Abstract
We applied a metagenomics approach to screen for transcriptional regulators that sense aromatic compounds. The library was constructed by cloning environmental DNA fragments into a promoter-less vector containing green fluorescence protein. Fluorescence-based screening was then performed in the presence of various aromatic compounds. A total of 12 clones were isolated that fluoresced in response to salicylate, 3-methyl catechol, 4-chlorocatechol and chlorohydroquinone. Sequence analysis revealed at least 1 putative transcriptional regulator, excluding 1 clone (CHLO8F). Deletion analysis identified compound-specific transcriptional regulators; namely, 8 LysR-types, 2 two-component-types and 1 AraC-type. Of these, 9 representative clones were selected and their reaction specificities to 18 aromatic compounds were investigated. Overall, our transcriptional regulators were functionally diverse in terms of both specificity and induction rates. LysR- and AraC- type regulators had relatively narrow specificities with high induction rates (5-50 fold), whereas two-component-types had wide specificities with low induction rates (3 fold). Numerous transcriptional regulators have been deposited in sequence databases, but their functions remain largely unknown. Thus, our results add valuable information regarding the sequence–function relationship of transcriptional regulators.
Highlights
Bacteria that degrade aromatic compounds are widely distributed in the environment and are important for breaking down both natural and xenobiotic compounds
Aromatic compounds are transformed into aromatic vicinal diols, which is performed by a monoxygenase or dioxygenase [10]
Library construction and screening The metagenomic library was constructed using environmental DNA extracted from groundwater contaminated with crude oil, as described previously [18]
Summary
Bacteria that degrade aromatic compounds are widely distributed in the environment and are important for breaking down both natural and xenobiotic compounds. Attempts have been made to screen for microorganisms that degrade aromatic compounds [1,2,3], as well as genes responsible for degradation [1,3,4,5,6,7]. These studies revealed that the majority of reported bacterial aromatic degradation processes are aerobic [8] and comprise a series of enzymes that are usually categorized as either ‘upper’- or ‘lower’-pathway enzymes [9]. The subsequent metabolic steps are referred to as meta- or ortho- pathways
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