The PaaX-Type Repressor MeqR2 of Arthrobacter sp. Strain Rue61a, Involved in the Regulation of Quinaldine Catabolism, Binds to Its Own Promoter and to Catabolic Promoters and Specifically Responds to Anthraniloyl Coenzyme A

Abstract

The genes coding for quinaldine catabolism in Arthrobacter sp. strain Rue61a are clustered on the linear plasmid pAL1 in two upper pathway operons (meqABC and meqDEF) coding for quinaldine conversion to anthranilate and a lower pathway operon encoding anthranilate degradation via coenzyme A (CoA) thioester intermediates. The meqR2 gene, located immediately downstream of the catabolic genes, codes for a PaaX-type transcriptional repressor. MeqR2, purified as recombinant fusion protein, forms a dimer in solution and shows specific and cooperative binding to promoter DNA in vitro. DNA fragments recognized by MeqR2 contained a highly conserved palindromic motif, 5=-TGACGNNCGTcA-3=, which is located at positions ؊35 to ؊24 of the two promoters that control the upper pathway operons, at positions ؉4 to ؉15 of the promoter of the lower pathway genes and at positions ؉53 to ؉64 of the meqR2 promoter. Disruption of the palindrome abolished MeqR2 binding. The dissociation constants (K D ) of MeqR2-DNA complexes as deduced from electrophoretic mobility shift assays were very similar for the four promoters tested (23 nM to 28 nM). Anthraniloyl-CoA was identified as the specific effector of MeqR2, which impairs MeqR2-DNA complex formation in vitro. A binding stoichiometry of one effector molecule per MeqR2 monomer and a K D of 22 nM were determined for the effector-protein complex by isothermal titration calorimetry (ITC). Quantitative reverse transcriptase PCR analyses suggested that MeqR2 is a potent regulator of the meqDEF operon; however, additional regulatory systems have a major impact on transcriptional control of the catabolic operons and of meqR2.A rthrobacter sp. strain Rue61a, an isolate from sludge of the wastewater treatment plant of a coal tar refinery, is able to utilize quinaldine (2-methylquinoline) as a source of carbon and energy (1, 2). Methylquinolines and related N-heteroaromatic compounds are constituents of coal tar and shale oil. Quinolines are more water soluble than their homocyclic naphthalene analogs, and hence they are more readily transported to subsoil and groundwater if entering the environment, e.g., from abandoned coal processing facilities or from wood-creosoting activities. Many quinoline derivatives are considered toxic and/or mutagenic and thus are of environmental concern. However, quinaldine is used in aquaculture for anesthesia of fish (3). Interestingly, quinaldine is also a possible chemical signal in the urine of the male red fox (4) and the male ferret (5) and a component of the anal sac secretion of skunks (6). Quinoline derivatives of natural origin moreover include a plethora of alkaloids from microbial, animal, and plant sources, especially from the Rutaceae (7). A number of bacterial isolates, mainly aerobes from soil, with the ability to degrade quinoline derivatives have been described in the literature (reviewed in reference 8).Quinaldine degradation by Arthrobacter sp. Rue61a is initiated by two hydroxylation steps, catalyzed by the molybdenum enzyme quinaldine 4-oxidase...

Recent Developments in Using Advanced Sequencing Technologies for the Genomic Studies of Lignin and Cellulose Degrading Microorganisms

“…According to Niewerth et.al (2012; 2013) Arthrobacter strain Rue61a expresses genes encoding quinaldine catabolism which clustered on a linear plasmid known as pAL1 with two upper operon pathways called meqABC and meqDEF. These cluster of genes codes for the conversion of quinaldine to anthranilate 36 , 37 . The study also confirms that Arthrobacter s outstanding degradation of anthranilate exclusively continues through CoA-thioester pathway.…”

“…The study also confirms that Arthrobacter s outstanding degradation of anthranilate exclusively continues through CoA-thioester pathway. Apart from quinaldine utilization, strain Rue61a has a set of aromatic degradation pathways, enabling the utilization of 4-hydroxy-carboxylic acids, which are characteristic products of lignin depolymerization, via ortho cleavage of protocatechuate 36 , 37 .…”

Plasmid-Mediated Tolerance Toward Environmental Pollutants

“…The function of MeqR1 is currently unknown, while MeqR2 is able to bind the promoter region of mepC, meqD, orf1, and to its own promoter and shows high binding specificity for anthraniloyl-CoA. These results suggest that MeqR2 may be involved in the regulation of the pathway, although other regulatory systems exist that are known to govern the expression of the meq genes (65). Although conserved gene clustering has been observed in pAL1, suggesting a modular structure, only one insertion sequence has been detected (63).…”