Physiological role for the membrane bound ascorbate-TMPD oxidase in pseudomonas putida.

The activity of the membrane-bound ascorbate-TMPD oxidase in Pseudomonas putida varies with growth conditions and age of the culture. A comparison of the effects of cyanide and azide on the oxidation of various substrates suggests that ascorbate-TMPD oxidase is not the terminal oxidase for NADH or succinate oxidation. However, it does have a role in the oxidation of nicotinate, and may act as an additional terminal oxidase under certain other growth conditions.     

Physiological role of the novel salicylaldehyde dehydrogenase NahV in mineralization of naphthalene by Pseudomonas putida ND6

The classical salicylaldehyde dehydrogenases found in naphthalene-degrading bacteria are denoted as NahF. In addition to NahF, NahV, and its corresponding gene nahV, were found here in multiple naphthalene-degrading bacteria isolated from industrial wastewater polluted with polycyclic aromatic hydrocarbons (PAHs). In this study, we described for the first time the biological function and regulation model of NahV for the mineralization of naphthalene by P. putida ND6 via the construction of nahF-, nahV- and regulatory gene nahR-deficient strains. The two mutants of salicylaldehyde dehydrogenase genes and wild-type Pseudomonas ND6 were compared with respect to growth rate, naphthalene degradation efficiency, protein expression level, and salicylaldehyde dehydrogenase activity. The data showed that the presence of NahV conferred a physiological advantage on P. putida ND6 for the catabolism of naphthalene in the presence of NahF. NahV could facilitate naphthalene degradation by increasing total salicylaldehyde dehydrogenase activity when both dehydrogenases are present and it could replace the function of NahF when nahF gene is deleted or mutated, thus ensuring mutants could survive in naphthalene-containing environments. To investigate regulation model of NahV, we detected the expression levels and salicylaldehyde dehydrogenase activity in the wild-type and the nahR mutant strains following cultivation in the presence of glucose±salicylate. The data demonstrated that just like the classical salicylaldehyde dehydrogenases, NahF, NahV was induced by salicylate in the presence of NahR.     

Physiological role of phosphatidylcholine in the Pseudomonas putida A ATCC 12633 response to tetradecyltrimethylammonium bromide and aluminium

Aims:  To evaluate the effect of tetradecyltrimethylammonium bromide (TTAB) and aluminium stresses on the phospholipid (PL) composition of Pseudomonas putida A ATCC 12633.
Methods and Results:  Pseudomonas putida were grown with TTAB in the presence or absence of AlCl₃, and the PL composition was analysed. The presence of TTAB resulted in an increase in phosphatidylglycerol and phosphatidic acid levels (6- and 20-fold, respectively) with respect to the levels in cells grown without the surfactant. With AlCl₃, phosphatidylcholine (PC) increased (threefold) and cell-free extracts contained approximately threefold more phosphatidylcholine synthase activities than extracts without AlCl₃, indicating that the PC level is dependent upon activation of this enzyme.
Conclusions:  The negative charges of the headgroups of PL are the primary membrane-associated factors for the response to TTAB. PC are involved in cellular responses to binding Al³⁺ and should be viewed as a temporary reservoir of available Al³⁺ to allow a more efficient utilization of TTAB by Ps. putida .
Significance and Impact of the Study:  The changes in the PL of Ps. putida in the presence of TTAB and AlCl₃ indicate that different responses are utilized by bacteria to maintain optimal PL composition in the presence of such environmental pollutants.     

Dual role of NER in mutagenesis in Pseudomonas putida

Nucleotide excision repair (NER) is one of the most important repair systems which counteracts different forms of DNA damage either induced by various chemicals or irradiation. At the same time, less is known about the functions of NER in repair of DNA that is not exposed to exogenous DNA-damaging agents. We have investigated the role of NER in mutagenesis in Pseudomonas putida. The genome of this organism contains two uvrA genes, uvrA and uvrA2. Genetic studies on the effects of uvrA, uvrA2, uvrB and UvrC in mutagenic processes revealed that all of these genes are responsible for the repair of UV-induced DNA damage in P. putida. However, uvrA plays more important role in this process than uvrA2 since the deletion of uvrA2 gene had an effect on the UV-tolerance of bacteria only in the case when uvrA was also inactivated. Interestingly, the lack of functional uvrB, uvrC or uvrA2 gene reduced the frequency of stationary-phase mutations. The contribution of uvrA2, uvrB and uvrC to the mutagenesis appeared to be most significant in the case of 1-bp deletions whose emergence is dependent on error-prone DNA polymerase Pol IV. These data imply that NER has a dual role in mutagenesis in P. putida-besides functioning in repair of damaged DNA, NER is also important in generation of mutations. We hypothesize that NER enzymes may initiate gratuitous DNA repair and the following DNA repair synthesis might be mutagenic.     

Physiological characterization of Pseudomonas putida DOT-T1E tolerance to p-hydroxybenzoate

Pseudomonas putida DOT-T1E was isolated as a toluene-tolerant strain. We show that it is also able to grow on high concentrations (up to 17 g/liter [123 mM]) of p-hydroxybenzoate (4HBA). Tolerance to this aromatic carboxylic acid (up to 30 g/liter [217 mM]) is improved by preexposing the cells to low 4HBA concentrations; the adaptation process is caused by the substrate itself rather than by products resulting from its metabolism. The mechanisms of 4HBA tolerance seem to involve increased rigidity of the cell membrane as a result of a decrease in the cis/trans ratio of unsaturated fatty acids. In addition, energy-dependent efflux systems seem to operate in the exclusion of 4HBA from the cell membranes.     

Physiological Characterization of Pseudomonas putida DOT-T1E Tolerance to p-Hydroxybenzoate

Pseudomonas putida DOT-T1E was isolated as a toluene-tolerant strain. We show that it is also able to grow on high concentrations (up to 17 g/liter [123 mM]) of p-hydroxybenzoate (4HBA). Tolerance to this aromatic carboxylic acid (up to 30 g/liter [217 mM]) is improved by preexposing the cells to low 4HBA concentrations; the adaptation process is caused by the substrate itself rather than by products resulting from its metabolism. The mechanisms of 4HBA tolerance seem to involve increased rigidity of the cell membrane as a result of a decrease in the cis/trans ratio of unsaturated fatty acids. In addition, energy-dependent efflux systems seem to operate in the exclusion of 4HBA from the cell membranes.     

Physiological stress in batch cultures of Pseudomonas putida 54G during toluene degradation

Physiological stress associated with toluene exposure in batch cultures of Pseudomonas putida 54G was investigated. P. putida 54G cells were grown using a continuous vapor phase feed stream containing 150 ppmv or 750 ppmv toluene as the sole carbon and energy source. Cells were enumerated on non-selective (R2A agar plates) and a selective minimal medium incubated in the presence of vapor phase toluene (HCMM2). Differential recovery on the two media was used to evaluate bacterial stress, culturability and loss of toluene-degrading capability. A majority of the bacteria were reversibly stressed and could resume active colony formation on selective medium after passage on non-selective medium. A small fraction of the bacterial cells suffered an irreversible loss of toluene degradation capability and were designated as Tol⁻ variants. Numbers of stressed organisms increased with duration of toluene exposure and toluene concentration and coincided with accumulation of metabolic intermediates from incomplete toluene degradation. Respiring cell numbers in the batch cultures decreased as injury increased, indicating a possible relationship between respiring and injured cells. Rate expressions for injury, for formation of Tol⁻ variants and for growth of Tol⁻ variants were determined by calibrating a theoretical model to the results obtained. These rate expressions can be used to calibrate bioreactor models, and provide a basis for better design and control of bioremediation systems. Received 01 July 1996/ Accepted in revised form 25 March 1997     

Physiological response of Pseudomonas putida S12 subjected to reduced water activity

Abstract The effect of osmotic stress, given as decreased water activity (a_w), on growth and the accumulation of potassium and the compatible solute betaine by Pseudomonas putida S12 was investigated. Reduced a_w was imposed by addition of sodium chloride, sucrose, glycerol or polyethylene glycol to the growth medium. Accumulation of potassium and betaine was established when sodium chloride and sucrose were used to cause osmotic stress. No accumulation of these solutes was found in the presence of glycerol. Addition of polyethylene glycol to the medium strongly decreased the growth rate in comparison with the other osmolytes tested at the corresponding a_w. Although polyethylene glycol did decrease the a_w, neither potassium nor betaine was accumulated by the cells.     

Physiological and Molecular Biological Characterization of Ammonia Oxidation of the Heterotrophic Nitrifier Pseudomonas putida

The heterotrophic nitrifier Pseudomonas putida aerobically oxidized ammonia to hydroxylamine, nitrite, and nitrate. Product formation was accompanied by a small but significant release of NO, whereas N₂O evolution could not be detected under the assay conditions employed. The isolate reduced nitrate to nitrite and partially further to NO under anaerobic conditions. Aerobically grown cells utilized γ-aminobutyrate as a carbon source and as a N-source by ammonification. The physiological experiments, in particular the inhibition pattern by C₂H₂, indicated that P. putida expressed an ammonia monooxigenase. DNA-hybridization with an amoA gene probe coding for the smaller subunit of the ammonia monooxigenase of Nitrosomonas europaea allowed us to identify, to clone, and to sequence a region with an open reading frame showing distinct sequence similarities to the amoA gene of autotrophic ammonia oxidizers. Received: 9 April 1998 / Accepted: 15 May 1998     

Chromium reduction in Pseudomonas putida

Reduction of hexavalent chromium (chromate) to less-toxic trivalent chromium was studied by using cell suspensions and cell-free supernatant fluids from Pseudomonas putida PRS2000. Chromate reductase activity was associated with soluble protein and not with the membrane fraction. The crude enzyme activity was heat labile and showed a Km of 40 microM CrO4(2-). Neither sulfate nor nitrate affected chromate reduction either in vitro or with intact cells.     

Overexpression and characterization of medium-chain-length polyhydroxyalkanoate granule bound polymerases from Pseudomonas putida GPo1

Background  

Polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA biosynthesis are PHA polymerases, which catalyze the covalent linkage of 3-hydroxyacyl coenzymeA thioesters by transesterification with concomitant release of CoA. Pseudomonas putida GPo1 and many other Pseudomonas species contain two different class II polymerases, encoded by phaC1 and phaC2. Although numerous studies have been carried out on PHA polymerases and they are well characterized at the molecular level, the biochemical properties of the class II polymerases have not been studied in detail. Previously we and other groups purified the polymerases, however, the activities of the purified enzymes were several magnitude lower than the granule-bound enzymes. It is problematic to study the intrinsic properties of these enzymes with such low activities, although they are pure.     

Glucolysis in Pseudomonas putida: Physiological Role of Alternative Routes from the Analysis of Defective Mutants

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.     

Aromatic acids are chemoattractants for Pseudomonas putida

A quantitative capillary assay was used to show that aromatic acids, compounds that are chemorepellents for Escherichia coli and Salmonella sp., are chemoattractants for Pseudomonas putida PRS2000. The most effective attractants were benzoate; p-hydroxybenzoate; the methylbenzoates; m-, p-, and o-toluate; salicylate; DL-mandelate; beta-phenylpyruvate; and benzoylformate. The chemotactic responses to these compounds were inducible. Taxis to benzoate and m-toluate was induced by beta-ketoadipate, a metabolic intermediate formed when benzoate is dissimilated via enzymes specified by chromosomal genes. Benzoylformate taxis was induced by benzoylformate and L(+)-mandelate. Taxis to mandelate, benzoylformate, and beta-phenylpyruvate was exhibited by cells grown on mandelate, but not by cells grown on benzoate. Cells grown on benzoate were chemotactic to benzoate, the toluates, p-hydroxybenzoate, and salicylate. These results show that P. putida synthesizes at least two distinct chemoreceptors for aromatic acids. Although DL-mandelate was an effective attractant in capillary assays, additional experiments indicated that the cells were actually responding to benzoylformate, a metabolite formed from mandelate. With the exception of mandelate taxis, chemotaxis to aromatic acids was not dependent on the expression of pathways for aromatic degradation. Therefore, the tactic responses exhibited by cells cannot be attributed to an effect of the oxidation of aromatic acids on the energy metabolism of cells.     

Role of Pseudomonas putida tol-oprL gene products in uptake of solutes through the cytoplasmic membrane

Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane. Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain. Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain. The pattern and amount of outer membrane protein in the P. putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane. Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P. putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level. Generation of a proton motive force appeared to be unaffected in these mutants. To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants. These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane.