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Applied and Environmental Microbiology, October 2002, p. 4965-4970, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.4965-4970.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Accumulation of 2-Aminophenoxazin-3-one-7-Carboxylate during Growth of Pseudomonas putida TW3 on 4-Nitro-Substituted Substrates Requires 4-Hydroxylaminobenzoate Lyase (PnbB)

Michelle A. Hughes,1 Michael J. Baggs,2 Juma'a al-Dulayymi,2 Mark S. Baird,2 and Peter A. Williams1*

School of Biological Sciences,1 Department of Chemistry, University of Wales Bangor, Bangor, Gwynedd LL57 2UW, Wales, United Kingdom2

Received 22 January 2002/ Accepted 1 July 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During growth of Pseudomonas putida strain TW3 on 4-nitrotoluene (4NT) or its metabolite 4-nitrobenzoate (4NB), the culture medium gradually becomes yellow-orange with a {lambda}max of 446 nm. The compound producing this color has been isolated and identified as a new phenoxazinone, 2-aminophenoxazin-3-one-7-carboxylate (APOC). This compound is formed more rapidly and in greater quantity when 4-amino-3-hydroxybenzoate (4A3HB) is added to growing cultures of strain TW3 and is also formed nonbiologically when 4A3HB is shaken in mineral salts medium but not in distilled water. It is postulated that APOC is formed by the oxidative dimerization of 4A3HB, although 4A3HB has not been reported to be a metabolite of 4NT or a product of 4NB catabolism by strain TW3. Using the cloned pnb structural genes from TW3, we demonstrated that the formation of the phenoxazinone requires 4-hydroxylaminobenzoate lyase (PnbB) activity, which converts 4-hydroxylaminobenzoate (4HAB) to 3,4-dihydroxybenzoate (protocatechuate) and that 4-nitrobenzoate reductase (PnbA) activity, which causes the accumulation of 4HAB from 4NB, does not on its own result in the formation of APOC. This rules out the possibility that 4A3HB is formed abiotically from 4HAB by a Bamberger rearrangement but suggests that PnbB first acts to effect a Bamberger-like rearrangement of 4HAB to 4A3HB followed by the replacement of the 4-amino group by a hydroxyl to form protocatechuate and that the phenoxazinone is produced as a result of some misrouting of the intermediate 4A3HB from its active site.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
During the metabolism of 4-nitrotoluene (4NT) as the sole carbon and nitrogen source by Pseudomonas putida strain TW3, the nitro group is retained during the initial sequential oxidations of the methyl group to form 4-nitrobenzoate (4NB) (9, 10, 15). This is then further metabolized to 3,4-dihydroxybenzoate (protocatechuate [PCA]) with release of the nitro group as NH4+ (15) through the sequential action of 4NB reductase (PnbA) and 4-hydroxylaminobenzoate lyase (PnbB), the genes for which have been cloned and characterized (Fig. 1) (8). During growth of strain TW3 on either 4NT or 4NB, the growth medium progressively becomes yellow to orange as the degradation proceeds. In this paper we demonstrate that this is due to accumulation of the novel phenoxazinone 2-aminophenoxazin-3-one-7-carboxylate (APOC). The results suggest that APOC is formed as a by-product during the conversion of 4-hydroxylaminobenzoate (4HAB) to PCA by PnbB.



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  		FIG. 1. Metabolic pathway and genes for conversion of 4NB to PCA in P. putida TW3 (8). (A) Metabolic pathway involving 4-nitrobenzoate reductase (PnbA) and 4-hydroxylaminobenzoate lyase (PnbB). The brackets around 4-nitrosobenzoate imply that it is an intermediate, but there is no experimental evidence to support its presence. (B) Arrangement of pnb genes in TW3. pnbR is a regulator gene, and orf1 and orf2 have no known function. The inserts in plasmids pRK3.14 and pRK3.15 are shown at the bottom of the diagram.

 

   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and plasmids.
The bacterial strains and plasmids used in this study are listed in Table 1 and shown in Fig. 1B.


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  		TABLE 1. Bacterial strains and plasmids

 
Chemicals and growth media.
Aromatic and aliphatic substrates were obtained from Aldrich Chemical Co. P. putida TW3 was grown on minimal salts medium supplemented with either solid 4NT (0.5 g/liter), the sodium salt of 4-nitrobenzoic acid (5 mM), or sodium succinate (10 mM). Escherichia coli strains were routinely grown on Luria-Bertani (LB) medium. Where appropriate, ampicillin was added at 100 µg/ml, streptomycin was added at 100 µg/ml, or tetracycline was added at 20 µg/ml.

Triparental matings for transfer of DNA into P. putida PaW340.
The donor strain, the recipient strain, and E. coli HB101 carrying pRK2013 as a helper plasmid were grown in LB medium until they reached an A600 of 0.6. Five hundred microliters of each culture was mixed and centrifuged, and the pellets were washed in minimal medium. The pellets were finally resuspended in 50 µl of minimal medium and dispensed onto a sterile nylon membrane (Bio-Rad) laid on the surface of an LB plate. Following incubation overnight at 30°C, the cells were washed off the filter into 2 ml of minimal medium, and appropriate dilutions were spread onto selective medium. Donor-only and recipient-only controls were treated in the same way.

Spectrophotometric analysis of the orange compound.
To analyze bacteria containing recombinant plasmids for the accumulation of the orange compound, they were grown in minimal salts medium containing 0.01% yeast extract, 0.01% Casamino Acids, and 10 mM glucose supplemented with 5 mM 4NB. Cultures of TW3 were grown in minimal medium containing 5 mM 4NB. Aliquots of the cultures were centrifuged, the supernatants were diluted 1:5 with minimal medium, and the absorption spectra between 400 and 600 nm were measured.

Isolation of the orange compound.
The yellow-orange supernatant with the characteristic absorption spectrum ({lambda}max, 430 and 446 nm) (Fig. 2A) was extracted from cultures (2 liters) with ethyl acetate (four extractions with 400 ml each time), which removed none of the colored material. Upon acidification of the material to pH 1 with concentrated HCl, the color changed to bronze, the characteristic peak became broader, and the {lambda}max shifted to a 5- to 10-nm-higher wavelength. This material was extracted again with ethyl acetate (four extractions with 400 ml each time), which produced a bright-orange organic layer with an absorption peak slightly displaced to a lower wavelength ({lambda}max, 416 and 436 nm) and which left a colorless residual aqueous layer with no major absorption peak. The solvent was evaporated from the organic layer at 14 mm Hg to give a brownish-black solid. A 1H nuclear magnetic resonance (NMR) spectrum of this solid in d6-dimethyl sulfoxide (d6-DMSO) showed one major set of signals in the aromatic region but more complex signals between {delta}-1 and {delta}-3, indicative of impurities. The solid was washed first with 10 ml of ethyl acetate and then with 5 ml of methanol. Comparisons of the visible spectra of the two organic washings at standard dilution showed that neither contained more than 5% of the compound, with a {lambda}max of 436 nm. The remaining solid (~20 mg) was thoroughly freed of residual solvent under a stream of N2 and then dissolved in d6-DMSO (1 ml). Dilution of the solution in methanol showed that it contained the bulk (~80%) of the product, with a {lambda}max of 436 nm and an overall absorbance matching that of the original ethyl acetate extracts. The 1H and 13C NMR spectra of the compound indicated that it was pure by both criteria and that the washings had removed the signals between {delta}-1 and {delta}-3: the 13C spectrum showed only the 13 carbons, and the 1H spectrum contained only signals assigned to the molecule and these were appropriately correlated to the 13C spectrum in a 1H-13C two-dimensional (2D) experiment. Column chromatography on silica, with methanol as eluting solvent, or ion-exchange chromatography on Dowex 50W-X8(H) did not lead to an increase in purity. A similar extraction and washing procedure performed on the culture supernatants of a 4NT-grown culture or on solutions of 4-amino-3-hydroxybenzoate (4A3HB) which had been shaken at 30°C gave products with the same 1H NMR spectrum as that of a 4NB-grown culture.



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  		FIG. 2. (A) Absorption spectrum of supernatants from P. putida TW3 grown on 4NT or 4NB minimal medium. (B) Measurements of turbidity (•, OD600), indicating the accumulation of yellow color ({blacksquare}, A446), and the calculated concentration ({blacksquare}, concentration) of APOC causing the yellow color during growth of TW3 on 4NB minimal medium.

 
Analytical methods.
A Bruker AC250 NMR spectrometer was used at 250 MHz for measuring proton NMR and at 62.9 MHz for 13C NMR, and the tests were broad band or gated decoupled. Spectral analyses were carried out in d6-DMSO; proton spectral analyses were run with a 2H lock, and carbon spectral analyses were standardized against the solvent shift. Mass spectra were determined at the Swansea Mass Spectrum Service (University of Swansea, Swansea, United Kingdom).

Determination of the molar extinction coefficient of APOC.
Solutions containing from 0.1 to 0.5 mM commercial 4A3HB in minimal salts medium were shaken at 30°C for 280 h until the A446 ceased to increase. The molar extinction coefficient was calculated as 22,000 M-1, assuming complete conversion of 1 mol of APOC from 2 mol of 4A3HB.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Growth of strain TW3.
During growth of P. putida TW3 on 4NT or 4NB, but not on any other substrate, cultures first became yellow and then became progressively more orange. The spectra of the supernatants throughout had a broad double peak consisting of two adjacent maxima at 430 and 446 nm (Fig. 2A). Growth of TW3 on 4NB and accumulation of the compound were monitored over 168 h. The A446 of the supernatant was measured, and growth was assessed by resuspending the centrifuged cell pellets in minimal medium and determining the optical density at 600 nm. Whereas growth reached a maximum at approximately 24 h, the orange compound continued to accumulate for up to 7 days (Fig. 2B), which suggests that it is not a metabolic intermediate but may be a slowly produced dead-end product of the metabolism. If the cells were completely removed by filtration after growth ceased, the culture supernatant continued to become progressively more yellow (data not shown).

Identification of the orange by-product.
In addition to broad signals at 7.1 and 4.1 (probably arising from HO and H2N subgroups), the 1H NMR spectrum of the purified compound in d6-DMSO showed just seven resonances. The spectrum showed {delta}H signals at 7.9 (1H, broad singlet), 7.87 (1H, d, J = 8.8 Hz), 7.75 (1H, d, J = 8.8 Hz), 6.4 (1H, s), and 6.35 (1H, s). The signals at 7.87 and 7.75 were tented towards each other, as is typical of an AB pattern. The 13C NMR spectrum showed eight quaternary and five tertiary carbons which ranged from a {delta}c of 98 to 180 and showed two carbonyl carbons at 180 and 166 (Table 2): the proton and carbon signals were correlated by 1H-13C 2D spectroscopy (Table 2). This is extremely close to the spectrum of 2-aminophenoxazin-3-one (Table 2) (7), apart from the additional carbon at a {delta}c of 166 and the replacement of a tertiary carbon by a quaternary carbon at a {delta}c of 129/130. The mass spectrum of the compound was rather difficult to obtain and suggested that some impurities were present. The major molecular ion that appeared in the electron impact spectrum was measured as m/z 256.0484 (C13H8N2O4 requires 256.0484). The spectra and mass are consistent with the structure of the compound APOC (Fig. 3, compound II). The same compound was isolated from both 4NB- and 4NT-grown cells and from the abiotic dimerization of 4A3HB (see below).


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  		TABLE 2. Comparison of 13C NMR signals

 


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  		FIG. 3. Proposed reactions leading to the formation of APOC (compound II) during the oxidative dimerization of 4A3HB. For comparison, the structures of 2-aminophenoxazin-3-one (compound III) formed from dimerization of 2-aminophenol (7, 20) and of 2-amino-4a,7-dimethyl-4,4a-dihydrophenoxazin-3-one (compound IV) formed by the condensation of 4-amino-3-hydroxytoluene (19) are shown.

 
The role of 4A3HB.
Since aminophenoxazinones can form spontaneously by dimerization of aminophenols (17), and since hydroxylaminobenzenes can undergo Bamberger rearrangements to form aminophenols (1, 18), the most likely precursor of APOC was 4A3HB, which in theory could be formed abiotically from the metabolite 4HAB. Cells of TW3 were induced by overnight growth on 4NB and centrifuged, and the cell pellets were resuspended in fresh medium containing 5 mM 4NB with 4A3HB added at either 0.1 or 0.5 mM. Cells grown in 5 mM 4NB with no added 4A3HB were included for comparison and gave an A446 of 0.57 after 96 h of incubation. With the added 0.1 mM 4A3HB, the same characteristic spectrum was produced but the A446 was increased to 0.7, and with 0.5 mM 4A3HB, it was increased to approximately 1.6 (Fig. 4A). Incubation of induced TW3 cells in 0.5 mM 4A3HB alone produced a higher A446 than when cells were grown in 5 mM 4NB (Fig. 4). These results suggest that 4A3HB is involved in the formation of APOC during the growth of TW3 on 4NB, although previous work has shown that it is not an intermediate of 4NB degradation (8, 15).



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  		FIG. 4. Biological formation of APOC from 4A3HB by 4NB-induced cells of P. putida TW3. , cells grown on 5 mM 4NB; ----, cells grown on 5 mM 4NB plus 0.1 mM 4A3HB; x, cells grown on 5 mM 4NB plus 0.5 mM 4A3HB; {square}, cells incubated with 0.5 mM 4A3HB alone.

 
Two flasks of minimal medium containing 0.5 mM 4A3HB were incubated at 30°C, and induced TW3 cells were added to one of these flasks. After 24 h, the absorption spectra of the supernatants showed that both flasks contained APOC, but the A446 produced in the presence of TW3 was about 40% higher than that produced without TW3 (data not shown). The abiotic formation of APOC from 4A3HB is a slow reaction that takes place in minimal medium at 30°C over at least 5 days (Fig. 5), with an estimated half-life of 40 to 50 h. It does not occur in distilled water (data not shown) and hence must be catalyzed by some component of the minimal salts medium.



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  		FIG. 5. Abiotic formation of APOC from 4A3HB by shaking 0.5 mM 4A3HB in minimal medium for 0 h (), 24 h (----), 48 h (x), and 72 h (•).

 
Investigation into the role of the pnb genes.
When plasmids carrying the complete cloned pnb genes were transferred into P. putida PaW340 (which cannot metabolize 4NB but can metabolize PCA), not only was the ability to grow on 4NB conferred (8), but also APOC was formed, as determined from the characteristic absorption spectra of culture supernatants. To investigate a possible role of PnbA and PnbB in APOC formation, pRK3.14 (which carries both pnbA and pnbB) and pRK3.15 (which lacks pnbB) (Fig. 1 and Table 1) were mobilized into strain PaW340 by triparental mating. Transconjugants were incubated in minimal medium containing succinate and 4NB, and the absorption spectra of the culture supernatants were measured. PaW340(pRK3.15) supernatants were yellow, but the spectra were different from that of APOC and were identical to that of 4HAB, with a {lambda}max of 262 nm (data not shown). Further incubation of the culture supernatants for periods of up to 72 h caused no detectable change in their spectra from one resembling 4HAB to one characteristic of APOC. By contrast, the supernatants of PaW340(pRK3.14) did acquire the characteristic spectrum of APOC (Fig. 6). This occurs not only in the P. putida host, but also in E. coli(pRK3.14), although in both cases the peaks are broader due to the simultaneous accumulation of PCA, which was detected by its lower absorption maxima, 254 and 290 nm, and by its reaction with p-toluidine (8). E. coli, unlike P. putida, cannot further metabolize PCA, which therefore accumulates. In E. coli(pRK3.15) containing pnbA but without pnbB, no characteristic APOC spectrum was observed (Fig. 6) and only the accumulation of 4HAB was observed (data not shown), as occurs with P. putida. This suggests that 4A3HB is not being formed from an abiotic rearrangement of the hydroxylamino compound but that it is being formed as a direct result of the activity of PnbB.



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  		FIG. 6. Absorption spectrum of medium after the growth of heterologous hosts carrying cloned pnb genes in the presence of 4NB. , P. putida PaW340(pRK3.14); ----, E. coli(pRK3.14); x, E. coli(pRK3.15).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Phenoxazinones have been described as products or by-products of bacterial metabolism. There is a specific synthase responsible for the synthesis of the phenoxazinone core of actinomycin D in Streptomyces (2). Phenoxazinones have been reported as by-products of the aerobic biotransformation of nitroaromatics and are the result of the abiotic dimerization of 2-aminophenols: compound III (Fig. 3) was formed from 2-aminophenol (20), and compound IV (Fig. 3) was formed from 4-amino-3-hydroxytoluene (19). Similar reactions have been well authenticated in nonbiological contexts (17). We believe that the 2-amino-4a,7-dimethyl-4,4a-dihydrophenoxazinone (Fig. 3, compound IV), which is formed by a Mycobacterium strain degrading 4NB by a pathway different from that in strain TW3 (19), is formally analogous to the phenoxazinone formed by strain TW3 from the condensation of 4A3HB (Fig. 3, compound I). However, the angular carboxylate group on carbon 4a is likely to be unstable, and a further chemical reaction involving decarboxylation and oxidation leads to the compound we have isolated from the cultures, namely, APOC (Fig. 3, compound II). Whereas with the Mycobacterium strain, the precursor of the phenoxazinone, 4-amino-3-hydroxytoluene, is part of the metabolic pathway, the likely precursor in strain TW3, 4A3HB, is not on the published pathway (Fig. 1). We have presented strong circumstantial evidence that 4A3HB is the precursor of APOC in TW3, since APOC is formed rapidly and in increased quantity when the bacterium grows on 4NB in the presence of 4A3HB. Additionally, the same compound is formed slowly and nonbiologically upon incubation of 4A3HB in mineral salts medium, but not in distilled water, when incubated under the same conditions used for the cultures (30°C with shaking). Our hypothesis that some component of the inorganic salts in the medium catalyzes this abiotic reaction is supported by the reported O2-dependent chemical conversion of 2-aminophenol to 2-aminophenoxazin-3-one (Fig. 3, compound III), which is catalyzed by cobalt(II) (17). The formation of APOC during metabolism of 4NT and 4NB is not specific to strain TW3, since we have observed the same spectrum produced during culture of 4NT-grown Pseudomonas strain 4NT, an independently isolated strain with the same pathway from 4NT to PCA as that in strain TW3 (5), and a yellow compound with a very similar absorption spectrum ({lambda}max, 425 and 466 nm) has been reported in cultures of other strains grown on 4NT (13) or 4NB (14).

The question we have tried to address is how 4A3HB is formed in strain TW3. The obvious answer appeared to be that 4HAB, which is a metabolite of the pathway (Fig. 1), accumulates in the medium and a proportion of it undergoes an abiotic Bamberger-type rearrangement (1, 18) to form 4A3HB, which then condenses to form APOC. If this were so, then a mutation inactivating pnbB, the gene for 4-hydroxylaminobenzoate lyase, should cause stoichiometric accumulation of 4HAB, thereby leading indirectly to the formation of APOC. We had already cloned the genes for 4NB catabolism from TW3 (Fig. 1) (8) and demonstrated that in a heterologous P. putida host (strain PaW340), they would support the complete catabolism of 4NB by providing a metabolic route to PCA which the host could utilize via its ß-ketoadipate pathway. It was therefore more practical to use the cloned genes in the heterologous host and simply delete pnbB rather than try to work with the native genes in the TW3 genome. Expecting that PaW340(pRK3.15), carrying pnbA alone (Fig. 1), would thus produce APOC when grown in the presence of 4NT or 4NB, we were surprised to find that although the medium turned yellow, the color was due to 4HAB and not APOC. Furthermore, prolonged shaking of the medium under growth conditions did not cause detectable phenoxazinone production. However, when the same experiment was repeated with the pnbA+ pnbB+ plasmid pRK3.14, there was production of the phenoxazinone during growth on 4NB; this occurred even during growth of E. coli(pRK3.14) in the presence of 4NB, although in this case, there was also considerable accumulation of PCA, which complicated the spectrum and the color of the medium. This demonstrated (i) that under the conditions of incubation, an abiotic Bamberger rearrangement of 4HAB does not take place and (ii) that PnbB is an essential factor in the production of the phenoxazinone. The most likely explanation of this is that PnbB is itself responsible for the conversion of its substrate 4HAB to 4A3HB by a mechanism similar or analogous to a Bamberger rearrangement. If some of the 4A3HB escapes from the enzyme into the medium, where we have shown that it can form APOC abiotically, this would account for the production of APOC. The final A446 of a culture grown on 5 mM 4NB (Fig. 2B) indicates that about 0.15 mM APOC is produced, accounting for a significant amount (~6%) of the substrate being misrouted in this way. The low rate of the abiotic conversion of 4A3HB to APOC (half-life, 40 to 50 h) also explains the continued production of APOC in wild-type cultures grown on 4NT or 4NB well after growth of the culture ceased (Fig. 2B) or when the cells were removed from the culture after growth has ceased if 4A3HB is released into solution during the growth phase and is then abiotically converted to APOC. An alternative explanation involving the oxidative condensation of two molecules of 4A3HB on the PnbB active site during the formation of PCA would fit the data less well, since APOC formation in this case would be growth dependent. In terms of the mechanism of action of PnbB, 4A3HB is a possible reaction intermediate between 4HAB and PCA, and the second part of the reaction would simply involve the replacement of the 4-amino group of 4A3HB with a hydroxyl (Fig. 7). There are precedents in nitroaromatic catabolism for the first part of this reaction, since Bamberger-like mutase reactions on hydroxylaminobenzenes have been described previously (3, 6, 14, 16, 19), although recent work has suggested that the mechanism of the conversion to aminophenols might be different from that of the abiotic Bamberger rearrangement (6). Recent results by J. C. Spain and his coworkers have made a similar suggestion, based on 18O labeling experiments, i.e., that PnbB initially causes the migration of the OH from the 4-hydroxylamino group to become the 3-hydroxyl group en route to PCA (L. J. Nadeau, Z. He, and J. C. Spain, Abstr. 102nd Gen. Meet. Am. Soc. Microbiol. 2002, abstr. O-31, p. 352, 2002). In their mechanism, however, the intermediate is not 4A3HB directly. These differences might be reconciled if there was a common reaction intermediate both in the PnbB-catalyzed reaction and in the abiotic formation of APOC from 4H3AB.



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  		FIG. 7. Proposed mechanism for the action of 4-hydroxylaminobenzoate lyase (PnbB) through 4A3HB as an intermediate leading to the accumulation of APOC in the medium.

 


   FOOTNOTES
 
* Corresponding author. Mailing address: School of Biological Sciences, University of Wales Bangor, Bangor, Gwynedd LL57 2UW, Wales, United Kingdom Phone: (44)-1248-382363. Fax: (44)-1248-370731. E-mail: P.A.Williams{at}bangor.ac.uk. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, October 2002, p. 4965-4970, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.4965-4970.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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