Metabolism of amino acid amides in Pseudomonas putida ATCC 12633

The metabolism of the natural amino acid l-valine, the unnatural amino acids d-valine, and d-, l-phenyglycine (d-, l-PG), and the unnatural amino acid amides d-, l-phenylglycine amide (d-, l-PG-NH₂) and l-valine amide (l-Val-NH₂) was studied in Pseudomonas putida ATCC 12633. The organism possessed constitutive l-amidase activities towards l-PG-NH₂ and l-Val-NH₂, both following the same pattern of expression, suggesting the involvement of similarly regulated enzymes, or a common enzyme. Quite surprisingly, growth in mineral media with l-PG-NH₂ resulted in variable, long lag phases of growth and strongly reduced l-amidase activities. Conversion of d-PG-NH₂ into d-PG and l-PG also occurred and could be attributed to the presence of an inducible d-amidase and the racemization of the amino acid amide in combination with l-amidase activity, respectively. The further degradation of l-PG and d-PG involved constitutive l-PG aminotransferase and inducible d-PG dehydrogenase activities, respectively, both with a high degree of enantioselectivity. Amino acid racemase activity for d- and l-PG was not detected. Correspondence to: L. Dijkhuizen     

Purification and Characterization of an l-Aminopeptidase from Pseudomonas putida ATCC 12633

An l-aminopeptidase of Pseudomonas putida, used in an industrial process for the hydrolysis of d,l-amino acid amide racemates, was purified to homogeneity. The highly l-enantioselective enzyme resembled thiol reagent-sensitive alkaline serine proteinases and was strongly activated by divalent cations. It possessed a high substrate specificity for dipeptides and alpha-H amino acid amides, e.g., l-phenylglycine amide.     

Pseudomonas putida A ATCC 12633 oxidizes trimethylamine aerobically via two different pathways

The present study examined the aerobic metabolism of trimethylamine in Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine. In both conditions, the trimethylamine was used as a nitrogen source and also accumulated in the cell, slowing the bacterial growth. Decreased bacterial growth was counteracted by the addition of AlCl₃. Cell-free extracts prepared from cells grown aerobically on tetradecyltrimethylammonium bromide exhibited trimethylamine monooxygenase activity that produced trimethylamine N-oxide and trimethylamine N-oxide demethylase activity that produced dimethylamine. Cell-free extracts from cells grown on trimethylamine exhibited trimethylamine dehydrogenase activity that produced dimethylamine, which was oxidized to methanal and methylamine by dimethylamine dehydrogenase. These results show that this bacterial strain uses two enzymes to initiate the oxidation of trimethylamine in aerobic conditions. The apparent K_m for trimethylamine was 0.7 mM for trimethylamine monooxygenase and 4.0 mM for trimethylamine dehydrogenase, but both enzymes maintain similar catalytic efficiency (0.5 and 0.4, respectively). Trimethylamine dehydrogenase was inhibited by trimethylamine from 1 mM. Therefore, the accumulation of trimethylamine inside Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine may be due to the low catalytic efficiency of trimethylamine monooxygenase and trimethylamine dehydrogenase.     

Physical, kinetic and spectrophotometric studies of a NAD(P)-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633

The mandelate pathway of Pseudomonas putida ATCC 12633 comprises five enzymes and catalyzes the conversion of R- and S-mandelamide to benzoic acid which subsequently enters the beta-ketoadipate pathway. Although the first four enzymes have been extensively characterized the terminal enzyme, a NAD(P)(+)-dependent benzaldehyde dehydrogenase (BADH), remains largely undescribed. Here we report that BADH is a dimer in solution, and that DTT is necessary both to maintain the activity of BADH and to prevent oligimerization of the enzyme. Site-directed mutagenesis confirms that Cys249 is the catalytic cysteine and identifies Cys140 as the cysteine likely to be involved in inter-monomer disulfide formation. BADH can utilize a range of aromatic substrates and will also operate efficiently with cyclohexanal as well as medium-chain aliphatic aldehydes. The logV and logV/K pH-rate profiles for benzaldehyde with either NAD(+) or NADP(+) as the coenzyme are both bell-shaped. The pK(a) values on the ascending limb range from 6.2 to 7.1 while those on the descending limb range from 9.6 to 9.9. A spectrophotometric approach was used to show that the pK(a) of Cys249 was 8.4, i.e., Cys249 is not responsible for the pK(a)s observed in the pH-rate profiles.     

Modification of phospholipid composition in Pseudomonas putida A ATCC 12633 induced by contact with tetradecyltrimethylammonium

AIMS: The aim of this work was to establish if the response to tetradecyltrimethylammonium (TDTMA), a representative quaternary ammonium compound (QAC), involves changes in the phospholipid (PL) composition of Pseudomonas putida A ATCC 12633. METHODS AND RESULTS: Pseudomonas putida was exposed to 50 mg l(-1) of TDTMA for 15 min, and PL composition was analysed. With respect to control values, phosphatidic acid and phosphatidylglycerol increased by 140% and 120%, respectively; cardiolipin decreased about 60%. In TDTMA-adapted bacteria, the most significant change was a 380% increase in phosphatidic acid. Accompanying this change was a 130% increase in phosphatidylglycerol and a 70% decrease in cardiolipin. The changes in adapted cells were reverted after two subcultures without biocide. CONCLUSIONS: Pseudomonas putida responded to TDTMA through quantitative changes in PLs with specific variations in the content of phosphatidic acid, phosphatidylglycerol and cardiolipin. These modifications indicated that these PLs are involved in cellular responses to QACs, utilizing phosphatidic acid principally to neutralize the high positive charge density given for the ammonium quaternary moiety from TDTMA. SIGNIFICANCE AND IMPACT OF THE STUDY: The changes in PL composition give a new insight about the response inflicted by Ps. putida when these bacteria are exposed to QACs.     

Identification and characterization of a mandelamide hydrolase and an NAD(P)+-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633

The enzymes of the mandelate metabolic pathway permit Pseudomonas putida ATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source. The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment. Part of that fragment was shown to contain the genes coding for mandelate racemase, mandelate dehydrogenase, and benzoylformate decarboxylase arranged in an operon. Here we report the sequencing of the remainder of the restriction fragment, which revealed three further open reading frames, denoted mdlX, mdlY, and mdlD. All were transcribed in the opposite direction from the genes of the mdlABC operon. Sequence alignments suggested that the open reading frames encoded a regulatory protein (mdlX), a member of the amidase signature family (mdlY), and an NAD(P)(+)-dependent dehydrogenase (mdlD). The mdlY and mdlD genes were isolated and expressed in Escherichia coli, and the purified gene products were characterized as a mandelamide hydrolase and an NAD(P)(+)-dependent benzaldehyde dehydrogenase, respectively.     

Interaction of Pseudomonas putida ATCC 12633 and Bacteriophage gh-1 in Berea Sandstone Rock

Measurements of the passage of Pseudomonas putida ATCC 12633 and a phage-resistant mutant through Berea sandstone rock were made. When bacteriophage gh-1 was adsorbed within the rock matrix, a reduction in the passage of the susceptible but not the resistant cells through the rock was observed.     

Degradation of cationic surfactants using Pseudomonas putida A ATCC 12633 immobilized in calcium alginate beads

In this study, the degradation of tetradecyltrimethylammonium bromide (TTAB) by freely suspended and alginate-entrapped cells from the bacteria Pseudomonas putida (P. putida) A ATCC 12633 was investigated in batch cultures. The optimal conditions to prepare beads for achieving a higher TTAB degradation rate were investigated by changing the concentration of sodium alginate, pH, temperature, agitation rate and initial concentration of TTAB. The results show that the optimal embedding conditions of calcium alginate beads are 4 % w/v of sodium alginate content and 2 × 10⁸ cfu ml^?1 of P. putida A ATCC 12633 cells that had been previously grown in rich medium. The optimal degradation process was carried out in pH 7.4 buffered medium at 30 °C on a rotary shaker at 100 rpm. After 48 h of incubation, the free cells degraded 26 mg l^?1 of TTAB from an initial concentration of 50 mg l^?1 TTAB. When the initial TTAB concentration was increased to 100 mg l^?1, the free cells lost their degrading activity and were no longer viable. In contrast, when the cells were immobilized on alginate, they degraded 75 % of the TTAB after 24 h of incubation from an initial concentration of 330 mg l^?1 of TTAB. The immobilized cells can be stored at 4 °C for 25 days without loss of viability and can be reused without losing degrading capacity for three cycles.     

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.     

Degradation of tetradecyltrimethylammonium by Pseudomonas putida A ATCC 12633 restricted by accumulation of trimethylamine is alleviated by addition of Al 3+ ions

Aims: To establish if tetradecyltrimethylammonium (TDTMA) might be degraded by pure culture of Pseudomonas strains, and how the presence of a Lewis’ acid in the medium influences its biodegradability. Methods and Results: From different strains of Pseudomonas screened, only Pseudomonas putida A ATCC 12633 grows with 50 mg l^?1 of TDTMA as the sole carbon and nitrogen source. A monooxygenase activity catalyzed the initial step of the biodegradation. The trimethylamine (TMA) produced was used as nitrogen source or accumulated inside the cell. To decrease the intracellular TMA, the culture was divided, and 0·1 mmol l^?1 AlCl₃ added. In this way, the growth and TDTMA consumption increased. The internal concentration of TMA, determined using the fluorochrome Morin, decreased by the formation of Al³⁺ : TMA complex. Conclusions: Pseudomonas putida utilized TDTMA as its sole carbon and nitrogen source. The TMA produced in the initial step of the biodegradation by a monooxygenase activity was used as nitrogen source or accumulated inside the cell, affecting the bacterial growth. This effect was alleviated by the addition of AlCl₃. Significance and Impact of the Study: The use of Lewis’ acids to sequester intracellular amines offers an alternative to achieve an efficient utilization of TDTMA by Ps. putida.     

Formation of Filaments by Pseudomonas putida

When Pseudomonas putida 40 was grown on a variety of liquid media in which oxygen became a limiting factor during growth, the latter stages of growth involved the elongation of cells without septation, which can result in the complete filamentation of the culture (up to several hundred micrometers long). The filaments appeared to consist of a chain of protoplasts within a common sacculus. Later these filaments were capable of a rapid fragmentation by septation to give a population of ordinary rods with a corresponding increase in the number of viable particles but no appreciable change in total bacterial mass. Filamentation did not occur if slow growth rates were maintained by restriction of oxygen availability from the beginning of growth. In complex media filaments were not formed during growth on 1% peptone alone, but the addition of 0.1 M phosphate or 6.6 × 10⁻⁴ M EDTA induced extensive filamentation that was reversed by the addition of 6.6 × 10⁻⁴ M Mg²⁺. In minimal media a much higher Mg²⁺ concentration than that required for active growth or present in the complex media was usually required for filamentation. A very narrow range of Mg²⁺ concentration promoted filamentation, and this optimum differed markedly depending on the carbon source used. Other medium variations which influenced the level of filamentation are reported. We found that most strains of P. putida (including the neotype strain) and P. fluorescens gave filaments under the conditions developed with strain 40, whereas several strains of P. aeruginosa failed to give filaments on the same media.     

Caffeine-inducible enzyme activity in Pseudomonas putida ATCC 700097

Caffeine (1,3,7-trimethylxanthine), a ubiquitous component of human diet has been suggested as a chemical indicator of ecosystem impacts of sewage spills and treated effluent discharges because it is not sufficiently metabolized by wastewater microorganisms. This study identified enzymes responsible for caffeine metabolism in sewage bacteria. Pseudomonas putida biotype A (ATCC 700097) originally isolated as a rare caffeine-degrading organism in domestic wastewater exhibited diauxic growth on caffeine, concomitant with the expression of a P450-type cytochrome and peroxidase enzyme activities. Initial growth phase lasted 13.8 ± 1.4 h with a growth rate that was five times slower than the secondary growth phase that lasted 5.5 ± 1.2 h. Molecular and enzymatic characteristics of the cytochrome P450-type enzyme differ from the previously described cytochrome P450 (P450_cam) of P. putida (ATCC 17453) involved in camphor metabolism. The caffeine-inducible cytochrome P450-type enzyme exhibited a carbon monoxide difference spectrum peak at 450 nm, but does not allow growth on camphor. Caffeine induced production of haem-associated peroxidase activity was confirmed with 3,3, 5,5-tetramethylbenzidine–H₂O₂ reaction in polyacrylamide gels. Polymerase chain reaction (PCR) primers derived from the gene for cytochrome P450_cam (camC) of P. putida (ATCC 17453) did not yield an amplification product when DNA extracted from P. putida strain ATCC 700097 was used as template. The data demonstrate that caffeine is metabolized through a specific biphasic pathway driven by oxygen-demanding enzymes.     

Stabilization and activity-enhancement of mandelate racemase from Pseudomonas putida ATCC 12336 by immobilization

Mandelate racemase [EC 5.1.2.2] from Pseudomonas putida ATCC 12336 was efficiently immobilized through ionic binding onto DEAE- and TEAE 23-cellulose. The activity of the immobilized enzyme was significantly enhanced as compared to the native protein, i.e., 2.7- and 2.5-fold, respectively. DEAE-cellulose-immobilized mandelate racemase could be efficiently used in repeated batch reactions for the racemization of (R)-mandelic acid under mild conditions.     

Fluorene and phenanthrene uptake by Pseudomonas putida ATCC 17514: kinetics and physiological aspects

Pseudomonas putida ATCC 17514 was used as a model strain to investigate the characteristics of bacterial growth in the presence of solid fluorene and phenanthrene. Despite the lower water-solubility of phenanthrene, P. putida degraded this polycyclic aromatic hydrocarbon (PAH) at a maximum observed rate of 1.4 +/- 0.1 mg L(-1) h(-1), higher than the apparent degradation rate of fluorene, 0.8 +/- 0.07 mg L(-1) h(-1). The role of physiological processes on the biodegradation of these PAHs was analyzed and two different uptake strategies were identified. Zeta potential measurements revealed that phenanthrene-grown cells were slightly more negatively charged (-57.5 +/- 4.7 mV) than fluorene-grown cells (-51.6 +/- 4.9 mV), but much more negatively charged than glucose-grown cells (-26.8 +/- 3.3 mV), suggesting that the PAH substrate induced modifications on the physical properties of bacterial surfaces. Furthermore, protein-to-exopolysaccharide ratios detected during bacterial growth on phenanthrene were typical of biofilms developed under physicochemical stress conditions, caused by the presence of sparingly water-soluble chemicals as the sole carbon and energy source for growth, the maximum value for TP/EPS during growth on phenanthrene (1.9) being lower than the one obtained with fluorene (5.5). Finally, confocal laser microscopy observations using a gfp-labeled derivative strain revealed that, in the presence of phenanthrene, P. putida::gfp cells formed a biofilm on accessible crystal surfaces, whereas in the presence of fluorene the strain grew randomly between the crystal clusters. The results showed that P. putida was able to overcome the lower aqueous solubility of phenanthrene by adhering to the solid PAH throughout the production of extracellular polymeric substances, thus promoting the availability and uptake of such a hydrophobic compound.     

Degradation of waste waters from olive oil mills by Yarrowia lipolytica ATCC 20255 and Pseudomonas putida

Waste waters from olive oil processing may cause severe pollution in the Mediterranean area, since they have a high level of chemical oxygen demand (COD) (100–200 g/l) and contain other organic and inorganic compounds. In all olive oil producing countries, the reduction of pollution in olive oil mill waste waters at reasonable costs and using techniques suitable for most industrial applications is an unsolved problem. For this paper, the yeast Yarrowia lipolytica ATCC 20255 was grown on waste waters from an olive oil mill in a 3.5 1 fermenter under batch culture conditions. The results showed that the yeast was capable of reducing the COD value by 80% in 24 h. In this way, a useful biomass of 22.45 g/l as single cell protein (SCP) and enzyme lipase were produced. During this process, most of the organic and inorganic substances were consumed, only aromatic pollutants were still present in the fermentation effluents. Therefore, we used a phenol degrader, namely Pseudomonas putida, to reduce phenolic compounds in the fermentation effuents after removing Yarrowia lipolytica cells. P. putida was effective in reducing phenols in only 12 h.     

Transformation of o-toluate in Pseudomonas putida isolate 1065 and Rhizopus japonicus ATCC 24794

Summary 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.     

FTIR Spectroscopic Study of Biogenic Mn-Oxide Formation by Pseudomonas putida GB-1

Biomineralization in heterogeneous aqueous systems results from a complex association between pre-existing surfaces, bacterial cells, extracellular biomacromolecules, and neoformed precipitates. Fourier transform infrared (FTIR) spectroscopy was used in several complementary sample introduction modes (attenuated total reflectance [ATR], diffuse reflectance [DRIFT], and transmission) to investigate the processes of cell adhesion, biofilm growth, and biological Mn-oxidation by Pseudomonas putida strain GB-1. Distinct differences in the adhesive properties of GB-1 were observed upon Mn oxidation. No adhesion to the ZnSe crystal surface was observed for planktonic GB-1 cells coated with biogenic MnO _x , whereas cell adhesion was extensive and a GB-1 biofilm was readily grown on ZnSe, CdTe, and Ge crystals prior to Mn-oxidation. IR peak intensity ratios reveal changes in biomolecular (carbohydrate, phosphate, and protein) composition during biologically catalyzed Mn-oxidation. In situ monitoring via ATR-FTIR of an active GB-1 biofilm and DRIFT data revealed an increase in extracellular protein (amide I and II) during Mn(II) oxidation, whereas transmission mode measurements suggest an overall increase in carbohydrate and phosphate moieties. The FTIR spectrum of biogenic Mn oxide comprises Mn-O stretching vibrations characteristic of various known Mn oxides (e.g., “acid” birnessite, romanechite, todorokite), but it is not identical to known synthetic solids, possibly because of solid-phase incorporation of biomolecular constituents. The results suggest that, when biogenic MnO _x accumulates on the surfaces of planktonic cells, adhesion of the bacteria to other negatively charged surfaces is hindered via blocking of surficial proteins.     

Purification and characterisation of TOL plasmid-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase of Pseudomonas putida

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two enzymes of the xylene degradative pathway encoded by the plasmid TOL of a Gram-negative bacterium Pseudomonas putida, were purified and characterized. Benzyl alcohol dehydrogenase catalyses the oxidation of benzyl alcohol to benzaldehyde with the concomitant reduction of NAD+; the reaction is reversible. Benzaldehyde dehydrogenase catalyses the oxidation of benzaldehyde to benzoic acid with the concomitant reduction of NAD+; the reaction is irreversible. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase also catalyse the oxidation of many substituted benzyl alcohols and benzaldehydes, respectively, though they were not capable of oxidizing aliphatic alcohols and aldehydes. The apparent Km value of benzyl alcohol dehydrogenase for benzyl alcohol was 220 microM, while that of benzaldehyde dehydrogenase for benzaldehyde was 460 microM. Neither enzyme contained a prosthetic group such as FAD or FMN, and both enzymes were inactivated by SH-blocking agents such as N-ethylmaleimide. Both enzymes were dimers of identical subunits; the monomer of benzyl alcohol dehydrogenase has a mass of 42 kDa whereas that of the monomer of benzaldehyde dehydrogenase was 57 kDa. Both enzymes transfer hydride to the pro-R side of the prochiral C4 of the pyridine ring of NAD+.