Deoxycholic acid degradation by a Pseudomonas species. Acidic intermediates from the initial part of the catabolic pathway.

The microbial catabolism of deoxycholic acid by a Pseudomonas species was studied, and three acidic products were isolated as their methyl esters. Evidence is presented that the compounds are methyl 3 alpha,12 alpha-dihydroxy-23,24-dinor-5 beta-cholan-22-oate, methyl 12 alpha-hydroxy-3-oxo-5 beta-cholan-24-oate and methyl 12 alpha-hydroxy-3-oxo-23,24-dinor-5 beta-cholan-22-oate.     

The bacterial degradation of flavonoids. Oxidative fission of the A-ring of dihydrogossypetin by a Pseudomonas sp

Cell-free extracts prepared from a Pseudomonas sp., grown on (+)-catechin, oxidized dihydrogossypetin (3',4',5,7,8-pentahydroxyflavanonol) by cleaving the A-ring to form oxaloacetic acid from C-5, C-6, C-7 and C-8 together with 5-(3,4-dihydroxyphenyl)-4-hydroxy-3-oxovalero-delta-lactone. The structure of this lactone was confirmed by synthesis of related phenylvalerolactones.     

Kinetics of p-cresol degradation by an immobilized Pseudomonas sp.

A p-cresol (PCR)-degrading Pseudomonas sp. was isolated from creosote-contaminated soil and shown to degrade PCR by conversion to protocatechuate via p-hydroxybenzaldehyde (PBA) and p-hydroxybenzoate (PHB). Cells of the Pseudomonas sp. were immobilized in calcium alginate beads and in polyurethane foam. The relationship between the PCR concentration and the PCR transformation rate was investigated in batch and continuous culture bioreactors. The biodegradation kinetics of PBA and PHB also were investigated. In batch culture reactors, the maximum PCR degradation rate (Vmax) for the alginate-immobilized Pseudomonas sp. cells was 1.5 mg of PCR g of bead-1 h-1 while the saturation constant (Ks) was 0.22 mM. For PHB degradation, the Vmax was 0.62 mg of PHB g of bead-1 h-1 while the Ks was 0.31 mM. For polyurethane-immobilized Pseudomonas sp. cells, the Vmax of PCR degradation was 0.80 mg of PCR g of foam-1 h-1 while the Ks was 0.28 mM. For PHB degradation, the Vmax was 0.21 mg of PHB g of foam-1 h-1 and the Ks was 0.22 mM. In a continuous column alginate bead reactor, the Vmax for PCR transformation was 2.6 mg g of bead-1 h-1 while the Ks was 0.20 mM. The Vmax and Ks for PBA transformation in the presence of PCR were 0.93 mg g of bead-1 h-1 and 0.063 mM, respectively. When PHB alone was added to a reactor, the Vmax was 1.48 mg g of bead-1 h-1 and the Ks was 0.32 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

The degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590.

The microbial degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590 was studied, and two major products were isolated and identified as 7 alpha, 12 beta-dihydroxyandrosta-1,4-diene-3,17-dione and 7 alpha, 12 alpha-dihydroxy-3-oxopregna-1,4-diene-20-carboxylic acid. Four minor products were isolated and evidence is given for the following structures: 7 alpha, 12 alpha-dihydroxyandrosta-1,4-diene-3,17-dione, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 7 alpha, 12 beta, 17 beta-trihydroxyandrosta-1,4-dien-3-one and 7 alpha, 12 alpha-dihydroxy-3-oxopregn-4-ene-20-carboxylic acid. The significance of the production of the steroid products is discussed, along with the possible enzymic mechanisms responsible for their production.     

Enhancement of phenol degradation by soil bioaugmentation with Pseudomonas sp. JS150

Aims: To test whether bioaugmentation with genetically modified Pseudomonas sp. JS150 strain could be used to enhance phenol degradation in contaminated soils. Methods and Results: The efficiency of phenol removal, content of humic carbon, survival of inoculant, number of total culturable autochthonous bacteria and changes in fatty acid methyl esters (FAME) profiling obtained directly from soils were examined. Bioaugmentation significantly accelerated phenol biodegradation rate in tested soils. Phenol applied at the highest concentration (5·0 mg g^?1 soil) was completely degraded in clay soil (FC) within 65 days, whereas in sand soil (FS) within 72 days. In comparison, phenol biodegradation proceeded for 68 and 96 days in nonbioaugmented FC and FS soils, respectively. The content of humic carbon remained at the same level at the beginning and the end of incubation time in all soil treatments. The number of introduced bacteria (2·50 × 10⁹ g^?1 soil) markedly decreased during the first 4 or 8 days depending on contamination level and type of soil; however, inoculant survived over the experimental period of time. Analysis of FAME patterns indicated that changes in the percentages of cyclopropane fatty acids 17:0 cy and 19:0 cyω10c and branched fatty acids might be useful markers for monitoring the progress of phenol removal from soil. Conclusions: It was confirmed that soil bioaugmentation with Pseudomonas sp. JS150 significantly enhanced soil activity towards phenol degradation. Cyclopropane and branched fatty acids were sensitive probes for degree of phenol utilization. Significance and Impact of the Study: In future, genetically modified Pseudomonas sp. JS150 strain could be of use in the bioaugmentation of phenol‐contaminated areas.     

Development of a continuous system for the degradation of a cyanuric acid by adsorbed Pseudomonas sp. NRRL B-12228

Cyanuric acid in high concentrations (15.5 mm) was degraded completely by Pseudomonas sp. NRRL B-12228 independently of glucose concentration. In the batch fermentations there was a relation between the glucose concentration, on the one hand, and the liberation of ammonia or production of protein, on the other. The greater the supply of carbon, the more biomass was produced, and fewer NH _inf4 ^sup+ ions were released. Continuous fermentations using adsorbed cells could be performed to degrade cyanuric acid. In spite of different glucose feeding there was only a negligible difference in residues of s-triazine. In a one-step continuous system with dilution rates between 0.021 h^–1 and 0.035 h^–1, even a ratio of 0.65 between glucose and cyanuric acid was not sufficient to degrade the cyanuric acid supplied (320–540 mol l^–1 h^–1) completely. When a continuous two-step system was applied with dilution rates between 0.035 h^–1 and 0.056 h^–1, the consumption of carbon source could be minimized while s-triazine degradation up to 860 mol l^–1 h^–1 was complete. In this way the ratio between glucose and cyanuric acid could be increased to 0.25 (molar C:N ratio = 0.33:1). Thereby the process was made considerably more economic.     

Monooxygenase-mediated 1,2-dichloroethane degradation by Pseudomonas sp. strain DCA1.

A bacterial strain, designated Pseudomonas sp. strain DCA1, was isolated from a 1,2-dichloroethane (DCA)-degrading biofilm. Strain DCA1 utilizes DCA as the sole carbon and energy source and does not require additional organic nutrients, such as vitamins, for optimal growth. The affinity of strain DCA1 for DCA is very high, with a Km value below the detection limit of 0.5 microM. Instead of a hydrolytic dehalogenation, as in other DCA utilizers, the first step in DCA degradation in strain DCA1 is an oxidation reaction. Oxygen and NAD(P)H are required for this initial step. Propene was converted to 1,2-epoxypropane by DCA-grown cells and competitively inhibited DCA degradation. We concluded that a monooxygenase is responsible for the first step in DCA degradation in strain DCA1. Oxidation of DCA probably results in the formation of the unstable intermediate 1,2-dichloroethanol, which spontaneously releases chloride, yielding chloroacetaldehyde. The DCA degradation pathway in strain DCA1 proceeds from chloroacetaldehyde via chloroacetic acid and presumably glycolic acid, which is similar to degradation routes observed in other DCA-utilizing bacteria.     

Polycyclic aromatic hydrocarbon degradation by biosurfactant-producing Pseudomonas sp. IR1

We characterized a newly isolated bacterium, designated as IR1, with respect to its ability to degrade polycyclic aromatic hydrocarbons (PAHs) and to produce biosurfactants. Isolated IR1 was identified as Pseudomonas putida by analysis of 16S rRNA sequences (99.6% homology). It was capable of utilizing two-, three- and four-ring PAHs but not hexadecane and octadecane as a sole carbon and energy source. PCR and DNA hybridization studies showed that enzymes involved in PAH metabolism were related to the naphthalene dioxygenase pathway. Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by IR1 during growth on both water miscible and immiscible substrates. The biosurfactants lowered the surface tension of medium from 54.9 dN cm(-1) to 35.4 dN cm(-1) and formed a stable and compact emulsion with an emulsifying activity of 74% with diesel oil, when grown on dextrose. These findings indicate that this isolate may be useful for bioremediation of sites contaminated with aromatic hydrocarbons.     

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.     

Involvement of plasmid in degradation of pentachlorophenol by Pseudomonas sp. from a chemostat

Pseudomonas sp. strain IST103 obtained from a stable bacterial consortium was capable of utilizing pentachlorophenol (PCP) as sole carbon and energy source. The consortium was developed by continuous enrichment in a chemostat. The degradation of PCP by bacterial strain proceeded through an oxidative route as indicated by accumulation of tetrachloro-p-hydroquinone and chlorohydroquinone determined by high performance liquid chromatography (HPLC), and chloride molecules released in culture medium. Two different molecular size plasmids, of approximately 80 and 4 kilobase, were found to be responsible for carrying genes for degradation of PCP. This was evidenced by mutants produced by curing of plasmid by treatment of ethidium bromide. The derivatives were not able to utilize PCP, however, transformation of low molecular size plasmid of Pseudomonas sp. strain 103 into E. coli JM109 utilized PCP, indicated a possible involvement of plasmid in degradation of pentachlorophenol.     

Kinetics of p-cresol degradation by an immobilized Pseudomonas sp

A p-cresol (PCR)-degrading Pseudomonas sp. was isolated from creosote-contaminated soil and shown to degrade PCR by conversion to protocatechuate via p-hydroxybenzaldehyde (PBA) and p-hydroxybenzoate (PHB). Cells of the Pseudomonas sp. were immobilized in calcium alginate beads and in polyurethane foam. The relationship between the PCR concentration and the PCR transformation rate was investigated in batch and continuous culture bioreactors. The biodegradation kinetics of PBA and PHB also were investigated. In batch culture reactors, the maximum PCR degradation rate (Vmax) for the alginate-immobilized Pseudomonas sp. cells was 1.5 mg of PCR g of bead-1 h-1 while the saturation constant (Ks) was 0.22 mM. For PHB degradation, the Vmax was 0.62 mg of PHB g of bead-1 h-1 while the Ks was 0.31 mM. For polyurethane-immobilized Pseudomonas sp. cells, the Vmax of PCR degradation was 0.80 mg of PCR g of foam-1 h-1 while the Ks was 0.28 mM. For PHB degradation, the Vmax was 0.21 mg of PHB g of foam-1 h-1 and the Ks was 0.22 mM. In a continuous column alginate bead reactor, the Vmax for PCR transformation was 2.6 mg g of bead-1 h-1 while the Ks was 0.20 mM. The Vmax and Ks for PBA transformation in the presence of PCR were 0.93 mg g of bead-1 h-1 and 0.063 mM, respectively. When PHB alone was added to a reactor, the Vmax was 1.48 mg g of bead-1 h-1 and the Ks was 0.32 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-metabolic degradation of chlorinated hydrocarbons by Pseudomonas sp. strain DCA1

Pseudomonas sp. strain DCA1, which is capable of utilizing 1,2-dichloroethane (DCA) as sole carbon and energy source, was used to oxidize chlorinated methanes, ethanes, propanes, and ethenes. Chloroacetic acid, an intermediate in the DCA degradation pathway of strain DCA1, was used as a co-substrate since it was readily oxidized by DCA-grown cells of strain DCAI and did not compete for the monooxygenase. All of the tested compounds except tetrachloroethylene (PER) were oxidized by cells expressing DCA monooxygenase. Strain DCAI could not utilize any of these compounds as a growth substrate. Co-metabolic oxidation during growth on DCA was studied with 1,2-dichloropropane. Although growth on this mixture occurred, 1,2-dichloropropane strongly inhibited growth of strain DCAI. This inhibition was not caused by competition for the monooxygenase. It was shown that the oxidation of 1,2dichloropropane resulted in the accumulation of 2,3-dichloro-1-propanol and 2-chloroethanol.     

Novel intermediates of acenaphthylene degradation by Rhizobium sp. strain CU-A1: evidence for naphthalene-1,8-dicarboxylic acid metabolism

The acenaphthylene-degrading bacterium Rhizobium sp. strain CU-A1 was isolated from petroleum-contaminated soil in Thailand. This strain was able to degrade 600 mg/liter acenaphthylene completely within three days. To elucidate the pathway for degradation of acenaphthylene, strain CU-A1 was mutagenized by transposon Tn5 in order to obtain mutant strains deficient in acenaphthylene degradation. Metabolites produced from Tn5-induced mutant strains B1, B5, and A53 were purified by thin-layer chromatography and silica gel column chromatography and characterized by mass spectrometry. The results suggested that this strain cleaved the fused five-membered ring of acenaphthylene to form naphthalene-1,8-dicarboxylic acid via acenaphthenequinone. One carboxyl group of naphthalene-1,8-dicarboxylic acid was removed to form 1-naphthoic acid which was transformed into salicylic acid before metabolization to gentisic acid. This work is the first report of complete acenaphthylene degradation by a bacterial strain.     

Transformation of isopropylamine to L-alaninol by Pseudomonas sp. strain KIE171 involves N-glutamylated intermediates

Pseudomonas sp. strain KIE171 was able to grow with isopropylamine or L-alaninol [S-(+)-2-amino-1-propanol] as the sole carbon source, but not with D-alaninol. To investigate the hypothesis that L-alaninol is an intermediate in the degradation of isopropylamine, two mini-Tn5 mutants unable to utilize both isopropylamine and L-alaninol were isolated. Whereas mutant KIE171-BI transformed isopropylamine to L-alaninol, mutant KIE171-BII failed to do so. The two genes containing a transposon insertion were cloned, and the DNA regions flanking the insertions were sequenced. Two clusters, one comprising eight ipu (isopropylamine utilization) genes (ipuABCDEFGH) and the other encompassing two genes (ipuI and orf259), were identified. Comparisons of sequences of the deduced Ipu proteins and those in the database suggested that isopropylamine is transported into the cytoplasm by a putative permease, IpuG. The next step, the formation of gamma-glutamyl-isopropylamide from isopropylamine, ATP, and L-glutamate, was shown to be catalyzed by IpuC, a gamma-glutamylamide synthetase. gamma-Glutamyl-isopropylamide is then subjected to stereospecific monooxygenation by the hypothetical four-component system IpuABDE, thereby yielding gamma-glutamyl-L-alaninol [gamma(L-glutamyl)-L-hydroxy-isopropylamide]. Enzymatic hydrolysis by a hydrolase, IpuF, was shown to finally liberate L-alaninol and to regenerate L-glutamate. No gene(s) encoding an enzyme for the next step in the degradation of isopropylamine was found in the ipu clusters. Presumably, L-alaninol is oxidized by an alcohol dehydrogenase to yield L-2-aminopropionaldehyde or it is deaminated by an ammonia lyase to propionaldehyde. Genetic evidence indicated that the aldehyde formed is then further oxidized by the hypothetical aldehyde dehydrogenases IpuI and IpuH to either L-alanine or propionic acid, compounds which can be processed by reactions of the intermediary metabolism.     

The degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590 under anaerobic conditions.

The bacterial degradation of cholic acid under anaerobic conditions by Pseudomonas sp. N.C.I.B. 10590 was studied. The major unsaturated neutral compound was identified as 12 beta-hydroxyandrosta-4,6-diene-3,17-dione, and the major unsaturated acidic metabolite was identified as 12 alpha-hydroxy-3-oxochola-4,6-dien-24-oic acid. Eight minor unsaturated metabolites were isolated and evidence is given for the following structures: 12 alpha-hydroxyandrosta-4,6-diene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-4,6-dien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 alpha-hydroxyandrosta-1,4-diene-3,17-dione, 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione, 3,12-dioxochola-4,6-dien-24-oic acid and 12 alpha-hydroxy-3-oxopregna-4,6-diene-20-carboxylic acid. In addition, a major saturated neutral compound was isolated and identified as 3 beta,12 beta-dihydroxy-5 beta-androstan-17-one, and the only saturated acidic metabolite was 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholan-24-oic acid. Nine minor saturated neutral compounds were also isolated, and evidence is presented for the following structures: 12 beta-hydroxy-5 beta-androstane-3,17-dione, 12 alpha-hydroxy-5 beta-androstane-3,17-dione, 3 beta,12 alpha-dihydroxy-5 beta-androstan-17-one, 3 alpha,12 beta-androstan-17-one, 3 alpha,12 alpha-dihydroxy-5 beta-androstan-17-one, 5 beta-androstane-3 beta,12 beta,17 beta-triol, 5 beta-androstane-3 beta,12 alpha,17 beta-triol, 5 beta-androstane-3 alpha,12 beta,17 beta-triol and 5 beta-androstane-3 alpha,12 alpha,17 beta-triol. The induction of 7 alpha-dehydroxylase and 12 alpha-dehydroxylase enzymes is discussed, together with the significance of dehydrogenation and ring fission under anaerobic conditions.