grew well in the addition of 200 and 400 mg l^?1 of octadecane, which showed that physical contact between substrate and bacteria was important in the substrate biodegradation. Meanwhile, Pseudomonas sp. DG17 produced rhamnolipids biosurfactant that contains 10 congeners, thus causing the surface tension of the culture medium decline and facilitating the contact between hydrocarbon and bacteria. Scanning‐electron‐microscopy results showed that a disruption of the surface membranes in certain zones was observed in some of the cells grown in 400 mg l^?1 octadecane at 176 h compared with the cells in exponential phase at 72 h due to the production of biosurfactant‐rhamnolipid. Conclusions: These results indicated the possibility that the direct contact with insoluble octadecane droplets occurred before the contact with pseudosolubilization smaller oil droplets. Significance: This report throws more light on the uptake mechanisms of octadecane by bacteria, and proposes the possibility that role of biosurfactant is to increase the contact between hydrocarbon and bacteria by changing the cell membrane structure which needs studied in depth. Impact of Study: Results of this study are useful in the bioremediation of petroleum polluted soil.     

Metabolism of benzalphthalide by Pseudomonas sp.

A Pseudomonas sp. degraded benzalphthalide to o-phthalate and benzoate. A tentative pathway for the metabolism of benzalphthalide in this Pseudomonas sp. is proposed on the basis of isolated metabolites, oxygraphic assay and enzymatic studies.     

Isolation and Characterization of Pseudomonas sp. Strain ONBA-17 Degrading o-Nitriobenzaldehyde

Aerobic bacteria degrading o-nitrobenzaldehyde (ONBA) were isolated from activated sludges. One of the isolates, ONBA-17, was identified as Pseudomonas sp. The isolate could grow on ONBA as its sole source of carbon and nitrogen. Further studies demonstrated that the strain was a moderately halophilic bacterium and capable of degrading benzoic acid, 2-nitrophenol, 2-aminophenol, 4-hydroxybenzoic acid, and 4-dimetylaminobenzaldehyde. It could completely degrade 100 mg L⁻¹ ONBA at a range of pH 6–8 in 48 h at 30°C, and up to 400 mg L⁻¹ after 288 h. The strain showed potential to be a good candidate for biotreatment of industrial wastewaters containing ONBA due to its salt-tolerance ability, multiresistance to some heavy metals and antibiotics, and the abilities of degradation of aromatic compounds. These findings may help in developing a process for ONBA-containing industrial wastewater treatment.     

Catabolism of Naphthalenesulfonic Acids by Pseudomonas sp. A3 and Pseudomonas sp. C22

Naphthalene and two naphthalenesulfonic acids were degraded by Pseudomonas sp. A3 and Pseudomonas sp. C22 by the same enzymes. Gentisate is a major metabolite. Catabolic activities for naphthalene, 1-naphthalenesulfonic acid, and 2-naphthalenesulfonic acid are induced by growth with naphthalene, 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid, methylnaphthalene, or salicylate. Gentisate is also an inducer in strain A3. Inhibition kinetics show that naphthalene and substituted naphthalenes are hydroxylated by the same naphthalene dioxygenase. Substrates with nondissociable substituents such as CH₃, OCH₃, Cl, or NO₂ are hydroxylated in the 7,8-position, and 4-substituted salicylates are accumulated. If CO₂H, CH₂CO₂H, or SO₃H are substituents, hydroxylation occurs with high regioselectivity in the 1,2-position. Thus, 1,2-dihydroxy-1,2-dihydronaphthalene-2-carboxylic acids are formed quantitatively from the corresponding naphthalenecarboxylic acids. Utilization of naphthalenesulfonic acids proceeds by the same regioselective 1,2-dioxygenation which labilizes the C—SO₃⁻ bond and eliminates sulfite.     

Cloning of genes involved in myo-inositol transport in a Pseudomonas sp.

A soil isolate of a Pseudomonas sp. can utilize myo-inositol (MI) as the sole carbon source. In this strain, MI is transported through the membrane by a high-affinity transport system in which a periplasmic binding protein is involved. Mutants impaired in the transport system were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and subsequently identified by their slow growth rate at low MI concentrations. Strains with a low linear initial rate of MI uptake were analyzed. Using a broad-host-range cosmid cloning system, we have constructed a gene bank of the wild-type Pseudomonas sp. in an Escherichia coli recA-host. A rapid mating technique enabled us to screen the gene library for clones which are able to restore the active transport of MI in the mutant. An 11.5-kilobase segment containing genes involved in the MI transport has been isolated, and its restriction enzyme cleavage map has been determined.     

Transport of methylamine by Pseudomonas sp. MA.

Pseudomonas sp. MA grows on methylamines as a sole source of carbon, nitrogen, and energy. The transport of methylamine into the organism was investigated. It was found that this organism possesses an inducible transport system for methylamine having the following physical parameters: pH optimum, 7.2; temperature optimum, 30 to 35 degrees C; Km, 1 to 30 mM; Vmax, 90 to 120 nmol/min per mg (dry weight) of cells. Methylamine uptake was curtailed by azide, cyanide, and carbonyl cyanide-m-chlorophenylhydrazone; osmotic shock treatment reduced the uptake by 50%. The uptake was not effectively inhibited by ammonium ion, amino acids, or amides, but was competitively inhibited by short-chain alkylamines. Cells grown on succinate-ammonium chloride did not possess the transport system, but it could be induced in such cells by methylamine in 20 h. Cells grown with methylamine as a sole nitrogen, but not carbon, source transported methylamine at a reduced rate.     

Degradation of bromacil by a Pseudomonas sp.

A gram-negative rod, identified as a Pseudomonas sp., was isolated from soil by using bromacil as the sole source of carbon and energy. During growth on bromacil or 5-bromouracil, almost stoichiometric amounts of bromide were released. The bacterium was shown to harbor two plasmids approximately 60 and 100 kilobases in size. They appeared to be associated with the ability to utilize bromacil as a sole source of carbon and also with resistance to ampicillin. This microorganism also showed the potential to decontaminate soil samples fortified with bromacil under laboratory conditions.     

Biodegradation of p-toluenesulphonamide by a Pseudomonas sp.

A bacterium capable of utilising p-toluenesulphonamide was isolated from activated sludge. The isolated strain designated PTSA was identified as a Pseudomonas sp. using chemotaxonomic and genetic studies. Pseudomonas PTSA grew on p-toluenesulphonamide in a chemostat with approximately 90% release of sulphate and 80% release of ammonium. The isolate was also able to grow on 4-carboxybenzenesulphonamide and 3,4-dihydroxybenzoate but did not grow on p-toluenesulphonate. The transient appearance of 4-hydroxymethylbenzenesulphonamide and 4-carboxybenzenesulphonamide during p-toluenesulphonamide degradation proves oxidation of the methyl group is the initial attack in the biodegradation pathway. Both metabolites of p-toluenesulphonamide degradation were identified by high-performance liquid chromatography-mass spectrometry. 4-Carboxybenzenesulphonamide is probably converted into 3,4-dihydroxybenzoate and amidosulphurous acid. The latter is a chemically unstable compound in aqueous solutions and immediately converted into sulphite and ammonium. Both sulphite and ammonium were formed during degradation of 4-carboxybenzenesulphonamide.     

Carbohydrates Oxidation by Pseudomonas albosesamae nov. sp.

An oxidation product of glucose, gluconate and 2-ketogluconate by Pseudomonas albosesamae was isolated and examined for its chemical structure. Shaking culture is available for the high yield production. Paper chromatographic analysis of fermented broth for the product gives one spot as “an reducing acid.” The acid was isolated as the unstable powder of calcium salt by methanol precipitation. On periodate oxidation, oxalic and glycolic acids were formed, but formaldehyde was hardly detected. On Ruff’s oxidation of the reduced product prepared by sodium borohydrate or catalytic reduction, D-arabinose and a small amount of L-xylose were detected. On catalytic reduction using Raney nickel, 2-ketogluconic acid was isolated as the main reductant. Bis-2, 4-dinitrophenylhydrazone of the product was obtained as analytically pure yellow needle crystals, m.p. dec. 156~157°C, 5+57.0, pyridine, 1 dm. From the above results, the product was certified to be 2,5-diketogluconic acid.     

Carbohydrates Oxidation by Pseudomonas albosesamae nov. sp.

Taxonomic behaviors of a newly isolated bacterium which produces 2,5-diketogluconic acid in high yield were examined in this paper. The bacterium was isolated from sesame seed. It is aerobic, rod-shaped bacilli (0.6~0.8 × 1.0~3.0 microns) with rounded ends. It occurs singly or as a small mass and shows motility with a polar flagellum. Gram staining and acid-fast staining are both negative. Endospore and capsule are not observed. It does not possess photosynthetic and usual pigments the cell. Glucose and other sugars are attacked with acid production, but no gas formation. Polysaccharides and sucrose are not attacked. This bacterium does not produce acetic acid from ethanol. The above behaviors and other physiological properties which are described in the text lead to the conclusion that the bacterium is a new species situated in the genus Pseudomonas. So, it was named Pseudomonas albosesamae, nov. sp. (Wakisaka) in relation with its isolated origin.     

Uranium accumulation by Pseudomonas sp. EPS-5028

Summary Pseudomonas sp. EPS-5028 was examined for the ability to accumulate uranium from solutions. The uptake of uranium by this microorganism is very rapid and is affected by pH but not by temperature, metabolic inhibitors, culture time and the presence of various cations and anions. The amount of uranium absorbed by the cells increased as the uranium concentration of the solution increased up to 55 mg uranium/g cell dry weight. Electron microscopy indicated that uranium accumulated intracellularly as needle-like fibrils. Uranium could be removed chemically from the cells, which could then be reused as a biosorbent. Offprint requests to: A. M. Marqués     

Biopolymer flocculant produced by an Pseudomonas sp.

A biopolymer flocculant produced by Pseudomonas sp. A-99 had flocculating activity both in inorganic suspensions containing Ca2, Mg2 or Fe3 and in organic suspensions containing Fe2, Fe3 or Al3. The flocculant was an acidic protein and contained a small amount of an acidic polysaccharide consisting of galacturonic acid, glucose and galactose. Productivity of the flocculant was about 450 mg/l medium. © Rapid Science Ltd. 1998     

Anti-herpesvirus activities of Pseudomonas sp. S-17 rhamnolipid and its complex with alginate

The rhamnolipid biosurfactant PS-17 and its complex with the polysaccharide alginate, both produced by the Pseudomonas sp. S-17 strain, were studied for their antiviral activity against herpes simplex virus (HSV) types 1 and 2. They significantly inhibited the herpesvirus cytopathic effect (CPE) in the Madin-Darby bovine kidney (MDBK) cell line. The investigations were carried out according to the CPE inhibition assay protocol. The suppressive effect of the compounds on HSV replication was dose-dependent and occurred at concentrations lower than the critical micelle concentration of the surfactant. The 50% inhibitory concentration (IC50) of rhamnolipid PS-17 was 14.5 microg/ml against HSV-1 and 13 microg/ml against HSV-2. The IC50 values of the complex were 435 microg/ml for HSV-1 and 482 microg/ml for HSV-2. The inhibitory effects of the substances were confirmed by measuring the infectious virus yields with the multicycle virus growth experimental design as well: deltalog CCID50 of 1.84-2.0 against the two types of herpes simplex viruses by rhamnolipid PS-17 (20 microg/ml), and a strong reduction of the HSV-2 virus yield under the effect of the alginate complex at a concentration of 450 microg/ml. The results indicate that rhamnolipid PS-17 and its alginate complex may be considered as promising substances for the development of anti-herpetic compounds.     

Transport of mevalonate by Pseudomonas sp. strain M.

Pseudomonas sp. M, isolated from soil by elective culture on R,S-mevalonate as the sole source of carbon, possessed an inducible transport system for mevalonate. This high-affinity system had a pH optimum of 7.0, a temperature optimum of 30 degrees C, a Km for R,S-mevalonate of 88 microM, and a V max of 26 nmol of mevalonate transported per min/mg of cells (dry weight). Transport was energy dependent since azide, cyanide, or m-chlorophenylhydrazone caused complete cessation of transport activity. Transport of mevalonate was highly substrate specific. Of the 16 structural analogs of mevalonate tested, only acetoacetate, mevinolin, and mevaldehyde significantly inhibited transport. Growth of cells on mevalonate induced transport activity by 40- to 65-fold over that observed in cells grown on alternate carbon sources. A biphasic pattern for cell growth, as well as for induction of mevalonate transport activity, was observed when mevalonate was added to a culture actively growing on glucose. The induction of transport activity under these conditions began within 30 min after the addition of mevalonate and reached 60% of maximal activity during phase I. A further increase in mevalonate transport activity occurred during phase II of growth. Glucose was the preferred carbon source for growth during phase I, whereas mevalonate was preferred during phase II. Only one isomer of the R,S-mevalonate mixture appeared to be utilized, since growth ceased after 45 to 50% of the total mevalonate was depleted from the medium. However, nearly 30% of the preferred mevalonate isomer was depleted from the medium during phase I without significant metabolism to CO2. These results suggest that mevalonate or a mevalonate catabolite may accumulate in cells of Pseudomonas sp. M during phase I and that glucose metabolism may inhibit or repress the expression of enzymes further along the mevalonate catabolic pathway.     

Phenanthrene mineralization by Pseudomonas sp. UG14

A phenanthrene-mineralizing Pseudomonas sp., designated UG14, was isolated from creosote-contaminated soil. It contained two plasmids, of approximately 77 kb and 76 kb, the smaller of which contained DNA sequences that hybridized with probes specific for ndoB and xylE, genes involved in catabolism of aromatic hydrocarbons. At initial phenanthrene concentrations of 10, 50, 200 and 1000 mg/l broth, 27%, 19%, 7.7% and 3.3%, respectively, of the [9-¹⁴C]phenanthrene was recovered as ¹⁴CO₂ after 36 days' incubation at 30°C. Most ¹⁴C-label was converted to a water-soluble metabolite tentatively identified as 1-hydroxy-2-naphthoic acid. Rhamnolipid biosurfactants produced by P. aeruginosa UG2 enhanced mineralization of 50 mg phenanthrene/l by Pseudomonas sp. UG14. With the biosurfactant at 0, 25 and 250 mg rhamnose equivalents/l, 6.5%, 8.2% and 9.8%, respectively, of the phenanthrene was mineralized after 35 days.M.A. Providenti, H. Lee and J.T. Trevors are with the Department of Environmental Biology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada; C.W. Greer is with the National Research Council Canada, Biotechnology Research Institute, 6100 Royalmount Ave, Montreal, Quebec, H4P 2R2, Canada.     

Utilization of d-carnitine by Pseudomonas sp. AK 1

Abstract The degradation of chlorophenols by Alcaligenes eutrophus JMP134 (pJP4) was studied. The strain grew on 2,4,6-trichlorophenol or 2,4,6-tribromophenol as the sole carbon and energy source. Complete degradation of 2,4,6-trichlorophenol was confirmed by chloride release and gas chromatography analysis of supernatants from growth cultures. The 2,3,5-, 2,3,4-, 2,3,6-and 2,4,5-isomers of trichlorophenol did not support growth. However, up to 40% of 2,4,5-trichlorophenol was mineralized during growth of A. eutrophus on chemostats fed with either phenol (0.4 mM) or 2,4,6-trichlorophenol (0.4 mM) plus 2,4,5-trichlorophenol (0.1 mM). Growth on 2,4,6-trihalophenols was also observed in A. Eutrophus JMP222, the strain lacking pJP4, suggesting that this new degradative ability reported for A. eutrophus is not related to pJP4 encoded catabolic functions.     

Cometabolism of DDT analogs by a Pseudomonas sp.

A Pseudomonas sp. capable of growth on several nonchlorinated and mono-p-chloro-substituted analogs of DDT as a sole carbon source degraded bis(p-chlorophenyl)methane and 1,1-bis(p-chlorophenyl)ethane only in the presence of diphenylethane. The products p-chlorophenylacetic acid and 2-(p-chlorophenyl)-propionic acid were not further metabolized by the bacterium. Other chlorinated analogs of DDT were found to be recalcitrant to cometabolic degradation with diphenylethane.     

Metabolism of DDT analogues by a Pseudomonas sp.

A Pseudomonas sp. rapidly metabolized several nonchlorinated analogues of DDT, with the exception of 2,2-diphenylethanol, as the sole carbon source. Several of the mono-p-chloro-substituted diphenyl analogues were also metabolized as the sole carbon source by the bacterium. The resulting chlorinated aromatic acid metabolites were not further metabolized. The isolate was unable to metabolize p,p'-dichlorodiphenyl analogues as the sole carbon source.     

Degradation of 3-phenylbutyric acid by Pseudomonas sp.

Pseudomonas sp. isolated by selective culture with 3-phenylbutyrate (3-PB) as the sole carbon source metabolized the compound through two different pathways by initial oxidation of the benzene ring and by initial oxidation of the side chain. During early exponential growth, a catechol substance identified as 3-(2,3-dihydroxyphenyl)butyrate (2,3-DHPB) and its meta-cleavage product 2-hydroxy-7-methyl-6-oxononadioic-2,4-dienoic acid were produced. These products disappeared during late exponential growth, and considerable amounts of 2,3-DHPB reacted to form brownish polymeric substances. The catechol intermediate 2,3-DHPB could not be isolated, but cell-free extracts were able only to oxidize 3-(2,3-dihydroxyphenyl)propionate of all dihydroxy aromatic acids tested. Moreover, a reaction product caused by dehydration of 2,3-DHPB on silica gel was isolated and identified by spectral analysis as (--)-8-hydroxy-4-methyl-3,4-dihydrocoumarin. 3-Phenylpropionate and a hydroxycinnamate were found in supernatants of cultures grown on 3-PB; phenylacetate and benzoate were found in supernatants of cultures grown on 3-phenylpropionate; and phenylacetate was found in cultures grown on cinnamate. Cells grown on 3-PB rapidly oxidized 3-phenylpropionate, cinnamate, catechol, and 3-(2,3-dihydroxyphenyl)propionate, whereas 2-phenylpropionate, 2,3-dihydroxycinnamate, benzoate, phenylacetate, and salicylate were oxidized at much slower rates. Phenylsuccinate was not utilized for growth nor was it oxidized by washed cell suspensions grown on 3-PB. However, dual axenic cultures of Pseudomonas acidovorans and Klebsiella pneumoniae, which could not grow on phenylsuccinate alone, could grow syntrophically and produced the same metabolites found during catabolism of 3-PB by Pseudomonas sp. Washed cell suspensions of dual axenic cultures also immediately oxidized phenylsuccinate, 3-phenylpropionate, cinnamate, phenylacetate, and benzoate.     

Microbial transformation of quinoline by a Pseudomonas sp.

A Pseudomonas sp. isolated from sewage by enrichment culture on quinoline metabolized this substrate by a novel pathway involving 8-hydroxycoumarin. During early growth of the organism on quinoline, 2-hydroxyquinoline accumulated as the intermediate; 8-hydroxycoumarin accumulated as the major metabolite on further incubation. 2,8-Dihydroxyquinoline and 2,3-dihydroxyphenylpropionic acid were identified as the other intermediates. Inhibition of quinoline metabolism by 1 mM sodium arsenite led to the accumulation of pyruvate, whereas inhibition by 5 mM arsenite resulted in the accumulation of 2-hydroxyquinoline as the major metabolite and 2,8-dihydroxyquinoline as the minor metabolite. Coumarin was not utilized as a growth substrate by this bacterium, but quinoline-grown cells converted it to 2-hydroxyphenylpropionic acid, which was not further metabolized. Quinoline, 2-hydroxyquinoline, 8-hydroxycoumarin, and 2,3-dihydroxyphenylpropionic acid were rapidly oxidized by quinoline-adapted cells, whereas 2,8-dihydroxyquinoline was oxidized very slowly. Quinoline catabolism in this Pseudomonas sp. is therefore initiated by hydroxylation(s) of the molecule followed by cleavage of the pyridine ring to yield 8-hydroxycoumarin, which is further metabolized via 2,3-dihydroxyphenylpropionic acid.