Cloning and genetic organization of the pca gene cluster from Acinetobacter calcoaceticus

Abstract

The lB-ketoadipate pathway of Acinetobacter caloaceticus comprises two parallel metabolic branches. One branch, mediated by six enzymes encoded by the cat genes, converts catechol to succinate and acetyl coenzyme A (acetyl-CoA); the other branch, catalyzed by products of the pca genes, converts protocatechuate to succinate and acetyl-CoA by six metabolic reactions analogous or identical to those of the catechol sequence. We used the expression plasmid pUC18 to construct expression libraries of DNA from an A. caloaceticus mutant strain from which the cat genes had been deleted. Immunological screening with antiserum to the pcaE gene product, ,B-ketoadipate:succinyl-CoA transferase I, resulted in the isolation of a cloned 11-kilobase-pair (kbp) fragment which inducibly expressed all six pea genes under control of the lac promoter on pUC18. The induced Escherichia coli cells formed the six pea gene products at levels 10-to 30-fold higher than found in fully induced A. calcoaceicus cultures, although protocatechuate 3,4-dioxygenase (the iron-containing product of the pcaA gene) from the recombinant strain possessed a relatively low turnover number. An E. coli culture expressing the cloned pea genes quantitatively converted protocatechuate to (-ketoadipate; failure of the organism to metabolize the latter compound can be most readily ascribed to relatively low pool levels of succinyl-CoA, a required substrate for 0-ketoadipate:succinyl-CoA transferase, in E. coli. The gene order and direction of transcription were determined to be pcACBDFE by identification of enzymes expressed in subclones, by using natural transformation to identify subclones carrying DNA corresponding to dysfunctional alleles in mutant A. calcoaceticus strains, and by restriction mapping of both the ll-kbp fragment and derivatives of the ll-kbp fragment containing TnS in the pcaA, pcaB, peaC, pcaD, and peaE genes. The fragment containing the pea gene hybridized strongly and specifically to a previously cloned fragment containing A. caloaceticus cat genes.The pca structural genes encode six enzymes that convert protocatechuate to citric acid cycle intermediates via Pketoadipate ( Fig. 1; 27). Protocatechuate induces coordinate synthesis of the enzymes in Acinetobacter calcoaceticus (3), and their unified transcriptional control has been suggested by their constitutive formation in regulatory mutants (4,22). The physical organization of the A. calcoaceticus pca genes has not been explored. The A. calcoaceticus cat genes encode six enzymes that convert catechol to citric acid cycle intermediates by reactions analogous or identical to those catalyzed by the pca gene products (Fig. 1) To establish a basis for analysis of possible interaction between cat and pca genes in A. calcoaceticus, we constructed expression libraries of A. calcoaceticus DNA and used immunological screening to identify a clone that expressed the pcaE gene. The cloned DNA was an 11-kbp EcoRI fragment that hybridized with the 5-kbp EcoRI fragment containing the catBCDE genes and pos...

Supraoperonic clustering of pca genes for catabolism of the phenolic compound protocatechuate in Agrobacterium tumefaciens

“…Disproportionately low PcaHG activity, relative to the level of PcaHG protein produced, was noted for cloned Acinetobacter calcoaceticus pcaHG genes expressed in E. coli (10). Very low levels of PcaHG were also observed when the expression of cloned P. putida pcaHG genes was driven by the lac, tac, or T7 promoter in E. coli (14).…”

Cloning and expression in Escherichia coli of Acinetobacter calcoaceticus genes for benzoate degradation

“…The analogous component purified from P. arvilla is a 38-kDa flavoprotein (45), and the Pseudomonas cepacia phthalate oxygenase reductase is a 34-kDa protein containing flavin mononucleotide and a ferredoxiniron-sulfur type center (3). (9,22,31), the only protein that appears to demonstrate discernable host-dependent properties is protocatechuate 3,4-dioxygenase, the product of the pcaA gene. This enzyme is relatively inactive when the gene is expressed in E. coli at extremely high levels (9).…”

mucK, a gene in Acinetobacter calcoaceticus ADP1 (BD413), encodes the ability to grow on exogenous cis,cis-muconate as the sole carbon source

“…An additional result which adds some extra weight to a physiological role for mucK involving some substrate other than muconate is its chromosomal location. Whereas the ben-cat genes involved in benzoate, catechol, and muconate utilization are clustered on the ADP1 chromosome (29,32), mucK has been shown to map within 10 kbp of the pca-qui-pob cluster (2,8,9), encoding the quinate and p-hydroxybenzoate branch of the ␤-ketoadipate pathway, but about 300 kbp distant from the ben-cat cluster (13).…”

“…The genes for the two convergent, biochemically and genetically homologous branches of the pathway are found in two supraoperonic clusters, the ben-cat cluster (29,32), comprising the genes for the conversion of benzoate to tricarboxylic acid cycle intermediates, and the pca-qui-pob cluster (2,8,9), comprising the genes for the catabolism of p-hydroxybenzoate and quinate. Both clusters are Ͼ20 kbp and yet are separated on the chromosome by about 290 kbp (13).…”