Complete Nucleotide Sequence of the 113-Kilobase Linear Catabolic Plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a and Transcriptional Analysis of Genes Involved in Quinaldine Degradation

Abstract

The nucleotide sequence of the linear catabolic plasmid pAL1 from the 2-methylquinoline (quinaldine)-degrading strain Arthrobacter nitroguajacolicus Rü61a comprises 112,992 bp. A total of 103 open reading frames (ORFs) were identified on pAL1, 49 of which had no annotatable function. The ORFs were assigned to the following functional groups: (i) catabolism of quinaldine and anthranilate, (ii) conjugation, and (iii) plasmid maintenance and DNA replication and repair. The genes for conversion of quinaldine to anthranilate are organized in two operons that include ORFs presumed to code for proteins involved in assembly of the quinaldine-4-oxidase holoenzyme, namely, a MobA-like putative molybdopterin cytosine dinucleotide synthase and an XdhC-like protein that could be required for insertion of the molybdenum cofactor. Genes possibly coding for enzymes involved in anthranilate degradation via 2-aminobenzoyl coenzyme A form another operon. These operons were expressed when cells were grown on quinaldine or on aromatic compounds downstream in the catabolic pathway. Single-stranded 3 overhangs of putative replication intermediates of pAL1 were predicted to form elaborate secondary structures due to palindromic and superpalindromic terminal sequences; however, the two telomeres appear to form different structures. Sequence analysis of ORFs 101 to 103 suggested that pAL1 codes for one or two putative terminal proteins, presumed to be covalently bound to the 5 termini, and a multidomain telomere-associated protein (Tap) comprising 1,707 amino acids. Even if the putative proteins encoded by ORFs 101 to 103 share motifs with the Tap and terminal proteins involved in telomere patching of Streptomyces linear replicons, their overall sequences and domain structures differ significantly.Arthrobacter nitroguajacolicus Rü61a, formerly assigned to Arthrobacter ilicis, is able to utilize quinaldine (2-methylquinoline) as a sole source of carbon and energy (27; for a review, see reference 13). The "upper pathway" of quinaldine degradation, the conversion of quinaldine to anthranilate, is encoded by a gene cluster containing genes encoding quinaldine 4-oxidase (Qox) (41), 1H-4-oxoquinaldine 3-monooxygenase (Moq), a 2,4-dioxygenase (Hod) catalyzing heterocyclic ring cleavage of 1H-3-hydroxy-4-oxoquinaldine to carbon monoxide and N-acetylanthranilate (14, 15), and an aryl-acylamidase (Amq) that forms anthranilate and acetate (32) (Fig. 1A). The "lower pathway" (i.e., the mineralization of anthranilate) has been suggested to involve catechol formation and ortho ring cleavage (27) (Fig. 1B). Recently, we found that the ability to convert quinaldine to anthranilate is conferred by the conjugative plasmid pAL1, which was identified as a linear replicon with proteins attached to its 5Ј ends (40).Linear plasmids of gram-positive bacteria were first described in Streptomyces rochei in 1979 (20). Since then, they have been reported to occur in many Streptomyces spp., several rhodococci and mycobacteria, Planobispora rosea, the plant pathogen ...

A Novel Replicative Enzyme Encoded by the LinearArthrobacterPlasmid pAL1

“…The adjacent gene pAL1.101 codes for a putative telomere-associated protein, which, however, is much larger than the Tap proteins of Streptomyces linear replicons. We hypothesized that the pAL1.101 protein might play a different, or more versatile, role in the replication of linear DNA than the Tap proteins (36). In this study, we show that the protein interacts with TP pAL1 in vivo.…”

“…The 113-kb conjugative plasmid pAL1, which confers on Arthrobacter nitroguajacolicus Rü61a the ability to utilize the N-heteroaromatic compound 2-methylquinoline, is so far the only described linear plasmid within this genus. The termini of pAL1 show the inverted-repeat sequence 5Ј-CCTGC…GCAGG-3Ј, and its 5Ј ends are capped with proteins (34,36). The terminal protein TP pAL1 is encoded by the pAL1.102 locus, which is located close to the "right" terminus of pAL1 (30).…”

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

“…Previous sequence analysis of the promoter regions of meqC (formerly, qox), meqD (formerly, moq), and ORF19 had suggested that meqCp and meqDp likely are 70 -dependent promoters, whereas ORF19p possibly is recognized by 38 -polymerase (13). As a first step to characterize the meqR2 promoter, the transcriptional start of meqR2 was mapped by 5=-RACE analysis to the position 250 nucleotides upstream of the translation initiation codon.…”

“…1B), show significant similarity to those of 2-aminobenzoate CoA ligase and 2-aminobenzoyl-CoA monooxygenase/reductase of Azoarcus evansii, respectively. Transcription of the three catabolic operons on pAL1 is upregulated in cells grown on quinaldine or on aromatic compounds downstream in the degradation pathway (13); however, the molecular basis of transcriptional control has remained unresolved.…”

“…CP003205.1) (Fig. 1B) (2,13). Whereas strictly aerobic bacteria described to degrade anthranilate usually oxidize anthranilate to catechol, which is subjected to dioxygenolytic ring cleavage, Arthrobacter sp.…”