Modulation of glucose transport causes preferential utilization of aromatic compounds in Pseudomonas putida CSV86

Pseudomonas putida CSV86 utilizes aromatic compounds in preference to glucose and coutilizes aromatics and organic acids. Protein analysis of cells grown on different carbon sources, either alone or in combination, revealed that a 43-kDa periplasmic-space protein was induced by glucose and repressed by aromatics and succinate. Two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis identified this protein as closely resembling the sugar ABC transporter of Pseudomonas putida KT2440. A partially purified 43-kDa protein showed glucose binding activity and was specific for glucose. The results demonstrate that the aromatic- and organic acid-mediated repression of a periplasmic-space glucose binding protein and consequent inhibition of glucose transport are responsible for this strain's ability to utilize aromatics and organic acids in preference to glucose.     

Preferential utilization of aromatic compounds over glucose by Pseudomonas putida CSV86

Pseudomonas putida CSV86, a naphthalene-degrading organism, exhibited diauxic growth on aromatic compounds plus glucose, with utilization of aromatics in the first log phase and of glucose in the second log phase. Glucose supplementation did not suppress the activity of degrading enzymes, which were induced upon addition of aromatic compounds. The induction was inhibited by chloramphenicol, suggesting that de novo protein synthesis was essential. Cells showed cometabolism of aromatic compounds and organic acids; however, organic acids suppressed glucose utilization.     

Sequential utilization of substrates by Pseudomonas putida CSV86: signatures of intermediate metabolites and online measurements

Pseudomonas putida CSV86 preferentially utilizes aromatics over glucose and co-metabolizes them with organic acids. On aromatics plus glucose, CSV86 utilized aromatics first with concomitant appearance of transient metabolites such as salicylate, benzaldehyde and benzoate. Citrate was the main extracellular metabolite observed during glucose uptake. The strain showed simultaneous utilization of organic acids and aromatic compounds. Based on the metabolite analysis and growth profiles, we hypothesize that the repression of glucose utilization could be due to organic acid intermediates generated from aromatic compound metabolism. The online measurements indicate the instantaneous metabolic state of the culture. For example, the CO₂ evolution and agitation speed show peak values during the two growth phases in the diauxic growth while dissolved oxygen values show decrease at the corresponding durations. These measurements correlated well with the offline measurements but provided a better time resolution of the process.     

Preferential Utilization of Aromatic Compounds over Glucose by Pseudomonas putida CSV86

Pseudomonas putida CSV86, a naphthalene-degrading organism, exhibited diauxic growth on aromatic compounds plus glucose, with utilization of aromatics in the first log phase and of glucose in the second log phase. Glucose supplementation did not suppress the activity of degrading enzymes, which were induced upon addition of aromatic compounds. The induction was inhibited by chloramphenicol, suggesting that de novo protein synthesis was essential. Cells showed cometabolism of aromatic compounds and organic acids; however, organic acids suppressed glucose utilization.     

Metabolism of benzyl alcohol via catechol ortho-pathway in methylnaphthalene-degrading Pseudomonas putida CSV86

Pseudomonas putida CSV86 metabolizes 1- and 2-methylnaphthalene through distinct catabolic and detoxification pathways. In spite of the similarity in the steps involved in the methylnaphthalene detoxification and the toluene side-chain hydroxylation pathways, the strain failed to utilize toluene or xylenes. However, it could grow on benzyl alcohol, 2- and 4-hydroxybenzyl alcohol. Metabolic studies suggest that the benzyl alcohol metabolism proceeds via the benzaldehyde, benzoate, and catechol ortho-cleavage pathway, in contrast to the well established catechol meta-cleavage pathway. Carbon source-dependent enzyme activity studies suggest that the degradation of aromatic alcohol involves two regulons. Aromatic alcohol induces the upper regulon, which codes for aromatic alcohol- and aromatic aldehyde-dehydrogenase and converts alcohol into acid. The aromatic acid so generated induces the specific lower regulon and is metabolized via either the ortho- or the meta-cleavage pathway. CSV86 cells transform 1- and 2-methylnaphthalene to 1- and 2-hydroxymethyl naphthalene, which are further converted to the respective naphthoic acids due to the basal level expression and broad substrate specificity of the upper regulon enzymes.     

Eco-physiological portrait of a novel Pseudomonas sp. CSV86: an ideal host/candidate for metabolic engineering and bioremediation

Pseudomonas sp. CSV86, an Indian soil isolate, degrades wide range of aromatic compounds like naphthalene, benzoate and phenylpropanoids, amongst others. Isolate displays the unique and novel property of preferential utilization of aromatics over glucose and co-metabolizes them with organic acids. Interestingly, as compared to other Pseudomonads, strain CSV86 harbours only high-affinity glucokinase pathway (and absence of low-affinity oxidative route) for glucose metabolism. Such lack of gluconate loop might be responsible for the novel phenotype of preferential utilization of aromatics. The genome analysis and comparative functional mining indicated a large genome (6.79 Mb) with significant enrichment of regulators, transporters as well as presence of various secondary metabolite production clusters, suggesting its eco-physiological and metabolic versatility. Strain harbours various integrative conjugative elements (ICEs) and genomic islands, probably acquired through horizontal gene transfer events, leading to genome mosaicity and plasticity. Naphthalene degradation genes are arranged as regulonic clusters and found to be part of ICE_CSV86nah. Various eco-physiological properties and absence of major pathogenicity and virulence factors (risk group-1) in CSV86 suggest it to be an ideal candidate for bioremediation. Further, strain can serve as an ideal chassis for metabolic engineering to degrade various xenobiotics preferentially over simple carbon sources for efficient remediation.     

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.     

Hyperinduction of enzymes of the phosphorylative pathway of glucose dissimilation inPseudomonas cepacia

Glucose-dehydrogenase-deficient (Gcd^–) strains ofPseudomonas cepacia 249 compensated for loss of operation of the direct oxidative pathway by expanding the phosphorylative pathway. When grown on glucose, they had between two- and fourfold higher than normal levels of glucokinase and NAD-linked glucose-6-phosphate dehydrogenase activity and a comparable increase in capacity to transport glucose. Similar expansion of the phosphorylative pathway was noted when the wild type was grown on cellobiose or trehalose. Gcd^– strains grew normally on cellobiose and trehalose, but not if also deficient in glucokinase; this indicates that the disaccharides were converted to glucose and metabolized via the phosphorylative pathway. The expansion of the phosphorylative pathway during growth of the wild type on disaccharides or of Gcd^– mutants on glucose was a consequence of hyperinduction of pathway enzymes. Other compounds that promoted such hyperinduction included aromatic conjugates of glucose such as arbutin and salicin, and mannose. Under conditions leading to expansion of the phosphorylative pathway, enzymes related to the direct oxidative pathway, such as gluconate dehydrogenase and the 6-phosphogluconate dehydrogenase active with NAD, were not formed. The results indicate that intracellular glucose and extracellular glucose are metabolized to 6-phosphogluconate via different routes.     

Simultaneous Growth on Citrate Reduces the Effects of Iron Limitation during Toluene Degradation in Pseudomonas

Rhizoremediation has been suggested as an attractive bioremediation strategy for the effective breakdown of pollutants in soil. The presence of plant root exudates such as organic acids, sugars, and amino acids that may serve as carbon sources or biosynthetic building blocks and the limited bioavailability of iron may influence the degradation of pollutants in the rhizosphere. To test the effect of such compounds on hydrocarbon degradation, trace concentrations of yeast extract or mixtures of organic acids and amino acids were added to continuous cultures of Pseudomonas putida mt2 and P. putida WCS358 (TOL) growing on toluene. By addition of these compounds increased growth yields and higher specific growth rates on toluene were obtained. The effects of iron limitation on the substrate utilization pattern of both strains were tested by growing the strains on a mixture of toluene and the readily degradable carbon source citrate while the iron concentration was varied. Simultaneous use of both substrates under carbon-limited as well as iron-limited conditions was observed. Growth yields were less reduced and iron requirement was lower during iron-limited growth in the toluene + citrate grown cultures compared to cultures in which toluene was used as the sole carbon source. The kinetic properties of the cells for toluene degradation were less hampered by the lack of iron when citrate was used as an additional carbon source. The results indicate that the availability of low concentrations of natural organic compounds, such as produced in the rhizosphere, may positively influence the degradative performance of hydrocarbon-degrading bacteria.     

Induction and quantification of phenylacyl-CoA ligase enzyme activities in Pseudomonas putida CA-3 grown on aromatic carboxylic acids

Three phenylacyl-CoA ligase activities were detected in extracts of Pseudomonas putida CA-3 cells grown with a variety of aromatic carboxylic acids. The three phenylacyl-CoA enzyme activities measured were phenylpropyl-CoA ligase (acting on both phenylpropanoic acid and cinnamic acid), a phenylacetyl-CoA ligase, and a medium chain length phenylalkanoyl-CoA ligase acting on aromatic substrates with 5 or more carbons in the acyl moiety. The rate of each enzyme activity detected in extracts of P. putida CA-3 cells is dependent on the growth substrate supplied. High rates of phenylpropyl-CoA ligase activity were observed with extracts of cells grown on phenylpropanoic acid, cinnamic acid or medium chain length phenylalkanoic acids with an uneven number of carbons in the acyl moiety. Extracts of P. putida CA-3 cells exhibited high rates of phenylacetyl-CoA ligase activity when grown on phenylacetic acid or medium chain length phenylalkanoic acids with an even number of carbons in the acyl moiety. In addition, high rates of medium chain length phenylalkanoyl-CoA ligase activity, towards phenylvaleric acid and phenylhexanoic acid, were exhibited by extracts of cells grown on all medium chain length phenylalkanoic acids. Low levels of the various phenylacyl-CoA ligase activities were found in extracts of cells grown on benzoic acid and glucose. Benzoyl-CoA ligase activity was not detected in any cell free extracts generated in this study.     

PcaK, a high-affinity permease for the aromatic compounds 4-hydroxybenzoate and protocatechuate from Pseudomonas putida.

PcaK is a transporter and chemoreceptor protein from Pseudomonas putida that is encoded as part of the beta-ketoadipate pathway regulon for aromatic acid degradation. When expressed in Escherichia coli, PcaK was localized to the membrane and catalyzed the accumulation of two aromatic substrates, 4-hydroxybenzoate and protocatechuate, against a concentration gradient. Benzoate inhibited 4-hydroxybenzoate uptake but was not a substrate for PcaK-catalyzed transport. A P. putida pcaK mutant was defective in its ability to accumulate micromolar amounts of 4-hydroxybenzoate and protocatechuate. The mutant was also impaired in growth on millimolar concentrations of these aromatic acids. In contrast, the pcaK mutant grew at wild-type rates on benzoate. The Vmax for uptake of 4-hydroxybenzoate was at least 25 nmol/min/mg of protein, and the Km was 6 microM. PcaK-mediated transport is energized by the proton motive force. These results show that although aromatic acids in the undissociated (uncharged) form can diffuse across bacterial membranes, high-specificity active transport systems probably also contribute to the ability of bacteria to grow on the micromolar concentrations of these compounds that are typically present in soil. A variety of aromatic molecules, including naturally occurring lignin derivatives and xenobiotics, are metabolized by bacteria and may be substrates for transport proteins. The characterization of PcaK provides a foundation for understanding active transport as a critical step in the metabolism of aromatic carbon sources.     

Taxonomic identification,phenanthrene uptake activity,and membrane lipid alterations of the PAH degrading <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. strain Sphe3

This report describes phenanthrene uptake as well as the effect of phenanthrene on the membrane phospholipid and fatty acid composition in a newly isolated bacterial strain, Sphe3, that we taxonomically identified as Arthrobacter sp. Strain Sphe3 is able to utilize phenanthrene as a carbon source at high rates and appears to internalize phenanthrene with two mechanisms: a passive diffusion when cells are grown on glucose, and an inducible active transport system when cells are grown on phenanthrene as a sole carbon source. Active transport followed Michaelis-Menten kinetics, and it was amenable to inhibition by 2,4-dinitrophenol and sodium azide. Evidence provided here indicates that apart from inducing an active PAH uptake, the presence of phenanthrene elicits significant changes in membrane fluidity.     

Microbial cooxidation of naphthalene for the production of 1,2-dihydroxy-1,2-dihydronaphthalene

A microbial cooxidation process for 1,2-dihydroxy-1,2-dihydronaphthalene from naphthalene has been demonstrated. A Pseudomonas putida it119 mutant strain grown with glucose as the sole carbon and energy source was used to oxidize naphthalene. Growth characteristics of the P. putida mutant strain were studied in both batch and continuous fermentation experiments. The rate of product formation was found to depend on naphthalene particle sizes, initial naphthalene and glucose concentrations. Kinetic models were developed to quantify the microbial cooxidation process and a two-stage fermentation process is proposed for further studies.     

Role of nitrogen and carbon transport, regulation, and metabolism genes for Saccharomyces cerevisiae survival in vivo

Saccharomyces cerevisiae is both an emerging opportunistic pathogen and a close relative of pathogenic Candida species. To better understand the ecology of fungal infection, we investigated the importance of pathways involved in uptake, metabolism, and biosynthesis of nitrogen and carbon compounds for survival of a clinical S. cerevisiae strain in a murine host. Potential nitrogen sources in vivo include ammonium, urea, and amino acids, while potential carbon sources include glucose, lactate, pyruvate, and fatty acids. Using mutants unable to either transport or utilize these compounds, we demonstrated that no individual nitrogen source was essential, while glucose was the most significant primary carbon source for yeast survival in vivo. Hydrolysis of the storage carbohydrate glycogen made a slight contribution for in vivo survival compared with a substantial requirement for trehalose hydrolysis. The ability to sense and respond to low glucose concentrations was also important for survival. In contrast, there was little or no requirement in vivo in this assay for any of the nitrogen-sensing pathways, nitrogen catabolite repression, the ammonium- or amino acid-sensing pathways, or general control. By using auxotrophic mutants, we found that some nitrogenous compounds (polyamines, methionine, and lysine) can be acquired from the host, while others (threonine, aromatic amino acids, isoleucine, and valine) must be synthesized by the pathogen. Our studies provide insights into the yeast-host environment interaction and identify potential antifungal drug targets.     

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.     

Maltose uptake and its regulation in Bacillus subtilis

Extracts prepared from cultures of Bacillus subtilis, grown on maltose as the sole carbon source, lacked maltose phosphotransferase system activity. There was, however, evidence for a maltose phosphorylase activity, and such extracts also possessed both glucokinase and glucose phosphotransferase system activities. Maltose was accumulated by whole cells of B. subtilis by an energy-dependent mechanism. This uptake was sensitive to the effects of uncouplers, suggesting a role for the proton-motive force in maltose transport. Accumulation of maltose was inhibited in the presence of glucose, and there was no accumulation of maltose by a strain carrying the ptsI6 null-mutation. A strain carrying the temperature-sensitive ptsI1 mutation accumulated maltose normally at 37 degrees C but, in contrast to the wild-type, was devoid of maltose transport activity at 47 degrees C. The results indicate a role for the phosphotransferase system in the regulation of maltose transport activity in this organism.     

Bacterial cis-dihydrodiol dehydrogenases: comparison of physicochemical and immunological protperties.

Cells of Pseudomonas putida NP, Pseudomonas species (NCIB 9816), and a Nocardia species, after growth on naphthalene as sole source of carbon and energy, contain a nicotinamide adenine dinucleotide (NAD+)-dependent enzyme that oxidizes cis-dihydrodiols of mono- and polycyclic aromatic compounds. Similarly, cells of a strain of P. putida biotype A, when grown either on toluene or benzene vapors, were found to contain a dehydrogenase that oxidized dihydrodiols of aromatic hydrocarbons with cis stereochemistry and required NAD+ as an electron acceptor. In all these cases, no enzymatic activity was detected when trans-naphthalene dihydrodiol was used as a substrate. Purified cis-naphthalene dihydrodiol dehydrogenase was injected into rabbits to obtain antibodies. Physiocochemical and immunological properties of cis-dihydrodiol:NAD+ oxidoreductases from four different organisms were examined. Kinetic analysis showed that, in all the cases, enzymes exhibited higher affinity for cis-dihydrodiols than for NAD+ and had pH optima between 8.8 and 9.0. except in the case of the enzyme from Nocarida sp., which showed maximum activity at pH 8.4. Molecular-weight determination of the dehydrogenases from the four different organisms by gel filtration on a Sephadex G-200 column gave values ranging from 92,000 for the enzyme from Nocardia sp. to 160,000 for that from P. putida biotype A. All the dehydrogenases, except the one from Nocardia sp., exhibited immunological cross-reaction with the antibodies prepared against the enzyme purified from P. putida NP.     

para-Aminobenzoic acid as a sole source of carbon and energy for Pseudomonas desmoliticum]

A strain of Psuedomonas desmoliticum has been isolated from soil. It utilizes, as a source of carbon and energy, p-aminobenozic (PABA), p-fluorobenzoic acids, and some other aromatic compounds. The strain has been isolated by inoculating soil suspensions onto Petri plates with a solid mineral medium containing 0.1% PABA as a carbon source. The preparatory metabolism of PABA was studied in this work; p-hydroxybenzoic and protocatechuic acids were found to be its intermediate products. Enzyme systems catalysing oxidation of aromatic compounds and glucose are inducible.