Regulation of benzoate degradation in Acinetobacter sp. strain ADP1 by BenM, a LysR-type transcriptional activator

Abstract

In Acinetobacter sp. strain ADP1, benzoate degradation requires the ben genes for converting benzoate to catechol and the cat genes for degrading catechol. Here we describe a novel transcriptional activator, BenM, that regulates the chromosomal ben and cat genes. BenM is homologous to CatM, a LysR-type transcriptional activator of the cat genes. Unusual regulatory features of this system include the abilities of both BenM and CatM to recognize the same inducer, cis,cis-muconate, and to regulate some of the same genes, such as catA and catB. Unlike CatM, BenM responded to benzoate. Benzoate together with cis,cis-muconate increased the BenM-dependent expression of the benABCDE operon synergistically. CatM was not required for this synergism, nor did CatM regulate the expression of a chromosomal benA::lacZ transcriptional fusion. BenM-mediated regulation differs significantly from that of the TOL plasmid-encoded conversion of benzoate to catechol in pseudomonads. The benM gene is immediately upstream of, and divergently transcribed from, benA, and a possible DNA binding site for BenM was identified between the two coding regions. Two mutations in the predicted operator/promoter region rendered ben gene expression either constitutive or inducible by cis,cis-muconate but not benzoate. Mutants lacking BenM, CatM, or both of these regulators degraded aromatic compounds at different rates, and the levels of intermediary metabolites that accumulated depended on the genetic background. These studies indicated that BenM is necessary for ben gene expression but not for expression of the cat genes, which can be regulated by CatM. In a catM-disrupted strain, BenM was able to induce higher levels of catA expression than catB expression.

FIG. 1

The β-ketoadipate pathway of Acinetobacter sp. strain ADP1. Compounds and genes relevant to these studies are indicated by boldfaced and italicized text, respectively.

FIG. 2

Restriction map of the chromosomal ben-cat region. The locations of genes, and their transcriptional directions, are shown relative to some of the known restriction endonuclease sites: EcoRI (E), EcoRV (RV), KpnI (K), SalI (S), HindIII (H), BsaAI (B), PvuII (P), NsiI (N), and SphI (Sp). Triangles mark the insertion sites of ΩS (29), ΩK (6), and the lacZ::Km^r cassette (18) of strains listed in Table 1. Below the map, the DNA sequence of the benM-benA intergenic region was aligned with the corresponding catM-catB intergenic region, with identical nucleotides indicated (:). The known CatM binding site (30) was used to predict a possible BenM binding site that has the consensus sequence (T-11 nt-A) and dyad symmetry (ATAC, GTAT) involved in binding LysR-type activators. An adjacent region with similar sequences is underlined. Circled nucleotides indicate mutations identified in strains ACN147 and ACN149 (G→A) and in strain ACN146 (T→A).

FIG. 3

Protein sequence alignment of homologous LysR-type transcriptional activators: ClcR (3), TcbR (36), TfdR (15), BenM, CatM (30), and CatR (31). Aligned residues identical or similar to those of BenM are in black or gray boxes, respectively. The underlined region, BenM residues 109 to 162, may be involved in inducer recognition.

FIG. 4

β-Galactosidase (LacZ) activity resulting from expression of a chromosomal benA::lacZ fusion in strains ACN32, ACN39, ACN41, ACN47, ACN47(pBAC14), and ACN42(pBAC14) (Table 1). Genotypes are noted above bars. Strains were cultured in LB with the inducers indicated. Activities are averages of at least three independent repetitions, and the corresponding standard deviations were <20% of the average value.

FIG. 5

CatA (catechol 1,2-dioxygenase) activity in strains ADP1, ISA36, ISA35, and ISA35(pBAC14) (Table 1). Genotypes are noted above bars. Strains were cultured in LB with the inducers indicated. Activities are the averages of at least three independent repetitions, and the corresponding standard deviations were <20% of the average value. In ADP1, but not the other strains, benzoate can be metabolized to cis,cis-muconate.

FIG. 6

Northern hybridization analysis of ben gene transcripts. Regions of benA and benE were labeled to make probes A and E, respectively, as described in Materials and Methods. Arrows indicate transcripts detected in RNA from the wild-type ADP1 grown with benzoate (A) or from ACN32, with a transcriptional terminator following the benA::lacZ fusion, grown with benzoate and cis,cis-muconate (B). Total wild-type RNA in one sample (A, lane 1) was stained with methylene blue to demonstrate the positions of the rRNA species.

FIG. 7

Metabolites detected by MO-NMR following the addition, at time zero, of 3 mM benzoate (A and B) or 3 mM benzoate and 3 mM p-hydroxybenzoate (A and C) to deuterated cultures of ISA36 or ACN9. Concentrations of benzoate (ben), p-hydroxybenzoate (pob), catechol (cat), and cis,cis-muconate (ccm) were calculated by integration of NMR spectral peaks.