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

Abstract

Benzyl alcohol, benzaldehyde, benzoate, and anthranilate are metabolized via catechol, cis,cis-muconate, and the ␤-ketoadipate pathway in Acinetobacter calcoaceticus ADP1 (BD413). Mutant strain ISA25 with a deletion spanning catBCIJF and unable to metabolize muconate further will not grow in the presence of an aromatic precursor of muconate. Growth on fumarate as the sole carbon source with added benzyl alcohol or benzaldehyde selected spontaneous mutants of ISA25. After repair of the cat deletion by natural transformation with linearized plasmid pPAN4 (catBCIJF) 10 mutants were unable to grow on benzoate or cis,cis-muconate but could still grow on anthranilate. Transformation with wild-type chromosomal DNA demonstrated the presence of two unlinked mutations in each strain, one in the benABCD region, encoding the conversion of benzoate to catechol, and the other in a gene determining the ability to grow on exogenous cis,cis-muconate. The wild-type gene, named mucK, was cloned into pUC18, and its nucleotide sequence was determined. It encodes a 413-residue protein of M r ‫؍‬ 45,252 which is a member of a superfamily of membrane transport proteins and which is within a subgroup involved in the uptake of organic acids. Five of the mutant alleles were cloned, and the mutations were determined by nucleotide sequencing. All the mutations were in the mucK coding region and consisted of three deletions, one duplication, and a substitution. Insertional inactivation of mucK resulted in the loss of the ability to utilize exogenous muconate. The location of mucK on the chromosome appeared to be unique for genes associated with the benzoate branch of the ␤-ketoadipate pathway in being close to the pca-qui-pob gene cluster (for p-hydroxybenzoate utilization) and distant from the functionally related ben-cat cluster. Downstream of mucK and transcribed in the same direction is an open reading frame encoding a protein of 570 residues (M r ‫؍‬ 63,002) which shows considerable homology with a mammalian electron transport protein; its insertional inactivation had no detectable phenotypic effect.

The LysR-type PcaQ protein regulates expression of a protocatechuate-inducible ABC-type transport system in Sinorhizobium meliloti

“…Transport systems involved in the uptake of aromatic acids primarily belong to the major facilitator superfamily (MFS) of transporter proteins (Harwood et al, 1994;Collier et al, 1997;Williams & Shaw, 1997;Leveau et al, 1998;D'Argenio et al, 1999;Chaudhry et al, 2007) and have been described predominantly in the c-proteobacteria. Within the a-proteobacteria, protocatechuate (and p-hydroxybenzoate) uptake has been described in R. leguminosarum, yet the genetic identity of the transport system has never been established (Wong et al, 1991).…”

“…While systems involved in the active transport of aromatic acids such as protocatechuate have been described in many species, including Pseudomonas putida (Harwood et al, 1994;Nichols & Harwood, 1997), a protocatechuate transport system has yet to be identified in S. meliloti or any other member of the a-proteobacteria. Furthermore, S. meliloti appears to lack protein homologues of the PcaK major facilitator superfamily of aromatic acid transport proteins (Collier et al, 1997;Williams & Shaw 1997;D'Argenio et al, 1999;Ledger et al, 2009).…”

3-Chlorobenzoate is taken up by a chromosomally encoded transport system in Cupriavidus necator JMP134

“…A few of these permease-type transport proteins have been biochemically characterized, and the corresponding gene has been described. This category includes benK for benzoate transport, vanK for vanillate, hcaK for hydroxycinnamate, and mucK for muconate, all found in Acinetobacter baylyi ADP-1 (Collier et al, 1997;D'Argenio et al, 1999;Parke & Ornston, 2003;Williams & Shaw, 1997), and pcaK for 4-HB and protocatechuate in P. putida PRS2000 (Harwood et al, 1994).…”

The benPK operon, proposed to play a role in transport, is part of a regulon for benzoate catabolism in Acinetobacter sp. strain ADP1

“…strain CA10 are encoded in genetic regions involved in the degradation of the heterocyclic aromatic compound carbazole via the catechol branch of the β-ketoadipate pathway (Nojiri et al, 2001). Moreover, the putative porin-encoding genes are often located near genes encoding proteins resembling inner-membrane permeases such as BenK, PcaK and MucK that are involved in the uptake of compounds degraded via the β-ketoadipate pathway (Nichols & Harwood, 1997 ;Williams & Shaw, 1997). Taken collectively, the data support a model in which a porin, such as BenP, and a permease, such as BenK, function together to facilitate the cellular entry of some substrates of bacterial aromatic compound catabolic pathways.…”