Metabolism of dibenzothiophene and naphthalene in Pseudomonas strains: complete DNA sequence of an upper naphthalene catabolic pathway.

From a soil isolate, Pseudomonas strain C18, we cloned and sequenced a 9.8-kb DNA fragment that encodes dibenzothiophene-degrading enzymes. Nine open reading frames were identified and designated doxABDEFGHIJ. Collectively, we refer to these genes as the DOX pathway. At the nucleotide level, doxABD are identical to the ndoABC genes that encode naphthalene dioxygenase of Pseudomonas putida. The DoxG protein is 97% identical to NahC (1,2-dihydroxynaphthalene dioxygenase) of P. putida. DoxE has 37% identity with cis-toluene dihydrodiol dehydrogenase. DoxF is similar to the aldehyde dehydrogenases of many organisms. The predicted DoxHIJ proteins have no obvious sequence similarities to known proteins. Gas chromatography with a flame ionization detector and mass spectroscopy confirmed that the DOX proteins convert naphthalene to salicylate and converting phenanthrene to 1-hydroxy-2-naphthoic acid. doxI mutants convert naphthalene to trans-o-hydroxybenzylidenepyruvate, indicating that the DoxI protein is similar to NahE (trans-o-hydroxybenzylidenepyruvate hydratase-aldolase). Comparison of the DOX sequence with restriction maps of cloned naphthalene catabolic pathway (NAH) genes revealed many conserved restriction sites. The DOX gene arrangement is identical to that proposed for NAH, except that the NAH equivalent of doxH has not been recognized. DoxH may be involved in the conversion of 2-hydroxy-4-(2'-oxo-3,5-cyclohexadienyl)-buta-2,4-dienoat e to cis-o-hydroxybenzylidenepyruvate. doxJ encodes an enzyme similar to NahD (isomerase). Our findings indicate that a single genetic pathway controls the metabolism of dibenzothiophene, naphthalene, and phenanthrene in strain C18 and that the DOX sequence encodes a complete upper naphthalene catabolic pathway similar to NAH.     

Biodegradation of naphthalene by free and alginate entrapped Pseudomonas sp

Naphthalene degradation by freely suspended and immobilized cells of Pseudomonas sp. isolated from contaminated effluents has been investigated in batch cultures and continuously in a packed bed reactor. Naphthalene concentration was varied from 25 mM to 75 mM, the temperature (30 degrees C) and pH (7.0) were kept constant. The results showed good acclimation of the strain to carbon source and degradation rate was highly affected by initial concentration. Alginate-entrapped cells have given good yields although initial rates were not as high as those encountered with free cells. A first order exponential decay kinetic model was proposed with values of parameters for each initial concentration. A laboratory scale packed-bed bioreactor was designed using parameters calculated above and continuous experiments were realized at different flow rates (100 to 200 ml/h), with different feed concentrations and operating during 30 days. The conversion at low feed concentrations and low flow rates was complete whereas at high flow rates and high concentrations it was less efficient because of diffusional limitations and short residence time.     

两株假单胞菌对吡啶和喹啉的生物降解

【目的】探究2株假单胞菌(Pseudomonas)对吡啶和喹啉的降解。【方法】基于16S rRNA序列同源性和基因间区分析,对分离菌株进行分类鉴定。通过分光光度法和电喷雾电离质谱法(Electrospray Ionisation/Mass Spectrometry,ESI/MS)确定分离菌株对吡啶和喹啉的降解性能。通过质粒消除验证降解质粒的存在,同时克隆了可能的降解基因。【结果】鉴定结果表明,两株分离细菌隶属于Pseudomonas,并将其命名为XJUHX-1和XJUHX-12。降解数据表明,2株菌株分别耐受吡啶和喹啉,同时分别检测到4种和2种吡啶和喹啉的可能降解产物。结果还表明,消除质粒后的菌株对吡啶和喹啉的降解能力降低。扩增的编码NADH还原酶部分的降解喹啉oxoR基因和编码硝酸还原酶的降解吡啶的nifH基因,同时在E.coli中表达了43kDa和16kDa的蛋白。【结论】2株Pseudomonas具有降解吡啶和喹啉的能力。     

石油烃和酚类物质在土中的生物降解与土壤酶活性

本文通过模拟实验,研究了不同条件下石油烃和酚类物质在土中的降解进程及其与土壤酶活性的关系,并在此基础上,对所述污染物的土地处理提出了若干建议。     

Biodegradation of petroleum hydrocarbons by filamentous fungi (Aspergillus ustus and Purpureocillium lilacinum) isolated from used engine oil contaminated soil

The use of microorganisms for remediation and restoration of hydrocarbons contaminated soils is an effective and economic solution. The current study aims to find out efficient telluric filamentous fungi to degrade petroleum hydrocarbons pollutants. Six fungal strains were isolated from used engine (UE) oil contaminated soil. Fungi were screened for their ability to degrade crude oil, diesel and UE oil using 2.6-dichlorophenol indophenol (DCPIP). Two isolates were selected, identified and registered at NCBI as Aspergillus ustus HM3.aaa and Purpureocillium lilacinum HM4.aaa. Fungi were tested for their tolerance to different concentration of petroleum oils using radial growth diameter assay. Hydrocarbons removal percentage was evaluated gravimetrically. The degradation kinetic of crude oil was studied at a time interval of 10 days. A.ustus was the most tolerant fungi to high concentration of petroleum oils in solid medium. Quantitative analysis showed that crude oil was the most degraded oil by both isolate; P. lilacinium and A. ustus removed 44.55% and 30.43% of crude oil, respectively. The two fungi were able to degrade, respectively, 27.66 and 21.27% of diesel and 14.39 and 16.00% of UE oil. As compared to the controls, these fungi accumulated high biomass in liquid medium with all petroleum oils. Likewise, crude oil removal rate constant (K) and half-lives (t_1/2) were 0.02 day⁻¹, 34.66 day and 0.015 day⁻¹, 46.21 day for P. lilacinium and A. ustus, respectively. The selected fungi appear interesting for petroleum oils biodegradation and their application for soil bioremediation require scale-up studies.     

Expression and transfer of engineered catabolic pathways harbored by Pseudomonas spp. introduced into activated sludge microcosms.

Two genetically engineered microorganisms (GEMs), Pseudomonas sp. strain B13 FR1(pFRC20P) (FR120) and Pseudomonas putida KT2440(pWWO-EB62) (EB62), were introduced into activated sludge microcosms that had the level of aeration, nutrient makeup, and microbial community structure of activated sludge reactors. FR120 contains an experimentally assembled ortho cleavage route for simultaneous degradation of 3-chlorobenzoate (3CB) and 4-methyl benzoate (4MB); EB62 contains a derivative TOL plasmid-encoded degradative pathway for toluene experimentally evolved so that it additionally processes 4-ethyl benzoate (4EB). Experiments assessed survival of the GEMs, their ability to degrade target substrates, and lateral transfer of plasmid-encoded recombinant DNA. GEMs added at initial densities of 10(6) to 10(7) bacteria per ml of activated sludge declined to stable population densities of 10(4) to 10(5) bacteria per ml. FR120 degraded combinations of 3CB and 4MB (1 mM each) following 3 days of adaptation in the microcosms. Indigenous microorganisms required an 8-day adaptation period before degradation of 4MB was observed; 3CB was degraded only after the concentration of 4MB was much reduced. The indigenous microbial community was killed when both compounds were present at concentrations of 4.0 mM. However, in parallel microcosms containing FR120, the microbial community maintained a normal density of viable cells. Indigenous microbes readily degraded 4EB (2 mM), and EB62 did not significantly increase the observed rate of degradation. In filter matings, transfer of pFRC20P, which specifies mobilization but not transfer functions, from FR120 to P. putida UWC1 was not detectable (< 10(-7) transconjugants per donor cell).(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and transfer of engineered catabolic pathways harbored by Pseudomonas spp. introduced into activated sludge microcosms.

Two genetically engineered microorganisms (GEMs), Pseudomonas sp. strain B13 FR1(pFRC20P) (FR120) and Pseudomonas putida KT2440(pWWO-EB62) (EB62), were introduced into activated sludge microcosms that had the level of aeration, nutrient makeup, and microbial community structure of activated sludge reactors. FR120 contains an experimentally assembled ortho cleavage route for simultaneous degradation of 3-chlorobenzoate (3CB) and 4-methyl benzoate (4MB); EB62 contains a derivative TOL plasmid-encoded degradative pathway for toluene experimentally evolved so that it additionally processes 4-ethyl benzoate (4EB). Experiments assessed survival of the GEMs, their ability to degrade target substrates, and lateral transfer of plasmid-encoded recombinant DNA. GEMs added at initial densities of 10(6) to 10(7) bacteria per ml of activated sludge declined to stable population densities of 10(4) to 10(5) bacteria per ml. FR120 degraded combinations of 3CB and 4MB (1 mM each) following 3 days of adaptation in the microcosms. Indigenous microorganisms required an 8-day adaptation period before degradation of 4MB was observed; 3CB was degraded only after the concentration of 4MB was much reduced. The indigenous microbial community was killed when both compounds were present at concentrations of 4.0 mM. However, in parallel microcosms containing FR120, the microbial community maintained a normal density of viable cells. Indigenous microbes readily degraded 4EB (2 mM), and EB62 did not significantly increase the observed rate of degradation. In filter matings, transfer of pFRC20P, which specifies mobilization but not transfer functions, from FR120 to P. putida UWC1 was not detectable (< 10(-7) transconjugants per donor cell).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative biochemical and genetic analysis of naphthalene degradation among Pseudomonas stutzeri strains.

Of a 49-strain collection of Pseudomonas stutzeri species, 11 isolates were able to degrade naphthalene and 1 isolate was able to use m- and p-toluate as sole carbon and energy sources. Of these 12 strains, 10 shared a highly homologous set of naphthalene catabolic genes, even though they belong to four different genomovars. These genes differed from those present in plasmid NAH7. In only one of these degraders could a plasmid-encoded pathway be demonstrated, and a chromosome-encoded pathway is proposed for the remaining strains. meta cleavage of catechol was only observed in those strains able to metabolize alkyl derivatives of catechol.     

Biodegradation of selected UV-irradiated and non-irradiated polycyclic aromatic hydrocarbons (PAHs)

Biodegradation of UV-irradiated anthracene, pyrene,benz[a]anthracene,and dibenz[a,h]anthracene was comparedto that of the non-irradiated samples, individuallyand in synthetic mixtures with enrichment cultures.Combined treatment was repeated for individual anthraceneand for the PAH mixture with Sphingomonas sp.strain EPA 505 and Sphingomonas yanoikuyae.Enrichment culture studies were performed on the PAHmixtures in the presence of the main photoproduct ofanthracene, pure 9,10-anthracenedione. Photochemicallypretreated creosote solutions were also subjected tobiodegradation and the results were compared tothose of the non-irradiated solutions. The primaryinterest was on 16 polycyclic aromatic hydrocarbons(PAHs) listed as priority pollutants by European Union(EU) and the United States Environmental ProtectionAgency (USEPA). Irradiation accelerated thebiodegradation onset for anthracene, pyrene, andbenz[a]anthracene when they were treatedindividually. The biodegradation of irradiatedpyrene started with no lag phase andwas complete by 122 h whereas biodegradation of thenon-irradiated sample had a lag of 280 h andresulted in complete degradation by 720 h. Biodegradation ofPAHs was accelerated in synthetic mixtures, especiallyin the presence of pure 9,10-anthracenedione.In general, irradiation had no effect on the biodegradation of PAHsincubated in synthetic mixtures or with pure cultures. Undercurrent experimental conditions, the UV-irradiation invariablyreduced the biodegradation of PAHs in creosote. Based onthe results of the present and previous photochemical-biologicalstudies of PAHs, the influence of the photochemical pretreatmenton the biodegradation is highly dependent on the compoundsbeing treated and other process parameters.     

The alkane oxidation system of Pseudomonas oleovorans: induction of the alk genes in Escherichia coli W3110(pGEc47) affects membrane biogenesis and results in overexpression of alkane hydroxylase in a distinct cytoplasmic membrane subtraction

The alkane hydroxylase system of Pseudomonas oleovorans, which catalyses the initial oxidation of aliphatic substrates, is encoded by three genes. One of the gene products, the alkane hydroxyiase AlkB, is an integral cytoplasmic membrane protein. Induction leads to the synthesis of 1.5–2% AlkB relative to the total cell protein, both in P. oleovorans and in recombinant Escherichia coli DH1. We present a study on the Induction and localization of the alkane hydroxylase in E. coli W3110, which appears to be an interesting host strain because it permits expression levels of AlkB of up to 10–15% of the total cell protein. This expression level had negative effects on cell growth. The phospholipid content of such cells was about threefold higher than that of wild-type W3110. Freeze-fracture electron microscopy showed that induction of the alk genes led to the appearance of membrane vesicles in the cytoplasm; these occurred much more frequently in cells expressing alkB than in the negative control, which contained all of the alk genes except for alkB. Isolation and separation of the membranes of cells expressing alkB by density gradient centrifugation showed the customary cytoplasmic and outer membranes, as well as a low-density membrane fraction. This additional fraction was highly enriched in AlkB, as shown both by SDS-PAGE and enzyme activity measurements. A typical cytoplasmic membrane protein, NADH oxidase, was absent from the low-density membrane fraction, alkB expression in W3110 changed the composition of the phospholipid headgroup in the membrane, as well as the fatty acid composition of the membrane. The major changes occurred in the unsaturated fatty acids: C_16:1 and C_18:1 increased at the expense of C_17:0cyc and C_19:0cyc*     

Metabolism of naphthalene, 2-methylnaphthalene, salicylate, and benzoate by Pseudomonas PG: regulation of tangential pathways.

Naphthalene is metabolized by Pseudomonas PG through 1,2-dihydroxynaphthalene and salicylate to catechol, which is then degraded by the meta pathway. 2-Methylnaphthalene, but not 1-methylnaphthalene, also serves as a growth substrate and is metabolized by the same route, through 4-methylcatechol. The same nonspecific meta pathway enzymes appear to be induced by growth on either naphthalene or 2-methylnaphthalene. The level to which 2-hydroxymuconic semialdehyde hydrolase is induced is low and probably of no metabolic significance. Growth on salicylate or catechol, both intermediates of naphthalene degradation, or benzoate results in induction of the ortho pathway, the alternative route for catechol dissimilation. No induction of 1,2-dihydroxynaphthalene oxygenase was found in salicylate-grown cells. Anaerobic growth on a succinate-nitrate medium in the presence of various inducers indicates that cis, cis-muconate, or one of its metabolites is the inducer of the ortho pathway enzymes. The inducer or inducers of the early enzymes of naphthalene degradation and of the meta pathway enzymes must be an early intermediate of the naphthalene pathway above salicylate.     

Biodegradation of individual and multiple chlorinated aliphatic hydrocarbons by methane-oxidizing cultures.

The microbial degradation of chlorinated and nonchlorinated methanes, ethanes, and ethanes by a mixed methane-oxidizing culture grown under chemostat and batch conditions is evaluated and compared with that by two pure methanotrophic strains: CAC1 (isolated from the mixed culture) and Methylosinus trichosporium OB3b. With the exception of 1,1-dichloroethylene, the transformation capacity (Tc) for each chlorinated aliphatic hydrocarbon was generally found to be in inverse proportion to its chlorine content within each aliphatic group (i.e., methanes, ethanes, and ethenes), whereas similar trends were not observed for degradation rate constants. Tc trends were similar for all methane-oxidizing cultures tested. None of the cultures were able to degrade the fully chlorinated aliphatics such as perchloroethylene and carbon tetrachloride. Of the four cultures tested, the chemostat-grown mixed culture exhibited the highest Tc for trichloroethylene, cis-1,2-dichloroethylene, tetrachloroethane, 1,1,1-trichloroethane, and 1,2-dichloroethane, whereas the pure batch-grown OB3b culture exhibited the highest Tc for all other compounds tested. The product toxicity of chlorinated aliphatic hydrocarbons in a mixture containing multiple compounds was cumulative and predictable when using parameters measured from the degradation of individual compounds. The Tc for each chlorinated aliphatic hydrocarbon in a mixture (Tcmix) and the total Tc for the mixture (sigma Tcmix) are functions of the individual Tc, the initial substrate concentration (S0), and the first-order rate constant (k/Ks) of each compound in the mixture, indicating the importance of identifying the properties and compositions of all potentially degradable compounds in a contaminant mixture.     

Biodegradation of hexazinone by two isolated bacterial strains (WFX-1 and WFX-2)

Two hexazinone-degrading bacterial strains were isolated from soil by enrichment culture technique, and identified as Pseudomonas sp. and Enterobacter cloacap, respectively. The two purified isolates, designated as WFX-1 and WFX-2, could rapidly degrade hexazinone with half-lives of 3.08 and 2.95 days in mineral salts medium (hereafter referred to as MSM). In contrast, their mixed bacterial culture (herein abbreviated as MBC) was found to degrade hexazinone, at an initial concentration of 50 mg l⁻¹, by enhancing 2.3-fold over that when the isolates were used alone. The degradation of hexazinone by MBC in MSM clearly decreased concomitant with the increase of initial concentration, and the level of hexazinone that was toxic enough to totally inhibit degradation was in the range of 150–200 mg l⁻¹. The appropriately combined conditions for hexazinone degradation by MBC in MSM were studied, and found to be pH 5.5, 30 °C and at agitation of 120 rpm. The addition of MBC to soil had a greater impact on disappearance of hexazinone, which nearly increased fivefold over that of the control set. As a result, findings in the present investigation provide useful information for soil and water decontamination of hexazinone.     

Degradation of n-alkanes and polycyclic aromatic hydrocarbons in petroleum by a newly isolated Pseudomonas aeruginosa DQ8

A bacterial isolate, designated as DQ8, was found capable of degrading diesel, crude oil, n-alkanes and polycyclic aromatic hydrocarbons (PAHs) in petroleum. Strain DQ8 was assigned to the genus Pseudomonas aeruginosa based on biochemical and genetic data. The metabolites identified from n-docosane as substrate suggested that P. aeruginosa DQ8 could oxidize n-alkanes via a terminal oxidation pathway. P. aeruginosa DQ8 could also degrade PAHs of three or four aromatic rings. The metabolites identified from fluorene as substrate suggested that P. aeruginosa DQ8 may degrade fluorene via two pathways. One is monooxygenation at C-9 of fluorene, and the other is initiated by dioxygenation at C-3 and C-4 of fluorene. P. aeruginosa DQ8 should be of great practical significance both in bioremediation of oil-contaminated soils and biotreatment of oil wastewater.     

Convergent and divergent points in catabolic pathways involved in utilization of fluoranthene, naphthalene, anthracene, and phenanthrene by Sphingomonas paucimobilis var. EPA505

Catabolic pathways for utilization of naphthalene (NAP), anthracene (ANT), phenanthrene (PHE), and fluoranthene (FLA) by Sphingomonas paucimobilis EPA505 were identified. Accumulation of catabolic intermediates was investigated with three classes of Tn5 mutants with the following polycyclic aromatic hydrocarbon (PAH)-negative phenotypes; (class I NAP(-) PHE(-) FLA(-), class II NAP(-) PHE(-), and class III FLA(-)). Class I mutant 200pbhA had a Tn5 insertion within a meta ring fission dioxygenase (pbhA), and a ferredoxin subunit gene (pbhB) resided directly downstream. Mutant 200pbhA and other class I mutants lost the ability to catalyze the initial dihydroxylation step and did not transform NAP, ANT, PHE, or FLA. Class I mutant 401 accumulated salicylic acid, 2-hydroxy-3-naphthoic acid, 1-hydroxy-2-naphthoic acid, and hydroxyacenaphthoic acid during incubation with NAP, ANT, PHE, or FLA, respectively. Class II mutant 132pbhC contained the Tn5 insertion in an aldolase hydratase (pbhC) and accumulated what appeared to be meta ring fission products: trans-o-hydroxybenzylidene pyruvate, trans-o-hydroxynaphylidene pyruvate, and trans-o-hydroxynaphthyl-oxobutenoic acid when incubated with NAP, ANT, and PHE, respectively. When mutant 132pbhC was incubated with 1-hydroxy-2-naphthoic acid, it accumulated trans-o-hydroxybenzylidene pyruvate. Class III mutant 104ppdk had a Tn5 insertion in a pyruvate phosphate dikinase gene that affected expression of a FLA-specific gene and accumulated a proposed meta ring fission product; trans-o-hydroxyacenaphyl-oxobutenoic acid during incubation with FLA. Trans-o-hydroxyacenaphyl-oxobutenoic acid was degraded to acenaphthenone that accumulated with class III mutant 611. Acenaphthenone was oxidized via incorporation of one molecule of dioxygen by another oxygenase. 2,3-Dihydroxybenzoic acid was the final FLA-derived catabolic intermediate detected. Analysis of PAH utilization mutants revealed that there are convergent and divergent points involved in NAP, ANT, PHE, and FLA utilization by S. paucimobilis EPA505.     

Effect of transposons on expression of genes for naphthalene biodegradation in Pseudomonas putida BS202(NPL-1) and derivative strains

NPL-1 and its derivative plasmid pBS106, which control the degradation of naphthalene and salicylate, were found to contain class II transposons of the Tn3 family. These transposons are involved in intraplasmid rearrangements, such as deletions and inversions, and can influence the expression of the catabolic and regulatory genes borne by biodegradation plasmids. The formation of a strong NahR-independent constitutive promoter by the inversion of a DNA fragment may be responsible for changing the character of naphthalene dioxygenase synthesis from inducible (in the case of plasmid NPL-1) to constitutive (in the case of plasmid NPL-41). The stability of plasmids NPL-1 and NPL-41 in the Pseudomonas putida strains grown on different substrates depends on the expression of the nah and tnp genes.     

Synthesis of poly(3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains

We have studied the accumulation kinetics and physical characteristics of the poly(3-hydroxyalkanoates) (PHAs) formed by several Pseudomonas strains, mutants and recombinants. Although PHA synthesis generally begins only after an essential nutrient such as N, P, S or Mg becomes limiting, we have identified at least one strain (P. putida KT2442) that begins producing PHA during the exponential growth phase. This PHA is chemically and physically identical to that produced by P. oleovorans GPol, the strain in which we first identified PHA. Analysis of the PHA formed by a mutant strain defective in PHA degradation (P. oleovorans GPo500) revealed that the molecular mass (M_w), the monomer composition and thermal characteristics were similar to that of the PHA of the wild-type parent strain P. oleovorans GPo1. The pha locus of P. oleovorans encodes enzymes that are involved in PHA biosynthesis and degradation. It has been subcloned to study the two PHA polymerases separately in a PHA^– mutant (GPp104) derived from P. putida KT2442. The recombinant strains accumulated lower PHA levels than the wild-type strains, and the M_w of these polymers were lower than those produced by the wild-type P. oleovorans and parent strain. The monomer composition of the two PHAs formed by the two PHA polymerases differed, indicating that the PHA polymerases have different substrate specificities for the incorporation of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers into PHA. Despite these differences, the PHAs formed were essentially indistinguishable from wild-type PHAs with respect to their thermal characteristics.Correspondence to: B. Witholt     

Relationships between the ethanol utilization (alc) pathway and unrelated catabolic pathways in Aspergillus nidulans.

The ethanol utilization pathway in Aspergillus nidulans is a model system, which has been thoroughly elucidated at the biochemical, genetic and molecular levels. Three main elements are involved: (a) high level expression of the positively autoregulated activator AlcR; (b) the strong promoters of the structural genes for alcohol dehydrogenase (alcA) and aldehyde dehydrogenase (aldA); and (c) powerful activation of AlcR by the physiological inducer, acetaldehyde, produced from growth substrates such as ethanol and l-threonine. We have previously characterized the chemical features of direct inducers of the alc regulon. These studies allowed us to predict which type of carbonyl compounds might induce the system. In this study we have determined that catabolism of different amino acids, such as L-valine, L-isoleucine, L-arginine and L-proline, produces aldehydes that are either not accumulated or fail to induce the alc system. On the other hand, catabolism of D-galacturonic acid and putrescine, during which aldehydes are transiently accumulated, gives rise to induction of the alc genes. We show that the formation of a direct inducer from carboxylic esters does not depend on alcA-encoded alcohol dehydrogenase I or on AlcR, and suggest that a cytochrome P450 might be responsible for the initial formation of a physiological aldehyde inducer.