Effects of iron limitation on the degradation of toluene by Pseudomonas strains carrying the tol (pWWO) plasmid

Most aerobic biodegradation pathways for hydrocarbons involve iron-containing oxygenases. In iron-limited environments, such as the rhizosphere, this may influence the rate of degradation of hydrocarbon pollutants. We investigated the effects of iron limitation on the degradation of toluene by Pseudomonas putida mt2 and the transconjugant rhizosphere bacterium P. putida WCS358(pWWO), both of which contain the pWWO (TOL) plasmid that harbors the genes for toluene degradation. The results of continuous-culture experiments showed that the activity of the upper-pathway toluene monooxygenase decreased but that the activity of benzyl alcohol dehydrogenase was not affected under iron-limited conditions. In contrast, the activities of three meta-pathway (lower-pathway) enzymes were all found to be reduced when iron concentrations were decreased. Additional experiments in which citrate was used as a growth substrate and the pathways were induced with the gratuitous inducer o-xylene showed that expression of the TOL genes increased the iron requirement in both strains. Growth yields were reduced and substrate affinities decreased under iron-limited conditions, suggesting that iron availability can be an important parameter in the oxidative breakdown of hydrocarbons.     

Effects of Iron Limitation on the Degradation of Toluene by Pseudomonas Strains Carrying the TOL (pWWO) Plasmid

Most aerobic biodegradation pathways for hydrocarbons involve iron-containing oxygenases. In iron-limited environments, such as the rhizosphere, this may influence the rate of degradation of hydrocarbon pollutants. We investigated the effects of iron limitation on the degradation of toluene by Pseudomonas putida mt2 and the transconjugant rhizosphere bacterium P. putida WCS358(pWWO), both of which contain the pWWO (TOL) plasmid that harbors the genes for toluene degradation. The results of continuous-culture experiments showed that the activity of the upper-pathway toluene monooxygenase decreased but that the activity of benzyl alcohol dehydrogenase was not affected under iron-limited conditions. In contrast, the activities of three meta-pathway (lower-pathway) enzymes were all found to be reduced when iron concentrations were decreased. Additional experiments in which citrate was used as a growth substrate and the pathways were induced with the gratuitous inducer o-xylene showed that expression of the TOL genes increased the iron requirement in both strains. Growth yields were reduced and substrate affinities decreased under iron-limited conditions, suggesting that iron availability can be an important parameter in the oxidative breakdown of hydrocarbons.     

Evaluation of novel assays to assess the influence of different iron sources on the growth of Clostridium difficile

The ability of four Clostridium difficile strains to utilize various exogenous organic and inorganic iron sources for growth under iron-depleted (250 μM DPP) and iron-limited (75 μM DPP) conditions was analyzed in liquid broth cultures grown in tubes and in microtiter plates, and data compared with results from a bioassay developed on solid media. The growth profile of C. difficile varied depending on the iron source and availability. Addition of FeSO(4), FeCl(3), Fe citrate and ferritin allowed growth in an iron-depleted environment whereas glycoproteins (iron-saturated and low-iron lactoferrin, apo- and holo-transferrin) and heme proteins (hemoglobin, hematin and hemin) did not. All iron sources, except lactoferrin, were able to restore bacterial growth under iron-limited conditions to varying extents. The results demonstrated that the broth microtiter assay developed here was reproducible, reliable and convenient for high-throughput analysis of the growth of C. difficile compared to alternative traditional methods.     

Substrate inhibition kinetics for toluene and benzene degrading pure cultures and a method for collection and analysis of respirometric data for strongly inhibited cultures

We present an evaluation of the qualitative and quantitative effects that high concentrations of benzene and toluene have on the growth rate of several pure cultures that use these compounds as their sole carbon and energy source. The cultures employed were five widely studied environmental isolates: Pseudomonas putida F1, P. putida mt2, P. mendocina KR, Ralstonia pickettii PKO1, and Burkholderia cepacia G4. Three cultures degraded toluene following a pattern consistent with the kinetic model of Wayman and Tseng (1976) while the other two followed a modification of this model introduced by Alagappan and Cowan (2001). The pattern followed for benzene degradation was different than that for toluene degradation for all four capable pure cultures and consistent with that described by the model of Luong (1987). Mechanisms of substrate inhibition and solvent toxicity are discussed, used to conceptually evaluate the reasons for the differences in inhibition behavior, and used to support a call for more widespread use of the empirical, terminal substrate concentration inhibition models employed here. We also present the methodology developed to overcome a limitation commonly encountered when attempting to collect oxygen uptake data for use in quantifying substrate inhibition kinetics. The experimental method was effective for use in the collection of high quality data and the substrate inhibition models most useful in representing the growth of bacteria on these solvents are those that show a complete loss of activity at high concentration rather than the more popular asymptotic inhibition models.     

Anaerobic degradation of toluene and o-xylene by a methanogenic consortium.

Toluene and o-xylene were completely mineralized to stoichiometric amounts of carbon dioxide, methane, and biomass by aquifer-derived microorganisms under strictly anaerobic conditions. The source of the inoculum was creosote-contaminated sediment from Pensacola, Fla. The adaptation periods before the onset of degradation were long (100 to 120 days for toluene degradation and 200 to 255 days for o-xylene). Successive transfers of the toluene- and o-xylene-degrading cultures remained active. Cell density in the cultures progressively increased over 2 to 3 years to stabilize at approximately 10(9) cells per ml. Degradation of toluene and o-xylene in stable mixed methanogenic cultures followed Monod kinetics, with inhibition noted at substrate concentrations above about 700 microM for o-xylene and 1,800 microM for toluene. The cultures degraded toluene or o-xylene but did not degrade m-xylene, p-xylene, benzene, ethylbenzene, or naphthalene. The degradative activity was retained after pasteurization or after starvation for 1 year. Degradation of toluene and o-xylene was inhibited by the alternate electron acceptors oxygen, nitrate, and sulfate. Degradation was also inhibited by the addition of preferred substrates such as acetate, H2, propionate, methanol, acetone, glucose, amino acids, fatty acids, peptone, and yeast extract. These data suggest that the presence of natural organic substrates or contaminants may inhibit anaerobic degradation of pollutants such as toluene and o-xylene at contaminated sites.     

腐殖质在环境污染物生物降解中的作用研究进展

腐殖质物质在地球的生态环境中大量存在,它不仅可以在有毒化合物的生物降解和生物转化过程中起到氧化还原中间体的作用,加速有毒物质的降解和转化。也可以作为唯一末端电子受体,接受来自一些有机酸或者甲苯等环境中有毒物质提供的电子,偶联能量的产生,支持菌体的生长,形成一种新的细菌厌氧呼吸形式——腐殖质呼吸。因此,对腐殖质在环境有毒物质的生物降解和生物转化过程中的作用进行研究,不仪对于深入理解细菌呼吸的本质具有重要的理论意义,而且对于环境有毒物质的降解和转化以及元素的生物地球化学循环具有重要的生态学意义,同时对地球表面的有毒物质进行更有效的生物降解具有重要的现实意义。     

Physiological and Proteomic Analysis of Escherichia coli Iron-Limited Chemostat Growth

Iron bioavailability is a major limiter of bacterial growth in mammalian host tissue and thus represents an important area of study. Escherichia coli K-12 metabolism was studied at four levels of iron limitation in chemostats using physiological and proteomic analyses. The data documented an E. coli acclimation gradient where progressively more severe iron scarcity resulted in a larger percentage of substrate carbon being directed into an overflow metabolism accompanied by a decrease in biomass yield on glucose. Acetate was the primary secreted organic by-product for moderate levels of iron limitation, but as stress increased, the metabolism shifted to secrete primarily lactate (∼70% of catabolized glucose carbon). Proteomic analysis reinforced the physiological data and quantified relative increases in glycolysis enzyme abundance and decreases in tricarboxylic acid (TCA) cycle enzyme abundance with increasing iron limitation stress. The combined data indicated that E. coli responds to limiting iron by investing the scarce resource in essential enzymes, at the cost of catabolic efficiency (i.e., downregulating high-ATP-yielding pathways containing enzymes with large iron requirements, like the TCA cycle). Acclimation to iron-limited growth was contrasted experimentally with acclimation to glucose-limited growth to identify both general and nutrient-specific acclimation strategies. While the iron-limited cultures maximized biomass yields on iron and increased expression of iron acquisition strategies, the glucose-limited cultures maximized biomass yields on glucose and increased expression of carbon acquisition strategies. This study quantified ecologically competitive acclimations to nutrient limitations, yielding knowledge essential for understanding medically relevant bacterial responses to host and to developing intervention strategies.     

Utilization of Iron Gallate and Other Organic Iron Complexes by Bacteria from Water Supplies

The degradation of four soluble organic iron compounds by bacteria isolated from surface waters and the precipitation of iron from these complexes by the isolates was studied. All eight isolates brought about the precipitation of iron when grown on ferric ammonium citrate agar. Three isolates were able to degrade ferric malonate, and three others degraded ferric malate with iron precipitation. Only three isolates, two strains of Pseudomonas and one of Moraxella, were able to degrade gallic acid when this was supplied as the sole carbon source. One strain of Pseudomonas was found to be active in degrading ferric gallate. Electron microscopy of cells of this bacterium after growth in ferric gallate as the sole carbon source yielded results indicating uniform deposition of the iron on or in the bacterial cells. Seven of the isolates could degrade the iron gallate complex if supplied with additional carbon in the form of yeast extract.     

Toluene Elicits a Carbon Starvation Response in Pseudomonas putida mt-2 Containing the TOL Plasmid pWW0

Pseudomonas putida mt-2(pWWO) exhibited a carbon starvation response in the presence of toluene, a utilizable carbon source. When growth-supporting (4-mg/liter), inhibitory (130-mg/liter), and lethal (267-mg/ liter) levels of toluene were provided as the sole carbon source, P. putida responded by rapidly inhibiting protein synthesis and by producing 26 new proteins, 22 of which overlapped with those induced by carbon starvation. P. putida produced the same proteins when cultures were starved by depleting their carbon source or were downshifted into a carbon-free medium. Carbon supplementation of toluene-exposed cells suppressed the production of the toluene-induced proteins. The level of toluene provided as the sole carbon source influenced the length of time that this response was observed. Following 1.5 to 3 h in a basal salts medium with 4 mg of toluene per liter, protein synthesis increased, the production of the majority of the toluene-induced proteins ceased, and the cells began to grow. In cells provided with 130 mg of toluene per liter, protein synthesis remained inhibited over a 6.5-h experimental period. At this concentration, the production of 15 toluene-induced proteins was prolonged, with nine still detectable in the profiles at 6.5 h. In cells provided with 267 mg of toluene per liter, there was a rapid loss of viability and the toluene-induced proteins were detected prior to death. In cells provided with 4 mg of toluene per liter, the carbon starvation response is transient and likely reflects a period of induction and/or adaptation prior to growth on toluene. At the toluene concentrations which inhibit growth, P. putida exhibits a prolonged starvation response despite the presence of an excess of a utilizable carbon source.     

Repression of Pseudomonas putida phenanthrene-degrading activity by plant root extracts and exudates

The phenanthrene-degrading activity (PDA) of Pseudomonas putida ATCC 17484 was repressed after incubation with plant root extracts of oat (Avena sativa), osage orange (Maclura pomifera), hybrid willow (Salix alba x matsudana), kou (Cordia subcordata) and milo (Thespesia populnea) and plant root exudates of oat (Avena sativa) and hybrid poplar (Populus deltoides x nigra DN34). Total organic carbon content of root extracts ranged from 103 to 395 mg l(-1). Characterization of root extracts identified acetate (not detectable to 8.0 mg l(-1)), amino acids (1.7-17.3 mg l(-1)) and glucose (1.6-14.0 mg l(-1)), indicating a complex mixture of substrates. Repression was also observed after exposure to potential root-derived substrates, including organic acids, glucose (carbohydrate) and glutamate (amino acid). Carbon source regulation (e.g. catabolite repression) was apparently responsible for the observed repression of P. putida PDA by root extracts. However, we showed that P. putida grows on root extracts and exudates as sole carbon and energy sources. Enhanced growth on root products may compensate for partial repression, because larger microbial populations are conducive to faster degradation rates. This would explain the commonly reported increase in phenanthrene removal in the rhizosphere.     

Conjugal transfer of a TOL-like plasmid and extension of the catabolic potential of Pseudomonas putida F1

Strain mX was isolated from a petrol-contaminated soil, after enrichment on minimal medium with 0.5% (v/v) meta-xylene as a sole carbon source. The strain was tentatively characterized as Pseudomonas putida and harboured a large plasmid (pMX) containing xyl genes involved in toluene or meta-xylene degradation pathways via an alkyl monooxygenase and a catechol 2,3-dioxygenase. This new TOL-like plasmid was stable over two hundred generations and was self-transferable. After conjugal transfer to P. putida F1, which possesses the Tod chromosomal toluene biodegradative pathway, the transconjugant P. putida F1(pMX) was able to grow on benzene, toluene, meta-xylene, para-xylene, and ethylbenzene compounds as the sole carbon sources. Catechol 2,3-dioxygenases of the transconjugant cells presented a more relaxed substrate specificity than those of parental cells (strain mX and P. putida F1).     

Toluene-degrading bacteria are chemotactic towards the environmental pollutants benzene, toluene, and trichloroethylene

The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis. Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant. Three of the five strains (Pseudomonas putida F1, Ralstonia pickettii PKO1, and Burkholderia cepacia G4) were attracted to toluene. In each case, the response was dependent on induction by growth with toluene. Pseudomonas mendocina KR1 and P. putida PaW15 did not show a convincing response. The chemotactic responses of P. putida F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined. Compounds that are growth substrates for P. putida F1, including benzene and ethylbenzene, were chemoattractants. P. putida F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by P. putida F1. Mutant strains of P. putida F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected. The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in P. putida F1, were required for the response. This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.     

A Study of Iron Acquisition Mechanisms of Legionella pneumophila Grown in Chemostat Culture

We recently demonstrated that the virulence of a clinical isolate of Legionella pneumophila is significantly attenuated when cultured in an iron-limited environment. In this study the influence of iron limitation on the expression of enzyme activities and iron-transport mechanisms was investigated. Expression of the important pathogenicity factor, the zinc metalloprotease, was reduced fivefold in response to iron limitation. Ferric citrate reductase activity was demonstrated in both iron-limited and replete cell fractions. Activity was located principally in the cytoplasm and periplasm, and was not enhanced by iron restriction. Optimum activity was observed with NADPH as reductant. Siderophores were not elaborated under these culture conditions. Iron-loaded transferrin enhanced the growth of steady-state, iron-limited cultures, demonstrating that transferrin represents a potentially important iron source for L. pneumophila in vivo. Although cell surface transferrin receptors were not detected, in vitro experiments demonstrated digestion of transferrin by the zinc metalloprotease activity of culture supernatants. Received: 3 July 1996 / Accepted: 3 October 1996     

Competition in chemostat culture between Pseudomonas strains that use different pathways for the degradation of toluene.

Pseudomonas putida mt-2, P. cepacia G4, P. mendocina KR1, and P. putida F1 degrade toluene through different pathways. In this study, we compared the competition behaviors of these strains in chemostat culture at a low growth rate (D = 0.05 h-1), with toluene as the sole source of carbon and energy. Either toluene or oxygen was growth limiting. Under toluene-limiting conditions, P. mendocina KR1, in which initial attack is by monooxygenation of the aromatic nucleus at the para position, outcompeted the other three strains. Under oxygen limitation, P. cepacia G4, which hydroxylates toluene in the ortho position, was the most competitive strain. P. putida mt-2, which metabolizes toluene via oxidation of the methyl group, was the least competitive strain under both growth conditions. The apparent superiority of strains carrying toluene degradation pathways that start degradation by hydroxylation of the aromatic nucleus was also found during competition experiments with pairs of strains of P. cepacia, P. fluorescence, and P. putida that were freshly isolated from contaminated soil.     

Regulation of Iron on Bacterial Growth and Production of Thermostable Direct Hemolysin by Vibrio parahaemolyticus in Intraperitoneal Infected Mice

Pathogenesis of Vibrio parahaemolyticus is not clearly understood. Effects of iron on the bacterial proliferation and production of thermostable direct hemolysin (TDH) in intraperitoneal infected mice were studied. Injection of bacterial culture in the presence of ferric ammonium citrate (100 μg/ml) significantly enhanced the lethality for mice, and simultaneously activated bacterial proliferation in vivo. The iron-limited cultures showed better proliferation than those iron-rich cultures in response to the addition of supplementary iron source. Production of TDH by the hemolytic strains ST550 and D62 was higher in the iron-limited cultures than the iron-rich cultures. Production of TDH by both the iron-limited or iron-rich cultures was inhibited by the addition of iron. In conclusion, the virulence enhancement effect of iron in V. parahaemolyticus was probably by activating bacterial proliferation in vivo and not by stimulating the production of TDH. V. parahaemolyticus precultured in iron-limited condition may be more adaptable to in vivo environment.     

Utilization of different carbon sources by bacteria isolated from the roots of pine seedlings (Pinus silvestris L.) inoculated with root-free, rhizosphere and mycorrhizosphere soil

Studies were carried out on the utilization of different sugars (glucose, fructose, arabinose, xylose, sucrose) and organic acids (acetic, citric, fumaric, propionic, succinic) by fast and slow growing bacteria isolated from the roots of pine seedlings (P. silvestris L.) inoculated with root-free, rhizosphere and mycorrhizosphere soil from nursery and mature pine forest. Sucrose among sugars and propionic acid among organic acids were the less frequently utilized compounds. Sugars were better carbon sources than organic acids. Proportion of isolates utilizing respective sugars or organic acids was, in general, significantly higher among fast growing bacteria as compared with slow growing ones. No significant differences in number of strains assimilating the appropiate sugars depending on their original habitat or kind of soil were observed. Such differences were noted for utilization of some organic acids. 3-factor ANOVA confirmed that the growth speed of bacteria at the moment of their isolation had the strongest effect on utilization of the compounds studied.     

Revealing Sulfate-Reducing Microorganisms in Oilfield Waters (Flysch Carpathians,South-eastern Poland)

Sulphidogenous microorganism communities were isolated from selected oilfield waters in the Flysch Carpathians of south-eastern Poland. Organisms were incubated using the microcosms method with application of two media: minimal medium and modified Postgate C medium with yeast extract or trisodium citrate or monocyclic hydrocarbons from the BTEX group (benzene, toluene, ethylbenzene, and xylene) as the sole carbon source. Activity of sulphidogenic, autochthonous microorganism communities was noted only on the Postgate medium. Beside active sulphate reduction – max. 70%, ca. 74% biodegradation of organic compounds was also observed in the cultures. The highest content of sulphate-reducing bacteria (SRB) in the COD (ca. 83%) was noted in cultures, in which trisodium citrate and yeast extract were applied as the sole carbon source. Molecular analysis indicated not only the presence of SRB such as Desulfobacterium autothrophicum, Desulfovibrio desulfuricans, but also other microorganisms, e.g., Geobacter metallireducens. All these taxa are obligatory or facultative anaerobes, with metabolism linked mostly with elemental sulphur and/or its oxidized forms, as well as iron. Analysis of the mineral composition of the residues confirmed the presence of elemental sulphur, testifying for the active reduction of sulphates by incompletely oxidizing sulphate reducers assigned to the SRB group. Based on the obtained results, it is concluded that the physical and chemical properties of the oilfield waters are favorable for the growth and development of sulphidogenic microorganism assemblages and mineral-forming processes conducted by them.     

Isolation and characterization of a Pseudomonas putida strain able to grow with trimethyl-1,2-dihydroxy-propyl-ammonium as sole source of carbon, energy and nitrogen

Trimethyl-1,2-dihydroxypropyl-ammonium (TM) originates from the hydrolysis of the parent esterquat surfactant, which is widely used as softener in fabric care. Based on test procedures mimicking complex biological systems, TM is supposed to degrade completely when reaching the environment. However, no organisms able to degrade TM were isolated nor has the degradation pathway been elucidated so far. We isolated a Gram-negative rod able to grow with TM as sole source of carbon, energy and nitrogen. The strain reached a maximum specific growth rate of 0.4(h-1) when growing with TM as the sole source of carbon, energy and nitrogen. TM was degraded to completion and surplus nitrogen was excreted as ammonium into the growth medium. A high percentage of the carbon in TM (68% in continuous culture and 60% in batch culture) was combusted to CO2 resulting in a low yield of 0.54 mg cell dry weight per mg carbon during continuous cultivation and 0.73 mg cell dry weight per mg carbon in batch cultures. Choline, a natural structurally related compound, served as a growth substrate, whereas a couple of similar other quaternary aminoalcohols also used in softeners did not. The isolated bacterium was identified by 165-rDNA sequencing as a strain of Pseudomonas putida with a difference of only one base pair to P. putida DSM 291T. Despite their high identity, the reference strain P. putida DSM 291T was not able to grow with TM and the two strains differed even in shape when growing on the same medium. This is the first microbial isolate able to degrade a quaternary ammonium softener head group to completion. Previously described strains growing on quaternary ammonium surfactants (decyltrimethylammonium, hexadecyltrimethylammonium and didecyldimethylammonium) either excreted metabolites or a consortium of bacteria was required for complete degradation.     

Ecophysiological Characterization of Rhizosphere Bacterial Communities at Different Root Locations and Plant Developmental Stages of Cucumber Grown on Rockwool

Bacterial communities from the rhizosphere of cucumber were characterized with respect to growth rates and carbon source utilization, in order to develop a selection strategy for biocontrol agents against Pythium aphanidermatum. Rhizosphere samples were collected from different root regions (root tips, the root base, and the intermediate region where lateral roots emerge) and developmental stages (the seedling, vegetative, and generative stage) from plants cultivated on reused rockwool. By colony counts on 1/10 strength TSA on subsequent days after plating, percentages of fast- and slow-growing isolates (i.e., forming visible colonies within 2 days, or after 3 or more days, respectively) were determined for each rhizosphere sample. At all plant developmental stages, root tips had the highest percentages of fast growing isolates, and root bases the lowest. During plant growth, the relative amounts of slowly growing bacteria increased. Community-level carbon source utilization was determined for the different rhizosphere samples with Biolog GN plates. Principal component analysis showed that rhizosphere samples from different developmental stages and root locations had distinct carbon source utilization patterns. Communities from root tips of seedlings showed the highest utilization of several monosaccharides. Communities from tips and intermediate regions of plants in the vegetative stage utilized relatively many amino acids and several organic acids, and in the generative stage, more di- and polysaccharides were used. Root base samples scored low with respect to carbon source utilization, except for some disaccharides. From the different rhizosphere samples, 826 bacteria, randomly collected from 1/10 strength TSA plates, were screened on the utilization of 9 carbon sources. The 9 selected carbon sources were chosen because they are reported to occur in the rhizosphere, to be used by the zoospores of Pythium in the infection process, or appeared to be discriminant in the analysis of community-level carbon source utilization performed in this study. It appeared that monosaccharides (glucose and fucose), amino acids (alanine and asparagine), and organic acids (galacturonic, succinic, and linoleic acid) were used for growth mainly by bacteria from the root tips, and to a lesser extent from the intermediate region, of young plants. Disaccharides were predominantly utilized by isolates from plants in the vegetative stage. Overall, the results indicated that growth rates and carbon source utilization reflect the adaptation of bacteria to the rhizosphere environment. The possibility of using these characteristics to screen for rhizosphere competent biocontrol agents that compete for substrates with P. aphanidermatum is discussed.     

Inducible uptake and metabolism of glucose by the phosphorylative pathway in Pseudomonas putida CSV86

Pseudomonas putida CSV86 utilizes glucose, naphthalene, methylnaphthalene, benzyl alcohol and benzoate as the sole source of carbon and energy. Compared with glucose, cells grew faster on aromatic compounds as well as on organic acids. The organism failed to grow on gluconate, 2-ketogluconate, fructose and mannitol. Whole-cell oxygen uptake, enzyme activity and metabolic studies suggest that in strain CSV86 glucose utilization is exclusively by the intracellular phosphorylative pathway, while in Stenotrophomonas maltophilia CSV89 and P. putida KT2442 glucose is metabolized by both direct oxidative and indirect phosphorylative pathways. Cells grown on glucose showed five- to sixfold higher activity of glucose-6-phosphate dehydrogenase compared with cells grown on aromatic compounds or organic acids as the carbon source. Study of [14C]glucose uptake by whole cells indicates that the glucose is taken up by active transport. Metabolic and transport studies clearly demonstrate that glucose metabolism is suppressed when strain CSV86 is grown on aromatic compounds or organic acids.