Metagenomic Screening for Aromatic Compound-Responsive Transcriptional Regulators

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.

Molecular engineering of the salicylate-inducible transcription factor Sal7AR for orthogonal and high gene expression in Escherichia coli

“…The pSAL7A vector contains a metagenomic insert fragment of approximately 4 kb in length from the promoter-trapping vector p18GFP [ 5 ], in which orf3 codes for the salicylate-responsive transcription factor [ 8 ] ( Fig 1 ). The transcription factor, designated Sal7AR, is highly specific to salicylate (2-hydroxybenzoate); virtually no cross-reactivity was observed for the following aromatic compounds: phenol, catechol, o-cresol, m-cresol, p-cresol, 3-methylcatechol, 4-methylcatechol, 4-chlorocatechol, benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, 2-chlorobenzoate, 3-chlorobenzoate, 4-chlorobenzoate, hydroquinone, chlorohydroquinone, and 4-chlororesorcinol [ 8 ]. Although ORF3 has been clarified to be responsible for the salicylate-dependent induction [ 8 ], it remains to be elucidated how flanking regions affect the induction activity.…”

“…The transcription factor, designated Sal7AR, is highly specific to salicylate (2-hydroxybenzoate); virtually no cross-reactivity was observed for the following aromatic compounds: phenol, catechol, o-cresol, m-cresol, p-cresol, 3-methylcatechol, 4-methylcatechol, 4-chlorocatechol, benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, 2-chlorobenzoate, 3-chlorobenzoate, 4-chlorobenzoate, hydroquinone, chlorohydroquinone, and 4-chlororesorcinol [ 8 ]. Although ORF3 has been clarified to be responsible for the salicylate-dependent induction [ 8 ], it remains to be elucidated how flanking regions affect the induction activity. To identify the core regions necessary for the expression, I first employed a deletion analysis.…”

“…Although such degraders are widespread in the environment, E . coli usually lacks the ability to sense and degrade such aromatic compounds [ 5 – 8 ]. Previously, through a functional (promoter activity) screen of a metagenomic library, we have isolated several clones that contained transcription factors that were responsive to aromatic compounds [ 5 , 7 ].…”

“…Previously, through a functional (promoter activity) screen of a metagenomic library, we have isolated several clones that contained transcription factors that were responsive to aromatic compounds [ 5 , 7 ]. One of the clones, pSAL7A, was highly specific to salicylate (2-hydroxybenzoate), but the induction rate was poor [ 8 ]. I considered that the transcriptional regulator, Sal7AR included in pSAL7A, would be potentially useful to develop a novel genetic switch.…”

Rhizosphere Metagenomics of Paspalum scrobiculatum L. (Kodo Millet) Reveals Rhizobiome Multifunctionalities

“…Microbial capabilities for utilization of aromatic compound through degradation of xenobiotic chemicals is essential for detoxification of natural habitats [81,82]. We identified sequence reads linked with different pathways involved in the anaerobic degradation of aromatic and xenobiotic compounds, metabolism of aromatic intermediates, and peripheral pathways for catabolism of aromatic molecules ( Figure 5).…”