Schematic diagram of ORFs on the 112.992-bp linear plasmid pAL1. Genes and predicted ORFs are indicated by arrows, and the arrowheads indicate the directions of transcription. The ORFs are grouped on the basis of (hypothetical) function, as follows: blue, ORFs involved in quinaldine or anthranilate catabolism; yellow, ORFs involved in DNA mobilization by conjugation or transposition; red, ORFs involved in DNA replication, repair, and modification and in plasmid maintenance; green, ORFs involved in diverse functions; gray, ORFs encoding conserved hypothetical proteins, white, ORFs coding for hypothetical proteins.

Quinaldine degradation by A. nitroguajacolicus Rü61a (13, 27, 40, 41). (A) Quinaldine conversion to anthranilate. 1, quinaldine (2-methylquinoline); 2, 1H-4-oxoquinaldine; 3, 1H-3-hydroxy-4-oxoquinaldine; 4, N-acetylanthranilic acid; 5, anthranilic acid; Qox, quinaldine 4-oxidase; Moq, 1H-4-oxoquinaldine 3-monooxygenase; Hod, 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase; Amq, N-acetylanthranilate amide hydrolase. (B) Hypothetical initial steps in anthranilate degradation by strain Rü61a. 6, catechol; 7, 2-aminobenzoyl-CoA; 8, 2-amino-5-oxo-cyclohex-1-ene-carbonyl-CoA. For details, see the text.

Nucleotide sequences upstream of qoxL (A), moq (B), and ORF 19 (C) and putative promoters. Translation initiation codons for the qoxL and moq genes and for ORF 19 (indicated by arrows) are in bold type; putative ribosome binding sites (RBS) are indicated by italics. The transcriptional start sites (+1) of qoxL, moq, and ORF 19 are indicated by bold type and italics; the direction of transcription is indicated by bent arrows. Hypothetical −10 and −35 regions of each promoter are underlined. The dotted arrows indicate the sequence corresponding to the PaaX binding motif (see text).

Termini of pAL1. (A) Predicted secondary structures formed by 3′ single-stranded DNA of the left and right ends of pAL1. (B) Nucleotide sequences of the left and right termini. Palindromic sequences are enclosed in boxes in panel B and are labeled with roman numerals in both panels; the palindromes that were predicted to form stem-loop structures are indicated by gray shading in both panels. Superpalindromic sequences indicated by different colors in panel A are indicated by arrows that are in the corresponding colors below the sequence in panel B. Note that sequences of 3′ overhangs shown in panel A are reverse complementary to the sequence in panel B. In panel B, the central motif 5′-GCTNCGC-3′ that is conserved in many actinomycete linear replicons (see text) is indicated by bold type and underlining; trinucleotides proposed to form hairpins with sheared G-A pairs in the corresponding 3′ overhang are indicated by white type with a black background; and asterisks indicate nonpairing bases within palindromes.

Operon structure of ORFs 3 to 24 (nucleotides 4,079 to 30,130) of pAL1 and RT-PCR analysis of transcripts generated from this region. (A) Schematic diagram of ORFs and RT-PCR strategy. For descriptions of ORFs 3 to 24, see Table 1 and the text. Bars I to XIV indicate the regions amplified in the RT-PCR analysis (for the primers and lengths of PCR products, see Table S1 in the supplemental material). Arrows with the same pattern indicate ORFs belonging to the same operon. (B) RT-PCR analysis of qoxM, amq, ORF 23, and ORF 16. Total RNA from A. nitroguajacolicus Rü61a grown on different carbon sources was used as a template for RT. The carbon sources for growth of cells were as follows: lanes 1 and 2, quinaldine; lanes 3 and 4, 1H-4-oxoquinaldine; lanes 5 and 6, 1H-3-hydroxy-4-oxoquinaldine; lanes 7 and 8, N-acetylanthranilate; lanes 9 and 10, anthranilate; and lanes 11 and 12, succinate. In negative controls (lanes 2, 4, 6, 8, 10, and 12), reverse transcriptase was omitted from the cDNA synthesis reaction mixture. As a positive control for the PCR, total DNA was used as a template for PCR (lanes 13). The roman numerals indicate the transcripts (see panel A), as follows: I, qoxM; II, amq; III, ORF 23; and IV, ORF 16. Lane M contained a size marker. (C) RT-PCR analysis using primer pairs to amplify intergenic regions between ORF 3 and qoxS (V), between amq and ORF 10 (IX), and between ORF 21 and ORF 22 (XIII). RNA used for cDNA synthesis was isolated from cells grown on quinaldine (lanes 1 and 2) and succinate (lanes 3 and 4). In the negative controls (lanes 2 and 4), reverse transcriptase was omitted from the reaction mixture. Lane 5 contained a positive control for PCR performed with total DNA as the template. Lane M contained a size marker. The results for amplification of regions VI (qoxSML), VII (moq-hod), VIII (hod-amq), and X (ORF 10 and ORF 11) were similar to the results for amplification of regions V and IX. Amplification of regions XI (ORF 19 and ORF 20), XII (ORF 20 and ORF 21), and XIV (ORF 22 and ORF 23) resulted in patterns and intensities of RT-PCR products corresponding to the patterns and intensities of RT-PCR products from region XIII.