Catabolism of biphenyl by Pseudomonas putida BS 893 strain containing the biodegradation plasmid pBS241

The isolation and identification of biphenyl catabolism products in Pseudomonas putida BS 893 (pBS241) showed the presence of benzoic, m-hydroxybenzoic and cinnamic acids. The two latter compounds were not found in biphenyl degradation by other bacterial strains. P. putida BS 893 (pBS241) differed from other biphenyl-positive Pseudomonas strains in the enzyme activity. These differences may stem from peculiarities in the pathway of biphenyl catabolism controlled by plasmid pBS241.     

Hybrid plasmid pBS251 containing genes for n-alkane degradation

The strain of Pseudomonas aeruginosa BS316 utilizing H-alkanes of the C6-C12 series (Alk+) harbours the 96 Md plasmid pBS250. The use of plasmid RP4 to mobilize Alk+ markers in conjugal transfer to Pseudomonas aeruginosa and Pseudomonas putida has resulted in isolation of transconjugants resistant to antibiotics (due to genes coded by plasmid RP4) and capable of growth on H-alkanes. A transconjugant from this series harbours plasmid pBS251, a derivative of plasmid RP4 containing the genes for octane and octanol catabolism. A fragment of DNA inserted into RP4 has a mol mass 3.8 Md, possesses two restriction sites for EcoRI, one site for PstI, is not restricted by SmaI and BamHI restriction endonucleases, and is localized in the region 4.5-5.7 Md on the physical map of plasmid RP4.     

Naphthalene oxidation by a Pseudomonas putida strain carrying a mutant plasmid

Naphthalene oxidation by a parent and a mutant strain of Pseudomonas putida was studied. The parent strain contained a plasmid NPL-1 which controlled oxidation of naphthalene to salicylic acid and was capable of oxidizing salicylate. The mutant strain did not oxidize salicylate because of a mutation in salicylate hydroxylase; it contained also a mutant plasmid NPL-41 which determined constitutive synthesis of naphthalene oxygenase. Salicylic acid which accumulated as a product of naphthalene catabolism in the cultural broth of the wild strain was found to undergo further oxidation by the population of growing cells. The content of salicylic acid in the cultural broth of the mutant strain reached maximum and then remained constant. An anion-exchange resin was tested in order to prevent the inhibition of naphthalene oxygenase by salicylate and to increase the yield of salicylic acid. The transmissible character of the mutant plasmid NPL-41 makes it possible, with the aid of conjugation, to construct Pseudomonas strains which would oxidize naphthalene to salicylic acid without further degradation of this compound.     

Degradation of long-chain n-alkanes (C36 and C40) by Pseudomonas aeruginosa strain WatG

Pseudomonas aeruginosa strain WatG was unable to utilize either n-hexatriacontane (C₃₆) or n-tetracontane (C₄₀), which are both insoluble in a mineral salts medium (MSM), as a sole carbon source. However, when C₃₆ and C₄₀ were added to MSM containing crude oil, more than 25% of each of the compounds was degraded by this strain after 2 weeks at 30 °C. These results demonstrate that P. aeruginosa strain WatG has the ability to degrade long-chain alkanes up to C₄₀, when they are solubilized by crude oil components.     

Characteristics of plasmid pBS271 controlling epsilon-caprolactam degradation by bacteria in the genus Pseudomonas

Conjugative plasmids control the ability of five Pseudomonas strains isolated from the rectifiers of chemical plants to grow on epsilon-caprolactam as a sole carbon and nitrogen source. All the plasmids have a high molecular mass of their DNA (ca. 300 MDa) and control epsilon-caprolactam degradation at least to succinate. One of the plasmids (pBS271) belongs to the incompatibility group P-2 and suppresses the growth of a broad spectrum of temperate and virulent P. aeruginosa bacteriophages as well as that of some P. putida bacteriophages.     

Oxidation of 1-Tetradecene by Pseudomonas aeruginosa

Pseudomonas aeruginosa strain Sol 20 was grown on 1-tetradecene as sole carbon source, and a vinyl-unsaturated 14-carbon monocarboxylic acid, 13-tetradecenoic acid, was identified from culture fluid. This acid was not produced when n-tetradecane served as substrate for growth. Oxidation of the methyl group represents one method of attack on the 1-alkene by this organism. Tentative identification of 2-tetradecanol indicates that an attack on the double bond is also occurring. α, ω-Dienes would not support growth.     

Nah-genes of Pseudomonas putida: molecular genetic analysis of the plasmid pBS286

The hybridization and restriction analysis of the plasmid pBS286 (73 Kb, the P-9 Inc group) as well as parental plasmids NPL-1, NPL-41 demonstrated that pBS286 plasmid (delta NPL-41::TnA) with the constitutive synthesis of naphthalene dioxygenase carried genes for naphthalene oxidation to salicylate and those participating in degradation of catechol. Restriction map of pBS286 using XhoI restriction endonuclease and that of the nah region using EcoRI, BamHI, SalI and XhoI were established. Structural peculiarities of nah genes from pBS286 are compared with previously described NAH7. Some nah genes were localized. An inverted DNA segment involved in nah gene regulation was shown to be closely linked to a proximal part of the nah1 operon or overlapped. Possible occurrence of a regulatory R locus in this region is suggested.     

Transformation of Pseudomonas aeruginosa strain PAO with bacteriophage and plasmid DNA.

A procedure has been developed which allows transformation of P. aeruginosa strain PAO with plasmid and bacteriophage DNA at a frequency of 10(-6) per recipient cell. The method is similar in outline to that developed for Escherichia coli. It involves growing the recipient cells to 3-5 x 10(8) per ml in nutrient broth, washing the cells with 0.1 M MgCl2, resuspending in 0.175 M CaCl2 for 20 min, exposing to DNA for 1 h and then heat pulsing at 42 degrees C for 1 min. Some plasmid markers are expressed immediately, whereas others require time for phenotypic expression.     

Oxidation of n-alkanes: Growth of Pseudomonas putida

Summary The induction of alkane hydroxylase activity was investigated in two strains of Pseudomonas putida with a view to the production of primary alcohols. n-Nonanol production rates (16.0 mol/g dry wt/h) with an alcohol dehydrogenase negative mutant P. putida PpS173 were considerably lower than might be expected from the growth of a wild type on n-alkane. Production of cells by fed-batch culture on n-nonane, with a specific alkane hydroxylase activity of 3.9 mmol/g/h, was considered most suitable for isolation of the alkane hydroxylase.     

铜绿假单胞菌SJTD-2降解长链烷烃与原油的特性

摘要:【目的】石油污染严重威胁生态系统和生物圈,微生物修复被认为是一种安全有效可代替物化方法来治理石油污染的办法。本文对我们从石油污染土壤中分离获得的一株可分解正烷烃和原油的革兰氏阴性菌SJTD-2的理化性质和降解效能进行了研究。【方法】利用菌株表型和生理性质、16S rRNA序列比较分析与进化树绘制,确定新分离菌株SJTD-2的种属;测定菌株SJTD-2的生长曲线,确定其利用不同长度烷烃和原油为单一碳源的效能;利用GC-MS检测烷烃类物质的残留量,确定菌株SJTD-2降解烷烃和原油SJTD-2的降解效率和降解周期。【结果】菌株表型与16S rRNA序列比较及进化树比对分析结果显示,菌株SJTD-2与假单胞菌属的亲缘关系十分接近,为铜绿假单胞菌。菌株SJTD-2 可有效分解C10到C26的中链和长链烷烃及原油,利用它们作为其单一碳源生长;该菌株对长链烷烃(C18－C22)的利用效果较中链烷烃好,其中正二十二烷被认为是其最佳碳源。48 h内,该菌株可完全降解500 mg/L正二十二烷;72h 后,2 g/L的正二十二烷可几乎被菌株全部分解利用。此外,菌株SJTD-2在7 d内可将2 g/L的原油分解88%以上。【结论】与现有其它烷烃降解菌相比,铜绿假单胞菌SJTD-2具有突出的长链烷烃与原油降解效能及耐受能力,该菌株的发现与研究将有助于烷烃降解机制的研究和环境修复的进程。     

FP2 plasmid curing in Pseudomonas aeruginosa

A procedure for the elimination of the IncP-8 plasmid FP2 from Pseudomonas aeruginosa strain 1 was developed. The procedure consists of freezing cells, competent for transformation, in 15% glycerol at -70 degrees C for at least 48 h and screening survivors for loss of mercuric chloride resistance. Curing frequencies of 0.5% were achieved only in host cells carrying a dht mutation (unable to convert thymine to dihydrothymine).     

Oxidation of D- and L-valine by enzymes of Pseudomonas aeruginosa

Norton, J. E. (University of Oklahoma School of Medicine, Oklahoma City), and J. R. Sokatch. Oxidation of d- and l-valine by enzymes of Pseudomonas aeruginosa. J. Bacteriol. 92:116-120. 1966.-Cell-free extracts prepared from Pseudomonas aeruginosa grown on dl-valine catalyzed the consumption of oxygen with several d-amino acids, but not with the corresponding l-amino acids. The product of d-valine oxidation was identified as 2-oxoisovalerate by the preparation and characterization of 2-oxoisovalerate 2,4-dinitrophenylhydrazone. The enzyme catalyzing d-amino acid oxidation was present in extracts of cells grown on valine, but not on glucose, had a pH optimum of approximately 9.0, consumed 1 atom of oxygen per mole of keto acid produced, and was not stimulated by any of the usual electron transport cofactors. It was not possible to demonstrate either the direct oxidation of l-valine or the conversion of l- to d-valine by these enzyme preparations. However, a possible route of l-valine metabolism by transamination with 2-oxoglutarate with regeneration of the amino group acceptor by glutamate oxidation was established by identification of the transaminase and l-glutamate dehydrogenase in these enzyme preparations.     

Regulation of the synthesis of the key enzymes for naphthalene catabolism in Pseudomonas putida and Pseudomonas fluorescens carrying the biodegradation plasmids NAH, pBS3, pBS2 and NPL-1

Regulation of the synthesis of key enzymes catalysing naphthalene catabolism was studied in Pseudomonas strains containing different plasmids of naphthalene biodegradation. The synthesis of naphthalene oxygenase, salicylate hydroxylase, catechol-1,2-oxygenase and cathechol-2,3-oxygenase was shown to be regulated in both the coordinated and non-coordinated manner.     

Purification and properties of two enzymes of meta-cleaving the aromatic ring controlled by the biphenyl biodegradation plasmid pBS 241 from Pseudomonas putida

It was shown that two metapyrocatechases (EC 1.13.11.2) function in Pseudomonas putida BS893. Biphenyl degradative plasmid pBS241 carries the genes of these enzymes. The basic properties of the both enzymes, i. e., MPC1 and MPC2, were investigated. It was found that MPC1 is an enzyme with a molecular mass of 135 kD and has a heterotetrameric subunit structure (alpha 2 beta 2), being made up of two non-identical polypeptides with Mr of 34 and 22.5 kD; pI is 5.15, the pH optimum is at 8.0, a temperature optimum is at 54 degrees C. MPC2 has a molecular mass of 154 kD and possesses a homotetrameric subunit structure (alpha 4); it consists of identical polypeptides with Mr of 41 kD and has a pI of 4.95, a pH optimum at 7.5 and a temperature optimum at 60 degrees C. The substrate specificity of the enzymes was studied, and the Km and Vmax values for substituted catechols were determined. MPC1 shows a high affinity for 2.3-dihydroxybiphenyl and hydrolyzes 3-methylcatechol and catechol (but not 4-methylcatechol) at a low rate. MPC2 has a moderate affinity for catechol, 3- and 4-methylcatechols, but is incapable of cleaving 2.3-dihydroxybiphenyl. Both enzymes share in common some typical properties of metapyrocatechases. The different role of MPC1 and MPC2 in biphenyl catabolism is discussed.