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Applied and Environmental Microbiology, April 2006, p. 2651-2660, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2651-2660.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The Locus Coding for the 3-Nitrobenzoate Dioxygenase of Comamonas sp. Strain JS46 Is Flanked by IS1071 Elements and Is Subject to Deletion and Inversion Events

Miguel A. Providenti,* Rachel E. Shaye, Krista D. Lynes, Neil T. McKenna, Jason M. O'Brien, Sarah Rosolen, R. Campbell Wyndham,{dagger} and Iain B. Lambert

Department of Biology, Carleton University, Ottawa, Ontario, Canada K1S 5B6

Received 18 October 2005/ Accepted 2 February 2006


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ABSTRACT
 
In Comamonas sp. strain JS46, 3-nitrobenzoate (3Nba) is initially oxidized at the 3,4 position by a dioxygenase, which results in release of nitrite and production of protocatechuate. The locus coding for the 3Nba dioxygenase (designated mnb, for m-nitrobenzoate) was mobilized from strain JS46 using a plasmid capture method, cloned, and sequenced. The 3Nba dioxygenase (MnbA) is a member of the phthalate family of aromatic oxygenases. An open reading frame designated mnbB that codes for an NAD(P)H-dependent class IA aromatic oxidoreductase is downstream of mnbA. MnbB is tentatively identified as the oxidoreductase that transfers reducing equivalents to MnbA in strain JS46. The mnb locus is flanked by IS1071 elements. The upstream element is interrupted by a novel insertion sequence designated ISCsp1, and the transposase genes of the flanking insertion elements are transcribed in the direction opposite the direction of mnbA transcription. Spontaneous deletion of mnb occurs because of homologous recombination between the directly repeated flanking IS1071 elements. In addition, in ~0.007 to 0.2% of any population of JS46 cells growing on 3Nba, alternative orientations of mnb relative to the flanking IS1071 elements are detected. These alternative forms are the result of inversions of mnb and the flanking IS1071 elements. Inversions appear to occur because of homologous recombination between the inverted repeats that flank the IS1071 elements.


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INTRODUCTION
 
Nitroaromatic compounds are used extensively in industry (e.g., for the synthesis of pesticides, dyes, and explosives) and are the by-products of natural processes (e.g., nitration during incomplete combustion of organic matter). Many nitroaromatic compounds are widely dispersed pollutants, and because of their toxicity and/or carcinogenicity, their presence in the environment is a cause for concern. Biodegradation of the simpler nitroaromatic compounds is a well-recognized phenomenon, and this natural process is currently being exploited for bioremediation of contaminated sites (34).

Bacterial degradation pathways for aerobic catabolism of nitrobenzoic acids have been described for all three isomers. With 2-nitrobenzoic acid (2Nba) and 4-nitrobenzoic acid (4Nba), the NO2 group initially undergoes reduction prior to final ring cleavage (6, 13-16, 27, 40). In contrast, with 3-nitrobenzoic acid (3Nba), O2 is added directly at the 3,4 position of the aromatic ring by a dioxygenase. The NO2 moiety is spontaneously released to produce protocatechuate (Pca) (20). Genes for the initial metabolism of 4Nba have been described (16, 40), but no genes for metabolism of 2Nba and 3Nba have been described.

Insertion elements have been implicated in the development and horizontal dissemination of various genetic elements in microorganisms. Various biodegradative genes are part of composite transposons, and IS1071 is an insertion sequence that flanks a range of catabolic operons, such as the operons for metabolism of various aromatic substrates and halogenated alkanes (25, 37). In this study, we showed that in Comamonas sp. strain JS46, which can grow on 3Nba but not on 2Nba or 4Nba (20), the locus coding for the dioxygenase that converts 3Nba to Pca (designated mnb, for m-nitrobenzoate) is flanked by IS1071 elements. We also showed that mnb is a genetically unstable element that is subject to both deletion and inversion events.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and growth conditions.
Strains and plasmids used in this study are listed in Table 1. Unless indicated otherwise, Escherichia coli was routinely grown at 30 to 37°C in Luria-Bertani medium (LB) (1% [wt/vol] tryptone, 0.5% [wt/vol] yeast extract, 0.5% [wt/vol] NaCl) supplemented with ampicillin (Amp) (100 mg liter–1), kanamycin (Kan) (50 mg liter–1), chloramphenicol (Cm) (50 mg liter–1), and isopropyl-ß-D-thiogalactopyranoside (IPTG) (1 mM), as required. Comamonas spp. were routinely grown at 30°C using minimal medium A (MMA) (36) supplemented with succinate (20 mM) or an aromatic substrate (1 to 4 mM). Kan (50 mg liter–1), Cm (100 mg liter–1), rifampin (150 mg liter–1), and tryptophan (Trp) (0.1 mM) were added as required. All chemicals and antibiotics were purchased from Sigma-Aldrich (Oakville, Ontario, Canada). When necessary, growth media were solidified by addition of agar (final concentration, 1.6% [wt/vol]).


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  		TABLE 1. Bacterial strains and plasmids used in this study

Molecular techniques.
All molecular manipulations, including preparation of plasmid and chromosomal DNA, agarose gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Southern blot transfer of DNA, were performed by using standard protocols (2, 32). Restriction enzymes were purchased from New England Biolabs (Mississauga, Ontario, Canada). DNA was blunt ended with T4 DNA polymerase (New England Biolabs) and was ligated with T4 DNA ligase (Invitrogen, Burlington, Ontario, Canada), both of which were performed according to the manufacturers' recommendations. DNA sequences were determined by the chain-terminating dideoxy method with ABI Prism automated sequencers at different commercial facilities (described below).

For PCR amplification of DNA, Taq polymerase (Invitrogen) was used as recommended by the manufacturer. Whole-cell suspensions were used as templates, and dimethyl sulfoxide (final concentration, 10% [vol/vol]) was included to ensure that cell lysis occurred. PCRs were routinely performed in 20-µl (total volume) mixtures without a mineral oil overlay, using the following conditions: initial denaturation at 95°C for 3 min; 25 to 40 cycles of 55°C for 35 s, 72°C for 1 to 2 min (0.2- to 2-kb amplification products) or for 3 to 5 min (2- to 4-kb amplification products), and 95°C for 35 s; and a final extension at 72°C for 10 min. PCRs were performed with a T3 thermocycler (Biometra, Montreal Biotech, Montreal, Quebec, Canada). Oligonucleotide primers were synthesized by SigmaGenosys (Oakville, Ontario, Canada). Various PCR products (described below) were cloned into pCR2.1-TOPO (Invitrogen) as recommended by the manufacturer.

Strains were probed for mnb, IS1071, and ISCsp1 by Southern blot analysis of restriction enzyme-digested chromosomal DNA (see Results for a description of the restriction enzymes used). Southern blot probes were generated by randomly labeling DNA with digoxigenin-11-dUTP, and probe-positive fragments were detected by chemiluminescence using an antidigoxigenin system, as recommended by the manufacturer (Roche Molecular Biochemicals, Laval, Québec, Canada). The limit of resolution for our Southern blots was ~23 kb as our extraction method sheared total DNA to fragments of that size. Probes for IS1071 (length, 511 bp), ISCsp1 (length, 414 bp), and the putative promoter of mnbA (PmnbA) (length, 253 bp) were generated by PCR with primers IS1071-F2 plus IS1071-R2, primers ISCsp1-F1 plus ISCsp1-R1, and primers Pmnb-F1 plus Pmnb-R2, respectively (Table 2). A probe specific for csj8' (and the downstream portion of csj7) was generated by digesting pUC-mnb#7 with PciI and HindIII and gel purifying the 625-bp fragment. The regions to which Southern blot probes hybridized are shown in Fig. 1A.


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  		TABLE 2. PCR primers used in this study


Figure 1
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  		FIG. 1. (A) Physical map of mnb, the locus coding for the 3Nba dioxygenase of Comamonas sp. strain JS46. mnb is flanked by IS1071 elements. The upstream IS1071 element is interrupted by a novel insertion element designated ISCsp1 and is designated IS1071::ISCsp1. In mnb 10 ORFs (black arrows) and a potential stem-loop structure (S-L) are indicated. The oxygenase that converts 3Nba to Pca is encoded by mnbA. An NAD(P)H-dependent oxidoreductase is encoded by mnbB. MnbB tentatively transfers reducing equivalents to MnbA. Uncharacterized ORFs in mnb are designated csj (for Comamonas sp. strain JS46). The following regions are also indicated on the maps: ORFs coding for the transposases (tnpA) of IS1071 (light gray arrows) and putative transposase of ISCsp1 (dark gray arrow), the left and right inverted repeats of IS1071, the positions of various restriction sites used for cloning or Southern blot analyses, and regions to which Southern blot probes hybridize. The more detailed map shown at the bottom indicates the positions of restriction sites used to generate pQE30-based constructs described in the text. (B) Sequence of the direct repeats flanking ISCsp1 (underlined nucleotides) and imperfect IRs adjacent to the direct repeats (nucleotides in boldface type; mismatches are indicated by lowercase letters).

Cloning and sequencing mnb and IS1071::ISCsp1.
mnb was mobilized from strain JS46 into pBRC45 and transferred to Comamonas testosteroni strain BR6025 in a two-step process (24). Standard filter mating procedures were used. pBRC45 was first transferred from strain BR6024 into strain JS46, and a transconjugant was recovered on MMA containing 3Nba and Kan. The resulting strain was then mated with strain BR6025, and a 3Nba-degrading transconjugant was recovered on MMA containing 3Nba, Trp, Kan, Cm, and rifampin. We confirmed that the transconjugant was a BR6025 derivative by testing for Trp auxotrophy. A large plasmid (designated pBRRD1) was detected in the transconjugant. The two novel NheI fragments from pBRRD1 (one ~4.4 kb long and harboring IS1071::ISCsp1, and the other ~9.3 kb long and harboring mnb [see Results]) were cloned into the XbaI site of pUC18Not.1, resulting in clones designated pUC-IS (for the 4.4-kb fragment) and pUC-mnb (for the 9.3-kb fragment) (Table 1). Both strands of the insert in pUC-mnb#6 were sequenced by a random transposon insertion method (QIAGEN Genomics Inc. Sequencing Services, Washington). For the insert in pUC-IS, both strands of the novel section that had inserted into IS1071 (see Results) were sequenced by primer walking (Biotechnology Research Institute, University of Ottawa, Ottawa, Canada). Very little DNA flanking the novel section was sequenced.

Bioinformatic analyses.
DNA sequences were compared to entries in the GenBank database to identify potential open reading frames (ORFs). Putative start codons for the ORFs were then selected based on visual inspection of the sequences and identification of potential ribosome binding sites. Conceptual translations of the ORFs were then compared to entries in the Protein Family (Pfam) database (3) and the Clusters of Orthologous Groups (COG) of Proteins database (35). All comparisons were done online using the BLAST (1) and Conserved Domains Database (19) network service of the National Center for Biotechnology Information (Bethesda, Md.) (http://www.ncbi.nlm.nih.gov).

Determining whether mnbA codes for the 3Nba oxygenase and mnbB codes for an oxidoreductase. (i) Colorimetric test.
E. coli JM109 containing pUC-mnb#6, pUC-mnb#7, or pUC-mnb3.7 (Table 1) was tested for the ability to convert 3Nba to Pca using a colorimetric assay for detection of vicinal diols (28), with some minor modifications (30). The negative control was E. coli containing pUC18Not.1.

(ii) Complementation.
A construct harboring mnbA and mnbB (carried on pUTCm-mnb3.7) (Table 1) was randomly inserted into the chromosome of strain JS46D via triparental filter mating (23). Transconjugants were recovered on solid MMA containing 4-hydroxybenzoate and Cm, and four arbitrarily selected colonies were tested for growth on 3Nba by patching them onto solid MMA containing 3Nba and Cm. Control mating preparations lacked E. coli CC118{lambda}pir(pUTCm-mnb3.7) and E. coli MM294(pRK2013).

(iii) Resting-cell assays with pQE30-mnbA1B and derivatives.
DNA spanning PmnbA to mnbB was cloned into pQE30 to generate pQE30-mnbA1B (Table 1). The latter plasmid was then manipulated to generate derivatives in which mnbA or mnbB was individually knocked out (pQE30-mnb{Delta}A1B and pQE30-mnbA1{Delta}B, respectively) or in which the region containing csj1 and mnbB was removed (pQE30-mnbA{Delta}[1B]) (Table 1). Deletions were confirmed by restriction analyses. Resting-cell assays of E. coli M15 containing these plasmids were conducted by growing cultures at 30°C to the early log phase (optical density at 600 nm, ~0.2) in 20 ml of MMA containing Amp, Kan, succinate, and LB (2%, vol/vol). Cultures were divided equally into separate tubes and then were treated with 1 mM 3Nba and 1 mM IPTG (induced cultures) or were not amended (uninduced), grown for an additional 3 h (optical density at 600 nm, ~0.6), centrifuged, washed once with MMA, resuspended in 1 ml MMA containing 20 mM succinate and 1 mM 3Nba, and incubated with shaking for an additional 18 h at 30°C. The 3Nba and Pca contents of cell-free culture supernatants were determined by high-performance liquid chromatography. Samples were acidified with H3PO4 (final concentration, 0.1 M) and analyzed using a Varian Prostar apparatus with a Polaris 5 C18-A reversed-phase column (250 by 4.6 mm). The solvent was 25% acetonitrile-75% phosphate-buffered water (0.01 M KPO4, pH 2.2), and the concentration of acetonitrile was increased to 40% over 10 min. The flow rate was 1.5 ml min–1, and aromatic compounds were detected at 230 nm.

(iv) In vitro assays with MnbB.
DNA coding for MnbB was PCR amplified using primers MnbB-FEx and MnbB-REx (Table 2). The amplicon was cloned (pCR2.1-mnbB), sequenced using vector-specific primers M13F and M13R (Macrogen, Seoul, South Korea) to ensure that mutations had not been introduced during PCR, and then subcloned into pQE30 as described in Table 1. N-terminally His-tagged MnbB (designated MnbBHis) was overproduced in E. coli M15 grown at 15°C using LB containing Kan, Amp, and IPTG amended with 0.2% (wt/vol) glucose, 50 mM phosphate, and 2% (wt/vol) ethanol (26), and it was affinity purified from cell-free crude extracts with Ni-nitrilotriacetic acid agarose (QIAGEN), using the manufacturer's recommendations. Cell extracts were prepared by sonication of cell suspensions, followed by centrifugation at 30,000 x g. The protein concentration was determined using the Bradford reagent (Bio-Rad, Mississauga, Ontario, Canada). The reductase activity of MnbBHis was measured by determining NAD(P)H-dependent reduction of 2,6-dichlorophenolindophenol (DCPIP) (17). As a control, we also determined DCPIP reduction by proteins extracted from M15(pQE30) prepared as described above.

PCRs with primers that amplify across the junctions of mnb and the flanking IS1071 elements.
The amplicons generated with the following primer pairs (the short names and the sizes of the amplicons are indicated in parentheses) were cloned (pCR2.1-mnbJ series of plasmids) (Table 1), and the ends were sequenced using M13F and M13R (Macrogen): Csj8-F1 plus IS1071-F1 (mnbJ1, 509 bp), Csj8-F1 plus IS1071-R1 (mnbJ2, 511 bp), Csj8-F1 plus ISCsp1-F1 (mnbJ3, 3963 bp), Csj8-F1 plus ISCsp1-R1 (mnbJ4, 1498 bp), Pmnb-R1 plus ISCsp1-R1 (mnbJ5, 442 bp), Pmnb-R1 plus IS1071-R1 (mnbJ6, 444 bp), and Pmnb-R1 plus IS1071-F1 (mnbJ8, 1427 bp). Except for mnbJ3, the inserts were completely sequenced.

The frequencies of alternative forms of mnb relative to the frequency of the flanking IS1071 elements were determined by PCR analysis of mid-log-phase cultures of strain JS46 growing in liquid MMA containing 3Nba. Cultures were started from single colonies on MMA agar containing 3Nba, and three independent cultures were analyzed. PCRs were performed as follows. Six to eight independent PCR tubes were prepared with the following sets of primer pairs: Pmnb-R1 plus IS1071-F1 and Pmnb-R1 plus IS1071-R1, Csj8-F1 plus IS1071-F1 and Csj8-F1 plus IS1071-R1, and Pmnb-R1 plus ISCsp1-R1 and Csj8-F1 plus ISCsp1-R1. Every six cycles, tubes were removed from the thermocycler. Aliquots of each reaction mixture were then electrophoretically resolved in 2% (wt/vol) agarose, the gels were stained with ethidium bromide, and subsaturation digital images of the gels were captured following UV transillumination with an AlphaImager 2200 (Alpha Innotech, San Leandro, CA). The yields of amplicons in each lane were determined by line densitometry analysis of images using software provided with the gel documentation system. The peak area versus the cycle number was plotted with Microsoft Excel, and x axis intercepts for PCRs with each primer pair were determined by inserting lines of best fit and extrapolating to zero. The relative proportion of alternative orientations was then estimated by assuming that each one-cycle difference between x axis intercepts indicated that one-half as many template molecules were initially present (for example, a one-cycle difference implied that there were one-half as many templates, a two-cycle difference implied that there were one-quarter as many templates, etc.).

To validate our approach, the six amplicons generated by the primer pairs described above were gel purified and quantified by UV spectroscopy, and known amounts of each were PCR amplified and analyzed as described above. With the similar-size amplicons generated by each set of primer pairs, the x axis intercepts were the same following analyses of equivalent concentrations of the templates. Furthermore, the amplification kinetics were virtually identical. This indicated that the products generated by each set of primer pairs amplified with the same efficiency and that in experiments with whole cells (see Results), variations in x axis intercepts were due to differences in the initial proportions of the templates.

Nucleotide sequence accession numbers.
The nucleotide sequences reported in this paper have been deposited in the GenBank database under accession number AY639948 for ISCsp1 and accession number AY639949 for the mnb locus.


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RESULTS
 
Cloning the mnb locus.
A spontaneous mutant of Comamonas sp. strain JS46 that could not grow on 3Nba was isolated and designated JS46D. Strain JS46D grew normally in minimal media containing Pca, indicating that the upper pathway for 3Nba degradation was defective. Strain JS46D was originally identified as a poorly growing colony on 3Nba agar that was likely growing on the small amounts of contaminating carbon present in the agar. Southern blots of total DNA from strains JS46 and JS46D digested with EcoRV (Fig. 2A) or BamHI (data not shown) and analyzed with an IS1071-specific probe indicated that at least seven copies of IS1071 were present in JS46, while JS46D harbored one less copy of IS1071 (Fig. 2A). No large plasmids were detected in strain JS46, although a small ~2.5-kb cryptic plasmid was identified (Fig. 2B). The same plasmid was also present in strain JS46D (data not shown). The results of a study in which this plasmid was described are presented elsewhere (31).


Figure 2
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  		FIG. 2. Southern blot analyses of Comamonas sp. strains JS46 and JS46D. (A) The numbers of copies of IS1071 in strains JS46 and JS46D were determined by hybridizing an IS1071-specific probe to total DNA digested with EcoRV. The probe-positive band that is not present in strain JS46D is indicated by a solid arrowhead. (B) The relative arrangement of mnb and the flanking IS1071 elements in strain JS46 was determined by using blots of total DNA digested with BamHI, BspDI, or EcoRV. Blots of the digests were hybridized with probes targeting PmnbA, ISCsp1, and csj8'. Areas to which probes hybridized and the positions of restriction enzyme sites are shown in Fig. 1A, and a table summarizing the predicted sizes of probe-positive fragments for the structure shown in Fig. 1A is shown below the blots. For the sake of brevity, predicted sizes for alternative arrangements are not shown. Note that no ISCsp1-positive fragment was detected in EcoRV-digested DNA, indicating that the next upstream EcoRV site was >23 kb away. With BspDI-digested DNA probed for csj8', fragments that were ~4.4 and ~5 kb long were detected following overexposure of the blot (indicated by solid arrowheads). The 4.4-kb fragment represents the region spanning the BspDI site in csj3 to the site at the end of csj7, while the ~5-kb fragment represents a partially digested fragment resulting from the 4.4-kb fragment attached to the 625-bp fragment spanning the csj8'-IS1071 junction. Strain JS46 harbors a small plasmid designated pJS46. The positions of the relaxed circular (RC), linear (L), and supercoiled (SC) forms of pJS46 are indicated by solid arrowheads on the agarose gel. Blots in panel B were not analyzed with the IS1071-specific probe because the hybridization patterns were too complex to be informative with respect to mapping the physical structure of mnb and the flanking IS1071 elements.

From these preliminary data, we concluded that the mnb locus is probably on the chromosome of strain JS46 or on a megaplasmid that was not detected. We hypothesized that mnb is flanked by IS1071 elements and that mnb (and the 3Nba degradation phenotype) was lost because of homologous recombination between the IS1071 elements, a phenomenon that we have observed previously with the cba genes (21, 38). We transferred the ability to grow on 3Nba from strain JS46 to C. testosteroni strain BR6025 using pBRC45 (see Materials and Methods), and pBRRD1 (the large plasmid isolated from strain BR6025) harbored two novel NheI fragments that were ~4.4 and ~9.3 kb long which were not present in NheI-digested pBRC45. NheI cut within the inverted repeats (IRs) flanking IS1071 elements (Fig. 1A) and should therefore have released at least two bands regardless of the genetic context, a 3.1-kb fragment representing IS1071 and any novel DNA fragments flanked by IS1071 elements. The 9.3-kb fragment was within the range of sizes of other catabolic loci flanked by IS1071 elements (25); therefore, this fragment was cloned and sequenced. In addition, Southern blot analyses of NheI-digested pBRRD1 with the IS1071-specific probe revealed that a 3.1-kb IS1071-positive fragment was still present in pBRRD1 and that the novel 4.4-kb fragment was positive for IS1071 (data not shown). Therefore, the 4.4-kb fragment was also cloned and sequenced.

mnb locus.
The 9.3-kb NheI fragment harbored eight full-length ORFs (designated mnbA, csj1, mnbB, and csj3 through csj7), two truncated ORFs (designated csj2' and csj8'), and a potential stem-loop structure between csj6 and csj7 (possibly a transcriptional terminator) (Fig. 1A). Table 3 shows the lengths and molecular weights of putative proteins encoded by the ORFs, as well as the locations of various domains identified within the putative proteins. An excellent candidate for the 3Nba oxygenase was MnbA, a member of the phthalate family of aromatic oxygenases (12). The most similar functionally characterized homologue of this protein is PobA (39% identity) from Pseudomonas pseudoalcaligenes strain POB310 (7). Similarly, an excellent candidate for the MnbA oxidoreductase was MnbB, a member of class IA of the aromatic oxidoreductases (4). The most similar functionally characterized homologue is VanB (53% identity) from Acinetobacter sp. strain ADP1 (33).


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  		TABLE 3. Comparison of putative proteins encoded by mnb to entries in the Pfam and COG databases

Confirmation that mnbA codes for the 3Nba oxygenase and mnbB codes for an oxidoreductase.
Three preliminary experiments strongly suggested that the locus coding for the 3Nba dioxygenase had been cloned. (i) Southern blots of NheI-digested DNA with probes targeting PmnbA and csj8' showed that mnb is present in strain JS46 but is missing in strain JS46D (data not shown). This indicated that loss of the 3Nba degradation phenotype was associated with deletion of the mnb locus. (ii) Colorimetric assays with E. coli containing pUC-mnb#6 and pUC-mnb#7 (which harbor mnb in different orientations relative to the lac promoter [Plac]), as well as pUC-mnb3.7 (which harbors the region spanning PmnbA to the end of csj2') (Table 1), resulted in formation of a dark purple color. No color was produced in the control. This indicated that the inserts coded for enzymes that convert 3Nba to a vicinal diol. (iii) Following conjugation of pUTCm-mnb3.7 into JS46D, transconjugants designated JS46D::mnb3.7 were able to grow on 3Nba, while none of the Cmr strains recovered in control matings grew when they were patched onto 3Nba. This indicated that the region spanning PmnbA to the end of csj2' could complement the missing 3Nba degradation phenotype in JS46D.

To confirm that MnbA is involved in conversion of 3Nba to Pca, assays were conducted with resting cultures of E. coli M15 containing pQE30-mnbA1B (which carries a region spanning PmnbA to the end of mnbB) and pQE30-mnb{Delta}ARB (a derivative in which mnbA is knocked out). In addition, to determine if 3Nba metabolism is affected when mnbB is knocked out or removed, assays were conducted with cells containing pQE30-mnbA1{Delta}B and pQE30-mnbA{Delta}[1B] (Table 1). The medium turned purple with cells containing pQE30-mnbA1B, pQE30-mnbA1{Delta}B, and pQE30-mnbA{Delta}[1B] (indicative of Pca production). With cells containing pQE30-mnb{Delta}ARB, the medium remained colorless. The presence of Pca in the medium from cells containing pQE30-mnbA1B, pQE30-mnbA1{Delta}B, and pQE30-mnbA{Delta}[1B] was confirmed by high-performance liquid chromatography. No Pca was detected in the medium from cells containing pQE30-mnb{Delta}A1B. This indicated that mnbA codes for the 3Nba oxygenase. Induced cultures of cells containing pQE30-mnbA1B, pQE30-mnbA1{Delta}B, and pQE30-mnbA{Delta}[1B] degraded ~50% of the 3Nba. With uninduced cultures, ~25% of the 3Nba was degraded, possibly because of a low level of IPTG-independent expression driven from PmnbA.

As reported below, mnbB codes for a functional oxidoreductase, and we expected little or no conversion of 3Nba to Pca by cells carrying pQE30-mnbA1{Delta}B and pQE30-mnbA{Delta}[1B]. However, because the levels of 3Nba degradation were similar to those in cells with pQE30-mnbA1B, we concluded that oxidoreductases other than MnbB are capable of transferring reducing equivalents to MnbA, at least in E. coli M15 (the host strain used in these assays). Based on BLAST analysis, E. coli K-12 (the parent of M15) possesses five putative oxidoreductases with significant homology to MnbB (data not shown). One or more of these chromosomally encoded proteins may have complemented for MnbB in these assays. To our knowledge, this would be the first report of an aromatic oxygenase showing activity independent of its (putative) cognate oxidoreductase.

To confirm that mnbB codes for a functional oxidoreductase, affinity-purified MnbBHis (Fig. 3) was subjected to assays with the artificial electron acceptor DCPIP, and this substrate was reduced in an NAD(P)H-dependent manner (specific activity, ~70 mkat/kg protein). MnbBHis could be stored at –20°C in 50% glycerol for up to 2 months with ~10% loss of activity. No activity was observed with affinity-purified extracts from the control, indicating that the DCPIP reduction was because of MnbBHis and not because of contaminating proteins that had copurified with it (Fig. 3).


Figure 3
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  		FIG. 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of affinity-purified MnbBHis (predicted molecular mass, 35.3 kDa). Ten micrograms of protein was loaded. The positions of molecular mass standards (in kDa) are indicated on the right.

Identification of IS1071::ISCsp1.
The 4.4-kb NheI fragment from pBRRD1 that was positive for IS1071 (see above) harbored IS1071 interrupted at nucleotide 684 by a novel insertion element designated ISCsp1. The interrupted IS1071 was designated IS1071::ISCsp1. The putative transposase for ISCsp1 is transcribed in the same direction as the interrupted tnpAIS1071 (Fig. 1A) and belongs to the IS256 family of prokaryotic insertion sequences (18), a subgroup of the mutator family of transposases (Pfam00872). A 9-bp direct repeat flanks ISCsp1, and imperfect IRs (length, 28 bp; six mismatches) are adjacent to the direct repeats (sequences shown in Fig. 1B). Southern blots of NheI-digested DNA with a probe specific for ISCsp1 and PCR analyses with primers ISCsp-F1 and ISCsp-R1 showed that ISCsp1 is present in strain JS46 but is not present in strain JS46D (not shown). This indicated that IS1071::ISCsp1 was lost during homologous recombination between the IS1071 elements that flank mnb and that only one copy of IS1071::ISCsp1 is present in strain JS46.

Physical organization of mnb and flanking IS1071 elements.
All data indicated that in strain JS46, mnb is flanked by IS1071 elements, one of which is IS1071::ISCsp1. However, the arrangement and orientation of the IS1071 elements relative to mnb were not known. Southern blot analysis with probes targeting PmnbA, ISCsp1, and csj8' (Fig. 2B) indicated that IS1071::ISCsp1 is adjacent to mnbA, that mnbA is transcribed in the direction opposite the direction of transcription of the flanking tnpA genes, and that IS1071 is adjacent to csj8' (Fig. 1A).

Southern blot results were verified by PCR. The positions, orientations, and shorthand designations of the primers that were used are shown in Fig. 4A, and the predicted PCR results and shorthand designations of the eight possible structures of mnb and the flanking IS1071 elements are shown in Fig. 4B. Primer pairs 5 plus 2, 5 plus 4, and 6 plus 1 generated products that were the sizes (Fig. 4C) and had the sequences predicted for structure ABC, indicating that the structure suggested by Southern blot analysis was present.


Figure 4
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  		FIG. 4. PCR analyses with primer pairs that amplify across the junctions of mnb and the flanking IS1071 elements. (A) Diagram showing the arrangement of IS1071::ISCsp1, mnb, and IS1071. The shorthand designations of the three sections are A, B, and C, respectively, and the arrangement shown is designated ABC. The positions and directions of PCR primers used in these analyses are indicated, and these primers are referred to by their shorthand designations, 1 through 6, in panels B through D. The positions of the left and right inverted repeats that flank IS1071 elements are also indicated. (B) Table summarizing the predicted PCR results for the structure designated ABC and the seven other possible structures. Sections that are inverted relative to ABC are designated A', B', and C'. The size of the predicted amplicon is indicated for each primer pair. Note that the 3,892-bp product predicted for primers 5 and 3 was not observed (shaded region), indicating that structures A'BC' and A'BC do not exist or are selected against. (C) Agarose gel electrophoresis analysis of PCRs performed with different primer pairs. Amplicons indicative of additional structures are indicated by asterisks. (D) Summary of the procedure used to estimate the relative frequencies of alternative orientations, using PCRs performed with primer pairs 6 plus 1 and 6 plus 4 as examples. The top panel shows the results of the PCRs performed with the primer pairs indicated. The cycle at which the PCR was terminated is shown above each lane. The bottom panel shows plots relating the amplicon yield (area under the peak; determined by densitometric analysis of the agarose gel) to the cycle at which the PCR was terminated. In this example, the x axis intercepts of the two plots (dotted lines) differ by about nine cycles, indicating that the alternative form accounts for 1/29 or ~0.2% of the templates. (E) Sequence evidence supporting the hypothesis that inversions of A, B, and C likely occur because of homologous recombination between IR-L and IR-R. The IRs flanking IS1071 elements have distinct sequences (eight mismatches; shown in IR-R), and the comparable regions in mnbJ2 and mnbJ4 (amplicons generated with primer pairs 6 plus 4 and 6 plus 2, respectively) are hybrids of IR-L (indicated by boldface type) and IR-R (indicated by italics). The position of the NheI site is shown for reference.

Unexpectedly, PCRs performed with primer pairs 5 plus 1, 6 plus 2, 6 plus 3, and 6 plus 4 generated products that were the sizes (Fig. 4C) and had the sequences predicted for additional structures, and we concluded that there were five potential alternatives (ABC', AB'C, AB'C', A'B'C, and A'B'C') (Fig. 4B). Despite multiple attempts with four independent 3Nba-grown cultures of strain JS46, a 3,892-bp product was not generated with primers 5 and 3. However, a faint ~900-bp fragment was evident in some PCRs (Fig. 4C). Primers 5 and 3 worked in combination with other primers (Fig. 4C), indicating that each primer was functional. Furthermore, we were routinely able to amplify fragments as large as 6 kb from strain JS46 (data not shown), suggesting that the size of the predicted product obtained with primers 5 and 3 was not a limitation. We therefore concluded that structures A'BC and A'BC' do not exist. Alternatively, these two structures do exist, but the faint ~900-bp band reduced the yields of the predicted 3,892-bp product. No attempt was made to characterize the ~900-bp band.

Frequency of alternative structures and evidence that they arise because of homologous recombination between the IRs that flank IS1071.
In the PCRs described above, the yields of the amplicons generated with primers 5 plus 1, 6 plus 4, and 6 plus 2 were consistently lower than yields of amplicons generated with primers 5 plus 4, 6 plus 1, and 5 plus 2, respectively (Fig. 4C). This suggested that the alternative structures are less abundant. To estimate the relative frequencies of these structures, 3Nba-grown cultures of strain JS46 were analyzed using an approach similar to quantitative real-time PCR (see Materials and Methods and Fig. 4D). Relative to PmnbA (i.e., PCRs performed with primers 5 and 4 versus PCRs performed with primers 5 and 1) and relative to IS1071::ISCsp1 (i.e., PCRs performed with primers 5 and 2 versus PCRs performed with primers 6 and 2), alternative forms occurred in ~0.007% to 0.07% of the population. Relative to csj8' (i.e., PCRs performed with primers 6 and 1 versus PCRs performed with primers 6 and 4), alternative forms occurred in ~0.009% to 0.2% of the population.

We hypothesized that the alternative structures were the result of inversions of IS1071::ISCsp1, mnb, and/or IS1071 brought about by homologous recombination between the 110-bp IRs that flank IS1071 elements. The left IR (IR-L) and the right IR (IR-R) could be distinguished from each other because of sequence differences (Fig. 4E), and based on the orientation of tnpAIS1071 in amplicons mnbJ2 and mnbJ4 (see Materials and Methods), the inserts should have harbored IR-L. However, the amplicons harbored distinct sequences that were hybrids of IR-L and IR-R (sequences shown in Fig. 4E), which provided evidence that homologous recombination had occurred between the diagnostic nucleotides.


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DISCUSSION
 
mnb locus.
A functional mnbA is required for conversion of 3Nba to Pca, and assays with MnbBHis confirmed that MnbB is an NAD(P)H-requiring oxidoreductase. Furthermore, a construct harboring mnbA and mnbB complemented the missing 3Nba degradation phenotype in strain JS46D (see above). Based on these results, we concluded that MnbA is the oxygenase component of the 3Nba dioxygenase and propose that in strain JS46, MnbB is the oxidoreductase that transfers reducing equivalents to MnbA. We constructed pQE30-based vectors that attached an N-terminal His tag to mnbA translated from the first and second putative ATG codons, and even though proteins were overproduced, no soluble His-tagged proteins were present in extracts (data not shown). We were therefore unable to conduct in vitro assays that would have confirmed that MnbB is the oxidoreductase for MnbA.

The mnb locus appears to be part of an IS1071-based composite transposon that we tentatively designated "TnMnb1." Furthermore, it appears that csj8' and possibly csj7 are the remnants of another operon that was recruited during evolution of "TnMnb1." This phenomenon was proposed previously for Tn5271, the prototypic IS1071-based composite transposon, which harbors a truncated ORF at its extreme end that codes for the C-terminal region of an aryl-coenzyme A ligase-like protein (9). Interestingly, mnbA and mnbB are transcribed in the direction opposite the direction of transcription of the flanking tnpAIS1071 sequence. This contrasts with most other catabolic genes flanked by IS1071 elements, which are transcribed in the same direction as tnpAIS1071 (25).

The mnb locus does not harbor a gene coding for a cis-diol dehydrogenase (Pfam01408). Members of this group of enzymes rearomatize the dihydroxylated cyclic intermediates generated during dioxygenase-mediated modification of the aromatic nucleus (22). Because the NO2 moiety is an excellent leaving group, nonenzymatic rearomatization was originally proposed for the 3Nba degradation pathway (20), and the results presented here support this proposal.

ISCsp1.
In the course of cloning mnb, we also discovered IS1071::ISCsp1 (see Results). The direct repeats flanking ISCsp1 (Fig. 1B) indicate that staggered nicks were generated during insertion of ISCsp1 into IS1071. The imperfect IRs adjacent to the direct repeats (Fig. 1B) may be the sequences recognized by the putative transposase of ISCsp1. The first reported example of an interrupted IS1071 is IS1071::IS1471, which flanks the plasmid-encoded 2,4-dichlorophenoxyacetic acid genes of Burkholderia cepacia strain 2a. In this case, IS1071 is interrupted at nucleotide 864 (29, 39).

Deletion and inversion of mnb in Comamonas sp. strain JS46.
Spontaneous deletion of mnb and loss of the 3Nba degradation phenotype in strain JS46 appear to be the result of homologous recombination between the directly repeated flanking IS1071 elements. In strain JS46D, ISCsp1 is missing (see Results), indicating that the recombination event occurred in the 2.5-kb region downstream of ISCsp1. In 14 of 15 other independently isolated deletion mutants of strain JS46, ISCsp1 is also missing (data not shown), suggesting that recombination preferentially occurs in this region, probably because it is 3.7 times longer than the alternative area where homologous recombination could occur (the 0.68-kb region upstream of ISCsp1) (Fig. 1A).

Inversion events that lead to alternative structures of "TnMnb1" appear to be the result of homologous recombination between the IRs that flank the IS1071 elements (see Results and Fig. 4E). Even though there are five potential alternative structures (see Results), two sequential inversion events (ABC to AB'C to A'B'C) are sufficient to account for all the amplification products observed. Because there is no evidence for structures A'BC and A'BC', it appears that inversions leading to these structures are either restricted or selected against. It is noteworthy that in three of the five possible structures (ABC', A'B'C, and AB'C'), mnb is flanked by IS1071 elements that are inverted relative to each other. Therefore, inversion of mnb in these structures not only could be the result of homologous recombination between the IRs that flank mnb but also could be the result of homologous recombination between the flanking IS1071 elements. The overall abundance of alternative forms in any population of JS46 is low (≤0.2%), explaining why Southern blots indicated that there was only one structure.

This is the first report of inversions by IS1071 elements and loci flanked by IS1071, and it will be interesting to determine whether inversions like the ones described here occur in other bacteria harboring IS1071 elements and whether this phenomenon affects the evolution of IS1071-based catabolic transposons. In addition, it will be interesting to determine whether the proposed inversion events are, for example, mediated by the general recombinational machinery of strain JS46, whether topoisomerase is involved, or whether this phenomenon occurs because of a RecA-independent mechanism like reciprocal strand switching during DNA replication (5). Furthermore, it will be interesting to determine whether TnpAIS1071 is involved, for example, by binding to the two IRs and bringing them closer together, which could conceivably promote inversion by the mechanisms described above.


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ACKNOWLEDGMENTS
 
We acknowledge J. C. Spain for providing Comamonas sp. strain JS46, Suzanne Paterson for helpful discussions, and William Willmore for assistance with high-performance liquid chromatography analyses.

M.A.P. was the recipient of a Natural Sciences and Engineering Research Council (NSERC) of Canada postdoctoral fellowship. N.T.M. was the recipient of an NSERC summer scholarship. This work was supported by NSERC grants to R.C.W. and I.B.L.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6. Phone: (613) 520-2600, ext. 3893. Fax: (613) 520-3539. E-mail: miguel_providenti{at}carleton.ca. Back

{dagger} Deceased. Back


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Applied and Environmental Microbiology, April 2006, p. 2651-2660, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2651-2660.2006
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