Pseudomonas fluorescens orchestrates a fine metabolic-balancing act to counter aluminium toxicity


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Aluminium (Al), an environmental toxin, is known to disrupt cellular functions by perturbing iron (Fe) homeostasis. However, Fe is essential for such metabolic processes as the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, the two pivotal networks that mediate ATP production during aerobiosis. To counter the Fe conundrum induced by Al toxicity, Pseudomonas fluorescens utilizes isocitrate lyase and isocitrate dehydrogenase-NADP dependent to metabolize citrate when confronted with an ineffective aconitase provoked by Al stress. By invoking fumarase C, a hydratase devoid of Fe, this microbe is able to generate essential metabolites. To compensate for the severely diminished enzymes like Complex I, Complex II and Complex IV, the upregulation of a H2O-generating NADH oxidase enables the metabolism of citrate, the sole carbon source via a modified TCA cycle. The overexpression of succinyl-CoA synthetase affords an effective route to ATP production by substrate-level phosphorylation in the absence of O2. This fine metabolic balance enables P. fluorescens to survive the dearth of bioavailable Fe triggered by an Al environment, a feature that may have potential applications in bioremediation technologies.

Al toxicity and impact on essential metal nutrients

Although Al is the most widespread metal in the earth's crust, it is not known to be involved in any biological function yet (Soni et al., 2001). Thus, it seems that organisms may have deliberately circumvented the incorporation of this metallic element during evolution. This is not surprising as Al interferes with a variety of biological processes due to its ability to mimic numerous essential cellular metals such as Fe, Ca and Mg. Ca2+-mediated signalling pathways are markedly perturbed by Al (Mundy et al., 1997). In trace concentrations, this trivalent metal has been shown to interfere with the protein kinase C-mediated pathways (Quarles et al., 1994), cAMP homeostasis (Hartle et al., 1996) and glutamate-nitric oxide synthase-cGMP signalling network (Lajeunesse et al., 1998). ATP stabilization and membrane dynamics catalysed by Mg is also known to be affected by Al (Nayak, 2002). This interference in the homeostasis of these two essential divalent metals has severe implications on cellular metabolism.

Such Al-triggered perturbations of living systems have become a concern due to the enhanced bioavailability of this trivalent metal (Yokel and McNamara, 2001). Industrial pollution coupled with the widespread use of Al in a variety of consumer and medical products have indeed made this toxin a public health threat. Although the exact molecular details responsible for the toxicity of Al still need to be delineated, its involvement in a variety of diseases has been documented. We have recently shown how Al toxicity leads to enhanced lipid production and accumulation in hepatocytes, liver cells responsible for a variety of metabolic processes (Mailloux et al., 2006). Its ability to perturb cellular morphology in astrocytes has also been demonstrated (Lemire et al., 2009). These cells are critical for the proper functioning of the brain. This is important as the star-shaped astrocytes are essential for normal cerebral functions. The inability of astrocytes to interact with neurons due to Al toxicity may be a cause to various neurological abnormalities associated with Al (Nayak, 2002).

Al interferes with Fe homeostasis

As Al and Fe share numerous common physicochemical features, it is not surprising that Fe metabolism is severely affected during Al stress (Morgan and Redgrave, 1998). Al is known to impede proteins/enzymes that are dependent on Fe to function in an effective manner due to theirsimilar trivalent state; Al can readily substitute for Fe especially in environments where the former is easily accessible (Perez et al., 1999). However, its inability to undergo any redox manipulation compels Al to be associated with these essential biomolecules; hence, rendering them ineffective (Zatta et al., 2003; Middaugh et al., 2005). The substitution of Fe by Al leads to a dangerous intracellular event, namely the generation of an oxidative environment (Exley, 2004; Beriault et al., 2007). The free Fe can react in the biological milieu via the Fenton reaction to create a variety of reactive oxygen species (ROS) that can indeed initiate numerous cellular dysfunctions (Caillet et al., 2007).

Hence, it is clear that a dysfunctional Fe homeostasis is an important route via which Al can exert its toxic impact (Fig. 1). Although most organisms succumb to this assault, some living systems are known to elaborate intricate strategies to fend against the dangers associated with Al toxicity. Plants secrete such organic acids as citrate, malate and oxalate to sequester Al in an effort to minimize the negative influence of this metal on Fe homeostasis (Morita et al., 2008). The elimination of Al as silicate derivatives has also been documented (Epstein, 1999; Teien et al., 2006). Microbial systems are also known to devise various stratagems to nullify the toxicity of Al. The immobilization of the metal in the cell wall, its sequestration into exopolysaccharides, its entrapment intracellularly and its complexation with siderophores enable microbes to survive Al stress (Cervantes and Silver, 1996; Cornelis, 2008). The latter strategy may indeed further interfere with Fe metabolism. Although some of the detoxification pathways involved in rendering Al innocuous are known, the molecular mechanisms detailing how these systems adapt to the dearth of bioavailable Fe and the ineffective Fe proteins/enzymes provoked by Al toxicity have not been fully delineated. In this report, we provide a global view on the different molecular stratagems invoked by Pseudomonas fluorescens to fend the Fe conundrum evoked by an Al challenge.

Figure 1.

A link between Al toxicity and Fe homeostasis. ACN, aconitase; SDH, succinate dehydrogenase; FUM A, fumarase A; ETC, electron transport chain; TCA cycle, tricarboxylic acid cycle.

This soil microbe, with a very versatile nutritional habit, has tremendous biotechnological potential and is utilized in numerous commercial processes. As Al perturbs Fe homeostasis, P. fluorescens has to elaborate alternative pathways to deal with this dilemma. These molecular mechanisms conferring tolerance to Al will help pave the way for the application of P. fluorescens in the decontamination of this toxic metal. As organic acids are involved in the mobilization of metals in general and Al in particular, the tricarboxylic acid (TCA), citrate is an excellent candidate to probe the interaction between Al and P. fluorescens. The metabolic adaptations aimed at circumventing the dysfunctional Fe homeostasis evoked by Al are also discussed.

Disruption of Fe-S cluster by Al and citrate metabolism

The TCA cycle and the aerobic formation of ATP are heavily reliant upon Fe. Aconitase (ACN), succinate dehydrogenase (Complex II), fumarase A (FUM A), Complex I, Complex II, Complex III and Complex IV all require Fe in order to fulfil their biological function (Fig. 2). In these enzymes, Fe is usually associated as haem or sulfur clusters (Fontecave and Ollagnier-de-Choudens, 2008). Aconitase and FUM A require a 4Fe-4S cluster in the active site in order to catalyse the reversible hydration of citrate and fumarate respectively. Complexes I, II, III and IV require Fe-S and/or haem to safely transfer electrons through the respiratory chain. A perturbation in the availability of Fe is known to severely affect these proteins. Genetic defects in Fe synthesis and Fe transport have been linked to severe deficiencies in energy metabolism and aerobic ATP production (Heidari et al., 2009).

Figure 2.

Al-triggered disruption of Fe proteins involved in aerobic-ATP production in P. fluorescens (Hamel and Appanna, 2001; Middaugh et al., 2005; Beriault et al., 2007; Singh et al., 2007). ACN, aconitase; SDH, succinate dehydrogenase; FUM A, fumarase A; ETC, electron transport chain; TCA cycle, tricarboxylic acid cycle.

The exposure of P. fluorescens to Al results in the loss of the Fe-S cluster integrity. Perturbation of this moiety leads to ineffective ACN, an enzyme that catalyses the reversible isomerization of citrate to isocitrate through a cis-aconitate intermediate. The ability of Fe salts to restore the activity of this enzyme clearly provided further evidence that Al does indeed trigger abnormal Fe metabolism. The expression of ACN does not seem to be affected. Hence, Al renders ACN ineffective by disturbing the interaction of Fe with ACN. The disruption of ACN by Al has also been shown in a variety of other cellular models (Mailloux et al., 2006). However, this situation forces the organism to devise alternative routes for citrate degradation, when this TCA is the sole carbon source. As citrate lyase is not evident in these cultures, the microbe is compelled to utilize the TCA cycle despite a severely diminished ACN. To help solve this dilemma, P. fluorescens drastically upregulates the two downstream enzymes, namely isocitrate lyase (ICL) and isocitrate dehydrogenase-NADP (ICDH-NADP) that act on isocitrate. While the activity of ICL was increased 6–8-fold, ICDH-NADP was augmented two-fold. The Al-stressed cells are also characterized by an isoenzyme of ICDH-NADP. This arrangement helps facilitate the degradation of citrate even though ACN is able to only minimally isomerize the citrate (Fig. 4). Any isocitrate formed is rapidly degraded into glyoxylate, succinate and α-ketoglutarate. Such a molecular stratagem allows for a rapid turnover of the substrate and the products (Beriault et al., 2007; Mailloux et al., 2007). Indeed, the physical proximity of numerous enzymes referred to as metabolon is known to promote the rapid metabolism of a desired substrate (Alvarez et al., 2005). In this instance, ICDH-NADP appears to be in close association with ACN as activity bands of these enzymes are in very close proximity (Middaugh, 2004). Even though the activity of ACN was drastically reduced in the Al-stressed cells, its association with ICDH-NADP may allow for the metabolism of citrate.

Figure 4.

Al-induced metabolic adaptation to counter Fe deprivation in P. fluorescens. inline image = increase in activity, inline image = decrease in activity. OCT, oxalyl-CoA transferase; AGODH, acylating glyoxylate dehydrogenase; SCS, succinyl-CoA synthase. Figure adapted from Mailloux and colleagues (2007); Chenier and colleagues (2008); Singh and colleagues (2009).

It is also important to note that the production of NADH is markedly decreased in P. fluorescens subjected to Al. Hence, ICDH-NAD and α-ketoglutarate deydrogenase (KGDH) are severely impeded when P. fluorescens is cultured in Al (Mailloux et al., 2007) (Fig. 3). FUM A is another key enzyme of the TCA cycle that has an Fe-S cluster in its active site. This enzyme is drastically reduced in Al-stressed cells. However, this situation is reversed when the microbe is grown in Al medium with added FeCl3 (Chenier et al., 2008). We have also shown that gallium, an Fe-mimetic, impedes the activity of this enzyme. To survive such a situation, the organism invokes the expression of FUM C, an Fe-independent enzyme (Park and Gunsalus, 1995; Chenier et al., 2008). This switch to a FUM devoid of Fe is in sharp contrast to the strategy utilized to adapt to an ineffective ACN. In numerous cellular systems, ACN with oxidized Fe-S, known as the iron-responsive protein is a post-transcriptional regulator that signals an oxidative environment (Cairo et al., 1998; Hantke, 2001). It is also quite possible that ACN with an improperly organized Fe centre has a signalling role. Hence, by upregulating ICL and ICDH-NADP dependent and invoking FUM C, an enzyme devoid of Fe, P. fluorescens is able to metabolize citrate via a modified TCA cycle.

Figure 3.

Enyzmatic profile of P. fluoresccens cultured in control and Al-stressed media. Activities are shown for inline image = ACNa, inline image = NAD-ICDHb, inline image = KGDHb, inline image = SDHc, inline image = FUMa, inline image = Complex Id, inline image = ICLb, inline image = MSb, inline image = MDHe, inline image = CSe.
a. double bonds were measured at 220 nm. 100% corresponds to 124 ± 10 nmol x min−1 mg X protein−1 for ACN and 3.0 ± 0.2 nmol • min−1• mg • protein−1 for FUM.
b.α-ketoglutarate and glyoxylate levels were measured using DNPH at 450 nm. 100% corresponds to 0.134 ± 0.033 µmol • min−1• mg • protein−1 for NAD-ICDH, 0.134 ± 0.033 µmol • min−1• mg • protein−1 for KGDH, 0.134 ± 0.033 µmol • min−1• mg • protein−1 for ICL, and 0.134 ± 0.033 µmol • min−1• mg • protein−1 for MS.
c. activity monitored at 600 nm using DCIP. 100% corresponds to 51 ± 4.1 nmol • min−1• mg • protein−1.
d. activity was assessed by quantifying BN-gel activity bands using Scion imaging software. 100% corresponds to 5528 arbitrary units.
e. activities were assessed via the production of NADH at 340 nm. 100% corresponds to 11 ± 2 nmol • min−1• mg • protein−1 for MDH and 24 ± 5 nmol • min−1• mg • protein−1. The activities of the controls are taken as 100%.
Figure adapted from (Middaugh et al., 2005; Beriault et al., 2007; Mailloux et al., 2007)

The electron transport chain and NADH metabolism

The electron transport chain (ETC) is composed of four respiratory complexes which are required to shuttle electrons from NADH and FADH2 to the terminal electron acceptor, the diatomic oxygen. The favourable transfer of the electron through the complexes is usually coupled to the formation of a proton gradient which is tapped by Complex V to produce ATP (Boyer, 1997; Fosslien, 2001). These respiratory complexes are dependent on Fe to perform the task of moving electrons to O2 with the concomitant generation of a proton motive force. This dependence on Fe makes these complexes an easy target during Al stress. When exposed to Al, Complex I, II, IV are severely impeded in P. fluorescens (Zatta et al., 2003; Hamel et al., 2004; Middaugh, 2004; Beriault et al., 2007; Chenier et al., 2008). Although Complex II does function albeit ineffectively, Complex IV appears to be almost absent in the cells subjected to Al. The activity band for this enzyme is barely discernable. The ETC has been shown to be affected in other organisms exposed to Al toxicity (Yamamoto et al., 2002). To effect the metabolism of citrate via the TCA cycle, it is essential that the limited NADH that is generated be oxidized. It appears that Al triggers the induction of NADH oxidase (NOX)-H2O generating in P. fluorescens (Chenier et al., 2008). This adaptation enables the oxidation of NADH with the concomitant generation of H2O (Chenier et al., 2008). The NAD produced is critical for the functioning of the modified TCA cycle and the utilization of citrate, the sole carbon source as the enzymes α-KGDH and malate dehydrogenase (MDH) requires this nicotinamide nucleotide (Fig. 4).

The TCA cycle is a source of ATP during Al stress

Al is usually referred to as a pro-oxidant due to its ability to increase the free Fe-burden of the cell (Exley, 2004; Beriault et al., 2007). This situation leads to increased ROS production. Hence, it is essential that if an organism is to survive Al toxicity, it has to limit the further generation of ROS. During aerobic respiration, the majority of ROS is formed when electrons from NADH and FADH2 are transported via the ETC to the O2. Hence, to limit this danger, the two key enzymes of the TCA cycle (ICDH-NAD and KGDH) involved in the production of NADH are sharply downregulated in the Al-stressed cells (Mailloux et al., 2007). However, this situation will impede the production of ATP via oxidative phosphorylation. Furthermore, formation of ATP promoted by the ETC is not favoured during Al stress due to the diminished bioavailability of Fe. To circumvent this problem, P. fluorescens elaborates an intricate system involving succinyl-CoA synthetase (SCS) and oxalate-CoA transferase (OCT) that work in tandem to produce ATP via substrate level phosphorylation (Singh et al., 2009) (Fig. 4). This metabolic manipulation not only fulfils the energy requirement, but also enables the microbe to synthesize oxalate, a key component that helps sequester Al.

Concluding remarks

Hence, this fine metabolic-balancing feat allows P. fluorescens to mitigate the Fe dysfunction triggered by Al toxicity. Even though ACN, a key critical enzyme of the TCA cycle is severely impeded, the microbe invokes the participation of ICL and ICDH, two downstream partners to facilitate the degradation of citrate. The upregulation of the NOX with the attribute of liberating H2O provides an effective route to regenerate NAD, a moiety pivotal to the functioning of the modified TCA cycle. As oxidative phosphorylation is hampered, ATP is synthesized via a substrate-level phosphorylation module orchestrated by SCS and OCT. This global view into the adaptation of P. fluorescens in response to the dysfunctional Fe homeostasis provoked by Al reveals the intriguing role metabolic networks play in enabling organisms survive environmental fluxes. In this instance, the microbe modifies its TCA cycle and the electron transport system to survive the dearth of bioavailable Fe triggered by an Al environment. This metabolic shift provides ATP, NADPH and oxalate; ingredients that help mediate the survival of P. fluorescens challenged by Al toxicity (Fig. 4). These molecular insights in metabolic adaptation may help in the development of bioremediation technologies for Al and other metal pollutants.


We thank Industry Canada, Human Resources Canada, NATO, Northern Ontario Heritage Fund, Ontario Centers of Excellence and the Ministry of Training, Colleges and Universities for funding research activities in our laboratory.