Dataset Information

Proteomics reveals a core molecular response of Pseudomonas putida F1 to acute chromate challenge.

ABSTRACT:

Background

Pseudomonas putida is a model organism for bioremediation because of its remarkable metabolic versatility, extensive biodegradative functions, and ubiquity in contaminated soil environments. To further the understanding of molecular pathways responding to the heavy metal chromium(VI) [Cr(VI)], the proteome of aerobically grown, Cr(VI)-stressed P. putida strain F1 was characterized within the context of two disparate nutritional environments: rich (LB) media and minimal (M9L) media containing lactate as the sole carbon source.

Results

Growth studies demonstrated that F1 sensitivity to Cr(VI) was impacted substantially by nutrient conditions, with a carbon-source-dependent hierarchy (lactate > glucose >> acetate) observed in minimal media. Two-dimensional HPLC-MS/MS was employed to identify differential proteome profiles generated in response to 1 mM chromate under LB and M9L growth conditions. The immediate response to Cr(VI) in LB-grown cells was up-regulation of proteins involved in inorganic ion transport, secondary metabolite biosynthesis and catabolism, and amino acid metabolism. By contrast, the chromate-responsive proteome derived under defined minimal growth conditions was characterized predominantly by up-regulated proteins related to cell envelope biogenesis, inorganic ion transport, and motility. TonB-dependent siderophore receptors involved in ferric iron acquisition and amino acid adenylation domains characterized up-regulated systems under LB-Cr(VI) conditions, while DNA repair proteins and systems scavenging sulfur from alternative sources (e.g., aliphatic sulfonates) tended to predominate the up-regulated proteome profile obtained under M9L-Cr(VI) conditions.

Conclusions

Comparative analysis indicated that the core molecular response to chromate, irrespective of the nutritional conditions tested, comprised seven up-regulated proteins belonging to six different functional categories including transcription, inorganic ion transport/metabolism, and amino acid transport/metabolism. These proteins might potentially serve as indicators of chromate stress in natural microbial communities.

SUBMITTER: Thompson DK

PROVIDER: S-EPMC2996968 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Perfluorinated carbon atoms in a diether linkage are common in commercial anesthetics, drugs, fungicides, and insecticides. An important chemical group comprising perfluorodiethers is the 2,2-fluoro-1,3-benzodioxole (DFBD) moiety. The fluorine atoms stabilize the molecule by mitigating against metabolism by humans and microbes, as used in drugs and pesticides, respectively. Pseudomonas putida F1 catalyzed defluorination of DFBD at an initial rate of 2,100 nmol/h per mg cellular protein. This is orders of magnitude higher than previously reported microbial defluorination rates with multiply fluorinated carbon atoms. Defluorination rates declined after several hours, and the medium darkened. Significant defluorination activity was observed with cells grown on toluene but not l-arginine. Defluorination required only toluene dioxygenase. Pseudomonas and recombinant Escherichia coli cells expressing toluene dioxygenase oxidized DFBD to DFBD-4,5-dihydrodiol. The dihydrodiol could be oxidized to 4,5-dihydroxy-DFBD via the dihydrodiol dehydrogenase from P. putida F1. The dihydrodiol dehydrated with acid to yield a mixture of 4-hydroxy-DFBD and 5-hydroxy-DFBD. All those metabolites retained the difluoromethylene group; no fluoride or dark color was observed. The major route of DFBD-4,5-dihydrodiol decomposition produced fluoride and 1,2,3-trihydroxybenzene, or pyrogallol, and that was shown to be the source of the dark colors in the medium. A mechanism for DFBD-4,5-dihydrodiol transformation to two fluoride ions and pyrogallol is proposed. The Pseudomonas genome database and other databases revealed hundreds of bacteria with enzymes sharing high amino acid sequence identity to toluene dioxygenase from P. putida F1, suggesting the mechanism revealed here may apply to the defluorination of DFBD-containing compounds in the environment. IMPORTANCE There are more than 9,000 polyfluorinated compounds developed for commercial use, some negatively impacting human health, and they are generally considered to be resistant to biodegradation. Only a limited number of studies have identified microbes with enzymes sufficiently reactive to defluorinate difluoromethylene carbon groups. The present study examined one important group of commercial fluorinated chemicals and showed its rapid defluorination by a bacterium and its key enzyme, a Rieske dioxygenase. Rieske dioxygenases are common in environmental bacteria, and those closely resembling toluene dioxygenase from Pseudomonas putida F1 are candidates for biodegradative defluorination of the common 2,2-fluoro-1,3-benzodioxole (DFBD) moiety.