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Philipps University, Institute of Physiological Chemistry, D-35043 Marburg, Germany
Correspondence
Klaus-Heinrich Röhm
roehm{at}staff.uni-marburg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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54-dependent two-component system AauRS. Binding of the response regulator AauR to the aat promoter was examined by gel mobility shift assays and DNase I footprinting. By in silico screening, the AauR-binding motif (the inverted repeat TTCGGNNNNCCGAA) was detected in further P. putida KT2440 genes with established or putative functions in acidic amino acid utilization, and also occurred in other pseudomonads. The products of these AauR-responsive genes include the H+/Glu symporter GltP, a periplasmic glutaminase/asparaginase, AnsB, and phosphoenolpyruvate synthase (PpsA), a key enzyme of gluconeogenesis in Gram-negative bacteria. Based on these findings, we propose that AauR is a central regulator of acidic amino acid uptake and metabolism in pseudomonads.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
ABC transporters are typically composed of four functional modules: two transmembrane permease domains, each of them spanning the membrane four to eight times, and two nucleotide-binding domains (NBDs). In eukaryotes, these modules are frequently fused to yield a single polypeptide chain, while bacterial ABC transporters are assembled from individual subunits. With most bacterial importers, a highly specific solute-binding protein is also part of the transport machinery, whereas ABC exporters lack the solute-binding protein (Fath & Kolter, 1993; Davidson & Chen, 2004). In Gram-negative organisms, solute receptors are dissolved in the periplasm, while in archaea and Gram-positive micro-organisms, they are membrane-anchored lipoproteins (Higgins, 1992; Hediger, 1994; Schneider & Hunke 1998; Saurin & Dassa, 1994). The specificity of ABC transporters mainly depends on the selective binding of the solute by the periplasmic receptor. They not only ensure effective solute capture but also act as essential activators of the cytoplasmic nucleotide-binding domains (Davidson & Chen, 2004; Saurin et al., 1999).
Pseudomonas putida KT2440 is a well-characterized Gram-negative soil micro-organism with potential applications in bioremediation and biocontrol. Like other pseudomonads, strain KT2440 is physiologically very versatile, growing on a wide range of carbon and nitrogen sources, including the acidic amino acids and their amides (Sonawane et al., 2003a). The ABC transporter characterized in the present study exhibits a marked selectivity for glutamate and aspartate. We therefore named the coding operon aat (for acidic amino acid transporter). P. putida KT2440 AatJMQP is encoded by genes PP1068–PP1071. Closely related orthologues exist in Pseudomonas aeruginosa (PA1339–PA1342), Pseudomonas fluorescens (PFl1048–PFl1051) and Pseudomonas syringae (PS4171–PS4174).
Like other bacterial ABC transporters, the aat system involves a periplasmic solute-binding protein AatJ (encoded by PP1071), two permease domains, AatQ and AatM (PP1070–PP1069), and an ATP-binding subunit, AatP (PP1068). In the P. putida KT2440 genome, aat is immediately adjacent to aau, an operon that encodes the two-component system AauRS (see Fig. 1). The aau system was recently identified as a regulator of acidic amino acid utilization in P. putida. As judged by sequence similarity, the response regulator AauR is a DNA-binding helix–turn–helix protein belonging to the Fis family (Morett & Bork, 1998). We have further shown that expression of aat depends on 54, an alternative sigma factor typically involved in the transcription of genes related to nitrogen metabolism (Sonawane et al., 2006).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. P. putida KT2440 was grown in LB broth (Gibco-BRL), in M9 minimal medium (Sambrook et al., 1989) or on LB agar plates at 30 °C.
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Construction of deletion mutants KTaatP and KTaatJ.
Genes aatP (PP1068) and aatJ (PP1071) were inactivated by a strategy described previously in detail (Sonawane et al., 2006). Briefly, internal gene fragments of about 300 bp were amplified from the KT2440 genome using primers that introduce single base-pair frameshifts and cloned into pK18, a suicide vector that cannot multiply inside P. putida unless integrated into the genome by homologous recombination. The resulting mutants contain fragments of the target gene separated by the plasmid sequence, i.e. (1) a short truncated fragment or (2) a fragment containing the frameshift mutation at the 5' end. The frameshift mutation was necessary, as in the second case, the transconjugants might contain a wild-type copy of the genes after recombination. In the case of aatP, a 261 bp fragment (408–147 bp relative to the translation start site) was amplified using primers PP1068_For and PP1068_Rev (Table 2). In the case of PP1071 (aatJ), a 284 bp internal fragment (189–473 bp) was generated using primers PP1071_For and PP1071_Rev. The amplified fragments were cloned into pK18 (Pridmore, 1987) and the resulting plasmids electroporated into P. putida KT2440, followed by selection of kanamycin-resistant clones. Successful recombination was verified by PCR using specific primers. All further work with these mutants was performed in the presence of kanamycin (40 µg ml–1).
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), a low-copy-number expression vector commonly used for complementation experiments with Pseudomonas spp. The sequenced vectors were electroporated into KTaatJ and KTaatP, and complemented clones were isolated by carbenicillin selection, yielding strains KTaatJ+ and KTaatP+.
Analysis of the aatJMPQ promoter region.
Various segments of the upstream promoter region of aatJMPQ (–83 to –343 bp relative to the transcription start site) were amplified from the KT2440 genome using forward primer PromFor and various reverse primers, as summarized in Table 2
. The amplified fragments were cloned in pUC19, and the resulting plasmids transformed into E. coli Top10 for multiplication. The vectors were then digested with EcoRI and HindIII and the excised fragment purified by agarose gel electrophoresis using the Qiagen gel extraction kit. The purified fragments were labelled with 33P in a 50 µl reaction volume containing 10 µl 5x forward reaction buffer, 5 pmol DNA, 25 U T4 Polynucleotide Kinase (Stratagene) and 5 µl [
-P33]ATP (10 µCi µl–1; 110 TBq mmol–1). After incubation at 37 °C for 1 h, the labelled fragment was separated from free [-P33]ATP on a Microspin S-300 HR column (GE Healthcare). An unspecific DNA fragment of about the same size was labelled accordingly. Specific radioactivities of the resulting fragments were estimated by liquid scintillation counting.
Gel mobility shift assay.
Binding of AauR to aatJMPQ promoter fragments was monitored by a published procedure (Banerjee & Kundu, 2003). Labelled DNA probe (5 fmol) was added to 10–100 nM of AauR in HGMKE buffer (25 mM HEPES, pH 8, with 5 %, v/v, glycerol, 10 mM MgCl2, 20 mM KCl and 0.1 mM EDTA). The mixtures were incubated at 37 °C for 20 min and separated in 6 % native polyacrylamide gels. The gels were dried and analysed by a Phosphorimager (Molecular Dynamics) after a 24 h exposure.
DNase I protection assay.
DNase I footprinting was performed according to Licht & Brantl (2006). The labelled DNA fragment was digested with BamHI to remove the 5' 33P label and purified by the phenol/chloroform method. Binding reactions were carried out in a final volume of 20 µl containing HGMKE buffer, 0.05 g l–1 salmon sperm DNA, 50 ng labelled probe and 20–100 nmol AauR. The reaction mixtures were incubated at 37 °C for 20 min and digested with 0.05 U DNase I (Fermentas) for 2 min at 37 °C. Digestion was stopped by extraction with phenol and subsequent ethanol precipitation. The pellets were separated on a polyacrylamide sequencing gel (8 % urea) along with standard sequencing ladders (G+A and C+A) produced by Maxam–Gilbert chemical sequencing, as described by Sambrook et al. (1989). The dried gel was exposed to a phosphor screen for 1 week and analysed by phosphor imaging.
Expression and purification of His-tagged AauR.
The gene encoding the response regulator AauR (PP1066) was amplified by PCR using primers aauRFor and aauRRev (Table 2) and ligated with the expression vector pTrcHis-A (Invitrogen) to generate pTrcaauR. After transformation into E. coli BL21, single colonies were grown in LB/Amp medium to OD595 0.8–0.9 before expression was induced by addition of 1 mM IPTG. After another 6 h, the cells were collected at 5000 r.p.m. for 10 min, washed with 100 ml cold His-tag protein-binding buffer (20 mM HEPES, pH 7.5, with 500 mM NaCl and 100 mM imidazole) and resuspended in 20 ml of the same buffer. The cell pellets were disrupted by sonication and the homogenate cleared by centrifugation (20 000 r.p.m., 30 min). The supernatant was loaded on an equilibrated Ni-NTA column (HisTrap-FF, GE Healthcare Biosciences) and protein was eluted with a linear imidazole gradient (100–500 mM). AauR-containing fractions were pooled and subjected to gel filtration on Superdex 200 (GE Healthcare Biosciences) equilibrated with the same buffer. Finally, the purified protein was dialysed against 50 mM HEPES, pH 7.5, containing 500 mM NaCl, and concentrated by a Centricon cartridge [10 kDa molecular weight cut-off (MWCO); Millipore].
Overexpression and purification of AatJ.
The aatJ gene is 918 bp long, with base pairs 1–72 encoding the signal peptide for periplasmic transport. A truncated form of aatJ lacking this leader sequence was amplified by using AatJ_For and AatJ_Rev (Table 2). The amplified fragment was cloned into expression vector pFLAG-ATS (Sigma). This vector already contains an ompA signal peptide coding gene and a small flag sequence that mediates transport of the expressed protein into the periplasmic space. E. coli BL21 cells containing the recombinant plasmid were grown in LB/Amp medium at 37 °C to OD595 0.8–1. Expression was induced by 1 mM IPTG and cultivation continued for another 6 h at 37 °C. The cells were harvested by centrifugation at 4 °C and the periplasmic protein fraction liberated by osmotic shock, as described by Harms et al. (1991). The periplasmic proteins were purified on a Q-Sepharose anion exchange column (GE Healthcare Biosciences) equilibrated with 50 mM Tris/HCl, pH 7.8, and eluted by a linear NaCl gradient (0–500 mM). AatJ-containing fractions were pooled and further purified by gel filtration on a Sephacryl S-100 HR column equilibrated with 50 mM Tris/HCl buffer, pH 7.8, containing 100 mM NaCl. The relevant fractions were pooled, concentrated using a Centricon cartridge (10 kDa MWCO), assayed for protein using a bicinchoninic acid (BCA) assay kit (Pierce) and stored at 4 °C.
[14C]Glu/[14C]Asp uptake assay.
The rates of Glu and Asp uptake were measured by a conventional filter assay (Hosie et al., 2001). Cells were grown overnight, washed twice with M9 salt solution, suspended in 50 ml M9 and stored at 4 °C until the start of the experiment. Then the cells were starved for amino acids by gentle agitation for 30 min at 30 °C. The uptake assay was initiated by adding 14C-labelled L-Glu or L-Asp (200 mCi mmol–1; Hartmann Analytic) to a final concentration of 5 µM. Then, 100 µl aliquots were removed at regular time intervals and added to 400 µl stop solution (M9 salt solution containing 50 mM of the respective unlabelled amino acid). In competition experiments, 50 µM unlabelled amino acids was added 2 min before addition of the labelled compounds. The cultures were filtered through nitrocellulose membranes (0.45 µm pore-size), and the filters were washed with 500 µl stop solution (M9 salt solution with 0.5 mM unlabelled amino acids) twice. After drying at room temperature for 12 h, the filter discs were immersed in 6 ml scintillation cocktail and counted on a Beckman Coulter LS 6500 scintillation counter. The protein content of the cells was measured by BCA assay kit (Pierce) using BSA as a standard. The kinetics of amino acid uptake from the medium was analysed by quantitative amino acid analysis, as described by Sonawane et al. (2006).
[14C]Glu/[14C]Asp binding assay by equilibrium microdialysis.
The binding of amino acids and other potential ligands to AatJ was studied by equilibrium dialysis. Purified AatJ was dialysed against ligand-binding buffer (50 mM HEPES, pH 7.5, 100 mM NaCl) and concentrated to a protein concentration of 10 µM using Centricon concentrators. The working solutions consisted of 5 µM AatJ protein and 0.1–25 µM [14C]L-Glu or [14C]L-Asp (200 mCi mmol–1; 7.4 GBq mmol–1) in 50 µl ligand-binding buffer. In competitive binding experiments, 50 µM unlabelled ligand was added together with the labelled amino acid. Disposable micro dialyser cells (DispoEquilibrium Dialyser, Harvard Apparatus) were loaded with sample solution on one side and buffer–ligand on the other, and allowed to equilibrate for 4–5 h at 25 °C with gentle rocking. Then, separately, the contents of the sample and buffer sides were mixed with 2 ml liquid scintillation cocktail and counted. The fraction of bound ligand was estimated from the difference of the two counts. As, in the present case, the ligand dissociation constants 
are comparable to the concentration of the receptor (AatJ), ligand depletion had to be taken into account. In such cases, the concentration of the ligand–receptor complex [RL] is given by:
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). Dissociation constants were estimated by fitting the above equation to the binding data. Binding constants 
of unlabelled ligands (Asn, Gln and dicarboxylates) were estimated from their competitive effects on Glu and Asp binding, based on the equation:
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The efficient utilization of these amino acids, however, depends on an intact AatJMQP system. Fig. 2 shows typical growth curves observed with wild-type and mutant strains of KT2440 cultured on minimal medium supplemented with these amino acids as sole organic nutrients. Quantitative growth data obtained with these and other amino acids are summarized in Table 3. The table shows that mutants lacking a functional solute-binding protein AatJ (KTaatJ) completely lost the ability to proliferate on Glu and Gln as sole organic nutrient, whereas growth on Asp and Asn was only slightly impaired. Interestingly, KTaatP (a strain defective in the Aat nucleotide-binding domain) was still able to grow on Glu or Gln, although with a markedly longer lag phase and reduced growth yields, while growth of KTaatP on Asp and Asn was almost unaffected. Growth on His, Lys, Pro, Arg was unaffected by aat inactivation (Table 3). Branched-chain amino acids do not support growth of P. putida KT2440 when supplied as the sole source of carbon and nitrogen. Glucose supplied as an additional carbon source restored normal growth of both Aat mutants on any of the amino acids examined (data not shown). As certain Glu uptake systems are coupled to a Na+ gradient (see below), we also examined whether growth of KTaatP on Glu was affected by increasing the Na+ concentration in the medium from 30 to 80 mM, but did not detect any positive growth effect (data not shown). As shown by Fig. 2, complementation of the mutations introduced into aatJ and aatP restored normal growth on any of the amino acids examined, indicating that the observed growth defects of KTaaJ and KTaatP were not caused by polar effects on downstream genes.
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, high-affinity uptake of [14C]Glu and [14C]Asp by wild-type KT2440 proceeded at the same velocity. With both mutants (KTaatP and KTaatJ), uptake rates were reduced to 10–15 % of the wild-type values. As expected, addition of an excess of unlabelled Glu or Asp (50 µM) inhibited the uptake of the respective labelled amino acid by 80–90 %, whereas 50 µM unlabelled amides (Gln and Asn) did not significantly compete with the import of [14C]Glu or [14C]Asp (data not shown).
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). Again, both amino acids were taken up from the medium at roughly the same rate. Under the conditions of the experiment, wild-type cells consumed the available amounts of Glu and Asp within 6–8 h, while the uptake by both mutants gradually slowed down to cease after 8–10 h.
Ligand-binding properties of the AatJ solute receptor
As already mentioned, the selectivity of ABC solute importers is principally dependent on the binding characteristics of the cognate solute-binding protein (Davidson & Chen, 2004). To examine the binding specificity of the Aat system, we therefore overexpressed and purified the solute receptor AatJ for equilibrium binding studies. The system used allowed the transport of the overexpressed protein into the periplasmic space, from where it was conveniently released by osmotic shock. Two subsequent purification steps (ion exchange chromatography on Q-Sepharose and gel filtration on Sephacryl-S100) provided 6–7 mg of highly enriched product per litre of culture (Fig. 4). By analytical gel filtration, the resulting preparation was found to be a monomer with the expected mass of about 34 kDa (data not shown).
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(a semilogarithmic plot is shown for clarity). As the AatJ concentration used was in the same range as the dissociation constant of the receptor–amino acid complex, ligand depletion had to be taken into account (see Methods). By fitting such a model to the data, dissociation constants, Kd, of 0.3 µM for Glu and 1.5–2 µM for Asp were obtained. Since Gln and Asn were not available in radiolabelled form, a competition method was applied to determine their binding characteristics. In this approach, the reduction in binding of labelled ligand in the presence of a large excess of unlabelled amino acid is used to estimate the dissociation constant of the competing unlabelled ligand. Constants calculated in this way are summarized in Table 4. In general, the values obtained by direct binding experiments and competition assays were in good agreement, with the exception of Asp, for which the competition assay yielded somewhat higher dissociation constants than the direct assay. Gln and the dicarboxylates fumarate and succinate were very weak ligands, with dissociation constants too high to be estimated with certainty.
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), it was of interest to delineate cis-acting elements in the aat promoter that are required for this activation. A promoter deletion series was prepared involving sequences between –83 and –202 relative to the transcription start site (+1). These fragments (schematically depicted in Fig. 6) were amplified from the KT2440 genome using primer PromFor and various PromRev primers (Table 2). The resulting PCR products were purified, labelled with 33P and used to analyse AauR binding by gel mobility shift assays. The results, summarized in Fig. 6, show that binding of AauR to the aat operon promoter is specific. A non-specific DNA fragment of about the same length did bind to AauR (data not shown). Only those DNA fragments that contained base pairs –117 to –136 were shifted, indicating that this partial sequence of the aat promoter interacts with AauR.
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, a 19 bp sequence located between –119 and –137 was efficiently and selectively protected by AauR. The footprint contains the conspicuous inverted repeat TTCGGNNNNCCGAA. The overall length and spacing of this motif exactly meet the requirements for binding sites of dimeric helix–turn–helix proteins, of which AauR is a member (Tzou & Hwang, 1999). In addition, a typical –12/–24 motif (Barrios et al., 1999), which is required for recruitment of the alternative sigma factor 54 to the initial transcription complex, was found in the aat promoter.
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). An especially intriguing hit was the monocistronic gene PP0137, which, by sequence homology, encodes a glutamate : H+ symporter of the GltP type (Raunser et al., 2006). Another putative AauR-binding motif was detected in the intergenic region of the operon braCDEFG (PP1141–PP1137), assigned as an ABC transporter for branched-chain amino acids. However, this assigned solute specificity needs to be substantiated by experiment, as the homologous Bra system of Rhizobium leguminosarum encodes a general amino acid permease with a broad range of solutes including L-Glu (Hosie et al., 2001). Two further genes with possible AauR-binding sites are not directly involved in amino acid uptake: the product of the dsbC gene (PP1469) assists in the periplasmic formation of disulfide bonds and has been shown to play an important role in the maturation of extracellular enzymes (Urban et al., 2001), while ppsA (PP2082) encodes phosphoenolpyruvate synthase (PpsA), a key enzyme in gluconeogenesis from 
-keto acids (Chao et al., 1993).
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the AauR-binding motif (TTCGGNNNNCCGAA) is also well conserved in the aat homologues of other pseudomonads. The same holds for further genes with a putative AauR recognition site (ansB, gltP, bra and dsbC; data not shown). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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as nitrogen sources or to glucose as carbon sources (Sonawane et al., 2003a). By contrast, E. coli is unable to proliferate on Glu as sole carbon source unless the capacity of its Glu uptake systems is increased by mutations (Booth et al., 1989). Again, contrary to P. putida, E. coli also grows more slowly on gluconeogenic carbon sources (e.g. succinate) than on glucose itself. Here, we show that the ability of P. putida KT2440 to proliferate on Glu or Gln as sole carbon and nitrogen source largely depends on the ABC transporter AatJMQP. The role of AatJMQP in Gln utilization is an indirect one, as P. putida KT2440 does not contain a Gln transporter; Gln is first hydrolysed by a periplasmic glutaminase/asparaginase (AnsB) to yield Glu, which is then taken up by the Aat system. P. putida mutants lacking a functional AnsB, therefore, are unable to grow on Gln (Sonawane et al., 2003a). Utilization of Asp and Asn by strain KT2440 does not depend on a functional Aat system, indicating that additional uptake systems for Asp and/or Asn exist. The presence of a selective Asn transporter in P. putida has not been established; it is known, however, that pseudomonads contain a cytosolic hydrolase with selectivity for Asn that could convert imported Asn to Asp (Sonawane et al., 2003a).
As judged by sequence similarity, AatJMQP is a typical receptor-dependent ABC transporter, belonging to the polar amino acid uptake transporter (PAAT) family (Saier, 2000). The closest orthologue of the P. putida Aat system in E. coli is the ABC transporter GltIJKLM (Linton & Higgins, 1998). Although this system is still poorly characterized, the available data suggest that its amino acid-binding properties are similar to those reported here for Aat. Furlong and co-workers reported apparent Kd values for the E. coli Glu/Asp-binding protein of 0.7 µM for Glu and 1.2 µM for Asp, respectively (Willis & Furlong, 1975; Schellenberg & Furlong, 1977), i.e. values that are in close agreement with our data (Table 4).
In addition to GltJKLM, E. coli contains at least two additional transport systems for Glu and/or Asp, i.e. an H+ symporter for both Glu and Asp (GltP) and an Na+-dependent Glu-specific uptake system (GltS). GltP from E. coli has been studied in considerable detail, and has been shown to catalyse an electrogenic cotransport of Glu and Asp with H+ ions (Tolner et al., 1995; Raunser et al., 2006), while GltS is a binding protein-independent Glu : Na+ symporter (Reizer et al., 1994) with selectivity for L- and D-glutamate, homocysteate and -methylglutamate (Kalman et al., 1991). Bacterial uptake systems with a strict selectivity for Asp seem to be uncommon. However, many organisms express C4-dicarboxylate carriers that not only import dicarboxylates such as succinate or fumarate but also Asp, although with lower affinity (Janausch et al., 2002). The E. coli genome encodes several such C4-dicarboxylate carriers, most of which are only expressed under anaerobic conditions. The main system responsible for aerobic Asp uptake, DctA, operates as an H+ or Na+ : dicarboxylate symporter and catalyses the uptake of succinate, fumarate, malate, aspartate and other carboxylic acids.
All of the Glu/Asp uptake systems mentioned above are also encoded by the P. putida KT2440 genome. A comparison of the respective loci in strain KT2440 and E. coli K12 is shown in Table 5. Mutual sequence identities of 50 % or more and similarities between 60 and 86 % suggest that the gene products of gltP, gltS and dctA in P. putida are functional counterparts of the respective E. coli systems. The growth characteristics and amino acid uptake properties of the P. putida strains described here indicate that AatJMQP is the most important Glu uptake system under the conditions in our experiments. Note that the growth curves shown in Fig. 2 and Table 3 and the data summarized in Fig. 3 were measured with cells pre-grown on 
/glucose, i.e. under conditions where the Aat system was not upregulated by amino acids. Thus, even at its basal level of activity, AatJMQP appears to account for about 85 % of the capacity for high-affinity Glu transport. Cells lacking a functional AatJ did not proliferate on Glu at all, while a mutant with a defective nucleotide-binding domain AatP did, although with a lag phase of 6–8 h and reduced cell yields (Fig. 2). An explanation for this unexpected finding is suggested by data indicating that nucleotide-binding domains of ABC transporters may interact not only with their cognate transporter but also with other ABC-type permeases. So Quentin et al. (1999) proposed that in Bacillus subtilis three ATPase subunits energize 10 different transporters. Similar observations have also been made with carbohydrate importers of the ABC type (Schneider, 2001). Thus, nucleotide-binding domains of other PAAT-family transporters of P. putida may partially compensate for the loss of AatP to sustain growth of strain KTaatP.
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). Here we provide evidence indicating that at least four additional genes are under the control of AauR, i.e. the Glu/Asp importer GltP, the enzyme phosphoenolpyruvate synthase (PpsA), a thiol–disulfide exchange protein (DscB) and the braCDEFG operon (PP1141–PP1137). Assuming that all of these genes are indeed regulated by aau, a consistent picture of Glu/Asp utilization by P. putida KT2440 and the regulatory role of the AauR–AauS two-component system begins to emerge (Fig. 9). Acidic amino acids and their amides present in the medium first activate the AauR–AauS system, which then upregulates (1) glutaminase/asparaginase [AnsB, enzyme (1)] to allow for the extracellular conversion of Asn and Gln to Asp and Glu, respectively; (2) Glu/Asp transporters (AatJMQP, and possibly also GltP and Bra; see below) to facilitate uptake of the Asp and/or Glu; and (3) phosphoenolpyruvate synthase [PpsA, enzyme 8] to stimulate gluconeogenesis from Asp and Glu (the gluconeogenetic pathway becomes essential for the synthesis of carbohydrates and lipids if amino acids are supplied as sole sources of carbon and nitrogen). The enzymes numbered 2–7 in Fig. 9 all catalyse reactions that channel the carbon skeletons of Glu or Asp into the tricarboxylic acid cycle or from there into pyruvate.
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). We have provided evidence to indicate that the BraCDEFG transporter of P. putida KT2440 might fulfil a similar role. While bra was down-regulated during growth on Glu, a Glu-grown KTaauR mutant lost Glu by efflux (Sonawane et al., 2006). We therefore hypothesize that binding of AauR to the P. putida bra promoter does not induce but rather inhibits expression of bra and thus helps to prevent losses of Glu via the overflow pathway. Further experiments are required to clarify these points.
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ACKNOWLEDGEMENTS |
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Edited by: M. A. Kertesz
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Received 12 September 2007;
revised 3 December 2007;
accepted 5 December 2007.
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OD595). 
, Wild-type; 
, KTaatJ; 
, KTaatJ+ (complemented); 
, KTaaP; 
) and KTaatP (
) at low and high amino acid concentrations. (a) 14C-labelled amino acids were added at zero time to a final concentration of 5 µM. Aliquots were removed from the cultures at 30 s intervals, mixed with stop solution and filtered through nitrocellulose membranes. The washed and dried filters were analysed by liquid scintillation counting. The data shown represent mean±SD of three experiments. (b) After adding unlabelled Glu or Asp to a final concentration of 5 mM at zero time, the decrease of amino acid concentration in the medium was followed by HPLC with pre-column derivatization (for details see text). Cumulative uptake is expressed as micromoles of amino acid consumed per millilitre of medium.
