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The Pseudomonas sp. ADP plasmid pADP-1 encodes the activities involved in the hydrolytic degradation of the s-triazine herbicide atrazine. Here, we explore the presence of a specific transport system for the central intermediate of the atrazine utilization pathway, cyanuric acid, in Pseudomonas sp. ADP. Growth in fed-batch cultures containing limiting cyanuric acid concentrations is consistent with high-affinity transport of this substrate. Acquisition of the ability to grow at low cyanuric acid concentrations upon conjugal transfer of pADP1 to the nondegrading host Pseudomonas putida KT2442 suggests that all activities required for this phenotype are encoded in this plasmid. Co-expression of the pADP1-borne atzDEF and atzTUVW genes, encoding the cyanuric acid utilization pathway and the subunits of an ABC-type solute transport system, in P. putida KT2442 was sufficient to promote growth at cyanuric acid concentrations as low as 50 μM in batch culture. Taken together, our results strongly suggest that the atzTUVW gene products are involved in high-affinity transport of cyanuric acid.
Pseudomonas sp. ADP (Mandelbaum et al., 1995) is the model organism for bacterial degradation of the s-triazine herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine). Pseudomonas sp. ADP uses atrazine as the sole nitrogen source via a six-step hydrolytic pathway, encoded in the 108 kbp catabolic plasmid pADP-1 (Martinez et al., 2001). The six genes involved in atrazine mineralization are localized in two distinct regions of the plasmid: atzA, atzB and atzC, responsible for atrazine conversion to cyanuric acid (2,4,6-trihydroxy-1,3,5-triazine), are harbored at three distant positions within a large (> 40 kbp) unstable region featuring multiple direct repeats and transposable elements. The genes involved in cyanuric acid cleavage and ammonium release are clustered at the stable portion of pADP-1 to form the atzDEF operon (reviewed by Wackett et al., 2002; Udiković-Kolić et al., 2012). The widespread use of atrazine and increasing concerns about its toxicological properties have boosted research aimed to bioremediation of polluted sites (Govantes et al., 2009). In addition, Pseudomonas sp. ADP atrazine degradation is a model system for the study of the evolution of new degradative pathways and the mechanisms of coordinated physiological gene regulation (Van der Meer, 2006; Govantes et al., 2010; Udiković-Kolić et al., 2012)
Our previous work showed that atrazine utilization by Pseudomonas sp. ADP is regulated by the general nitrogen control system, and the cyanuric acid utilization operon atzDEF is one of the targets for such regulation (García-González et al., 2003; García-Gonzalez et al., 2005). Expression of atzDEF is induced by the substrate of the pathway, cyanuric acid, and repressed in the presence of a preferential nitrogen source. The product of atzR, the gene transcribed divergently from atzDEF, is a LysR-type transcriptional regulator (LTTR) required for both nitrogen- and cyanuric acid-dependent control. Transcription of atzR is activated by the general nitrogen control protein NtrC and repressed by AtzR (García-Gonzalez et al., 2005). The regulation of the atrazine pathway and the characterization of the divergent PatzR-PatzDEF promoter region was recently reviewed (Govantes et al., 2009, 2010).
Cyanuric acid is a relatively hydrophilic compound (log POW = −0.2; Pichon et al., 1995), and it was previously shown that passive transport across the cell membrane limits its metabolization (Shiomi et al., 2006). However, no specific transport system for cyanuric acid or other s-triazines has been described so far. Recently, we characterized the transcriptional organization and regulation of a gene cluster, atzSTUVW, located immediately downstream from atzR, and encoding an outer membrane protein and an ABC-type solute transport system. The complete cluster is co-transcribed with atzR, although most of the transcription of the four distal genes is initiated at the internal PatzT promoter. Both promoters are similarly regulated in response to nitrogen availability and AtzR, suggesting a functional relation between the putative transporter and cyanuric acid metabolism (Platero et al., 2012).
In this work, we demonstrate the involvement of the atzTUVW genes in the utilization of cyanuric acid when provided at low concentration. Our results are fully consistent with the notion that AtzT, AtzU, AtzV and AtzW are the subunits of an ABC transport system required for high-affinity cyanuric acid uptake.
Materials and methods
Bacterial strains and growth conditions
Pseudomonas strains used in this work were Pseudomonas sp. ADP (Mandelbaum et al., 1995), its derivative lacking the unstable section of pADP-1 MPO101 (García-Gonzalez et al., 2005) and Pseudomonas putida KT2442 (Franklin et al., 1981). Escherichia coli DH5α (Hanahan, 1983) was used as a host for cloning procedures. Minimal medium containing 25 mM sodium succinate as the sole carbon source was used for growth assays (Mandelbaum et al., 1993). Nitrogen sources were 1 g L−1 ammonium chloride or cyanuric acid as indicated. Luria–Bertani (LB) medium was used as rich medium (Sambrook et al., 2000). Liquid cultures were grown in culture tubes or flasks with shaking (180–200 r.p.m.) at 30 °C. For solid media, Bacto-Agar (Difco) was added to a final concentration of 18 g L−1. Antibiotics were used, when required, at the following concentrations: ampicillin (100 mg L−1), kanamycin (20 mg L−1), carbenicillin (500 mg L−1), rifampicin (10 mg L−1) and tetracyclin (5 mg L−1). All reagents were purchased from Sigma-Aldrich.
All DNA manipulations were performed according to standard procedures (Sambrook et al., 2000). Restriction enzymes, DNA polymerases and T4 DNA ligase were purchased from Roche Applied Science. The Klenow fragment was routinely used to fill in recessed 3′ ends of incompatible restriction sites. Plasmid DNA preparation and DNA purification kits were purchased from Sigma-Aldrich, General Electric Healthcare or Macherey-Nagel and used according to the manufacturers specifications.
A 8.5 kbp BalI fragment from pADP-1 containing the atzDEF operon, atzR, atzT and part of atzU was cloned into XbaI-linearized pBluescript II-SK(+)(Apr) to yield pMPO216. The complete pADP-1 insert was subsequently excised from pMPO216 with SpeI and SacI and cloned into SpeI- and SacI-cleaved pBBR1-MCS3 (Tcr) (Kovach et al., 1995) to yield pMPO221. A 10.1 kbp HindIII fragment containing the complete atzRSTUVW operon was cloned into HindIII-cleaved pBluescript II-SK(+) to yield pMPO220. A 5.3 kbp ScaI fragment containing atzTUVW was excised from pMPO220 and cloned downstream from the 3-methyl benzoic acid-inducible Pm promoter in EcoRV-linearized pJB861 (Kmr)(Blatny et al., 1997) to yield pMPO880.
Cyanuric acid fed-batch growth assays
For cyanuric acid fed-batch growth assays, Pseudomonas sp. ADP or MPO101 was grown to saturation in minimal medium containing 3.3 mM cyanuric acid. Cells were washed three times in 1 × NaCl-P buffer (60 mM sodium-potassium phosphate, 0.5 g L−1 NaCl, pH 7.0) and resuspended at an A600 nm of 0.01 in minimal medium containing cyanuric acid as the sole nitrogen source at concentrations ranging between 0 and 100 μM. These cultures were incubated for 48 h, a period that we previously determined to be sufficient for the cultures to reach their maximum A600 nm, even at the highest cyanuric acid concentration. At this point, A600 nm of the cultures was determined, cyanuric acid was replenished at the initial concentration, and incubation was resumed for an additional 48-h period. This cycle was repeated for 10 days. Growth yields were calculated from the slopes of the linear segments of the A600 nm vs. time plots, assuming that cyanuric acid was exhausted in each cycle.
Conjugation of pADP-1
Mercury was used as selective agent for conjugal transfer of pADP-1 to P. putida KT2442. A drop dilution plate assay (Amador et al., 2010) was used to determine that the minimal inhibitory HgCl2 concentration in P. putida is 10 μM, while that of Pseudomonas sp. ADP is greater than 100 μM (data not shown). One milliliter aliquots of saturated cultures of Pseudomonas sp. ADP grown in cyanuric acid minimal medium and P. putida KT2442 grown in LB were washed with 1 × NaCl-P buffer, resuspended in a small volume and mixed. Mating mixtures were spotted on LB agar, incubated overnight and dilution plated on LB agar containing 20 μg mL−1 rifampicin and 15 μM HgCl2. Plasmid pADP-1 contains an unstable region bearing genes atzA, atzB and atzC as well as multiple repeated sequences and transposable elements, and spontaneous deletion of all or part of this DNA segment is common (García-González et al., 2003; Van der Meer, 2006; Devers et al., 2007; Changey et al., 2011). Thus, integrity of the pADP-1 unstable region in the exconjugants was verified by testing for growth and clear halo formation on atrazine minimal medium, PCR amplification of atzA, atzB and atzC using previously described primers (De Souza et al., 1998), and restriction analysis of pADP-1 plasmid DNA (data not shown). Strain MPO107, meeting all the above requirements, was selected for further analysis.
Results and discussion
Pseudomonas sp. ADP grows at extremely low cyanuric acid concentrations
To test the possible presence of a high-affinity transport system for cyanuric acid in Pseudomonas sp. ADP, we determined whether Pseudomonas sp. ADP is able to grow in the presence of low concentrations of this substrate. As growth is expected to be low at the smallest concentrations to be tested, we designed a fed-batch culture strategy in which Pseudomonas sp. ADP was grown in minimal medium in the presence of limiting (5–100 μM) cyanuric acid concentrations. Growth associated with the consumption of such amounts of cyanuric acid was monitored as A600 nm, after which cyanuric acid was replenished at the original concentrations to start a new growth cycle. The results are shown in Fig. 1a.
Growth of Pseudomonas sp. ADP was observed at all cyanuric acid concentrations, while the increase in A600 nm was negligible in the negative control. The increase in biomass was linear with time and proportional to the initial nitrogen source concentration for the first four cycles at all cyanuric acid concentrations between 5 and 50 μM. A consistent growth yield of 4.07 ± 0.37 A600 nm units mmole cyanuric acid−1 was observed at all concentrations in this interval, indicating that growth in each cycle proceeded until the limiting substrate was exhausted, and cyanuric acid concentration did not build significantly after each addition. The fact that cyanuric acid supports growth at concentrations in the low micromolar range strongly suggests that a concentrative, high-affinity cyanuric acid transporter is functional in Pseudomonas sp. ADP.
The Pseudomonas sp. ADP plasmid pADP-1 contains genes for three putative solute transport systems: orf46, encoding a putative xanthine/uracil permease, orf69, encoding a putative secondary magnesium/citrate transporter, and the atzTUVW cluster, encoding a putative ABC-type transporter (Martinez et al., 2001). A Pseudomonas sp. ADP derivative, MPO101, bearing a deleted version of pADP-1 that lacks most of the unstable region of this plasmid including orf46 and orf69, was previously described (García-González et al., 2003). To test the possible involvement of these genes in cyanuric acid transport, fed-batch growth of MPO101 in cyanuric acid-limited minimal medium was also tested (Fig. 1b). Growth of MPO101 was indistinguishable from that of Pseudomonas sp. ADP, indicating that orf46 and orf69 are dispensable for cyanuric acid utilization.
Conjugal transfer of pADP-1 to P. putida KT2442 promotes growth on cyanuric acid
The experiments described above suggest that Pseudomonas sp. ADP bears a high-affinity cyanuric acid transporter encoded either in the stable portion of pADP-1 or in its chromosome. To distinguish between these two possibilities, as Pseudomonas sp. ADP is not amenable to genetic manipulation, and therefore mutational inactivation of relevant genes in this organism is not feasible (García-Gonzalez et al., 2005), we determined whether transfer of pADP-1 to the heterologous, nondegrading host P. putida KT2442 confers the ability to grow at a low cyanuric acid concentration. For conjugal transfer of pADP-1 to KT2442, we took advantage of the presence of a mercury resistance operon in the degradative plasmid (orf50-57) (Martinez et al., 2001). The use of mercury as a selective agent for conjugal transfer of pADP-1 has not been previously reported.
Batch growth of MPO107 in minimal medium containing 25 mM succinate as the sole carbon source, and 1 mM or 100 μM cyanuric acid as the sole nitrogen source or no nitrogen source added, was determined by monitoring A600 nm over time. Control cultures of Pseudomonas sp. ADP and KT2442 bearing pMPO221, a pBBR1-MCS3-based plasmid expressing the cyanuric acid utilization operon atzDEF from its own promoter, were grown in the same conditions (Fig. 2). Cyanuric acid supported growth of Pseudomonas sp. ADP and the MPO107 at both concentrations tested, while growth in the absence of a nitrogen source was negligible (Fig. 2a and b). The growth rate and yield of MPO107 at the smaller cyanuric acid concentration were lower than that of the natural pADP-1 host, but distinguishable from that in the control condition lacking cyanuric acid (Fig. 2d). Cyanuric acid at the highest concentration (1 mM) supported growth of KT2442/pMPO221, albeit the growth rate and yield were lower than those of Pseudomonas sp. ADP and MPO107. No significant increase in A600 nm was observed in medium containing 100 μM cyanuric acid (Fig. 2c and d). These results are consistent with the notion that pADP-1 encodes a determinant in addition to the cyanuric acid degradative operon that promotes growth of P. putida KT2442 on cyanuric acid, and whose role is more prominent at a low substrate concentration. However, we cannot exclude that the differences in growth between MPO107 and KT2442/pMPO221 may be due to growth retardation of the latter because of the presence of the tetracycline-resistant plasmid pMPO221 (however, see below).
Co-expression of atzDEF and atzTUVW in P. putida is sufficient for growth at low cyanuric acid concentrations
To test directly the involvement of the atzRSTUVW operon in promoting growth in the presence of cyanuric acid, co-expression of the six genes and the atzDEF operon was attempted in P. putida KT2442. However, transfer of the constructs containing atzRSTUVW invariably resulted in slow growth and recombinant strain instability (data not shown), suggesting that production of one or more of the gene products from a plasmid vector is deleterious to the host strain. According to our previous work, atzS, encoding an outer membrane protein, is expressed at a low level from the weak PatzR promoter, while the distal atzTUVW genes, encoding the ABC transporter subunits, are expressed at higher levels from the internal PatzT promoter (Platero et al., 2012). We reasoned that AtzS overexpression may be responsible for the observed toxicity. On the other hand, being a relatively water-soluble molecule with a molecular weight of only 129 Da, cyanuric acid is expected to readily cross the outer membrane through the nonspecific, general porins (Davidson et al., 2008), and therefore, the role of AtzS in cyanuric acid transport, if any, is likely secondary. Accordingly, we set out to produce only the AtzTUVW transporter subunits, for which the atzTUVW cluster was cloned downstream from the Pm promoter of the xylene/toluene utilization pathway in pJB861 (Blatny et al., 1997) to yield pMPO880. This construct, along with the empty vector pJB861, was transferred by mating to P. putida KT2442 bearing pMPO221, and growth of the resulting strains in minimal medium containing 25 mM succinate as the sole carbon source and no nitrogen source or cyanuric acid at concentrations 50 μM to 1 mM as the sole nitrogen source. Addition of the Pm promoter inducer 3-methyl benzoic acid resulted in growth inhibition, and therefore, basal expression in the absence of inducer was used in these assays. Growth was monitored as the evolution of A600 nm for a 75-h period. The results are shown in Fig. 3.
The results obtained with KT2442 expressing atzDEF but bearing the control plasmid pJB861 are consistent with the observations above (Fig. 3a and c). Growth was observed with 1 mM cyanuric acid, but it was negligible at lower substrate concentrations. In contrast, KT2442 co-expressing atzDEF and atzTUVW exhibited increased growth rate and yield at 1 mM cyanuric acid, and significant growth with yields roughly proportional to the cyanuric acid concentration was observed at all concentrations used. As both strains in this assay bear the same plasmid backbones (a pBBR1-MCS3-derived Tcr plasmid and a pJB861-based Kmr plasmid), an artifact related to plasmid DNA burden or drug resistance as suggested above is ruled out. Taken together, our results strongly support the notion that atzTUVW encodes an ABC transporter involved in cyanuric uptake and required for cyanuric acid utilization when present at low (< 1 mM) concentration.
Physiological roles of the AtzTUVW cyanuric acid transporter
Membrane transport of s-triazines by degrading strains has not been studied in detail, and to the best of our knowledge, this is the first report of a s-triazine transport system. The presence of an inducible, metabolic energy-dependent cyanuric acid transporter in Pseudomonas sp. NRRL B-12227 was proposed based on the observation that the cyanuric acid degradation rate is limited by uptake in unacclimated but not in cyanuric acid-acclimated cells and stimulated by the presence of an energy source (Shiomi et al., 2006). The involvement of a high-affinity transport system in cyanuric acid uptake by Pseudomonas sp. ADP is also supported by our fed-batch growth experiments, in which cells metabolized cyanuric acid when present at concentrations below 5 μM (Fig. 1). On the other hand, Gebendinger and Radosevich (1999) first proposed the presence of a specific transport system for atrazine in Ralstonia brasilensis M91-3, based on a major discrepancy between the atrazine degradation rates in cell extracts and whole cells. Our previous work suggested that a nitrogen-regulated atrazine transport system is also present in Pseudomonas sp. ADP, based on the observation that atrazine degradation is inhibited in cells grown under nitrogen sufficiency while synthesis of the enzymes required for atrazine conversion to cyanuric acid is constitutive (García-González et al., 2003). Preliminary results suggest that Pseudomonas sp. ADP can use atrazine as a nitrogen source at concentrations as low as 10 μM. Given the high Km for atrazine (149 μM; Scott et al., 2009) of the atrazine chlorohydrolase AtzA, this observation is very suggestive of concentrative atrazine transport. However, poor growth of Pseudomonas sp. ADP on atrazine, along with the low water solubility of the herbicide (33 mg L−1 at 20 °C; log POW = 2.38; Nemeth-Konda et al., 2002), and the possible presence of impurities in our atrazine stock (technical grade, 99% purity; a gift from Novartis), precluded any further conclusions (data not shown), and we cannot at this point confirm or rule out this possibility.
Two cytoplasmic Pseudomonas sp. ADP proteins are known to interact with cyanuric acid. On the one hand, cyanuric acid is transformed into biuret by cyanuric acid amidohydrolase, which has a Km for its substrate of 23 μM (Seffernick et al., 2012). Cyanuric acid transport by AtzTUVW may facilitate optimal functioning of the degradative pathway by allowing the substrate concentration to build inside the cells to meet the requirements of the utilization pathway. It should be noted that an ABC-type transporter appears to be suitable for this purpose, as transporters in this family often operate in the low micromolar range or below and concentrate the transported solutes inside the cells (Davidson et al., 2008). In addition, cyanuric acid interacts with the LTTR AtzR to induce transcription of the atzDEF operon, encoding the cyanuric acid utilization enzymes (Porrúa et al., 2007). Although we have not analyzed the affinity of AtzR for cyanuric acid in vitro, we have determined that maximum in vivo induction of the AtzR-activated atzDEF operon in P. putida KT2442 not bearing a specific cyanuric acid transporter requires a concentration of 100 μM cyanuric acid in the growth medium (V. García-González and F. Govantes, unpublished results). Therefore, it is likely that intracellular cyanuric acid concentration build-up due to AtzTUVW-dependent transport promotes interaction with AtzR and subsequent activation of atzDEF activation, thus enabling growth in the presence of low cyanuric acid concentrations. Interestingly, concentrative transport of cyanuric acid may also be detrimental to growth on this substrate, as cyanuric acid was recently shown to inhibit the activity of cyanuric acid amidohydrolase in vitro when present at concentrations above 60 μM (Peat et al., 2013). In that regard, the negative regulation exerted by AtzR on the two promoters that drive transcription of the atzRSTUVW operon (PatzR and PatzT)( Porrúa et al., 2009; Platero et al., 2012) may be envisioned as a safety mechanism to prevent excessive cyanuric acid transport that may preclude its metabolization. Future analyses will hopefully provide further insights on how these processes are interlocked in the cell physiology.
We are indebted to Enrique Flores (Instituto de Biología Vegetal y Fotosíntesis, CSIC, Sevilla, Spain) for thoughtful discussions, advice on experimental design and critical review of the manuscript. We also thank all members of the Govantes and Santero laboratories at CABD for providing materials and critical discussion. This work was supported by grants BIO2007-63754, BIO2010-17853, CSD2007-0005 and BIO2011-24003, co-funded by the Spanish Ministerio de Educación y Ciencia and the European Regional Development Fund.