INTRODUCTION

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Pseudomonas aeruginosa is a gram-negative soil bacterium that can transiently colonize animal hosts. Host defenses are usually sufficient to prevent infections, but when these defenses are compromised (e.g., after burns, during immunosuppressive therapy, or in wounds), P. aeruginosa becomes an opportunistic pathogen that can cause serious life-threatening infections. During such infections, P. aeruginosa secretes or carries on its outer membrane several virulence factors that contribute to its pathogenicity. These include pili, ADP-ribosyltransferase toxins (exotoxin A [ETA] and exoenzyme S), rhamnolipids, hemolytic and nonhemolytic phospholipases, endotoxin, proteases, and the exopolysaccharide alginate (10). Of these, ETA is the most toxic (26).

Most of the known regulators of ETA synthesis control the synthesis of RegA, which has been proposed to interact with RNA polymerase holoenzyme and thereby facilitate open promoter complex formation (36, 37). Transcription of regA requires the products of vfr and pvdS. Vfr is a Pseudomonas homologue of the cyclic AMP receptor protein (38), and pvdS codes for an alternate sigma factor (31) which is also required for synthesis of the iron chelator pyoverdine (6). Finally, the iron-sensing ferric uptake regulator (Fur) represses pvdS transcription (31). Although several genes have been implicated in the control of ETA synthesis, there are some significant gaps in our knowledge. For example, a site for an activator of toxA (ETA-encoding) expression has been identified, but the activator and the factors that control its activity are not known (35). In fact, no protein that interacts with the toxA promoter region has been definitively identified.

Iron limitation is a major stimulus for ETA synthesis (3). Iron-dependent regulation is mediated, in part, by Fur, which controls PvdS synthesis. However, residual control by iron availability in Pseudomonas fur mutants suggests the existence of a Fur-independent mechanism of iron regulation (1). Two other environmental factors stimulate ETA synthesis: entry into stationary phase (2) and high levels of certain amino acids, such as glutamate (25). The relationships between these factors are poorly understood. Our primary purpose was to analyze the physiological function of supplemental amino acids. We made the new observations that glutamate, citrate, and isocitrate partially mimic the growth-stimulatory effect of exogenous iron; that iron limitation resulted in a reduction in the activity of aconitase, an iron-sulfur protein, thus accounting for the growth stimulation by glutamate; and that there was a reciprocal correlation between aconitase activity and ETA synthesis. We discuss the broader significance of the amino acid supplementation and the circumstantial evidence that a bacterial aconitase may have a regulatory function similar to that of the human iron-regulatory protein 1 (IRP-1) (32).

(This work is presented as partial fulfillment for the requirements for the Ph.D. degree at the University of Texas at Dallas for G. Somerville.)

	MATERIALS AND METHODS

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Bacterial strains. The P. aeruginosa strains used in this study were the prototrophic strain PA103 (27) and PA103-CM1, which contains a chromosomal toxA-lacZ fusion. The latter strain was constructed as follows. The toxA promoter region was removed from pRC362 (5) as a 760-bp PvuII-BamHI fragment and ligated into the SmaI and BamHI sites in the polylinker of pRS414 (34), which has lacZ immediately downstream of the BamHI site. The first 24 bp of the toxA gene were joined in frame to the N terminus of lacZ by linearizing the plasmid at the unique BamHI site, filling in with the Klenow fragment of Escherichia coli DNA polymerase, and religating. The toxA-lacZ fusion was removed as a 2.7-kb EcoRI-SacI fragment, which replaced the corresponding fragment of pUJ9 (8). This plasmid has a T7 terminator at the end of lacZ and allows excision of the toxA-lacZ fusion as a NotI fragment. The 4.2-kb NotI fragment was inserted into the transposon vector pUT, which contains the mini-Tn5 streptomycin/spectinomycine resistance gene cassette, and the plasmid was transformed into E. coli S17-1(pir) (19). The donor E. coli strain was grown overnight at 37°C; the recipient strain, PA103, was grown at 43°C. Mobilization of the toxA-lacZ-containing pUT derivative was achieved by mixing the two strains in a 1:3 ratio (donor to recipient), collecting the cells on a 0.45-µm-pore-size filter, and allowing the cells to mate on an LB plate at 32°C. The next day the cells were eluted and an appropriate dilution was plated on LB plates with streptomycin (500 µg/ml). This selects against E. coli, which is not resistant to such a high concentration of streptomycin, and selects for the transconjugates. This procedure selects for cells with a random insertion of the fusion onto the chromosomethe fusion does not replace the wild-type toxA gene. The P. aeruginosa strain designated PA103-CM1 was chosen based on a quantitative assay for -galactosidase that showed iron regulation.

Media and growth conditions. Pseudomonas cultures were grown in either 2xYT (29), deferrated trypticase soy broth dialysate (DTSB) (20), or Pseudomonas isolation agar (Difco) at 32°C. DTSB was supplemented with 1 mM MgSO₄ and with other compounds as noted. Liquid cultures were shaken at 250 rpm. The flask-to-medium volume ratio was always 20:1. The removal of iron from DTSB was not quantitative but was sufficient to limit growth (see Results). Most of the time, growth ceased at the same cell density, but there was some variation from batch to batch of medium. Occasionally the iron chelation was so efficient that it was necessary to add 1 µM FeSO₄ to obtain sufficient growth.

Biochemical assays. Aconitase activity was assayed by the method of Gruer and Guest (17). Briefly, 1.5-ml cultures were harvested at the indicated times, resuspended in Tris-citrate (20 mM, pH 8.0), and ultrasonically disrupted. The suspension was then centrifuged for 5 min at 12,000 rpm to remove cell debris. The cell-free lysate was then assayed for aconitase activity. Enzyme activity was determined spectrophotometrically by monitoring the formation of cis-aconitate from isocitrate at 240 nm according to the method of Kennedy et al. (21).

	RESULTS

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Effect of glutamate during iron-restricted growth. The first studies on ETA synthesis showed that the optimal medium was a broth that contained glycerol and albumin (7, 25). Because albumin and several amino acids stimulated growth, it was concluded that toxigenic medium was nitrogen limiting (25). It was subsequently shown that iron limitation induced ETA synthesis (2, 3). Glutamate is still routinely added to iron-limited toxigenic medium, but its function, if any, has not been reexamined. It is possible that glutamate counters an effect of iron limitation.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Iron-restricted growth of PA103. (A) Growth of PA103 in DTSB without supplemental iron or with 100 µM FeSO4. (B) Parallel cultures were grown in iron-limited DTSB for 6 h. Supplemental iron [100 µM Fe(NH4)2(SO4)2] was added to one () but not the other ().

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Additions to DTSB affect the growth yield but not the growth rate. PA103 cultures were grown in DTSB with no supplement or with the indicated supplement at 50 mM.

View this table: [in this window] [in a new window]   TABLE 1.   Additions to DTSB affect growth and the production of -galactosidase from a toxA-lacZ fusion

Function of glutamate. It was originally proposed that glutamate overcomes a deficiency of nitrogen (25). However, it is also possible that glutamate provides carbon and energy, as it does for Haemophilus influenzae, which lacks several citric acid cycle enzymes (11). To distinguish between these possibilities, we examined the effects of several citric acid cycle intermediates on the growth of iron-restricted cells (Table 1). Stimulation of growth by nonnitrogenous intermediates would imply that iron restriction imposes a carbon or energy limitation. Isocitrate and citrate increased the growth yield almost as well as glutamate (Table 1). Malate and -ketoglutarate stimulated growth but not as well as glutamate, whereas succinate did not affect growth (Fig. 2 and Table 1). None of these supplements altered the growth rate (Fig. 2). Since nonnitrogenous compounds stimulated growth, we conclude that glutamate primarily provides carbon or energy. The fact that glutamate is the best growth supplement suggests either that the provision of nitrogen stimulates growth slightly or that glutamate is transported more efficiently than the other compounds.

Iron limitation and aconitase activity. Since some citric acid cycle intermediates, but especially citrate, isocitrate, and glutamate, enhanced growth, we suspected that iron limitation inactivated a citric acid cycle enzyme that is required for the synthesis of -ketoglutarate, the precursor for glutamate. Therefore, we examined the effects of iron limitation on two citric acid cycle enzymes, aconitase and isocitrate dehydrogenase (Fig. 3).

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Effects of iron restriction on aconitase (A) and isocitrate dehydrogenase (B) activities over time in PA103 cultures grown in iron-limited DTSB or DTSB with 100 µM FeSO4.

Reciprocal relationship between aconitase activity and ETA synthesis. Iron availability affects both aconitase activity and ETA synthesis. Therefore, we examined how closely these two activities correlated as a function of exogenous iron. Cells were grown for 14 h and then assayed for aconitase and -galactosidase from PA103-CM1. A reciprocal correlation between aconitase and -galactosidase activities was readily apparent (Fig. 4A). Replotting -galactosidase activity as a function of the reciprocal of aconitase activity shows a linear relationship, which breaks down only when -galactosidase activity is nearly maximal (Fig. 4B). In other words, aconitase activity is low when toxA is expressed.

View larger version (15K): [in this window] [in a new window]   FIG. 4.   Transcription of toxA inversely correlates with aconitase activity in PA103. (A) Aconitase activity () and toxA expression (-galactosidase activity from a strain with a toxA-lacZ fusion) () in cultures of PA103-CM1 grown for 14 h in increasing concentrations of FeSO4. (B) Replot of the data from panel A, showing the inverse correlation between aconitase activity and toxA transcription.

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Isocitrate stabilizes aconitase and reduces toxA expression. (A) Aconitase activities of PA103-CM1 cultures grown in DTSB supplemented with 50 mM glutamate () or isocitrate (). (B) -Galactosidase activity of cultures from panel A.

A metabolic block in the citric acid cycle is not sufficient to induce ETA synthesis. A possible explanation for the reciprocal relationship between aconitase activity and ETA synthesis is that an iron limitation-dependent metabolic block in the citric acid cycle is sufficient to stimulate ETA synthesis. To test this possibility, we examined the effect of fluorocitrate, an aconitase-specific inhibitor, on ETA synthesis. Aconitase converts fluorocitrate to fluoro-cis-aconitate and then to 4-hydroxy-trans-aconitate, which binds tightly, but not covalently, to aconitase. The latter intermediate inhibits aconitase activity. At the same time that 4-hydroxy-trans-aconitate inhibits aconitase, it also stabilizes the [4Fe-4S] cluster and thus prevents aconitase's conversion into the enzymatically inactive [3Fe-4S] form (23). The inhibition by 4-hydroxy-trans-aconitate is reversible; therefore, when cell extracts are prepared and aconitase is saturated with substrate, aconitase activity can be measured. This leads to the apparently paradoxical situation in which an increase in aconitase activity from a crude extract, which results from stabilization of the [4Fe-4S] cluster, implies a proportional inhibition of aconitase activity in vivo. If fluorocitrate stimulates ETA synthesis, we would conclude that a metabolic block induces ETA synthesis. This result was not observed.

View larger version (17K): [in this window] [in a new window]   FIG. 6.   Fluorocitrate stabilizes aconitase and inhibits toxA expression. PA103-CM1, which contains a chromosomal toxA-lacZ fusion, was grown for 10 h in DTSB supplemented with citrate. The cells were then harvested and resuspended in prewarmed DTSB supplemented with either 2 mM citrate or 2 mM fluorocitrate. These cultures were grown for 4 h and then assayed for -galactosidase and aconitase activities. The data represents the averages of two independent experiments with each assay done in triplicate, with the standard deviation for each also presented.

Evidence for posttranslational aconitase inactivation. Two explanations could account for the diminished aconitase activity from iron-restricted cells. First, iron depletion could prevent aconitase synthesis, and subsequent cell growth could dilute aconitase activity. Alternately, iron restriction could posttranslationally inactivate aconitase by loss of iron from its essential [4Fe-4S] cluster. If iron depletion reversibly inactivates aconitase, then addition of iron to iron-limited cells would reactivate aconitase. To test this possibility, we incubated cells in the presence of a protein synthesis inhibitor, which should prevent an increase in newly synthesized aconitase. Figure 7B shows the growth of iron-limited cells to which exogenous iron was added. Without gentamicin, growth resumed at a faster rate, which implies that iron is being transported into the cell under the conditions of the experiment. With gentamicin, growth ceased, which implies substantial, although perhaps not complete, inhibition of protein synthesis. Under these conditions, exogenous iron increased aconitase activity fourfold (Fig. 7A).

View larger version (15K): [in this window] [in a new window]   FIG. 7.   In vivo reactivation of aconitase. (A) PA103 was grown in iron-limited DTSB for 6 h (A600 of ~2.0), at which time the culture was split into four equal volumes. Two of the cultures were treated with 20 µg/ml of gentamicin, (Gm), and two were untreated. The cultures were incubated for an additional 15 min, and then one treated and one untreated culture were supplemented with Fe(NH4)2(SO4)2 to a concentration of 100 µM. The cultures were incubated for an additional 45 min, harvested, and assayed for aconitase activity. Without gentamicin, the cell density increased by about 15% without iron and 30% with iron. The data represent the averages from two independent cultures done on different days. The assays were done in triplicate, with error bars indicating the standard deviations. (B) Growth of iron-limited PA103 with () or without gentamicin (). Both cultures were supplemented with 100 µM Fe(NH4)2(SO4)2 15 min after addition of gentamicin to one culture.

	DISCUSSION

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Glutamate and virulence factor synthesis. Our primary objective was to analyze glutamate's effect on ETA synthesis, which was first observed 25 years ago (25). We found that glutamate delayed the entry of iron-limited cells into stationary phase. Several citric acid cycle intermediates, especially citrate and isocitrate, had a similar effect. These results suggested that iron limitation impaired citric acid cycle metabolism, and we found that iron limitation specifically reduced aconitase activity. The growth stimulation by citrate, which is the substrate for aconitase, does not contradict this conclusion, since citrate may either stabilize aconitase or increase the reaction rate of residual aconitase.

Aconitases in E. coli and P. aeruginosa. E. coli has two, and possibly three, aconitases. AcnB is the major aconitase during exponential growth (16). A second aconitase, AcnA, is induced during stress in stationary phase, and genetic evidence suggests that its induction requires iron (17). A double mutant without AcnA and AcnB still has residual activity, which suggests that there may be a third aconitase (16). Analysis of the partially complete P. aeruginosa genome sequence suggests that there are three aconitase-specifying genes: two are homologous to E. coli acnA, and one is homologous to E. coli acnB. The multiplicity of aconitases can explain some of the results presented in this report if the P. aeruginosa aconitases are similar to the E. coli enzymes. For example, the 12-fold difference in total aconitase activity between iron-replete and iron-limited cells might be the result of differential effects on two different aconitases. Iron restriction might cause a fourfold reduction in the activity of the exponential phase aconitase and, in addition, failure to induce the iron-inducible stationary phase aconitase. This would also explain the extent of the apparent iron reactivation of aconitase from iron-limited cells. In the absence of the iron-inducible stationary phase aconitase, iron would be expected to increase aconitase activity only fourfold, and this is exactly what is observed (Fig. 7A).

Aconitase and virulence factor synthesis in other pathogens. The amino acid sequences of aconitases are highly conserved (15, 32), which might suggest that conditions that reduce aconitase activity in one organism probably do so in other organisms as well. Since oxidative stress, which inactivates E. coli aconitase activity (18), and iron restriction are frequently encountered by pathogens, it is possible that such environments are generally associated with a reduction in aconitase activity. If so, then the stimulatory effect of amino acids and citric acid cycle intermediates on virulence factor synthesis may be more general than is currently recognized. For example, since the same amino acids that stimulate ETA synthesis also stimulate cholera toxin synthesis in Vibrio cholerae (4), cholera toxin synthesis may also negatively correlate with aconitase activity. If this is true, then agents that stabilize aconitase, such as isocitrate and fluorocitrate, may also impede virulence factor synthesis in other pathogens.

Iron, aconitase, and ETA synthesis. Iron is essential for most bacteria, and complex and diverse mechanisms have evolved to sense and respond to iron restriction. Iron limitation was first implicated in microbial virulence over 50 years ago (33), and since then has been shown to be important for many pathogens.