Showing papers on "Pseudomonas putida published in 1973"
TL;DR: The capacity of Pseudomonas putida PpG7 to grow on naphthalene, phenotype Nah(+), is lost spontaneously, and the frequency is increased by treatment with mitomycin C, and this character can be transferred to cured or heterologous fluorescent pseudomonads lacking this capacity by conjugation.
Abstract: The capacity of Pseudomonas putida PpG7 (ATCC 17,485) to grow on naphthalene, phenotype Nah+, is lost spontaneously, and the frequency is increased by treatment with mitomycin C. The Nah+ growth character can be transferred to cured or heterologous fluorescent pseudomonads lacking this capacity by conjugation, or between phage pf16-sensitive strains by transduction. After mutagenesis, strains can be selected with increased donor capacity in conjugation. Clones which use naphthalene grow on salicylate and carry catechol 2,3-oxygenase, the initial enzyme of the aromatic α-keto acid pathway, whereas cured strains grow neither on salicylate nor naphthalene and lack catechol 2,3-oxygenase, but retain catechol 1,2-oxygenase and the aromatic β-keto adipate pathway enzymes.
376 citations
TL;DR: An hypothesis is presented of a general mechanism for the formation and maintenance of metabolic diversity, and two gene linkage groups are shown for the reactions before and after isobutyrate.
Abstract: Earlier papers demonstrated an extensive genetic exchange among fluorescent Pseudomonads; this one documents for genes specifying enzymes of peripheral dissimilation an extrachromosomal array, segregation, and frequent interstrain transfer. An hypothesis is presented of a general mechanism for the formation and maintenance of metabolic diversity. The example used, the path of oxidative cleavage of the carbocyclic rings of the bicyclic monoterpene D- and L-camphor, terminates in acetate release and isobutyrate chain debranching. By transduction, two gene linkage groups are shown for the reactions before and after isobutyrate. The group for reactions before isobutyrate is plasmid borne, contransferable by conjugation, mitomycin curable, and shows a higher segregation rate from cells that are multiplasmid rather than carrying a single plasmid. The genes that code for isobutyrate and essential anaplerotic and amphibolic metabolism are chromosomal. By conjugation plasmid-borne genes are transferred at a higher frequency than are chromosomal, and are transferred in homologous crosses more frequently than between heterologous species. Most isobutyrate-positive fluorescent pseudomonad strains will accept and express the camphor plasmid.
208 citations
TL;DR: Findings indicate that the strain mt-2 possesses the genetic capacity for enzymes of both the meta- and ortho-cleavage pathways of benzoate degradation, but its phenotypic expression is the meta pathway.
Abstract: Benzoate-grown cells of Pseudomonas putida(arvilla) mt-2 contain both metapyrocatechase and pyrocatechase activities, although the former activity is much higher than that of the latter. A spontaneous mutant deficient in metapyrocatechase and 2-hydroxymuconic semialdehyde hydrolyase, the first two enzymes in the meta-cleavage pathway of the ring of catechol, has been isolated from this strain. This mutant grows well on a minimal medium containing benzoate as a sole carbon source and has the high activity of pyrocatechase. These findings indicate that the strain mt-2 possesses the genetic capacity for enzymes of both the meta- and ortho-cleavage pathways of benzoate degradation, but its phenotypic expression is the meta pathway.
154 citations
124 citations
TL;DR: This strain was used to show that the products of meta ring fission of the cresols and phenol are metabolized as follows: ortho- and meta-cresol exclusively by a hydrolase; para- cresol entirely by a NAD(+)-dependent aldehyde dehydrogenase; phenol by both a NAD (+)- dependent dehydrogen enzyme and a Hydrolase in the approximate ratio of 5 to 1.
Abstract: Mutant strains of Pseudomonas putida strain U have been obtained which are deficient in enzymes of the degradative pathways of phenol and cresols. Mutant strains deficient in catechol 2, 3-oxygenase accumulated the appropriate catechol derivative from cresols. A mutant strain which would not grow on either phenol or a cresol was shown to be deficient in both 2-hydroxymuconic semialdehyde hydrolase and a nicotinamide adenine dinucleotide, oxidized form, (NAD+)-dependent aldehyde dehydrogenase. When this strain was grown in the presence of phenol or a cresol, the appropriate product of meta fission of these compounds accumulated in the growth medium. A partial revertant of this mutant strain, which was able to grow on ortho- and meta-cresol but not para-cresol, was shown to have regained only the hydrolase activity. This strain was used to show that the products of meta ring fission of the cresols and phenol are metabolized as follows: (i) ortho- and meta-cresol exclusively by a hydrolase; (ii) para-cresol exclusively by a NAD+-dependent aldehyde dehydrogenase; (iii) phenol by both a NAD+-dependent dehydrogenase and a hydrolase in the approximate ratio of 5 to 1. This conclusion is supported by the substrate specificity and enzymatic activity of the hydrolase and NAD+-dependent aldehyde dehydrogenase enzymes of the wild-type strain. The results are discussed in terms of the physiological significance of the pathway. Properties of some of the mutant strains isolated are discussed.
121 citations
TL;DR: It was demonstrated that l-(S)-4-hydroxy-2-oxohexanoate is the biologically active enantiomer of this hydroxy acid, and a mechanism is proposed which closely resembles that for 4-Hydroxy- 2-oxopentanoate aldolase.
Abstract: Two reactions in the catabolism of catechol by meta-fission, namely, hydration of 2-oxopent-4-enoate (vinylpyruvate) and aldol fission of the product, are catalyzed by stereospecific enzymes. The absolute configuration of this hydration product was shown to be l(S)-4-hydroxy-2-oxopentanoate. Vinylpyruvate hydratase, purified almost to homogeneity, had a molecular weight of about 287,000 and was dissociated in sodium dodecyl sulfate, without prior treatment with mercaptoethanol, into a species with an approximate molecular weight of 28,000. The hydratase was highly specific for its substrates; thus, although 2-oxo-cis-hex-4-enoate was also hydrated, structurally similar compounds such as the trans isomer, vinylacetic and crotonic acids, and the ring-fission products of catechol and methylcatechols were not attacked. Vinylpyruvate hydratase was activated by Mn(2+) ions. On the basis of these observations, a mechanism is proposed which closely resembles that for 4-hydroxy-2-oxopentanoate aldolase. A possible evolutionary connection between functionally related, divalent cation-activated hydro-lyases and aldolases is discussed. It was also demonstrated that l-(S)-4-hydroxy-2-oxohexanoate is the biologically active enantiomer of this hydroxy acid.
97 citations
TL;DR: The presence in the same reaction mixtures of either 4-hydroxy-2-oxovalerate or 2-hydroxypenta-2,4-dienoate was suggested by mass spectrometry and the degradative pathway of biphenyl is discussed.
Abstract: 1 2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid was isolated and identified from washed suspensions of Pseudomonas putida incubated in the presence of 2,3-dihydroxybiphenyl 2 Benzoic acid was isolated from reaction mixtures of crude cell-free extracts incubated with 2,3-dihydroxybiphenyl 3 The presence in the same reaction mixtures of either 4-hydroxy-2-oxovalerate or 2-hydroxypenta-2,4-dienoate was suggested by mass spectrometry 4 The degradative pathway of biphenyl is discussed
88 citations
88 citations
87 citations
TL;DR: The study of the behavior of mutants isolated from Pseudomonas putida shows that this organism possesses a single glucolytic pathway with physiological significance, and the Entner-Doudoroff enzymes, which catalyze the conversion of gluconate-6-phosphate into pyruvate and trioseph phosphate, appear to be essential to grow on glucose and also on glUconate and 2-ketogluconate.
Abstract: A number of mutants in which glucolysis is impaired have been isolated from Pseudomonas putida. The study of their behavior shows that this organism possesses a single glucolytic pathway with physiological significance. The first step of the pathway consists in the oxidation of glucose into gluconate. Two proteins with glucose dehydrogenase activity appear to exist in P. putida but the reasons for this duplicity are not clear. The process continues with the formation of 2-ketogluconate which is in turn converted into gluconate-6-phosphate. This is proved by the fact that mutants unable to form gluconate-6-phosphate from 2-ketogluconate show extremely slow growth on glucose or gluconate (generation times are increased more than 100 times). Other possible routes for the conversion of glucose into gluconate-6-phosphate, the glucose-6-phosphate pathway, or the direct phosphorylation of the gluconate formed by glucose oxidation are only minor shunts in P. putida. The Entner-Doudoroff enzymes, which catalyze the conversion of gluconate-6-phosphate into pyruvate and triosephosphate, appear to be essential to grow on glucose and also on gluconate and 2-ketogluconate. A significative role of the pentose route in the catabolism of these substrates is not apparent from this study. In contrast, P. putida strains showing no activity of the Entner-Doudoroff enzymes grow readily on fructose, although there is evidence that this hexose is at least partially catabolized via gluconate-6-phosphate.
78 citations
TL;DR: The interaction of substrates with a 4-methoxybenzoate O-demethylating enzyme system was studied by use of crude cell-free extracts and by the purified enzyme system, which requires NADH and oxygen as cofactors and acts on various substrates.
Abstract: The interaction of substrates with a 4-methoxybenzoate O-demethylating enzyme system was studied by use of crude cell-free extracts and also by the purified enzyme system. The two components of the enzyme system, an iron-containing flavoprotein and an iron-sulphur protein, were obtained in pure state from Pseudomonas putida grown on 4-methoxybenzoate as the sole carbon source. The purified enzyme system requires NADH and oxygen as cofactors and acts on various substrates. The highest affinity is found for 4-methoxybenzoate, but also N-methyl-4-aminobenzoate is demethylated and 4-alkylbenzoates are hydroxylated at the side chain. The enzyme is rather specific for para-substituted benzoic acid derivatives whereas 3-methoxybenzoate and 4-hydroxy-3-methoxybenzoate are demethylated slowly. The enzyme is also able to hydroxylate the aromatic ring. This is shown by the isolation of 3,4-dihydroxybenzoate as the hydroxylation product of 3-hydroxy- or 4-hydroxy-benzoate, respectively. Studies on substrate binding and oxygen consumption with substrate analogues showed an absolute requirement for the carboxy group at the aromatic ring. Benzoic acid derivatives without a suitable CH-bond uncouple oxygen uptake with a concomitant formation of hydrogen peroxide. Measurements of oxygen consumption indicate that the affinity towards oxygen is substrate dependent, probably due to steric alterations as a consequence of substrate binding.
TL;DR: The two degradative plasmids, CAM and OCT, are incompatible with each other and cannot coexist in the same cell, but by the use of UV irradiation and suitable selection techniques, they are fuse so they become part of the same replicon and coexist.
Abstract: The genes specifying enzymes responsible for the degradation of camphor and octane occur on transmissible plasmids in Pseudomonas putida strain PpG1 and P. oleovorans. Since the presence of the plasmids is vital for the oxidative metabolism of camphor or octane (by the cells) in the absence of other carbon sources, such naturally occurring, energy-generating plasmids have been designated as degradative plasmids. The two degradative plasmids, CAM and OCT, are incompatible with each other and cannot coexist in the same cell. By the use of UV irradiation and suitable selection techniques, it has been possible to fuse these two plasmids so they become part of the same replicon and coexist. Such a technique might be useful in introducing several degradative pathways in the same cell.
TL;DR: Strains of Pseudomonas producing fluorescin but no pyocyanin or pyorubrin were studied by biochemical and antibiotic sensitivity testing and a rapid nitrate test was found to be useful in distinguishing P. aeruginosa from P. putida.
Abstract: Strains of Pseudomonas producing fluorescin but no pyocyanin or pyorubrin were studied by biochemical and antibiotic sensitivity testing. A rapid nitrate test was found to be useful in distinguishing P. aeruginosa (positive) from P. fluorescens and P. putida (both negative). A shortened gelatin test differentiated P. fluorescens (positive) from P. putida (negative). P. fluorescens and P. putida were very sensitive to low levels of kanamycin and resistant to carbenicillin, a pattern just the opposite of that obtained with P. aeruginosa.
TL;DR: It is proposed that this clustering is a consequence of the selection, in natural populations of bacteria, of gene arrangements which permit simultaneous transfer of the genetic determinants of a variety of dissimilatory pathways.
Abstract: Two previously demonstrated linkage groups containing genes with catabolic function inPseudomonas putida have been shown to cotransduce with a third cluster of catabolic genes, namely those specifying enzymes of nicotinic acid dissimilation. Thus enzymes of the following dissimilatory pathways are coded by genes clustered in one small (10-15% of the chromosome) genetic region:p-hydroxy-benzoic, quinic, shikimic, benzoic, mandelic, phenylacetic, and nicotinic acids; and histidine, tyrosine and phenylalanine. We propose that this clustering is a consequence of the selection, in natural populations of bacteria, of gene arrangements which permit simultaneous transfer of the genetic determinants of a variety of dissimilatory pathways.
TL;DR: Two mutants which produce the histidine-dissimilitory enzymes constitutively have been isolated and appear to carry mutations in a regulatory locus, which maps in the same region as the structural genes of the pathway.
Abstract: In P. putida the first four enzymes involved in the dissimilation of histidine are induced by the first intermediate of the pathway, urocanic acid. The genes specifying these enzymes, hutH, hutU, hutF and (probably) hutI appear to be clustered on the chromosome between pcaE and pcaA (genes involved in p-hydroxybenzoic acid catabolism). Two mutants which produce the histidine-dissimilitory enzymes constitutively have been isolated. They appear to carry mutations in a regulatory locus, which maps in the same region as the structural genes of the pathway.
TL;DR: It is established that imidazolyl-lactate is metabolized via histidine whereas imidAZolylpropionate enters the histidine degradation pathway after conversion into urocanate.
Abstract: 1. Imidazol-5-ylpropionate and imidazol-5-yl-lactate are degraded by Pseudomonas testosteroni via inducible pathways. 2. Growth on either compound as the sole source of carbon results in the induction of the enzymes for histidine catabolism. 3. The pathway of histidine degradation in this organism, a non-fluorescent Pseudomonad, is shown to be the same as that operating in Pseudomonas fluorescens and Pseudomonas putida. It consists of the successive formation of urocanate, imidazol-4-on-5-ylpropionate, N-formimino-l-glutamate, N-formyl-l-glutamate and glutamate. 4. Whole cells of P. testosteroni accumulate urocanate in the reaction mixture when incubated with imidazolylpropionate, but only after an adaptive lag period which is removed by previous growth on imidazolylpropionate as the source of carbon. 5. Imidazolyl-lactate is oxidized to imidazolylpyruvate, which then gives rise to histidine by specific transamination with l-glutamate. 6. Cells grown on histidine, urocanate or imidazolylpropionate are also able to degrade imidazolyllactate. 7. Mutants lacking urocanase are unable to grow on imidazolylpropionate, imidazolyl-lactate, histidine or urocanate. One with impaired histidase activity cannot utilize histidine or imidazolyl-lactate, but grows normally on imidazolylpropionate or urocanate. A mutant unable to grow on imidazolylpropionate is indistinguishable from the wild-type with respect to growth on histidine, imidazolyl-lactate or urocanate. 8. Thus it is established that imidazolyl-lactate is metabolized via histidine whereas imidazolylpropionate enters the histidine degradation pathway after conversion into urocanate.
TL;DR: Recombinants selected for prototrophy are stable and most acquire the camphor (CAM) plasmid concomitantly; only a few of the Cam(+) recombinants inherit the donor's ability to transfer chromosomal genes at a high frequency.
Abstract: Camphor-utilizing strains of Pseudomonas putida have been shown to carry the genetic information required for camphor degradation on a plasmid. The plasmid-carrying strains can serve as donors of both plasmid-borne and chromosomal genes. As recipients, plasmid-deleted strains are much superior to those carrying the camphor pathway genes. The transfer frequency of chromosomal, but not plasmid-borne, genes is markedly enhanced if the donor cells are irradiated with ultraviolet light followed by 3-h of growth on a rich medium in the dark. Recombinants selected for prototrophy are stable and most acquire the camphor (CAM) plasmid concomitantly; only a few of the Cam+ recombinants inherit the donor's ability to transfer chromosomal genes at a high frequency. Transfer-defective mutations occur on the CAM plasmid, affecting both CAM and chromosomal gene transfer.
TL;DR: It is however possible for the α subunits from Escherichia coli and Pseudomonas putida to complement the β2 subunit of B. subtilis in this latter reaction and produce a level of activity equal to 30% of the homologous complementation.
TL;DR: Isolates of male and female Oedogonium cardiacum for which defined media had been established were subsequently found to be contaminated with a species of Corynebacterium which failed to grow in the nutrient broth used, to test for contamination.
Abstract: SUMMARY
Isolates of male and female Oedogonium cardiacum for which defined media had been established were subsequently found to be contaminated with a species of Corynebacterium which failed to grow in the nutrient broth used, to test for contamination. After the cultures were rendered, axenic through treatment with, penicillin G, they failed to develop oogonia or sperm except occasionally at a very low level. The addition of small amounts of the bacterium increased the development of the reproductive structures; however a much more striking increase was obtained by constantly infecting the algal cultures with Pseudomonas putida. Neither of the bacteria increased growth as measured by dry weight; however the P. putida resulted, in the growth of very long filaments in contrast to the short filaments characteristic of both the axenic cultures and those infected with Corynebacterium sp.
TL;DR: Wild-type strains of Pseudomonas putida, P. cepacia and P. acidovorans grew in minimal salts media with acetamide or phenylacetamide as the carbon or nitrogen source but utilized acetamide but not phenyl acetamide.
Abstract: SUMMARY: Wild-type strains of Pseudomonas putida, P. cepacia and P. acidovorans grew in minimal salts media with acetamide or phenylacetamide as the carbon or nitrogen source. Wild-type strains of P. aeruginosa utilized acetamide but not phenylacetamide. Pseudomonas putida A90 and mutants isolated from P. putida A87 and from P. cepacia 716 grew on phenylacetamide but not on acetamide. These activities were due to two types of amidase: (i) aliphatic amidases, which hydrolyse acetamide, produced by P. aeruginosa and strains of P. putida, P. cepacia and P. acidovorans; and (ii) phenylacetamidases, which hydrolyse phenylacetamide, produced by some strains of P. putida, P. cepacia and P. acidovorans.
TL;DR: Pseudomonas putidais a Gram-negative, aerobic, fluorescent pseudomonad that has multitrichous flagella, does not grow at 42 °C, and is characterized by variable acid productio...
Abstract: Excerpt To the editor:Pseudomonas putidais a Gram-negative, aerobic, fluorescent pseudomonad that has multitrichous flagella, does not grow at 42 °C, and is characterized by variable acid productio...
TL;DR: High specific activity d - and l -amino[6- 14 C]adipate(47 μCi/μmole) were isolated via sclective metabolism of the opposite isomer by Alcaligenes denitrificans and Pseudomonas putida to determinations of isomeric purity which was at least 99% for both isomers.
Journal Article•
TL;DR: Using a purified preparation of Pseudomonas putida cytochrome P-450, the number of reactive sulfHydryl groups by PMB titration is determined and the possibility that a sulfhydryl group is one of the axial ligands and the question of the identity of the otherAxial ligand is considered.
Abstract: I should like to discuss the ligands of ferric cytochrome P-450 and how these ligands change during substrate binding. The ferric ion of heme complexes is usually considered to be hexacoordinate. Four of these coordinate positions are occupied by the nitrogen atoms of the tetrapyrrole ring. The remaining two axial ligands are the ones which may change during normal hemoprotein functioning. First we will consider the possibility that a sulfhydryl group is one of the axial ligands and then we will consider the question of the identity of the other axial ligand. A few years ago, Dr. Mason (I) proposed that the unique EPR2 spectrum which was observed with liver microsomes, which he called microsomal Fey, was due to a heme iron atom which had a sulfide ligand. Later, a similar proposal was advanced by Jefcoate and Gaylor (2) based on their observation that the addition of propylmercaptan to metmyoglobin resulted in an EPR spectrum very much like that of low-spin cytochrome P-450. Tsai et a!. (3) came to a similar conclusion based upon their analysis of the EPR spectrum of camphor-free bacterial cytochrome P-450 and its similarity to model heme compounds. Using a purified preparation of Pseudomonas putida cytochrome P-450, we have determined the number of reactive sulfhydryl groups by PMB titration. In the presence of camphor, as shown in This work was supported inpart by U.S. Public Health Service Research Grants AM 13366 and GM 16488 and Research Grant -405 from The Robert A. Welch Foundation. Postdoctoral Fellow of The Robert A. Welch Founda-
TL;DR: Among various soil microorganisms tested only Pseudomonas putida isolate 1065 and Rhizopus japonicus ATCC 24794 were able to transform o-toluate, and both compounds accumulated during a one week growth period.
Abstract: Among various soil microorganisms tested only Pseudomonas putida isolate 1065 and Rhizopus japonicus ATCC 24794 were able to transform o-toluate. In P. putida o-toluate was quantitatively hydroxylated to give 2-hydroxymethyl-benzoate and in R. japonicus it was reduced to 2-hydroxymethyltoluene. Both compounds, which were identified on the basis of their physical properties, accumulated during a one week growth period.
01 Jan 1973
TL;DR: Mutantstrains ofPseudomonas putida strain U have been obtained which aredeficient inenzymes ofthedegradative pathways ofphenol andcresols, and the results are discussed intermsofthephysiological significance of the pathway.
Abstract: Mutantstrains ofPseudomonas putida strain U havebeenobtained which aredeficient inenzymes ofthedegradative pathways ofphenol andcresols. Mutantstrains deficient incatechol 2,3-oxygenase accumulated theappropriate catechol derivative fromcresols. A mutantstrain whichwould notgrow on either phenol or a cresol was shownto.bedeficient inboth2-hydroxymuconic semialdehyde hydrolase anda nicotinamide adenine dinucleotide, oxidized form, (NAD+)-dependent aldehyde dehydrogenase. Whenthis strain was grown inthepresenceofphenol oracresol, theappropriate product ofmetafission of these compounds accumulated inthegrowth medium.A partial revertant of this mutantstrain, which was abletogrow on ortho- andmeta-cresol butnot para-cresol, was showntohaveregained onlythehydrolase activity. This strain was usedtoshowthattheproducts ofmetaring fission ofthecresols and phenol aremetabolized asfollows: (i)ortho- andmeta-cresol exclusively bya hydrolase; (ii) para-cresol exclusively bya NAD+-dependent aldehyde dehydrogenase; (iii) phenolbybotha NAD+-dependent dehydrogenase anda hydrolase intheapproximate ratio of5to1.Thisconclusion issupported by thesubstrate specificity andenzymatic activity ofthehydrolase andNAD+dependent aldehyde dehydrogenase enzymes ofthewild-type strain. Theresults are discussed intermsofthephysiological significance ofthepathway. Properties ofsome ofthemutantstrains isolated arediscussed.