|dc.description.abstract||Multiple mechanisms regulate aromatic compound degradation in the soil bacterium Acinetobacter baylyi ADP1. Herein, complementary mechanisms controlling the consumption of benzoate are explored: 1) transcriptional regulation and 2) chromosomal amplification of catabolic genes. The former highlights BenM, a member of the LysR family of regulators controlling transcription of a complex regulon for benzoate degradation in ADP1. BenM activates gene expression in response to two effectors, benzoate and muconate. These effectors act synergistically to allow high-level gene expression, a feature not characterized in any other member of this broad family. Here, the unique effector profile of BenM is explored. Genetic studies, combined with structural work, elucidate the role of two distinct effector-binding sites in BenM. Targeted mutagenesis confirms the physiological relevance of a hydrophobic benzoate binding pocket and pinpoints critical residues, Arg160 and Tyr293, essential for benzoate-dependent gene expression and synergistic transcriptional activation. BenM variants that activate transcription in the absence of inducer are examined: BenM(R156H), BenM(R225H), and BenM(E226K). Structural studies of BenM variants reveal subtle conformational changes associated with transcriptional activation.
The recent discovery of gene amplification in ADP1 highlights an alternate strategy of genetic regulation. In strains lacking wild-type regulation, chromosomal amplification of catabolic genes can compensate for low transcript levels. Here, a wide spectrum of amplification events were studied using multiple approaches uniquely available in the tractable ADP1 model system. Approximately 50 amplification mutants with enhanced benzoate catabolism were characterized. Each harbors multiple copies of a genomic region, ranging from 12 kbp to more than 300 kbp, encompassing the ben and cat functional genes. Analysis of the precise sequence at amplicon endpoints identifies the DNA sequence involved in formation of the original duplication. In all cases examined, these sequences indicate genetic duplication was initiated by a homology-independent illegitimate recombination mechanism. Recurrence of exact duplication junctions in multiple mutants reveals site preference in duplication formation, a mechanism we designate position-specific illegitimate recombination. The novel experimental tools developed here will facilitate a rapid and thorough examination of the full spectrum of gene amplification events contributing to ADP1 genome plasticity, as well as enable studies into the relevant recombination mechanisms.||