Showing papers on "Pseudomonas putida published in 1972"

Genetic Basis of the Biodegradation of Salicylate in Pseudomonas

Abstract: The genetic basis of the biodegradation of salicylate in Pseudomonas putida R1 has been studied This strain utilizes the meta pathway for oxidizing salicylate through formation of catechol and 2-hydroxymuconic semialdehyde The enzymes of the meta pathway are induced by salicylate but not by catechol, and the genes specifying these enzymes are clustered The gene cluster can be eliminated from some salicylate-positive cells by treatment with mitomycin C and appears to exist inside the cell as an extrachromosomal element This extrachromosomal gene cluster, termed the SAL plasmid, can be transferred by conjugation from P putida R1 to a variety of other Pseudomonas species

The metabolic divergence in the meta cleavage of catechols by Pseudomonas putida NCIB 10015. Physiological significance and evolutionary implications.

Abstract: 1 A reinvestigation of the catabolic pathway(s) used by Pseudomonas putida NCIB 10015 (Dagley's strain) for the degradation of phenol and the cresols has proved the existence of a metabolic divergence after meta cleavage of the catechols formed by hydroxylation of the primary substrates. The ring-fission products of catechol and 4-methylcatechol are shown to be simultaneously catabolized by two different enzymic activities, an NAD+-dependent dehydrogenase and a cofactor-independent hydrolase. The metabolizing activities of both ring-fission products in extracts of cells grown on phenol and the cresols (o-, m- and p-cresol) were found to be non specific; thermal inactivation of extracts of phenol-grown cells has shown that this nonspecificity is attributable to only one enzyme expressing each activity and that the two activities are located on separate proteins. 2 Extracts of cells grown on all four substrates contain high induced levels of the meta cleavage suite of enzymes functional in the dissimilation of catechol, including both the 4-oxalocrotonate branch (NAD+-dependent 2-hydroxymuconic semialdehyde dehydrogenase, 4-oxalocrotonate tautomerase and 4-oxalocrotonate decarboxylase) and the hydrolytic branch (2-hydroxymuconic semialdehyde hydrolase). 3 The hydroxylase, oxygenase, dehydrogenase and hydrolase activities are shown to be nonspecific and can also act upon the methyl derivatives of their respective substrates. A constant pattern of specificity was found for these enzymes, independent of the monophenolic substrate used for growth. 4 From studies with a mutant lacking phenol hydroxylase, the entire suite of meta cleavage enzymes are shown to be coincidently induced from the top by the primary substrate (phenol or the cresols). 5 The evolutionary and physiological implications of the divergent pathway are discussed.

1 Tryptophan Synthetase

Abstract: Publisher Summary Tryptophan synthetase (L-serine hydrolyase) is a multimeric enzyme catalyzing the terminal reaction in the biosynthesis of L-tryptophan by microbes arid plants. In general, these enzymes in bacteria and plants appear to dissociate readily and reversibly into smaller subunits, while those of fungi do not. Several factors have made E. coli tryptophan synthetase an enzyme of choice for studies of transcription and translation and the regulation of these processes. The structural genes for α and β chains are adjacent to each other and are well mapped. They comprise the two most distal genes of the multigenic trp operon. All or portions of this operon have been translocated to the chromosome of a series of temperate bacteriophage mutants. DNA from these bacteriophages has been used to quantitate the amount of messenger RNA corresponding to various segments of the operon. The α chain of tryptophan synthetase has been isolated in pure form from Escherichia coli (K-12 and B strains), Salmonella typhimurium, Shigella dysenteriae, Aerobacter aerogenes, and Pseudomonas putida. The molecular weight of the α chain obtained from these sources is approximately 29,000.

Microorganisms having multiple compatible degradative energy-generating plasmids and preparation thereof

Abstract: Unique microorganisms have been developed by the application of genetic engineering techniques. These microorganisms contain at least two stable (compatible) energy-generating plasmids, these plasmids specifying separate degradative pathways. The techniques for preparing such multi-plasmid strains from bacteria of the genus Pseudomonas are described. Living cultures of two strains of Pseudomonas (P. aeruginosa [NRRL B-5472] and P. putida [NRRL B-5473]) have been deposited with the United States Department of Agriculture, Agricultural Research Service, Northern Marketing and Nutrient Research Division, Peoria, Ill. The P. aeruginosa NRRL B-5472 was derived from Pseudomonas aeruginosa strain 1c by the genetic transfer thereto, and containment therein, of camphor, octane, salicylate and naphthalene degradative pathways in the form of plasmids. The P. putida NRRL B-5473 was derived from Pseudomonas putida strain PpG1 by genetic transfer thereto, and containment therein, of camphor, salicylate and naphthalene degradative pathways and drug resistance factor RP-1, all in the form of plasmids.

Regulation of Valine Catabolism in Pseudomonas putida

Abstract: The generation time of Pseudomonas putida with l-leucine was 20 h in synthetic media but only 3 h with d-leucine. Slow growth in the presence of l-leucine was partially overcome by addition of 0.1 mM amounts of either d-valine, l-valine, or 2-ketoisovalerate. The activities of five enzymes which take part in the oxidation of leucine by P. putida were measured under various conditions of growth. Four enzymes were induced by growth with dl-leucine as sole source of carbon: d-amino acid dehydrogenase, branched-chain keto acid dehydrogenase, 3-methylcrotonyl-coenzyme A carboxylase, and 3-hydroxy-3-methylglutaryl-coenzyme A lyase. The segment of the pathway required for oxidation of 3-methylcrotonate was induced by growth on isovalerate or 3-methylcrotonate without formation of the preceding enzymes. The synthesis of carboxylase and lyase appeared to have been repressed by the addition of l-glutamate or glucose to cells growing on dl-leucine as the sole carbon source. Mutants unable to grow at the expense of isovalerate had reduced levels of carboxylase and lyase, whereas the levels of three enzymes common to the catabolism of all three branched-chain amino acids and those of two isoleucine catabolic enzymes were normal.

Genetic Control of Enzyme Induction in the β-Ketoadipate Pathway of Pseudomonas putida: Deletion Mapping of cat Mutations

Abstract: A number of spontaneous mutant strains of Pseudomonas putida, obtained by repeated selection for inability to grow with cis,cis-muconate, have been shown to carry deletions in catB, the structural gene for muconate lactonizing enzyme. These strains have been employed for deletion mapping of the genetic region containing catB and catC (the structural gene for muconolactone isomerase, the synthesis of which is coordinate with that of muconate lactonizing enzyme). All deletions that overlap mutant sites located on the left side of the genetic map, as well as the point mutations in that region, lead to a pleiotropic loss of both catB and catC activities. We propose that this region to the left of catB has a regulatory function. Although the details of regulation at the molecular level are unclear, our data indicate that catB and catC may well be controlled by a mechanism unlike any yet described by workers on enteric bacteria.

Kinetics of Deoxyribonucleic Acid Destruction and Synthesis During Growth of Bdellovibrio bacteriovorus Strain 109D on Pseudomonas putida and Escherichia coli

Abstract: During the growth of Bdellovibrio bacteriovorus on Pseudomonas putida or Escherichia coli in either 10−3m tris(hydroxymethyl)aminomethane or in dilute nutrient broth, the host deoxyribonucleic acid (DNA) was rapidly degraded, and by 30 to 60 min after the initiation of the bdellovibrio development cycle essentially all host DNA became nonbandable in CsCl gradients. At this stage the host DNA degradation products were nondiffusable, and there was no appreciable pool of low-molecular-weight (cold acid soluble) DNA fragments in the cells or in the suspending medium. Bdellovibrio DNA synthesis occurred only after degradation of host DNA to a nonbandable form was complete. The synthesis occurred in a continuous fashion with P. putida as the host and in two separate periods with E. coli as host. By using E. coli containing a 3H-thymidine label, it was shown that 73%, on the average, of the thymine residues of host DNA were incorporated into bdellovibrio DNA when E. coli was the only source of nutrient. In the presence of dilute nutrient broth, the host cells still served as the major source of precursors for bdellovibrio DNA synthesis, with only 20% of the precursors arising from the exogenous nutrients. The data indicate an efficient and controlled utilization of host DNA by the bdellovibrio. The host DNA is apparently degraded early in the developmental cycle to oligonucleotides of intermediate molecular weight from which the biosynthetic monomers are generated only as they become needed for bdellovibrio DNA synthesis.

Inducible Uptake System for β-Carboxy-cis, cis-muconate in a Permeability Mutant of Pseudomonas putida

Abstract: A procedure for the large-scale enzymatic synthesis of β-carboxymuconate is described. When used as a growth substrate, β-carboxymuconate selected for mutant strains of Pseudomonas putida that were permeable to polycarboxylic acid intermediates of the β-ketoadipate pathway. One mutant organism, strain PRS2110, was investigated in detail. It differed from the parental strain in that it possessed a β-carboxymuconate uptake system that was formed when the compound was supplied exogenously to the cells. The uptake system was not induced by β-carboxymuconate supplied endogenously during growth with p -hydroxybenzoate. These observations suggested that β-carboxymuconate was contained within a physical compartment of enzymes during growth with p -hydroxybenzoate. Support for this hypothesis came from the demonstration that enzymes of the β-ketoadipate pathway were held together by weak chemical interactions during the chromatography of crude extracts of benzoategrown P. putida on diethylaminoethyl-cellulose columns.

Proton magnetic resonance reveals high-spin iron (II) in ferrous cytochrome P450 cam from Pseudomonas putida.

Abstract: High-resolution proton nuclear magnetic resonance spectra were studied in D(+)-camphor-saturated solutions of ferric and ferrous cytochrome P450(cam) from Pseudomonas putida, and of the cyanide complex of ferric P450(cam). In all these compounds several hyperfineshifted resonances of the heme group could be detected. In the ferrous protein, these resonance lines, which exhibit a Curie-type temperature dependence in the range of 5-28 degrees , indicate the presence of high-spin iron (II). It is suggested that the iron (II) in ferrous P450(cam) might be pentacoordinated, as in other hemoproteins that can reversibly bind molecular oxygen and carbon monoxide.

Genetic Control of Enzyme Induction in the β-Ketoadipate Pathway of Pseudomonas putida: Two-Point Crosses with a Regulatory Mutant Strain

Abstract: Several mutant strains of Pseudomonas putida, selected on the basis of their inability to grow at the expense of benzoate, have been shown to be unable to form inducibly both muconate lactonizing enzyme and muconolactone isomerase. A secondary mutant strain derived from one of these pleiotropically negative strains forms these two enzymes and, in addition, catechol oxygenase in the absence of inducer. This constitutive mutant strain was used as a donor in transductionally mediated two-point crosses to determine the order of point mutations within the structural genes for muconate lactonizing enzyme and muconolactone isomerase (the catB and catC genes, respectively). The gene order conformed precisely with the one that has been established by deletion mapping.

Regulation of pathways degrading aromatic substrates in Pseudomonas putida. Enzymic response to binary mixtures of substrates

Abstract: 1. Induction constants (Kind) and repression constants (Krep), which are a measure of the affinity of the inducers or repressors for the induction systems, were measured for mandelate, benzoate and p-hydroxybenzoate in Pseudomonas putida. 2. From these results, the enzymic response of the organism to media containing pairs of these substrates was predicted. Nitrogen-limited chemostats, operated at high growth rates, were used to investigate these predictions in cells grown first on one aromatic substrate with the second added later. 3. In general, the values of Kind and Krep predicted quite accurately the response to substrate mixtures. Thus, in the presence of mandelate and either benzoate or p-hydroxybenzoate, the enzymes of mandelate metabolism were repressed almost completely, and the bacteria were fully induced for the alternative substrate (benzoate or p-hydroxybenzoate), which was preferentially utilized for growth. When benzoate and p-hydroxybenzoate were the two substrates in the mixture, the enzymes for metabolism of the latter were strongly repressed and growth took place mainly on benzoate. 4. The enzymic response to mixed substrates did not result in the metabolism of the better growth substrate, but in the substrate requiring the synthesis of fewer enzymes. Thus benzoate is used in preference to mandelate although the latter supports a faster growth rate. It is nevertheless considered that, with our present knowledge of the natural habitat of the organism, it is impossible to decide whether protein economy or growth rate was the factor determining the evolution of this control system.

Consequences of lysine oversynthesis in Pseudomonas mutants insensitive to feedback inhibition. Lysine excretion or endogenous induction of a lysine-catabolic pathway.

Abstract: Regulation of lysine biosynthesis was studied in Pseudomonas putida and Pseudomonas acidovorans. In both species, aspartokinase was subject to feedback inhibition by lysine and threonine and dihydrodipicolinate synthetase was inhibited by lysine. P. acidovorans could not degrade lysine whereas P. putida degraded lysine through an inducible lysine oxygenase. P. putida mutants with an aspartokinase insensitive to lysine inhibition were obtained. They did not excrete lysine but their lysine oxygenase was endogenously induced. However mutants obtained from a mutant lacking lysine oxygenase still did not excrete lysine. P. acidovorans mutants with a dihydrodipicolinate synthetase desensitized towards lysine inhibition were obtained. From them double mutants with both dihydrodipicolinate synthetase and aspartokinase desensitized were also obtained. All of these mutants excreted lysine.

Isolation, Identification, and Characterization of a Lipoate-Degrading Pseudomonad and of a Lipoate Catabolite

Abstract: A strain of bacteria that can degrade lipoic acid was isolated from soil. The bacterium, adapted to use 0.4% dl-lipoate as the sole organic substrate to supply carbon, sulfur, and energy, was identified morphologically and physiologically as a strain of Pseudomonas putida. Degradation of 1,6-14C-lipoic acid, synthesized from 1,6-14C-adipic acid, was evidenced by: (i) loss of approximately 50% of the total radioactivity from the medium after bacterial growth; (ii) appearance of 14C-degradation products upon paper and thin-layer chromatography of the culture medium; and (iii) oxygraphically measured utilization of O2 by cells in the presence of lipoate or other oxidizable substrates. Analyses of the benzene extract of culture medium by infrared, nuclear magnetic resonance, and mass spectrometry, and by gas-liquid chromatography after desulfuration, have characterized bisnorlipoic acid, or 4,6-dithiohexanoic acid, as the major catabolite present in the medium. β-Oxidation of the side chain is thus proven to be a pathway employed by the pseudomonad to degrade lipoic acid.

Regulation of a catabolic pathway. Lysine degradation in Pseudomonas putida.

Abstract: 1 The regulation of the pathway responsible for l-lysine degradation in Pseudomonas putida has been studied It has been shown that l-lysine is the actual inducer of the two first enzymes of the pathway, lysine oxygenase and 5-aminovaleramidase 2 No important repression of lysine oxygenase by various carbon sources and by intermediates of the lysine degradation pathway has been found 3 Activation of lysine oxygenase by its substrate, lysine, has been observed A physiological function for this phenomenom is proposed 4 Sodium and potassium ions are activators whereas magnesium ion, phosphate and phosphorylated compounds are inhibitors of lysine oxygenase Citrate also is inhibitory

A heat-stable nicotinamide-adenine dinucleotide glycohydrolase from Pseudomonas putida KB1. Partial purification and some properties of the enzyme and an inhibitory protein.

Abstract: A thermostable NAD(P)+ glycohydrolase (EC 3226) detected in cell-free extracts of Pseudomonas putida KB1 was purified to a single component on polyacrylamide-gel electrophoresis A heat-labile inhibitor of the enzyme was also partially purified Enzyme free of inhibitor is present in culture supernatants After an ultrasonic treatment enzyme–inhibitor complex and excess of inhibitor are present in both the cell-debris and soluble fractions The general properties of the enzyme and inhibitor are described The molecular weights of enzyme, inhibitor and enzyme–inhibitor complex, determined by gel filtration are about 23500, 15000 and 35000 respectively The binding of inhibitor and enzyme is inhibited by the presence of substrate

Purification and properties of 2-furoyl-coenzyme A hydroxylase from Pseudomonas putida F2

Abstract: 1. 2-Furoyl-CoA hydroxylase of Pseudomonas putida F2 has been purified 60-fold by a combination of (NH4)2SO4 fractionation, DEAE-cellulose chromatography and agarose chromatography. 2. The purified enzyme catalyses the formation of 5-hydroxy-2-furoyl-CoA, which tautomerizes to form 5-oxo-Δ2-dihydro-2-furoyl-CoA. 3. The enzyme has a requirement for an electron acceptor that can be satisfied by a membrane preparation from 2-furoate-grown Ps. putida F2 or by artificial electron acceptors, and so presumably the incorporated oxygen atom is derived from water rather than molecular oxygen. 4. The enzyme is a large protein with a molecular weight of 3.27×106 and is disrupted to form inactive subunits in the presence of 0.2% (w/v) sodium dodecyl sulphate. It has a pH optimum of 8.5–9.5, a Km for 2-furoyl-CoA of 20.2μm and an absorption spectrum with a trough at 265nm and a single peak at 273nm. No absorption peaks are detectable in the visible region of the spectrum. 5. The enzyme is resistant to the effects of a wide range of potential inhibitors, but is inhibited by the copper-chelating agents bathocuproin and cuprizone, though not by sodium diethyldithiocarbamate. 6. Flavins are absent and the iron content does not show a sustained increase during purification. The copper content of the protein increases in close correlation with the increase in specific activity during purification. 7. A catalytic sequence for the hydroxylation of 2-furoyl-CoA by a copper protein is proposed.

Photoactivation of urocanase in Pseudomonas putida: possible conformational change during activation.

Abstract: Earlier studies of urocanase indicated that it is converted to an inactive form in resting cells of Pseudomonas putida. When cell extracts are irradiated by ultraviolet (u.v.) light, the enzyme is activated[1]. The chromophore is closely associated with the enzyme [1,2]. The action spectrum for photoactivation showed that, although near-u.v. light was effective, the most efficient wavelengths were at 275–280 nm[3]. The study we present here suggests that urocanase undergoes a conformational change upon photoactivation.