p-cymene pathway in Pseudomonas putida: initial reactions.

Initial reactions of the p-cymene pathway induced in Pseudomonas putida PL have been reinvestigated. Oxidation of the methyl group attached to the nucleus occurs in three steps to give p-cumic acid. The substrate for the ring cleavage of 2,3-dihydroxy-p-cumate is formed from p-cumate in two reactions via a dihydrodiol intermediate (2,3-dihydroxy-4-isopropylcyclohexa-4,6-dienoate) and not as previously postulated via 3-hydroxy-p-cumate. There are three pieces of evidence for the physiological role of the dihydrodiol intermediate. (i) a mutant of P. putida PL-pT-11/43, which is unable to grow with p-cumate, accumulates a compound from p-cumate, which was identified as 2,3-dihydroxy-4-isopropylcyclohexa-4,6-dienoate. (II) This metabolite is enzymically oxidized by a nicotinamide adenine dinucleotide-dependent dehydrogenase that is present in crude extracts of the wild type and a revertant strain (PL-pT-11/43-R1) but not in the mutant. (iii) 3-Hydroxy-p-cumate does not support growth of P . putida PL-W, and it is not oxidized by cells or extracts. 3-Hydroxy-p-cumate was readily isolated as before from culture supernatants, due to its ready formation from the dihydrodiol in acid solution. Mass spectral analysis of the dihydrodiol accumulated in 18O2-enriched atmospheres showed that both hydroxyl atoms are derived from the same molecule of O2. The formation and absorbance maxima of dihydrodiols that accumulated during the growth of the mutant PL-pT-11/43 in the presence of various benzoates (or toluenes) that have substituents at the carbon 4 atom also is reported.     

Biotransformations catalyzed by cloned p-cymene monooxygenase from Pseudomonas putida F1

p-Cymene monooxygenase (CMO) from Pseudomonas putida F1 consists of a hydroxylase (CymA1) and a reductase component (CymA2) which initiate pcymene (p-isopropyltoluene) catabolism by oxidation of the methyl group to p-isopropylbenzyl alcohol (p-cumic alcohol). To study the possible diverse range of substrates catalyzed by CMO, the cymA1A2 genes were cloned in an Escherichia coli pT7-5 expression system and the cells were used in transformation experiments. The tested substrates include different substituents on the aromatic ring at the 2 (ortho), 3 (meta) or 4 (para) position relative to the methyl moiety. As a result, a distinct preference was observed for substrates containing at least an alkyl or heteroatom substituent at the para-position of toluene. The conversion rate of 4-chlorotoluene or 4-methylthiotoluene to the corresponding benzyl alcohol was found to be as good as the canonical substrate, p-cymene. But 3-chlorotoluene, 4-fluorotoluene and 4-nitrotoluene were relatively poor substrates. CMO is also capable of producing styrene oxide from styrene. However, the oxidation of 4-chlorostyrene to 4-chlorostyrene oxide was by far the fastest among the substrates used in this study. The various biotransformation products were identified by a combined solid phase microextraction/gas chromatographic-mass spectrometric analytical technique.     

Regulation of alkane oxidation in Pseudomonas putida.

We have studied the appearance of whole-cell oxidizing activity for n-alkanes and their oxidation products in strains of Pseudomonas putida carrying the OCT plasmid. Our results indicate that the OCT plasmid codes for inducible alkane-hydroxylating and primary alcohol-dehydrogenating activities and that the chromosome codes for constitutive oxidizing activities for primary alcohols, aliphatic aldehydes, and fatty acids. Mutant isolation confirms the presence of an alcohol dehydrogenase locus on the OCT plasmid and indicated the presence of multiple alcohol and aldehyde dehydrogenase loci on the P. putida chromosome. Induction tests with various compounds indicate that inducer recognition has specificity for chain length and can be affected by the degree of oxidation of the carbon chain. Some inducers are neither growth nor respiration substrates. Growth tests with and without a gratuitous inducer indicate that undecane is not a growth substrate because it does not induce alkane hydroxylase activity. Using a growth test for determining induction of the plasmid alcohol dehydrogenase it is possible to show that heptane induces this activity in hydroxylase-negative mutants. This suggests that unoxidized alkane molecules are the physiological inducers of both plasmid activities.     

Cloning of genes specifying carbohydrate catabolism in Pseudomonas aeruginosa and Pseudomonas putida.

A 6.0-kilobase EcoRI fragment of the Pseudomonas aeruginosa PAO chromosome containing a cluster of genes specifying carbohydrate catabolism was cloned into the multicopy plasmid pRO1769. The vector contains a unique EcoRI site for cloning within a streptomycin resistance determinant and a selectable gene encoding gentamicin resistance. Mutants of P. aeruginosa PAO transformed with the chimeric plasmid pRO1816 regained the ability to grow on glucose, and the following deficiencies in enzyme or transport activities corresponding to the specific mutations were complemented: glcT1, glucose transport and periplasmic glucose-binding protein; glcK1, glucokinase; and edd-1, 6-phosphogluconate dehydratase. Two other carbohydrate catabolic markers that are cotransducible with glcT1 and edd-1 were not complemented by plasmid pRO1816: zwf-1, glucose-6-phosphate dehydrogenase; and eda-9001, 2-keto-3-deoxy-6-phosphogluconate aldolase. However, all five of these normally inducible activities were expressed at markedly elevated basal levels when transformed cells of prototrophic strain PAO1 were grown without carbohydrate inducer. Vector plasmid pRO1769 had no effect on the expression of these activities in transformed mutant or wild-type cells. Thus, the chromosomal insert in pRO1816 contains the edd and glcK structural genes, at least one gene (glcT) that is essential for expression of the glucose active transport system, and other loci that regulate the expression of the five clustered carbohydrate catabolic genes. The insert in pRO1816 also complemented the edd-1 mutation in a glucose-negative Pseudomonas putida mutant but not the eda-1 defect in another mutant. Moreover, pRO1816 caused the expression of high specific activities of glucokinase, an enzyme that is naturally lacking in these strains of Pseudomonas putida.     

Expression of the argF gene of Pseudomonas aeruginosa in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli

R' plasmids carrying argF genes from Pseudomonas aeruginosa strains PAO and PAC were transferred to Pseudomonas putida argF and Escherichia coli argF strains. Expression in P. putida was similar to that in P. aeruginosa and was repressed by exogenous arginine. Expression in E. coli was 2 to 4% of that in P. aeruginosa. Exogenous arginine had no effect, and there were no significant differences between argR' and argR strains of E. coli in this respect.     

Growth of genetically engineered Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere.

The effect of the addition of a recombinant plasmid containing the pglA gene encoding an alpha-1,4-endopolygalacturonase from Pseudomonas solanacearum on the growth of Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere was determined. Despite a high level of polygalacturonase production by genetically engineered P. putida and P. aeruginosa, the results suggest that polygalacturonase production had little effect on the growth of these strains in soil or rhizosphere.     

Molecular characterization of Rifr mutations in Pseudomonas aeruginosa and Pseudomonas putida

The rpoB gene encoding for β subunit of RNA polymerase is a target of mutations leading to rifampicin resistant (Rif^r) phenotype of bacteria. Here we have characterized rpoB/Rif^r system in Pseudomonas aeruginosa and Pseudomonas putida as a test system for studying mutational processes. We found that in addition to the appearance of large colonies which were clearly visible on Rif selective plates already after 24 h of plating, small colonies grew up on these plates for 48 h. The time-dependent appearance of the mutant colonies onto selective plates was caused by different levels of Rif resistance of the mutants. The Rif^r clusters of the rpoB gene were sequenced and analyzed for 360 mutants of P. aeruginosa and for 167 mutants of P. putida. The spectrum of Rif^r mutations characterized for P. aeruginosa grown at 37 °C and that characterized for P. putida grown at 30 °C were dissimilar but the differences almost disappeared when the mutants of both strain were isolated at the same temperature, at 30 °C. The strong Rif^r phenotype of P. aeruginosa and P. putida was accompanied only with substitutions of these residues which belong to the putative Rif-binding pocket. Approximately 70% of P. aeruginosa mutants, which were isolated at 37 °C and expressed weak Rif^r phenotype, contained base substitutions in the N-terminal cluster of the rpoB gene. The differences in the spectra of mutations at 30 °C and 37 °C can be explained by temperature-sensitive growth of several mutants in the presence of rifampicin. Thus, our results imply that both the temperature for the growth of bacteria and the time for isolation of Rif^r mutants from selective plates are critical when the rpoB/Rif^r test system is employed for comparative studies of mutagenic processes in Pseudomonas species which are conventionally cultivated at different temperatures.     

Plasmid characteristics of naphthalene and salicylate biodegradation in Pseudomonas putida

The object of this work was to study the physico-chemical and biological properties of DNAs of the biodegradation plasmids NAH and SAL. A comparative analysis of the physico-chemical parameters for these DNAs made it possible to detect a number of identical properties in them: the same sedimentation profile for covalently-closed circular DNA forms, 68--70 S; the molecular weight of ca. 50 MD; a roughly equal number of fragments (up to 23) was found when the DNAs of NAH and SAL were restricted by EcoRI endonuclease. The transformation of the plasmidless strain PpGI was done.     

Degradation of Ethylbenzene by Pseudomonas putida Harboring OCT Plasmid

We have investigated the utilization of a variety of alkylbenzenes by P. putida strains and found that a strain harboring the OCT plasmid assimilated ethylbenzene. The linkage between the determinant for the degradation of ethylbenzene (Etb⁺ phenotype) and the OCT plasmid was inferred from conjugation experiments. The growth characteristics of the strains carrying mutations in the alk genes of the OCT plasmid which determine the assimilation of /t-alkanes indicated that alkB, alkA, and alkR should be responsible for the degradation of ethylbenzene. The exposure of ethylbenzene to the P. putida strain harboring the CAM-OCT plasmid resulted in the accumulation of β-phenylethyl alcohol. A possible degradation pathway for ethylbenzene including the terminal oxidation of the alkyl side chain was proposed.     

Plasmid modification of radiation and chemical-mutagen sensitivity in Pseudomonas aeruginosa.

The R factor pMG2 protects Pseudomonas aeruginosa against the lethal effects of ultraviolet (u.v.) and gamma irradiation, and methyl methanesulphonate and N-methyl-N'-nitro-N-nitrosoguanidine treatment. Enhanced survival occurs in strains of uvr+ rec+ (wild-type) genotype and a variety of uvr rec+ type mutants. No protection occurs in a rec A-type mutant. The plasmid also enhances u.v.-induced mutagenesis. These effects appear to be due to host-cell controlled plasmid-determined DNA repair function(s). Studies on P. aeruginosa strains deficient in DNA polymerase I (polyA) suggest that a plasmid-determined repair resynthesis function may be responsible for increased u.v.-survival and enhanced u.v.-mutability in pMG2-containing bacteria.     

Initial reactions in the oxidation of naphthalene by Pseudomonas putida.

A strain of Pseudomonas putida that can utilize naphthalene as its sole source of carbon and energy was isolated from soil. A mutant strain of this organism, P. putida 119, when grown on glucose in the presence of naphthalene, accumulates optically pure (+)-cis-1(R),2(S)-dihydroxy-1,2-dihydronaphthalene in the culture medium. The cis relative stereochemistry in this molecule was established by nuclear magnetic resonance spectrometry. Radiochemical trapping experiments established that this cis dihydrodiol is an intermediate in the metabolism of naphthalene by P. Fluorescens (formerly ATCC, 17483), P. putida (ATCC, 17484), and a Pseudomonas species (NCIB 9816), as well as the parent strain of P. putida described in this report. Formation of the cis dihydrodiol is catalyzed by a dioxygenase which requires either NADH or NADPH as an electron donor. A double label procedure is described for determining the origin of oxygen in the cis dihydrodiol under conditions where this metabolite would not normally accumulate. Several aromatic hydrocarbons are oxidized by cell extracts prepared from naphthalene-grown cells of P. putida. The cis dihydrodiol is converted to 1,2-dihydroxynaphthalene by an NAD+-dependent dehydrogenase. This enzyme is specific for the (+) isomer of the dihydrodiol and shows a primary isotope effect when the dihydrodiol is substituted at C-2 with deuterium.     

Plasmid Involvement in Acyclic Isoprenoid Metabolism by Pseudomonas putida

An organism identified as Pseudomonas putida was found to utilize citronellol or geraniol as the sole carbon and energy source. The ability to degrade these acyclic isoprenols was associated with pSRQ50, a 50-megadalton transmissible plasmid.     

Heterologous production of Pseudomonas aeruginosa EMS1 biosurfactant in Pseudomonas putida

A new bacterial strain isolated from activated sludge, identified as Pseudomonas aeruginosa EMS1, produced a biosurfactant when grown on acidified soybean oil as the sole carbon source. An optimum biosurfactant production of 5 g/L was obtained with the following medium composition: 2% acidified soybean oil, 0.3% NH4NO3, 0.03% KH2PO4, 0.03% K2HPO4, 0.02% MgSO4.7H2O and 0.025% CaCl2.2H2O, with shaking at 200 rpm for an incubation period of 100 h at 30 degrees C. The production of the biosurfactant was found to be a function of cell growth, with maximum production occurring during the exponential phase. Hemolysis of erythrocytes and thin-layer chromatography studies revealed that the secreted biosurfactant was rhamnolipid. To overcome the complex environmental regulation with respect to rhamnolipid biosynthesis, and to replace the opportunistic pathogen P. aeruginosa with a safe industrial strain, attempts were made to achieve rhamnolipid production in a heterologous host, Pseudomonas putida, using molecular cloning of rhlAB rhamnosyltransferase genes with the rhlRI quorum sensing system, assuming that a functional rhamnosyltransferase would catalyze the formation of rhamnosyl-6-hydroxydecanoyl-6-hydroxydecanoate (mono-rhamnolipid) in P. putida. It was shown that rhamnolipid can be produced in the heterologous strain, P. putida, when provided with the rhamnosyltransferase genes.     

Use of ureidopenicillins for selection of plasmid vector transformants in Pseudomonas aeruginosa and Pseudomonas putida.

Broad-host-range plasmids coding for beta-lactamase were successfully selected after transformation of Pseudomonas strains. Transformants of both Pseudomonas aeruginosa and Pseudomonas putida containing plasmid pRO1614 were isolated in media containing low concentrations of piperacillin. These strains were also susceptible to other ureidopenicillins. Similar selections of transformants with carbenicillin, ampicillin, or ticarcillin required high concentrations of antibiotics and yielded backgrounds of spontaneous resistant mutants.     

Growth of genetically engineered Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere

The effect of the addition of a recombinant plasmid containing the pglA gene encoding an alpha-1,4-endopolygalacturonase from Pseudomonas solanacearum on the growth of Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere was determined. Despite a high level of polygalacturonase production by genetically engineered P. putida and P. aeruginosa, the results suggest that polygalacturonase production had little effect on the growth of these strains in soil or rhizosphere.     

Paraffin oxidation in Pseudomonas aeruginosa. I. Induction of paraffin oxidation

The induction of paraffin oxidation in intact cells of Pseudomonas aeruginosa was investigated. Oxidation of (14)C-heptane by cell-free extracts of adapted cells showed that the activity of whole cells is a reliable reflection of the synthesis of the first enzyme in the degradation of n-alkanes. Induction was significantly affected by glucose and could be completely repressed by malate. The amino acids l-proline, l-alanine, l-arginine, and l-tyrosine exhibited a rather low repressor action. Malonate, a nonrepressive carbon source, allowed gratuitous enzyme synthesis. A number of compounds which did not sustain growth were found to be suitable substitutes for paraffins as an inducer. Among these were cyclopropane and diethoxymethane. The induction studied under conditions of gratuity with the latter compound as an inducer showed immediate linear kinetics only at saturating inducer concentrations. With n-hexane as the inducer, a lag time was always observed, even when high concentrations were used.     

High-throughput dilution-based growth method enables time-resolved exo-metabolomics of Pseudomonas putida and Pseudomonas aeruginosa

Understanding metabolism is fundamental to access and harness bacterial physiology. In most bacteria, nutrient utilization is hierarchically optimized according to their energetic potential and their availability in the environment to maximise growth rates. Low-throughput methods have been largely used to characterize bacterial metabolic profiles. However, in-depth analysis of large collections of strains across several conditions is challenging since high-throughput approaches are still limited – especially for non-traditional hosts. Here, we developed a high-throughput dilution-resolved cultivation method for metabolic footprinting of Pseudomonas putida and Pseudomonas aeruginosa. This method was benchmarked against a conventional low-throughput time-resolved cultivation approach using either a synthetic culture medium (where a single carbon source is present) for P. putida or a complex nutrient mixture for P. aeruginosa. Dynamic metabolic footprinting, either by sugar quantification or by targeted exo-metabolomic analyses, revealed overlaps between the bacterial metabolic profiles irrespective of the cultivation strategy, suggesting a certain level of robustness and flexibility of the high-throughput dilution-resolved method. Cultivation of P. putida in microtiter plates imposed a metabolic constraint, dependent on oxygen availability, which altered the pattern of secreted metabolites at the level of sugar oxidation. Deep-well plates, however, constituted an optimal cultivation set-up yielding consistent and comparable metabolic profiles across conditions and strains. Altogether, the results illustrate the usefulness of this technological advance for high-throughput analyses of bacterial metabolism for both biotechnological applications and automation purposes.     

Transduction and Genetic Homology Between Pseudomonas Species putida and aeruginosa

Genetic crosses occur by transduction between the species Pseudomonas aeruginosa and P. putida. The frequency relative to intraspecific transfer is reduced and varies among markers, suggesting that these genomes contain discrete regions of homology and nonhomology.     

Formate-stimulated oxidation of methanol by Pseudomonas putida 9816

It has been reported that Pseudomonas putida 9816 is able to grow on methanol, but it does not have methanol dehydrogenase or oxidase activity. To utilize methanol it requires yeast extract. The utilization of methanol can be accelerated by adding formate, which obviously helps oxidize methanol and win biologically useful energy. This pseudo-oxidation is catalyzed by a reverse formaldehyde dismutase. Thus, methanol can be both assimilated and dissimilated. Formate alone cannot replace yeast extract. The strain is auxotrophic.