No abstract

Transcriptional control of carbon source preferences by Acinetobacter sp. strain ADP1 was assessed with a pobA::lacZ fusion during growth on alternative substrates. The pobA-encoded enzyme catalyzes the first step in the degradation of 4-hydroxybenzoate, a compound consumed rapidly as a sole carbon source. If additional aromatic carbon sources are available, 4-hydroxybenzoate consumption is inhibited by unknown mechanisms. As reported here, during growth on aromatic substrates, pobA was not expressed despite the presence of 4-hydroxybenzoate, an inducer that normally causes the PobR regulator to activate pobA transcription. Growth on organic acids such as succinate, fumarate, and acetate allowed higher levels of pobA expression. In each case, pobA expression increased at the end of the exponential growth phase. Complex transcriptional regulation controlled 4-hydroxybenzoate catabolism in multisubstrate environments. Additional studies focused on the wild-type preference for benzoate consumption prior to 4-hydroxybenzoate consumption. These compounds are degraded via the catechol and protocatechuate branches of the ␤-ketoadipate pathway, respectively. Here, mutants were characterized that degraded benzoate and 4-hydroxybenzoate concurrently. These mutants lacked the BenM and CatM transcriptional regulators that normally activate genes for benzoate catabolism. A model is presented in which BenM and CatM prevent pobA expression indirectly during growth on benzoate. These regulators may affect pobA expression by lowering the PcaK-mediated uptake of 4-hydroxybenzoate. Consistent with this model, BenM and CatM bound in vitro to an operator-promoter fragment controlling the expression of several pca genes, including pcaK. These studies provide the first direct evidence of transcriptional cross-regulation between the distinct but analogous branches of the ␤-ketoadipate pathway.

BenM and CatM are distinct, but similar, LysR-type transcriptional regulators of the soil bacterium Acinetobacter sp. strain ADP1. Together, the two regulators control the expression of at least 14 genes involved in the degradation of aromatic compounds via the catechol branch of the β-ketoadipate pathway. In these studies, BenM and CatM were each purified to homogeneity to test the possibility that they regulate the expression of two additional genes, benP and benK, that are adjacent to benM on the chromosome. Each regulator bound to a DNA fragment containing the benP promoter region. Additional transcriptional studies suggested that benP and benK are co-transcribed as an operon, and a site of transcription initiation was identified. Alignment of this initiation site with those of several CatM-and BenM-regulated genes revealed common regulatory motifs. Mutants lacking both CatM and BenM failed to activate benP transcription. The ability of each protein to regulate gene expression was inferred from strains lacking either CatM or BenM that were still capable of increasing benP expression in response to cis,cis-muconate. This compound has previously been shown to induce all enzymes of the catechol branch of the β-ketoadipate pathway through a complex transcriptional circuit involving CatM and BenM. Thus, the regulated expression of the benPK operon in concert with other genes of the regulon is consistent with the model that BenP, a putative outer-membrane porin, and BenK, an inner-membrane permease, transport aromatic compounds in strain ADP1.

Fluorescence emission spectroscopy was used to investigate interactions between two effectors and BenM, a transcriptional regulator of benzoate catabolism. BenM had a higher affinity for cis,cis-muconate than for benzoate as the sole effector. However, the presence of benzoate increased the apparent dissociation constant (reduced the affinity) of the protein for cis,cis-muconate. Similar results were obtained with truncated BenM lacking the DNA-binding domain. High-level transcriptional activation may require that some monomers within a BenM tetramer bind benzoate and others bind cis,cis-muconate.

BenM, a member of the LysR‐type family of transcriptional regulators, controls genes for benzoate degradation in the Gram‐negative bacterium Acinetobacter sp. strain ADP1. Recent studies show that BenM activates benABCDE expression synergistically in response to two effector ligands: cis,cis‐muconate (CCM) and benzoate. As an initial step in investigating the structural basis of dual effector response, the effector‐binding domain of BenM (BenM‐EBD) was crystallized by the microbatch‐under‐oil technique with conditions optimized from high‐throughput screens performed by the Hauptman–Woodward Institute. Data‐collection quality crystals of BenM‐EBD belonged to space group P212121, diffracted to 2.3 Å and had unit‐cell parameters a = 65.64, b = 66.34, c = 117.46 Å. The influence of effector ligands on crystal formation was also evaluated. The presence of benzoate or CCM impaired the formation of crystals. The presence of both effectors together resulted in a dramatic decrease in the production of crystals. The effector‐binding domain of CatM, a homolog of BenM, was also crystallized.