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PubMed2000

Marked increase of venlafaxine enantiomer concentrations as a consequence of metabolic interactions: a case report.

On three occasions, unusually high trough plasma concentrations of venlafaxine were measured in a patient phenotyped and genotyped as being an extensive CYP2D6 metabolizer and receiving 450 mg/day of venlafaxine and multiple comedications. Values of 1.54 and of 0.60 mg/l of venlafaxine and O-desmethylvenlafaxine, respectively, were determined in the first blood sample, giving an unusually high venlafaxine to O-desmethylvenlafaxine ratio. This suggests an impaired metabolism of venlafaxine to O-desmethylvenlafaxine, and is most likely due to metabolic interactions with mianserin (240 mg/day) and propranolol (40 mg/day). Concentration of (S)-venlafaxine measured in this blood sample was almost twice as high as (R)-venlafaxine ((S)/(R) ratio: 1.94). At the second blood sampling, after addition of thioridazine (260 mg/day), which is a strong CYP2D6 inhibitor, concentrations of venlafaxine were further increased (2.76 mg/l), and concentrations of O-desmethylvenlafaxine decreased (0.22 mg/l). A decrease of the (S)/(R)-venlafaxine ratio (-20%) suggests a possible stereoselectivity towards the (R)-enantiomer of the enzyme(s) involved in venlafaxine O-demethylation at these high venlafaxine concentrations. At the third blood sampling, after interruption of thioridazine, concentrations of venlafaxine and O-desmethylvenlafaxine were similar to those measured in the first blood sample. This case report shows the importance of performing studies on the effects of either genetically determined or acquired deficiency of metabolism on the kinetics of venlafaxine.

Adult↗

PubMed2004

Potential beneficial metabolic interactions between tamoxifen and isoflavones via cytochrome P450-mediated pathways in female rat liver microsomes.

PURPOSE: This study aims to evaluate a cytochrome P450-based tamoxifen-isoflavone interaction and to determine the mechanisms responsible for inhibitory effects of isoflavones (e.g., genistein) on the formation of alpha-hydroxytamoxifen. METHODS: Metabolism studies were performed in vitro using female rat liver microsomes. The effects of genistein and an isoflavone mixture on tamoxifen metabolism and the inhibition mechanism were determined using standard kinetic analysis, preincubation, and selective chemical inhibitors of P450. RESULTS: Metabolism of tamoxifen was saturable with Km values of 4.9+/-0.6, 14.6+/-2.2, 25+/-5.9 microM and Vmax values of 34.7+/-1.4, 297.5+/-19.2, 1867+/-231 pmol min(-1) mg(-1) for a-hydroxylation, N-desmethylation, and N-oxidation, respectively. Genistein (25 microM) inhibited alpha-hydroxylation at 2.5 microM tamoxifen by 64% (p < 0.001) but did not affect the 4-hydroxylation, N-desmethylation, and N-oxidation. A combination of three (genistein, daidzein, and glycitein) to five isoflavones (plus biochanin A and formononetin) inhibited tamoxifen alpha-hydroxylation to a greater extent but did not decrease the formation of identified metabolites. The inhibition on alpha-hydroxylation by genistein was mixed-typed with a Ki, value of 10.6 microM. Studies using selective chemical inhibitors showed that tamoxifen alpha-hydroxylation was mainly mediated by rat CYP1A2 and CYP3A1/2 and that genistein 3'-hydroxylation was mainly mediated by rat CYP1A2, CYP2C6 and CYP2D1. CONCLUSIONS: Genistein and its isoflavone analogs have the potential to decrease side effects of tamoxifen through metabolic interactions that inhibit the formation of a-hydroxytamoxifen via inhibition of CYP1A2.

Algorithms↗

PubMed2001

Vascular factors and metabolic interactions in the pathogenesis of diabetic neuropathy.

Diabetes mellitus is a major cause of peripheral neuropathy, commonly manifested as distal symmetrical polyneuropathy. This review examines evidence for the importance of vascular factors and their metabolic substrate from human and animal studies. Diabetic neuropathy is associated with risk factors for macrovascular disease and with other microvascular complications such as poor metabolic control, dyslipidaemia, body mass index, smoking, microalbuminuria and retinopathy. Studies in human and animal models have shown reduced nerve perfusion and endoneurial hypoxia. Investigations on biopsy material from patients with mild to severe neuropathy show graded structural changes in nerve microvasculature including basement membrane thickening, pericyte degeneration and endothelial cell hyperplasia. Arterio-venous shunting also contributes to reduced endoneurial perfusion. These vascular changes strongly correlate with clinical defects and nerve pathology. Vasodilator treatment in patients and animals improves nerve function. Early vasa nervorum functional changes are caused by the metabolic insults of diabetes, the balance between vasodilation and vasoconstriction is altered. Vascular endothelium is particularly vulnerable, with deficits in the major endothelial vasodilators, nitric oxide, endothelium-derived hyperpolarising factor and prostacyclin. Hyperglycaemia and dyslipidaemia driven oxidative stress is a major contributor, enhanced by advanced glycation end product formation and polyol pathway activation. These are coupled to protein kinase C activation and omega-6 essential fatty acid dysmetabolism. Together, this complex of interacting metabolic factors accounts for endothelial dysfunction, reduced nerve perfusion and function. Thus, the evidence emphasises the importance of vascular dysfunction, driven by metabolic change, as a cause of diabetic neuropathy, and highlights potential therapeutic approaches.

Animals↗

PubMed1998

Metabolic interaction of n-hexane and methyl ethyl ketone in vitro in a head space rat liver S9 vial equilibration system.

Methyl ethyl ketone pretreatment induced rat liver cytochrome P450 and increased significantly the in vitro metabolism of n-hexane and the formation of 2,5-hexanedione in rat liver S9. No significant changes were, however, found in the levels of the intermediate metabolites 2-hexanol, 2,5-hexanediol or methyl n-butyl ketone. Methyl ethyl ketone added in vitro to untreated (non-induced) liver S9 inhibited in a non-competitive pattern the metabolism of n-hexane and decreased significantly and in a dose-dependent way the levels of methyl n-butyl ketone and 2,5-hexanedione. When methyl ethyl ketone and n-hexane were added in vitro to in vivo methyl ethyl ketone pretreated (induced liver S9, the significant increase in the formation of 2,5-hexanedione was maintained, an increase which was only to a minor extent influenced by the in vitro addition of methyl ethyl ketone. These findings are in agreement with an in vivo induction by methyl ethyl ketone of key enzyme(s) in a generally minor metabolic pathway for the conversion of n-hexane to 2,5-hexanedione in rat liver, a pathway which is not influenced by the presence of methyl ethyl ketone itself. The results obtained in this study indicate that the head space equilibration technique is well suited for screening studies of metabolic interactions between organic solvents.

Animals↗

PubMed1994

Metabolic interactions between anaerobic bacteria in methanogenic environments.

In methanogenic environments organic matter is degraded by associations of fermenting, acetogenic and methanogenic bacteria. Hydrogen and formate consumption, and to some extent also acetate consumption, by methanogens affects the metabolism of the other bacteria. Product formation of fermenting bacteria is shifted to more oxidized products, while acetogenic bacteria are only able to metabolize compounds when methanogens consume hydrogen and formate efficiently. These types of metabolic interaction between anaerobic bacteria is due to the fact that the oxidation of NADH and FADH2 coupled to proton or bicarbonate reduction in thermodynamically only feasible at low hydrogen and formate concentrations. Syntrophic relationships which depend on interspecies hydrogen or formate transfer were described for the degradation of e.g. fatty acids, amino acids and aromatic compounds.

Acetates↗

PubMed2006

In vitro evaluation of the anti-HIV activity and metabolic interactions of tenofovir and emtricitabine.

BACKGROUND: Tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) are nucleoside reverse transcriptase inhibitors (NRTIs) with once-daily dosing approved for use in HIV-1 infection. We evaluated this combination for anti-HIV activity and intracellular metabolic interactions in vitro. METHODS: Intracellular anabolism of combinations of FTC and tenofovir (TFV) were studied in the human T leukaemic cell line CEM and human peripheral blood mononuclear cells (PBMCs). The anti-HIV activity of the combination was studied in the human T leukaemic MT-2 cell line against wild-type or mutant virus (K65R and M184V) and in PBMCs against wild-type virus. Antiviral synergy was quantitated by isobologram and MacSynergy methods. RESULTS: Both TFV and FTC were efficiently converted to their active metabolites in PBMCs and CEM cells. In CEM cells, there was a statistically significant increase in the levels of TFV diphosphate (P=0.047) and FTC triphosphate (P=0.0069) following a 24 h incubation with the combination compared with the levels seen with the individual drugs. In PBMCs, similar levels of active metabolites were observed for each drug when they were incubated iindividually or in combination. The combination of TFV and FTC displayed additive to synergistic activity against HIV replication in PBMCs and resulted in strongly synergistic anti-HIV activity in MT-2 cells against both wild-type and mutant virus. CONCLUSIONS: The combination of TFV and FTC showed additive to synergistic anti-HIV activity in vitro, which correlated with the levels of intracellular phosphorylation observed. These results support the use of these drugs as a dual NRTI backbone in combination therapy for the treatment of HIV-1.

Adenine↗

PubMed1988

Metabolic interactions of parenchymal hepatocytes and dividing epithelial cells in co-culture.

When parenchymal hepatocytes isolated from adult liver are co-cultured with other epithelial cells, the production of various plasma proteins by the hepatocytes is preserved for much longer than in conventional culture. This study examines some of the metabolic interactions between parenchymal hepatocytes and epithelial cells maintained in co-culture. The leakage of lactate dehydrogenase by hepatocytes co-cultured with epithelial cells was lower than in conventional hepatocyte culture. The epithelial cells have a high glycolytic rate and provide the hepatocytes with a continual supply of lactate. The [lactate] was lower in co-cultures of hepatocytes and epithelial cells than in pure epithelial cultures of similar density, suggesting lactate clearance by the hepatocytes. Alanine uptake was higher in conventional hepatocyte cultures, which lack an exogenous supply of lactate, than in parenchymal hepatocytes in co-culture. Studies with pure parenchymal hepatocytes incubated with increasing [lactate] suggest that lactate is utilized in preference to alanine as a gluconeogenic substrate by hepatocytes co-cultured with epithelial cells. Ketogenesis and carnitine palmitoyltransferase activity declined more slowly in hepatocytes co-cultured with epithelial cells than in conventional culture. It is concluded that the co-culture model has potential for long-term studies of carbohydrate and lipid metabolism.

Adult↗

PubMed1998

In vitro screening for metabolic interactions among frequently occurring binary mixtures of volatile organic chemicals in Norwegian occupational atmosphere.

Surveys of Norwegian industrial occupational atmosphere between 1983 to 1996, have identified the 12 most frequent occurring binary combinations of volatile organic chemicals. These combinations were tested in vitro for mutual inhibition or enhancement of metabolism by the head space vial equilibration technique with liver S9 obtained from in vivo untreated or pretreated (with the binary mixture) rats. The in vitro system responded to in vivo pretreatment by increasing the metabolic rate of several potentially toxic organic chemicals such as toluene, xylene, styrene, and dichloromethane. In untreated liver S9, the metabolism of several of the tested binary pairs was inhibited when coexposed in vitro to their most prevalent follower as shown for instance for ethanol (with ethyl acetate), dichloromethane (with styrene) and mutually between toluene and xylene. This inhibitory effect disappeared, however, for several of the solvents when combined with the in vivo induced liver S9, a situation which may be the most relevant for occupational exposure. It is concluded that several metabolic interactions occur between low-molecular weight volatile chemicals found in occupational air. These are both inductive and inhibitory in nature and a further mechanistic evaluation including a higher number of differentiated dosage levels, must be performed before a possible health hazard can be confirmed or rejected for the investigated combinations.

Air Pollutants, Occupational↗

PubMed1989

Pharmacological and metabolic interactions between ethanol and methyl n-butyl ketone, methyl isobutyl ketone, methyl ethyl ketone, or acetone in mice.

Methyl n-butyl ketone (MnBK), methyl isobutyl ketone (MIBK), methyl ethyl ketone (MEK), and acetone are widely used industrial solvents to which certain groups of workers are exposed. Pharmacological and metabolic interactions between these solvents and ethanol were explored in male CD-1 mice. The effects of these solvents on the duration of ethanol-induced loss of righting reflex and on ethanol elimination in mice were studied. The solvents were dissolved in corn oil and injected intraperitoneally 30 min before ethanol 4 g/kg ip. The four solvents prolonged significantly the duration of ethanol-induced loss of righting reflex when given in the following doses (mmol/kg): MnBK, 3.75 and 5; MIBK, 5; MEK, 5 and 10, acetone, 20 and 40. This prolongation was dose related and increased as the dose of the solvent was increased. The concentrations of ethanol in blood or brain on return of the righting reflex were similar in solvent-treated and control animals, with the exception of the group of mice treated with 40 mmol/kg acetone in which the ethanol concentrations were significantly lower than in control animals. The mean elimination rate of ethanol was markedly reduced in mice treated with MnBK 5 mmol/kg, MEK 15 mmol/kg, and acetone 40 mmol/kg. All four solvents reduced the activity of mouse liver alcohol dehydrogenase in vitro. It is concluded that enhancement of the ethanol-induced loss of righting reflex by these solvents in mice is well correlated to reduced elimination rate of ethanol.

Acetone↗

PubMed2001

Physical and metabolic interactions of Pseudomonas sp. strain JA5-B45 and Rhodococcus sp. strain F9-D79 during growth on crude oil and effect of a chemical surfactant on them.

Methods to enhance crude oil biodegradation by mixed bacterial cultures, for example, (bio)surfactant addition, are complicated by the diversity of microbial populations within a given culture. The physical and metabolic interactions between Rhodococcus sp. strain F9-D79 and Pseudomonas sp. strain JA5-B45 were examined during growth on Bow River crude oil. The effects of a nonionic chemical surfactant, Igepal CO-630 (nonylphenol ethoxylate), also were evaluated. Strain F9-D79 grew attached to the oil-water interface and produced a mycolic acid-containing capsule. Crude oil emulsification and surface activity were associated with the cellular fraction. Strain JA5-B45 grew in the aqueous phase and was unable to emulsify oil, but cell-free supernatants mediated kerosene-water emulsion formation. In coculture, stable emulsions were formed and strain JA5-B45 had an affinity for the capsule produced by strain F9-D79. Igepal CO-630 inhibited F9-D79 cells from adhering to the interface, and cells grew dispersed in the aqueous phase as 0.5-microm cocci rather than 2.5-microm rods. The surfactant increased total petroleum hydrocarbon removal by strain JA5-B45 from 4 to 22% and included both saturated compounds and aromatics. In coculture, TPH removal increased from 13 to 40% following surfactant addition. The culture pH normally increased from 7.0 to between 7.5 and 8.5, although addition of Igepal CO-630 to F9-D79 cultures resulted in a drop to pH 5.5. We suggest a dual role for the nonylphenol ethoxylate surfactant in the coculture: (i) to improve hydrocarbon uptake by strain JA5-B45 through emulsification and (ii) to prevent strain F9-D79 from adhering to the oil-water interface, indirectly increasing hydrocarbon availability. These varied effects on hydrocarbon biodegradation could explain some of the known diversity of surfactant effects.

Biodegradation, Environmental↗

PubMed2001

[Metabolic interactions with statins].

BACKGROUND: Isoenzymes of the cytochrome P450 (CYP) system play a prominent role in drug metabolism, including the HMG-CoA reductase inhibitors (statins). Alteration in metabolic activity is often the underlying mechanism of clinically relevant interactions, thus it is important to make a rational assessment of the risk for metabolic interactions with statins. MATERIAL AND METHODS: Articles covering the field of statin metabolism and related interactions were mainly searched for via PubMed. RESULT: More than 50% of the overall CYP metabolism is mediated through the isoenzyme CYP3A4, which is the main elimination route of simvastatin, lovastatin and atorvastatin. Interaction studies reveal that simvastatin and lovastatin have the highest potential for clinically relevant interactions related to this isoenzyme, most often leading to increased efficacy/toxicity of the statin. Interactions with agents that alter the activity of CYP3A4 can also be expected for atorvastatin. Other statins are to a lesser extent dependent on CYP3A4 in their elimination. Thus, pravastatin, cerivastatin and fluvastatin have a minor potential for interactions linked to this isoenzyme. Interactions associated with CYP2C9-interference may, however, be present for fluvastatin. INTERPRETATION: Since lipid-lowering therapy most often is life-long, the use of statins will frequently be accompanied by the use of multiple drugs, hence the importance of considering the risk for interactions when selecting statins.

Anti-Bacterial Agents↗

PubMed1999

Quantitative drug interactions prediction system (Q-DIPS): a computer-based prediction and management support system for drug metabolism interactions.

OBJECTIVE: Drug biotransformation and interactions are a major source of variability in the response to drugs. The superfamily of cytochromes P450 plays a key role in this phenomenon but, because of the complexity of interactions between drugs and isozymes, it becomes more and more difficult for clinicians to master the knowledge required to predict the occurrence of such drug interactions. To predict and help manage the occurrence of cytochrome P450-dependent interactions, we developed an original computer application: Q-DIPS (quantitative drug interactions prediction system). METHODS: A multidisciplinary work team was created, associating clinical pharmacologists, pharmacists and a computer scientist. Major steps of investigation were: (1) the creation of a database to collect qualitative and quantitative data describing substrates, inhibitors and inducers of specific cytochrome P450 isozymes, with quality assessments; (2) the development of multi-access to these data and (3) their incorporation into extrapolation systems allowing the prediction of in vivo drug interactions on the basis of in vitro data. As an example, prediction and validation studies of CYP3A4 inhibition by ketoconazole and fluconazole will be discussed. RESULTS: Q-DIPS gives up-to-date information, in dynamic tables, describing which specific P450 isozymes metabolise a given drug, as well as which drugs may inhibit or induce a given isozyme. To better answer common clinical questions and help to rapidly evaluate the risk of interactions, it is possible to obtain an overview of substances causing interactions with a specific drug or to focus on drugs taken by a patient ("clinical case"). For each question, key references, relevant quantitative data and quality indices are easily accessible. Two modules allowing input with commercial names and the anatomical therapeutic chemical classification were also included. On the basis of enzymatic and pharmacokinetic data generated in vitro or collected in vivo, the extrapolation module integrates quantitative models to predict the impact of a treatment on enzymatic activities. The simplest model predicted a strong but fluctuating inhibition of CYP3A4 by ketoconazole, whereas the impact of fluconazole was lower. Validations with published in vivo data suggested an appropriate prediction of the risk. CONCLUSION: The current Q-DIPS prototype shows promising potential for helping to improve the management of drug interactions involving metabolism. Validation of extrapolation techniques need to be completed, in view of including important factors such as intrahepatocyte drug accumulation, contribution of metabolites to inhibition as well as in vitro non-specific binding to microsomal proteins. The final goal will be to help select the most judicious clinical studies to be performed so as to avoid useless, expensive and unethical investigations in man.

Antifungal Agents↗

PubMed1994

Metabolic interactions of methoxsalen and coumarin in humans and mice.

Methoxsalen (8-methoxypsoralen) is a very potent inhibitor of human cytochrome P450 2A6 (CYP2A6) and mouse Cyp2a-5-mediated coumarin 7-hydroxylation in vitro. To determine the effect of methoxsalen on coumarin 7-hydroxylation in humans in vivo, five subjects were given 45 mg of methoxsalen and 5 mg of coumarin. Methoxsalen inhibited in vivo coumarin metabolism by 47 +/- 9.2% (mean +/- SEM). Methoxsalen was metabolized in human liver microsomes at the rate of 50-100 pmol/mg protein/min (approx. 30% of the activity in mouse liver microsomes). Metabolism was not inhibited by the anti-Cyp2a-5 antibody in human liver microsomes. NIH 3T3 cells stably expressing catalytically active CYP2A6 enzyme did not metabolize methoxsalen, indicating that CYP2A6 does not accept methoxsalen as a substrate. In pyrazole-induced mouse liver microsomes, methoxsalen metabolism was inhibited by the anti-Cyp2a-5 antibody. Cyp2a-5 protein expressed in the yeast Saccharomyces cerevisiae was capable of metabolizing methoxsalen, indicating that methoxsalen is a substrate of Cyp2a-5. Although kinetic studies indicated that the inhibition of coumarin 7-hydroxylation by methoxsalen is competitive in human liver microsomes, methoxsalen does not appear to be a substrate for CYP2A6. Methoxsalen and coumarin have the potential of strong metabolic interactions in man.

Animals↗

PubMed1996

Metabolic interaction between fluoxetine and clomipramine: a case report.

An 83-year-old patient suffering from a depressive episode was treated with a combination of clomipramine and fluoxetine. As no marked improvement was observed and cardiac side-effects were observed, a blood sample was taken for therapeutic drug monitoring. The result showed that the concentrations of clomipramine and desmethylclomipramine were very high (i.e. in the subtoxic range). A metabolic interaction was suspected and both antidepressants were immediately discontinued. Over time, concentrations of clomipramine and its metabolite decreased slowly and, simultaneously, the mood improved and side-effects disappeared. When clomipramine and desmethylclomipramine concentrations reached the lower limit of the optimal therapeutic range, monotherapy was restarted with clomipramine, and the patient improved enough to be discharged from the hospital.

Aged↗

PubMed1988

Metabolic interaction between imipramine and carbamazepine in vivo and in vitro in rats.

The pharmacokinetic consequences of the combination of carbamazepine with imipramine in male Wistar rats have been investigated. It was found that a 2-week treatment with the combination resulted in the increase of the concentrations of the parent compounds and a simultaneous decrease in their metabolites in blood plasma i.e. carbamazepine inhibited imipramine demethylation in the side chain while imipramine inhibited carbamazepine 10,11-epoxidation. The velocity of imipramine 2-hydroxylation and 10,11-epoxy-carbamazepine hydration did not seem to be changed by the combination. On the basis of studies in vitro it is concluded that the observed metabolic interaction between carbamazepine and imipramine is due to the competition of the drugs for the active centre of cytochrome P 450 and to a certain qualitative alteration of the enzyme by imipramine as can be deducted from the decrease of carbamazepine binding to the cytochrome.

Animals↗

PubMed1994

Kinetics of biomethanation of solid tannery waste and the concept of interactive metabolic control.

Anaerobic digestion of calf skin collagenous waste was optimized for a batch process based on accelerated maximal methane yield per gram of input volatile solid. A kinetic analysis with respect to changes in the levels of volatile solid, collagen, amino sugars, amino acids, hydroxyproline, ammonium ions, and volatile fatty acid were followed for a period of 80 d. Distinct metabolic phases included an initial high rate collagenolysis for 4d, with 50% degradation and was followed by an acidogenic phase between 4-12 d with volatile fatty acids levels increasing to 215 mmol/L. Subsequently methanogenesis ensued and was maximal between 12-24 d when volatile fatty acids attained steady state levels. During the period of 80 d, the overall decrease in volatile solid level was 65%, whereas the collagen level declined by 85% with 0.45 L of methane yield/g of volatile solid degraded. Based on the levels of various metabolites detected, the concept of interactive metabolic control earlier proposed has been validated.

Anaerobiosis↗

PubMed2005

Prediction of herb-drug metabolic interactions: a simulation study.

In vitro and in vivo studies have indicated that the induction or inhibition of cytochrome P450 (CYP) is one of the major mechanisms for some clinically important pharmacokinetic herb-drug interactions. An attempt was made to simulate the effects of herbal preparation with single or multiple CYP-inhibiting constituents on the area of the plasma concentration-time curve (AUC) of coadministered drug that was either a low clearance drug by intravenous (i.v.) injection or a high clearance drug by oral route. Our simulation studies indicated that the expected increase (Rc) in the AUC of the coadministered drug by inhibiting herbal constituent(s) was dependent on the route of administration. For low clearance drug by i.v. injection, Rc was generally determined by inhibition constant (Ki), unbound inhibitor concentration ([I]), hepatic fraction (fh), number of inhibitory herbal constituents (n) and metabolic pathway fraction in hepatic metabolism (fm), while Rc for a high clearance drug by oral route, Rc was determined by Ki, [I], n and fm. By varying these parameters, Rc changed accordingly. It appeared likely to predict a herb-drug metabolic interaction, if the inhibiting herbal constituents could be quantitatively determined. However, many herb- and drug-related factors may cause difficulties with the prediction, and thus in vivo animal and human studies are always necessary.

Administration, Oral↗

PubMed1998

In situ gene expression in mixed-culture biofilms: evidence of metabolic interactions between community members.

Microbial communities growing in laboratory-based flow chambers were investigated in order to study compartmentalization of specific gene expression. Among the community members studied, the focus was in particular on Pseudomonas putida and a strain of an Acinetobacter sp., and the genes studied are involved in the biodegradation of toluene and related aromatic compounds. The upper-pathway promoter (Pu) and the meta-pathway promoter (Pm) from the TOL plasmid were fused independently to the gene coding for the green fluorescent protein (GFP), and expression from these promoters was studied in P. putida, which was a dominant community member. Biofilms were cultured in flow chambers, which in combination with scanning confocal laser microscopy allowed direct monitoring of promoter activity with single-cell spatial resolution. Expression from the Pu promoter was homogeneously induced by benzyl alcohol in both community and pure-culture biofilms, while the Pm promoter was induced in the mixed community but not in a pure-culture biofilm. By sequentially adding community members, induction of Pm was shown to be a consequence of direct metabolic interactions between an Acinetobacter species and P. putida. Furthermore, in fixed biofilm samples organism identity was determined and gene expression was visualized at the same time by combining GFP expression with in situ hybridization with fluorescence-labeled 16S rRNA targeting probes. This combination of techniques is a powerful approach for investigating structure-function relationships in microbial communities.

Acinetobacter↗