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Real-time solvent tolerance analysis of pseudomonas sp. strain VLB120{Delta}C catalytic biofilms.

ABSTRACT: Biofilms are ubiquitous surface-associated microbial communities embedded in an extracellular polymeric (EPS) matrix, which gives the biofilm structural integrity and strength. It is often reported that biofilm-grown cells exhibit enhanced tolerance toward adverse environmental stress conditions, and thus there has been a growing interest in recent years to use biofilms for biotechnological applications. We present a time- and locus-resolved, noninvasive, quantitative approach to study biofilm development and its response to the toxic solvent styrene. Pseudomonas sp. strain VLB120?C-BT-gfp1 was grown in modified flow-cell reactors and exposed to the solvent styrene. Biofilm-grown cells displayed stable catalytic activity, producing (S)-styrene oxide continuously during the experimental period. The pillar-like structure and growth rate of the biofilm was not influenced by the presence of the solvent. However, the cells experience severe membrane damage during styrene treatment, although they obviously are able to adapt to the solvent, as the amount of permeabilized cells decreased from 75 to 80% down to 40% in 48 h. Concomitantly, the fraction of concanavalin A (ConA)-stainable EPS increased, substantiating the assumption that those polysaccharides play a major role in structural integrity and enhanced biofilm tolerance toward toxic environments. Compared to control experiments with planktonic grown cells, the Pseudomonas biofilm adapted much better to toxic concentrations of styrene, as nearly 65% of biofilm cells were not permeabilized (viable), compared to only 7% in analogous planktonic cultures. These findings underline the robustness of biofilms under stress conditions and its potential for fine chemical syntheses.

SUBMITTER: Halan B

PROVIDER: S-EPMC3067263 | biostudies-other | 2011 Mar

REPOSITORIES: biostudies-other

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Real-time solvent tolerance analysis of pseudomonas sp. strain VLB120{Delta}C catalytic biofilms.

Halan Babu B Schmid Andreas A Buehler Katja K

Applied and environmental microbiology 20101230 5

Biofilms are ubiquitous surface-associated microbial communities embedded in an extracellular polymeric (EPS) matrix, which gives the biofilm structural integrity and strength. It is often reported that biofilm-grown cells exhibit enhanced tolerance toward adverse environmental stress conditions, and thus there has been a growing interest in recent years to use biofilms for biotechnological applications. We present a time- and locus-resolved, noninvasive, quantitative approach to study biofilm d ...[more]

PMID: 21193676

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Project description:The 4-hydroxybenzoyl-CoA (4-HB-CoA) thioesterase from Pseudomonas sp. strain CBS3 catalyzes the final step of the 4-chlorobenzoate degradation pathway, which is the hydrolysis of 4-HB-CoA to coenzyme A (CoA) and 4-hydroxybenzoate (4-HB). In previous work, X-ray structural analysis of the substrate-bound thioesterase provided evidence of the role of an active site Asp17 in nucleophilic catalysis [Thoden, J. B., Holden, H. M., Zhuang, Z., and Dunaway-Mariano, D. (2002) X-ray crystallographic analyses of inhibitor and substrate complexes of wild-type and mutant 4-hydroxybenzoyl-CoA thioesterase. J. Biol. Chem. 277, 27468-27476]. In the study presented here, kinetic techniques were used to test the catalytic mechanism that was suggested by the X-ray structural data. The time course for the multiple-turnover reaction of 50 ?M [(14)C]-4-HB-CoA catalyzed by 10 ?M thioesterase supported a two-step pathway in which the second step is rate-limiting. Steady-state product inhibition studies revealed that binding of CoA (K(is) = 250 ± 70 ?M; K(ii) = 900 ± 300 ?M) and 4-HB (K(is) = 1.2 ± 0.2 mM) is weak, suggesting that product release is not rate-limiting. A substantial D(2)O solvent kinetic isotope effect (3.8) on the steady-state k(cat) value (18 s(-1)) provided evidence that a chemical step involving proton transfer is the rate-limiting step. Taken together, the kinetic results support a two-chemical pathway. The microscopic rate constants governing the formation and consumption of the putative aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate were determined by simulation-based fitting of a kinetic model to time courses for the substrate binding reaction (5.0 ?M 4-HB-CoA and 0.54 ?M thioesterase), single-turnover reaction (5 ?M [(14)C]-4-HB-CoA catalyzed by 50 ?M thioesterase), steady-state reaction (5.2 ?M 4-HB-CoA catalyzed by 0.003 ?M thioesterase), and transient-state multiple-turnover reaction (50 ?M [(14)C]-4-HB-CoA catalyzed by 10 ?M thioesterase). Together with the results obtained from solvent (18)O labeling experiments, the findings are interpreted as evidence of the formation of an aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate that undergoes rate-limiting hydrolytic cleavage at the hydroxybenzoyl carbonyl carbon atom.

Dataset Information

Real-time solvent tolerance analysis of pseudomonas sp. strain VLB120{Delta}C catalytic biofilms.

Publications

Real-time solvent tolerance analysis of pseudomonas sp. strain VLB120{Delta}C catalytic biofilms.

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