Showing papers on "Pseudomonas putida published in 1982"

Plasmid gene organization: naphthalene/salicylate oxidation.

Abstract: Genes for naphthalene metabolism are localized on nah7, an 83-kilobase (kb) plasmid, in two gene clusters under salicylate control. Polar mutations formed by insertion of the transposon Tn5 permit detection of the transcription direction and the gene organization within two approximately 10-kb DNA segments separated by a approximately 7-kb regulatory gene region. The gene cluster specifying conversion of naphthalene to salicylate lies near the left initiation of a 25-kb DNA fragment A released by EcoRI; that for the salicylate pathway via catechol meta-fission lies near the right terminus with extension into the adjoining 5.9-kb fragment C. The genetic organization and regulation resemble the tol plasmid-encoded "upper" and "lower" pathways of toluene/xylene oxidation in Pseudomonas putida mt2.

Stereospecificity of 2-monochloropropionate dehalogenation by the two dehalogenases of Pseudomonas putida PP3: evidence for two different dehalogenation mechanisms

Abstract: SUMMARY: Pseudomonas putida PP3 grew on dl-2-monochloropropionate (2MCPA) with a release of chloride ions consistent with the dechlorination of both isomers. The organism grew on either d- or l-2MCPA. Dehalogenase activity in cell-free extracts showed that both d- and l-2MCPA were dehalogenated. Pseudomonas putida PP3 contains two dehalogenases, and studies with the separated enzymes revealed that the fraction I enzyme used both d- and l-2MCPA, the rate of dechlorination of l-2MCPA being 80% of the rate of d-2MCPA dechlorination. The product of the reaction, lactate, retained the same optical configuration as the substrate provided. The fraction II dehalogenase also dechlorinated d- and l-2MCPA, with the same difference in rates as for the fraction I dehalogenase, but the lactates produced were of the opposite configuration to their precursors. The two dehalogenases showed further differences with respect to inhibition by two sulphydryl-blocking agents, N-ethylmaleimide and p-chloromercuribenzoate. Fraction I dehalogenase was considerably more sensitive to these two reagents compared with the fraction II dehalogenase. Dithiothreitol partially protected the fraction I dehalogenase from N-ethylmaleimide inhibition. The results are discussed in terms of the possible evolutionary relationships of the two dehalogenases.

TOL plasmid pWW0 in constructed halobenzoate-degrading Pseudomonas strains: prevention of meta pathway.

Abstract: The hybrid pathway for chlorobenzoate metabolism was studied in WR211 and WR216, which were derived from Pseudomonas sp. B13 by acquisition of TOL plasmid pWW0 from Pseudomonas putida mt-2. Chlorobenzoates are utilized readily by these strains when meta cleavage of chlorocatechols is suppressed. When WR211 utilizes 3-chlorobenzoate (3CB), the expression of catechol 2,3-dioxygenase (C23O) and the catabolic activities for chloroaromatics via the ortho pathway coexist as a consequence of inactivation of the meta cleavage activity by 3-chlorocatechol. Utilization of 4-chlorobenzoate (4CB) by WR216 presupposes the suppression of C23O by a spontaneous mutation in the structural gene, so that 4-chlorocatechol is not misrouted into the meta pathway. Such C23O- mutants were also selected when WR211 was grown continuously on 3CB. Our data explain why the phenotypic characters 3CB+ and Mtol+ (m-toluate) are compatible, whereas 4CB+ and Mtol+ are incompatible.

Monoclonal antibodies against Pseudomonas aeruginosa outer membrane antigens: isolation and characterization.

Abstract: Hybridomas secreting monoclonal antibodies specific for Pseudomonas aeruginosa outer membrane antigens were isolated. One of the antibodies was highly specific for the O antigen of the lipopolysaccharide of International Antigen Typing Scheme serotype 5 strains, reacting only weakly with a serotype 17 strain and failing to react with the outer membranes of strains representing 15 other serotypes. This monoclonal antibody was able to agglutinate heat-killed bacterial cells as well as lipopolysaccharide-coated sheep erythrocytes. Two other monoclonal antibodies were able to interact with the outer membranes of strains representing all 17 serotypes, although they were unable to agglutinate heat-killed bacterial cells. One of these was shown to be specific for the major outer membrane lipoprotein H2. The antigenic site against which this monoclonal antibody reacted was present in the outer membranes of two Pseudomonas fluorescens strains, two Pseudomonas putida strains, a Pseudomonas anguilliseptica strain, and an Azotobacter vinelandii strain, but not in the outer membranes of five other bacterial species.

The Plasmid-coded Metabolism of Naphthalene and 2-Methylnaphthalene in Pseudomonas Strains: Phenotypic Changes Correlated with Structural Modification of the Plasmid pWW60-1

Abstract: SUMMARY: Pseudomonas sp. NCIB 9816 contains two plasmids: pWW60, an IncP9 plasmid of 87 kb encoding genes for the catabolism of naphthalene, and pWW61, a cryptic plasmid of about 65 kb. The ability to degrade naphthalene was transferred at low frequency by conjugation from strain NCIB 9816 into a plasmid-free strain of Pseudomonas putida, PaW340. A transconjugant, PaW701, containing the naphthalene plasmid pWW60-1, metabolized naphthalene and salicylate via the ortho pathway. 2-Methylnaphthalene was not a growth substrate but was partly metabolized with accumulation of a brown compound in the medium (λmax = 440 nm). Spontaneous mutants of PaW701 with the ability to grow on 2-methylnaphthalene arose at a frequency of about 10-5. These fell into two groups. Group A mutants had no detectable salicylate hydroxylase activity and accumulated salicylate from naphthalene in culture supernatants: they appeared to grow on the pyruvate released from oxidation of the first ring of both substrates. Their plasmids all contained a 16.7 kb insert in different sites within a small, limited region of the plasmid. Group B mutants used a meta pathway for catabolism of naphthalene and 2-methylnaphthalene. Their plasmids had undergone a small deletion of from 1.2 to 1.6 kb in a region of the plasmid close to the sites of the insertions in the group A mutants.

TOL plasmid pWW0 in constructed halobenzoate-degrading Pseudomonas strains: enzyme regulation and DNA structure

Abstract: WR211 and WR216 are derivatives of halobenzoate-degrading Pseudomonas sp. strain B13 into which the 117-kilobase TOL degradative plasmid pWW0 has been transferred from Pseudomonas putida mt-2. WR211 has lost the ability to grow on the TOL-specific substrate m-xylene but retains the ability to grow on its metabolite, m-toluate. An analysis of the induction of enzymes was consistent with WR211 carrying a nonfunctional regulatory gene, xy1R, WR216 is a spontaneous derivative of WR211 which grows on one of the TOL substrates and yet expresses the nonspecific toluate oxidase, which enables it to grow on the novel substrate 4-chlorobenzoate. In addition to the xy1R lesion inherited from WR211, WR216 appears to carry a mutation in the structural gene for catechol 2,3-oxygenase, xy1E. The plasmids in both strains were analyzed by restriction endonuclease digestion. pWW0-1211 in WR211 has a large deletion (39 kilobases) compared with pWW0 and appears to be identical to a previously described plasmid (pWW0-8) which encodes none of the TOL degradative functions. pWW0-1216 in WR216 has undergone a major structural reorganization relative to its parent, pWW0-1211. This plasmid has a smaller deletion (19 kilobases), which is staggered relative to the deletion in pWW0-1211, and in addition it has two 3-kilobase insertions of unknown origin, one of which appears to cause the xylE mutation.

The TOL Plasmid is Naturally Derepressed for Transfer

Abstract: SUMMARY: Pseudomonas putida mt-2, formerly known as Pseudomonas arvilla mt-2, which carries the wild-type TOL plasmid, and P. putida strain AC37 carrying TOL, were completely lysed by the pilus-adsorbing plasmid-specific bacteriophages PR4 and PRD1. Pseudomonas putida strain PpS388, also harbouring the plasmid, was not lysed. In a P. putida mt-2 host, TOL transferred 18-fold better on a surface (2·5 × 10-1 transconjugants per donor h-1) than in liquid; when P. putida PpS388 was the host, however, a frequency of only 2·3 × 10-4 transconjugants per donor h-1 was obtained. Thus, TOL was derepressed for transfer in P. putida mt-2 and P. putida AC37, but not in P. putida PpS388. Electron microscopy revealed that TOL determined thick (8·5--10 nm diameter) flexible pili in large numbers, suggesting constitutive expression in its derepressed state.

Method for protecting the growth of plants employing mutant siderophore producing strains of Pseudomonas Putida

Abstract: Plants susceptible to pathogenic fungi are contacted with a mutant strain of Pseudomonas putida, particularly Pseudomonas putida NRRL-B-12537 which produces iron complexing siderophores thereby affording protection from the fungi. The Pseudomonas competes with the fungi for iron found in the soil thereby inhibiting the fungi growth. The method is particularly effective in controlling Fusarium oxysporum Sp lycopersici on tomato plants.

The metabolism of cyclohexanecarboxylic acid and 3-cyclohexenecarboxylic acid by Pseudomonas putida

Abstract: A strain of Pseudomonas putida grew rapidly on cyclohexanecarboxylic acid as a sole source of carbon. A CoA-mediated β-oxidation pathway was induced for the metabolism of the compound. The organism...

Active transport of benzoate in Pseudomonas putida.

Abstract: SUMMARY: Benzoate uptake in Pseudomonas putida is mediated by an active transport system capable of accumulating benzoate against a 150-fold concentration gradient when subsequent metabolism is blocked by mutation. Initial benzoate transport rates are inhibited by CCCP, sodium azide, arsenate and DCCD. Uptake is stimulated by including a respirable carbon source during preincubation of the bacteria. The initial uptake rate and the ATP pool levels are not correlated and no periplasmic components were found to bind benzoate. These observations indicate that benzoate uptake is energized by the membrane potential, rather than by ATP hydrolysis.

Identification of chromosomally integrated TOL DNA in cured derivatives of Pseudomonas putida PAW1.

Abstract: Some plasmid-free Tol- strains derived from Pseudomonas putida PAW1 (which carries the TOL plasmid pWW0) have a segment of TOL DNA located chromosomally. Of three independently isolated strains, PAW86 had an integrated TOL segment of 16 kilobases and PAW85 had two copies of this segment in different chromosomal locations, whereas the chromosomal DNA of PAW82 showed no homology with the TOL plasmid. In cultures of the parental strain, it appears that a 56-kilobase TOL DNA segment is located chromosomally in some cells.

delta1-piperideine-2-carboxylate reductase of Pseudomonas putida.

Abstract: Pseudomonas putida metabolizes D-lysine to delta 1-piperideine-2-carboxylate and L-pipecolate. The second step of this catabolic pathway is catalyzed by delta 1-piperideine-2-carboxylate reductase. This enzyme was isolated and purified from cells grown on DL-lysine as substrate. The enzyme was very unstable, resulting in low recovery of activity and low purity after a six-step purification procedure. The enzyme had a pH optimum of 8.0 to 8.3. The Km values for delta 1-piperideine-2-carboxylate and NADPH were 0.23 and 0.13 mM, respectively. NADPH at concentrations above 0.15 mM was inhibitory to the enzyme. Delta 1-pyrroline-5-carboxylate, pyroglutamate, and NADH were poor substrates or coenzyme for delta 1-piperideine-2-carboxylate reductase. The enzyme reaction from delta 1-piperideine-2-carboxylate to L-pipecolate was irreversible. EDTA, sodium pyrophosphate, and dithiothreitol at concentrations of 1 mM protected the enzyme during storage. The enzyme was inhibited almost totally by Zn2+, Mn2+, Hg2+ Co2+, and p-chloromercuribenzoate at concentrations of 0.1 mM. The enzyme had a molecular weight of about 200,000. Both D-lysine and L-lysine were good inducers for the enzyme. Neither delta1-piperideine-2-carboxylate nor L-pipecolate was an effective inducer for the enzyme. P. putida cells grew on D-lysine only after a 5- to 8-h lag, which could be abolished by adding a supplement of 0.01% alpha-ketoglutarate or other readily metabolizable compounds. Such a supplement also converted the noncoordinate induction of this enzyme and pipecolate oxidase, both of the D-lysine pathway, to coordinacy. However, this effect was not observed if the enzyme pair was from different pathways of lysine metabolism in this organism (i.e., the D- and L-lysine pathways).

Spontaneous deletions in the TOL plasmid pWW20 which give rise to the B3 regulatory mutants of Pseudomonas putida MT20.

Abstract: Summary: The size of the TOL plasmid pWW20 from Pseudomonas putida MT20, as measured by analysis of agarose electrophoresis gels after restriction endonuclease hydrolysis, was 270-280 kilobase pairs (kb). During growth on benzoate, MT20 segregates strains carrying mutations in the plasmid regulatory gene xylS; these so-called B3 strains retain the ability to grow on m-xylene (Mxy+) but do not grow on its metabolite m-toluate (Mtol−) and have also lost the ability to transfer the plasmid (Tra−). Analysis of restriction digests of plasmid DNA from seven such segregants, independently isolated, showed that pWW20 had undergone extensive deletions of 90–100 kb. All the deleted plasmids had lost a common core of DNA, of about 72–80 kb, but in class A mutants the deletion extended at one end of this core and in class B mutants at the other end. Class A and B mutants also differed in their rate of growth on m-xylene as a result of differences in the level of expression of their plasmid-coded catabolic enzymes. This suggests that an additional gene, involved in regulating levels of gene expression, is located in the region uniquely deleted in the class B mutants.

Degradation of caffeine by immobilized cells of Pseudomonas putida strain C 3024

Abstract: A caffeine-resistant strain of Pseudomonas putida was isolated from soil and was grown with caffeine as the sole source of carbon, energy and nitrogen. Cells were immobilized in agar gel particles which were continuously supplied with a caffeine solution (0.52 g · l−1, D=1.0 h−1) in a homogeneously mixed aerated reaction vessel. In the presence of the ATPase inhibitor arsenate the caffeine was removed by the immobilized cells at an average rate of 0.25 mg caffeine · h−1 · (mg cell carbon)−1 during 6 days. Thereafter a rapid decline of activity was observed. From a similar system without arsenate supplied with a growth medium containing a limiting amount of caffeine (0.13 g · l−1) the caffeine was almost completely oxidized by the immobilized cells. The concentration of the remaining caffeine was 1.4 mg · l−1, which is much lower than the substrate constant for caffeine (9.7 mg · l−1) observed with freshly harvested suspended resting cells.

Assimilation of γ-butyrobetaine, and d -and l -carnitine by resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida

Abstract: The metabolic pattern of utilization of [1,2,3,4-14C, methyl-3H] γ-butyrobetaine and d-and l-[1-14C, methyl-3H]carnitine has been examined with variously grown resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida. Ps. putida grown on d, l-carnitine as the sole source of carbon, degraded only l-carnitine with stoichiometric accumulation of glycinebetaine. Alternatively, when grown on γ-butyrobetaine, Ps. putida rapidly metabolized γ-butyrobetaine, and to a lesser but significant extent, both d-and l-carnitine with equivalent formation of trimethylamine and degradation of the betaine carbon skeleton. Ac. calcoaceticus grown on either d,l-carnitine or γ-butyrobetaine, effectively utilized all three betaines at nearly the same rates. Disappearance of each of these quarternary ammonium compounds was accompanied by stoichiometric formation of trimethylamine and degradation of the carbon backbone. Utilization of the betaines and corresponding formation of trimethylamine by resting cell suspensions of appropriately grown Ac. calcoaceticus and Ps. putida, was essentially abolished under conditions of anaerobiosis and severely impaired in the presence of sodium cyanide, sodium azide, 2,4-dinitrophenol or 2,2′-bipyridine. The results of the present investigations with resting cell suspensions of both Ac. calcoaceticus and Ps. putida do not support an earlier suggestion that γ-butyrobetaine degradation in these organisms proceeds by its prior hydroxylation to l-carnitine. Indeed, disrupted cell-free preparations of Ac. calcoaceticus and Ps. putida grown on either d,l-carnitine or γ-butyrobetaine showed no detectable γ-butyrobetaine hydroxylase activity.

Biofilm build-up of Pseudomonas putida in a chemostat at different dilution rates

Abstract: A test system was set up where the build-up of a biofilm on a defined surface could be studied in a carbon source limited chemostat. The attachment of P. putida ATCC 11172 to glass when growing on L-asparagine was studied at different dilution rates (specific growth rates) from 0.1 to 1.5 h−1 The number of attached colony forming units (cfu) increased with dilution rate from 1×106 cfu/cm2 at 0.1 h−1 to 4×107 cfu/cm2 at 1.0 h−1 and then the attachment decreased to about 6×106 cfu/cm2 at higher dilution rates (1.1–1.5 h−1). The number of attached cfu was measured after 24 h exposure. The value of the maximum specific growth rate in batch culture was 0.6 h−1. The total amount of attached cell-mass followed roughly the same pattern as the viable count. The viable count of the cells suspended in the growth medium showed its lowest value at the same dilution rate as resulted in maximum adhesion. It was shown that the effect of growth rate on the biofilm build-up of P. putida is significant, and ought to be borne in mind when continuous culture systems are set up and results evaluated.

Oxidation of n-alkanes: Growth of Pseudomonas putida

Abstract: 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 μmol/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.

A model for noninhibitory microbial growth.

Abstract: A model for noninhibitory microbial growth has been developed which is superior to the Monod model in that it can predict the decline in steady-state growth yields at both the slow and the fast specific growth rates. The model parameters are evaluated from data obtained for steady-state, phenol-limited Pseudomonas putida growth using a conventional 1-dm(3) cheniostat. The model also has been successfully applied to Mor and Fiechter's data for cheniostat yeast cultures.

Pseudomonas degradation of hydrocarbons

Abstract: Selected new bacteria of the genus Pseudomonas, particularly the species Pseudomonas putida and Pseudomonas aeruginosa, which have the ability to utilize organic compounds from the generic groups aliphatic, cyclo aliphatic, aromatic and/or polynuclear aromatic hydrocarbons are described. The source of genetic materials facilitating degradation of the aromatic compounds are metabolic plasmids. In particular, Pseudomonas putida or other Pseudomonas obtained from soil samples and having a non-transmissible and stable ability to degrade hexane (as well as related aliphatic hydrocarbons) are used as starting strains to produce the new bacteria. Transconjugal mating and selection for these genetic traits resulted in the production of bacteria capable of utilizing representative compounds of all the generic groups of the previously listed organic compounds. The bacteria are useful for waste degradation.

[Integration of the genome of the Mu-like Pseudomonas aeruginosa bacteriophage D3112 into plasmid RP4 and its hybrid plasmid transfer into Pseudomonas putida and Escherichia coli C600 bacteria].

Abstract: The genome of a Mu-like bacteriophage D3112 specific for Pseudomonas aeruginosa was integrated in vivo into the RP4 plasmid. The fact of integration has been proved by two experiments: 1. The loss of RP4 plasmid is accompanied by loss of D3112 prophage; 2. Transfer of the plasmid by conjugation from Pseudomonas aeruginosa into bacteria of other species - P. putida PgG1 or Escherichia coli C600 leads to the occurrence of clones of these species which liberate phage capable of growing on the lawn of P. aeruginosa bacteria. The integrated state of D3112 inserted into RP4 is stable in P. aeruginosa, E. coli, P. putida. The transfer frequency of RP4 with integrated D3112 prophage into different bacteria which do not contain homoimmune prophage is essentially lower than that of the RP4 having no D3112 prophage. Specific manifestation of D3112 genome activity in E. coli cells is the sensitivity of cell growth to lower temperature (30 degrees C) of incubation.

Microorganism capable of degrading phenolics

Abstract: Mutant microorganism, Pseudomonas putida CB-173 degrading phenolics, and at a temperature as low as, e.g., about 1° to 4° C., at a faster rate than known Pseudomonas putida type strains, and process for treating wastewater containing phenolics using the mutant microorganism strain Pseudomonas putida CB-173.

Production of para-cresol

Abstract: The present invention provides a process for the production of p-cresol in a quantitative yield, which involves the acidification of an aqueous solution of 4-methylcyclohexa-3,5-diene-1,2-diol-1-carboxylic acid under ambient conditions of temperature and pressure to cause spontaneous decomposition of the starting material to p-cresol. The 4-methylcyclohexa-3,5-diene-1,2-diol-1-carboxylic acid is produced by the conversion of p-xylene with the microorganism, Pseudomonas putida Biotype A strain ATCC No. 39119.

[Expression of the genome of Mu-like phage D3112 specific for Pseudomonas aeruginosa in Escherichia coli and Pseudomonas putida cells].

Abstract: The behavior of Escherichia coli cells carrying RP4 plasmid which contains the genome of a Mu-like D3112 phage specific for Pseudomonas aeruginosa was studied. Two different types of D3112 genome expression were revealed in E. coli. The first is BP4-dependent expression. In this case, expression of certain D3112 genes designated as "kil" only takes place when RP4 is present. As a result, cell division stops at 30 degrees C and cells form filaments. Cell division is not blocked at 42 degrees C. The second type of D3112 genome expression is RP4-independent. A small number of phage is produced independently of RP4 plasmid but this does not take place at 42 degrees C. No detectable quantity of the functionally active repressor of the phage was determined in E. coli (D3112). It is possible that the only cause for cell stability of E. coli (D3112) or E. coli (RP4::D3112) at 42 degrees C in the absence of the repressor is the fact of an extremely poor expression of D3112. In another heterologous system, P. putida both ways of phage development (lytic and lysogenic) are observed. This special state of D3112 genome in E. coli cells is proposed to be named "conditionally expressible prophage" or, in short, "conex-phage", to distinguish it from a classical lysogenic state when stability is determined by repressor activity. Specific blockade of cell division, due to D3112 expression, was also found in P. putida cells. It is evident that the kil function of D3112 is not specific to recognize the difference between division machinery of bacteria belonging to distinct species or genera. Protein synthesis is needed to stop cell division and during a short time period this process could be reversible. Isolation of E. coli (D3112) which lost RP4 plasmid may be regarded as an evidence for D3112 transposition in E. coli. Some possibilities for using the system to look for E. coli mutants with modified expression of foreign genes are considered.