Preparation of protoplast-like structures from conidia ofFusarium culmorum

Protoplast-like structures have been formed by digestion of the cell walls ofFusarium culmorum conidia by lytic enzyme preparations ofMicromonospora AS. Under the test conditions extrusion of the protoplasts was not observed. It seems that digestion of the cell wall occurs in different stages. Digestion of the septa preceded the formation of protoplasts of the individual cells of the multicellularF. culmorum conidia. A few protoplasts survived the lytic enzyme treatment. “Protoplasts” obtained from conidia are much more stable than those obtained from young hyphae and were able to germinate with the formation of normal mycelium. Lysis of some of the protoplast bodies led to the formation of a membranous structure. The protoplasts derived from each of the constituent cells of the conidia could be isolated with the micromanipulator. No differences were found in the ability of the isolated cells to germinate.     

Preparation and analysis of Pseudomonas putida cells for elemental composition

A series of procedures including cultivation, harvesting, washing, drying, ashing, and chemical preparation were developed for the analysis of the elemental composition of Pseudomonas putida cells. The composition of P. putida grown in continuous culture in five separate experiments at 30°C, pH 7.5, in 0.1% peptone‐yeast extract medium adjusted to a salinity of 26.5 g/L was averaged in μg/g for 50 subsamples: C, 521,000 ± 10,200; N, 143,000 ± 2,100; H, 73,600 ± 2,000; P, 17,600 ± 4,600; S, 5,200 ± 200; Mg, 3,970 ± 1,360; Na, 2,730 ± 1,160; Ca, 2,560 ± 700; K, 2,400 ± 630; Fe, 170 ± 55; Zn, 97 ± 34; and Cu, 28 ± 7.

The critical steps in the procedure were washing three times with “pure”; 4°C water using mechanical agitation and homogenization of the dried cells with an agate mortar and pestle.     

Roles of cysteine sulfinate and transaminase on in vitro dark reversion of urocanase in Pseudomonas putida.

Urocanase is inactivated in intact cells of Pseudomonas putida and photoactivated by brief exposure of the cells to the UV radiation in sunlight. The dark reversion (inactivation) in vitro is explained by the formation of a sulfite-NAD adduct. Our objective was to investigate the dark reversion in vivo. Various compounds were added to P. putida cells, and the reversion was measured, after sonication, by comparison of the activity before and after UV irradiation. Sulfite, cysteine sulfinate, and hypotaurine enhanced the reversion of urocanase in resting cells. The reversion was time and concentration dependent. Sulfite modified the purified enzyme, but cysteine sulfinate and hypotaurine could not, indicating that those two substances had to be metabolized to support the reversion. Both of those compounds yielded sulfite when they were incubated with cells. Transaminases form sulfite from cysteine sulfinate. P. putida extract contained a transaminase whose activity involved as alpha-keto acid and either cysteine sulfinate or hypotaurine for (i) production of sulfite, (ii) disappearance of substrates, (iii) formation of corresponding amino acids, and (iv) urocanase reversion. Porcine crystalline transaminase caused reversion of highly purified P. putida urocanase with cysteine sulfinate and alpha-ketoglutarate. We conclude that in P. putida cysteine sulfinate or hypotaurine is catabolized in vivo by a transaminase reaction to sulfite, which modifies urocanase to a form that can be photoactivated. We suggest that this photoregulatory process is natural because it occurs in cells with the aid of sunlight and cellular metabolism.     

Pseudomonas putida KT2440 responds specifically to chlorophenoxy herbicides and their initial metabolites

Pseudomonas putida KT2440 is often used as a model to investigate toxicity mechanisms and adaptation to hazardous chemicals in bacteria. The objective of this paper was to test the impact of the chlorophenoxy herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-(2,4-dichlorophenoxy)propanoic acid (DCPP) and their metabolites 2,4-dichlorophenol (DCP) and 3,5-dichlorocatechol (DCC), on protein expression patterns and physiological parameters. Both approaches showed that DCC has a different mode of action and induces different responses than DCPP, 2,4-D and DCP. DCC was the most toxic compound and was active as an uncoupler of oxidative phosphorylation. It repressed the synthesis of ferric uptake regulator (Fur)-dependent proteins, e.g. fumarase C and L-ornithine N5-oxygenase, which are involved in oxidative stress response and iron uptake. DCPP, 2,4-D and DCP were less toxic than DCC. They disturbed oxidative phosphorylation to a lesser extent by a yet unknown mechanism. Furthermore, they repressed enzymes of energy-consuming biosynthetic pathways and induced membrane transporters for organic substrates. A TolC homologue component of multidrug resistance transporters was found to be induced, which is probably involved in the removal of lipophilic compounds from membranes.     

Revised structures of the pyoverdins from Pseudomonas putida CFBP 2461 and from Pseudomonas fluorescens CFBP 2392

Several suggestions for structures of the siderophores (pyoverdins) from Pseudomonas spp. can be found in the literature which are based on a FAB mass spectrometric analysis only. Availability of two original strains of two Pseudomonas spp. allowed to re-investigate the structure of their pyoverdins. In both cases the amino acid sequence had to be corrected. In addition, d- and l-amino acids could be identified and located in the peptide chain. The knowledge of the correct structures is important in view of an ongoing study to establish relationships between the nature of the peptide chains of pyoverdins and their recognition by outer membrane proteins.     

Binding of Germanium to Pseudomonas putida Cells

The binding of germanium to Pseudomonas putida ATCC 33015 was investigated by using whole intact cells grown in a medium supplemented with GeO₂ and catechol or acetate. Electron-microscopic examination of the control and metal-loaded samples revealed that germanium was bound within the cell envelope. A certain number of small electron-dense deposits of the bound element were found in the cytoplasm when the cells were grown in the presence of GeO₂ and catechol. The study of germanium distribution in cellular fractions revealed that catechol facilitated the intracellular accumulation of this element.     

D- and L-isoleucine metabolism and regulation of their pathways in Pseudomonas putida

Pseudomonas putida oxidized isoleucine to acetyl-coenzyme A (CoA) and propionyl-CoA by a pathway which involved deamination of d-isoleucine by oxidation and l-isoleucine by transamination, oxidative decarboxylation, and beta oxidation at the ethyl side chain. At least three separate inductive events were required to form all of the enzymes of the pathway: d-amino acid dehydrogenase was induced during growth in the presence of d-isoleucine; branched-chain keto dehydrogenase was induced during growth on 2-keto-3-methylvalerate and enzymes specific for isoleucine metabolism; tiglyl-CoA hydrase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase were induced by growth on isoleucine, 2-keto-3-methylvalerate, 2-methylbutyrate, or tiglate. Tiglyl-CoA hydrase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase were purified simultaneously by several enzyme concentration procedures, but were separated by isoelectric focusing. Isoelectric points, pH optima, substrate specificity, and requirements for enzyme action were determined for both enzymes. Evidence was obtained that the dehydrogenase catalyzed the oxidation of 2-methyl-3-hydroxybutyryl-CoA to 2-methylacetoacetyl-CoA. 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase catalyzed the oxidation of 3-hydroxybutyryl-CoA, but l-3-hydroxyacyl-CoA dehydrogenase from pig heart did not catalyze the oxidation of 2-methyl-3-hydroxybutyryl-CoA; therefore, they appeared to be different dehydrogenases. Furthermore, growth on tiglate resulted in the induction of tiglyl-CoA hydrase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase, but these two enzymes were not induced during growth on crotonate or 3-hydroxybutyrate.     

Study of morphology and genome structure of Pseudomonas putida bacteriophages for their classification

A group of 27 bacteriophages specific for Pseudomonas putida strains PpG1 and PpN has been isolated. The phages were characterized and compared with the previously described virulent (pf 16, af, tf and PMW) and temperate (PP56 and PP71) phages. The new phages belong to B1 and C1 morphotypes, according to Ackerman's classification. Phage DNAs were digested with several endonucleases; the molecular weights and homology of the DNAs were determined. All phages of P. putida isolated up to now were distributed into 10 species (groups), on the basis of particle morphology, genome size and the results of homology studies. Recombination processes are believed to participate in formation of phages belonging to certain species.     

Mannitol, a novel bacterial compatible solute in Pseudomonas putida S12.

The aim of this study was to identify the compatible solutes accumulated by Pseudomonas putida S12 subjected to osmotic stress. In response to reduced water activity, P. putida S12 accumulated Nalpha-acetylglutaminylglutamine amide (NAGGN) simultaneously with a novel compatible solute identified as mannitol (using 13C- and 1H-nuclear magnetic resonance, liquid chromatography-mass spectroscopy and high-performance liquid chromatography methods) to maximum concentrations of 74 and 258 micromol g (dry weight) of cells(-1), respectively. The intracellular amounts of each solute varied with both the type and amount of osmolyte applied to induce osmotic stress in the medium. Both solutes were synthesized de novo. Addition of betaine to the medium resulted in accumulation of this compound and depletion of both NAGGN and mannitol. Mannitol and NAGGN were accumulated when sucrose instead of salts was used to reduce the medium water activity. Furthermore, both compatible solutes were accumulated when glucose was substituted by other carbon sources. However, the intracellular quantities of mannitol decreased when fructose, succinate, or lactate were applied as a carbon source. Mannitol was also raised to high intracellular concentrations by other salt-stressed Pseudomonas putida strains. This is the first study demonstrating a principal role for the de novo-synthesized polyol mannitol in osmoadaptation of a heterotrophic eubacterium.     

Purification and characterization of cutinase from a fluorescent Pseudomonas putida bacterial strain isolated from phyllosphere

Cutinase, an extracellular enzyme, was induced by cutin in a fluorescent Pseudomonas putida strain that was found to be cohabiting with an apparently nitrogen-fixing Corynebacterium. This enzyme was purified from the culture fluid by acetone precipitation followed by chromatography on DEAE-cellulose, QAE-Sephadex, Sepharose 6B, and Sephadex G-100. The purified enzyme showed a single band when subjected to polyacrylamide electrophoresis and the enzymatic activity coincided with the protein band. Sodium dodecyl sulfate-polyacrylamide electrophoresis showed a single band at a molecular weight of 30,000 and gel filtration of the native enzyme through a calibrated Sephadex G-100 column indicated a molecular weight of 30,000, showing that the enzyme is a monomer. The amino acid composition of bacterial cutinase is distinctly different from that of fungal or plant cutinases. This bacterial cutinase showed a broad pH optimum between 8.5 and 10.5 with 3H-labeled apple cutin as the substrate. Linear rates of cutin hydrolysis were measured up to 20 min of incubation time and 4 mg/ml of cutin gave the maximum hydrolysis rate. This cutinase catalyzed hydrolysis of p-nitrophenyl esters of C4 to C16 fatty acids with decreasing V and increasing Km for the longer chain esters. It did not hydrolyze tripalmitoyl glycerol or trioleyl glycerol, indicating that this is not a general lipase. Active serine-directed reagents such as organophosphates and organoboronic acids severely inhibited the enzyme, suggesting that bacterial cutinase is an "active serine" enzyme. Neither thiol-directed reagents nor metal ion chelators had any effect on this enzyme. Antibody raised against purified enzyme gave a single precipitin line on Ouchterlony double diffusion analysis. Western blot analysis of the extracellular fluid of induced Ps. putida showed a single band at 30 kDa. No immunological cross-reactivity was detected between the present bacterial enzyme and the fungal enzyme from Fusarium solani pisi when rabbit antibodies against either enzyme was used.     

Structure of bacterial L forms and their parent bacteria

Weibull, Claes (Rocky Mountain Laboratory, Hamilton, Mont.). Structure of bacterial L forms and their parent bacteria. J. Bacteriol. 90:1467-1480. 1965.-Light and electron microscopic studies were done on normal cells and L forms of Proteus mirabilis, Staphylococcus aureus, and Corynebacterium sp. grown in liquid media. Under the prevailing growth conditions, the L forms studied were morphologically indistinguishable from one another. They appeared as approximately spherical elements occurring singly or more often connected with each other by thinner portions of cell material. In sections of large L forms, the following structures were seen: a peripheral, triple-layered ("unit") membrane, a granular cytoplasm, nuclear regions, and vacuoles limited by membranes. Small bodies often were present inside the vacuoles. These bodies also contained a peripheral membrane and a granular cytoplasm but usually no nuclear regions. The normal bacteria from which the L forms were derived differed markedly in structure from one another, especially in the surface layers of the cells.     

Alpha-hydroxyglutarate oxidoreductase of Pseudomonas putida

Oxidation of d-alpha-hydroxyglutarate to alpha-ketoglutarate is catalyzed by d-alpha-hydroxyglutarate oxidoreductase, an inducible membrane-bound enzyme of the electron transport particle [ETP; a comminuted cytoplasmic membrane preparation with enzymic properties and chemical composition resembling beef heart mitochondrial ETP (1)] of Pseudomonas putida P2 (P2-ETP). Treatment of P2-ETP with a nonionic detergent yields a preparation with the sedimentation characteristics of a soluble enzyme, but which retains an intact electron transport chain. Oxygen acts solely as a terminal electron acceptor and may be replaced by ferricyanide, 2,6-dichlorophenol indophenol, or mammalian cytochrome c. The oxidoreductase is specific for the d-isomer (K(m) = 4.0 x 10(-4)m for dl-alpha-hydroxyglutarate) and is distinct both from l- and d-malate dehydrogenases. Spectral studies suggest that the carrier sequence is substrate --> flavine or nonheme iron --> cyt b --> [cyt c] --> oxygen.