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Nuevos aspectos del control del metabolismo en Pseudomonas putida KT2440: metabolismo del ácido fenilacético y papel del gen apaH

AutorAgulló Carvajal, Loreine
DirectorDíaz Fernández, Eduardo; Nogales, Juan
Fecha de publicación7-feb-2014
EditorCSIC - Centro de Investigaciones Biológicas (CIB)
Universidad Complutense de Madrid
ResumenAromatic compounds are widely distributed in the environment and they represent a common carbon source for many microorganisms. The metabolic pathways for microbial degradation of aromatic compounds and the regulatory circuits that control their expression have been widely studied in the last years. Although most of the studies on the regulation of aromatic degradation pathways have focused on the transcriptional control of the cognate catabolic genes (transcriptional regulation), the posttranscriptional control of these genes is also a major regulatory checkpoint (Díaz y Prieto, 2000; Prieto et al., 2004; Rojo y Dinamarca, 2004; Tropel y van der Meer, 2004). When faced with a mixture of alternative carbon sources, bacteria usually strategically utilize the substrate that yields the highest energy return. The utilization of preferred carbon sources is controlled by global regulatory mechanisms, termed carbon catabolite repression, which depend on the energy status of the cell. This enables bacteria to optimize their growth rates in natural environments that provide complex mixtures of nutrients. The mechanisms that undertake catabolite repression are diverse and they have been well characterized in bacteria such as Escherichia coli and Bacillus subtillis (Deutscher et al., 2006; Park et al., 2006), but they are less well characterized in other bacteria of great environmental relevance as those of the Pseudomonas genus (Rojo, 2010). The repression that some aromatic compounds exert on the catabolism of other aromatic compounds that use different peripheral routes is a topic of great interest that has not been deeply studied so far. Bacteria use two basic strategies, that generally coexist in the same cell, for the aerobic degradation of aromatic compounds, i) diverse aerobic classic pathways, e.g., the β-ketoadipate pathway, which involve catecholic intermediates, and ii) some aerobic hybrid pathways, e.g., the phenylacetic acid (AFA) and the benzoyl-CoA pathways, that involve CoA-derived aromatic epoxydes as intermediates. Although there has been argued that classic and hybrid aerobic degradation routes might be profitable when bacteria drive in habitats with high and low oxygen availability, respectively, the biological significance of these two basic types of catabolism and the possible preference of one type over the other is unknown so far.
The few studies on the bacterial utilization of mixtures of aromatic compounds have focused on compounds that are degraded through aerobic classic pathways. Thus, in Acinetobacter baylyi (Bleichrodt et al. 2010), Cupriavidus necator JMP134 (Donoso et al., 2011), and in the model bacterium Pseudomonas putida (Nichols y Harwood, 1995), benzoate was shown to be a preferred carbon source over 4-hydroxybenzoate, although both compounds are degraded via the same β-ketoadipate central pathway. However, the possible cross-regulation between an aerobic hybrid route and an aerobic classic route had not been reported. Getting some light on this issue could contribute to understand why the specific hybrid routes co-exist with the more diverse and extended classic routes. In this thesis, we approach for the first time a study on the effect of the AFA pathway, a paradigm of hybrid pathway, over the metabolism of other aromatic carbon sources that use classic routes, such as the widely distributed β-ketoadipate intradiolic route (Harwood y Parales, 1996) or the gallic acid (GA) extradiolic route (Nogales, 2009). These studies, which constitute the first thematic chapter of this thesis, have been fundamental to reveal for the first time the key role of the apaH gene (that encodes the major hydrolase of the Ap4A allarmone) in the control of the bacterial metabolism, which constitutes the topic of the second thematic chapter of this thesis. All these studies have been carried out in P. putida KT2440 because this bacterium is considered to be a model of metabolic versatility and robustness, especially regarding to the catabolism of aromatic compounds, its physiology, biochemistry and genetics is well known, and a genome scale metabolic model (iJN1411) is available to integrate and understand omic-based studies. Taken these assumptions into account, the major objectives of this work were the following: OBJECTIVES I. To study the cross-regulation between a hybrid aerobic pathway, i.e., the phenylacetate (AFA) pathway, and classical aerobic pathways for the degradation of other aromatic compounds, e.g. 4-hydroxybenzoate (4HBA) and gallate (GA) in P. putida KT2440. II. To explore the physiological effects of the inactivation of the apaH gene, which encodes the major Ap4A hydrolase, in P. putida KT2440ΔapaH. III. To determine the molecular basis that accounts for the perturbations at the morphological, metabolic and stress programmes in P. putida KT2440ΔapaH.
IV. To study the signalling/regulatory network that controls the atypical behaviour of P. putida KT2440ΔapaH. RESULTS AND DISCUSSION To check the possible cross-regulation between a hybrid pathway and a classical aerobic pathway for degradation of aromatic compounds, we selected the AFA pathway and the highly distributed b-ketoadipate pathway, respectively. When P. putida KT2440 grows in the presence of AFA and 4HBA, both aromatics are co-metabolized. Although 4HBA becomes metabolized first, this degradation accelerates the initial consumption of AFA. Furthermore, the metabolism of AFA affects negatively the 4HBA consumption. The repressor effect of AFA was confirmed by showing the inhibition of the two key enzymes of the peripheral and central 4HBA degradation pathway, i.e. the 4-hydroxybenzoate-3-monooxygenase (PobA) and the protocatechuate 3,4-dioxygenase (PcaGH), respectively. The AFA effect was carried out at the transcriptional level since the expression of the pob and pca operons involved in 4HBA degradation was reduced when P. putida KT2440 grew in the AFA/4HBA mixture. Interestingly, neither the 4HBA transporter (PcaK) nor the transcriptional activator of the pob genes (PobR), which were shown previously to mediate benzoate-mediated repression of 4HBA, were involved in the AFA effect. Blockage of the AFA pathway by disruption of the gene encoding the first enzymatic step alleviates the repression effect on the 4HBA pathway, suggesting that the latter requires the efficient metabolism of AFA. The repressor effect of AFA was also tested with other aerobic classic pathways such as the extradiolic route for GA degradation. Again, the presence of AFA repressed the expression of the gal genes involved in GA degradation. All these results taken together confirm for the first time that the AFA hybrid pathway causes repression of other classical aerobic degradation pathways in P. putida. The iJN1411 metabolic model confirmed in silico that AFA and other aromatics degraded via phenylacetyl-CoA are indeed carbon sources preferred to aromatics degraded via aerobic classic pathways, which agrees with the previous observation that P. putida KT2440 is highly adapted to the efficient metabolism of compounds that use b-oxidation like mechanisms, as is the case of AFA. Random mini-Tn5 transposon mutagenesis was used to isolate five mutant strains that do not present repression of gal genes in the presence of AFA. One of these mutants, P. putida KTGAL-14, contained the mini-Tn5 transposon inserted within a putative operon containing the apaH gene. ApaH is the major hydrolase of the allarmone diadenosine tetraphosphate (Ap4A) and it catalyzes the symmetric cleavage of this molecule into two ADP molecules. This result constituted the first report showing the involvement of the apaH gene controlling the metabolism of aromatic compounds in bacteria. Since the lack of the apaH gene had not been described so far in P. putida, a P. putidaΔapaH strain was constructed. Several apaH-related phenotypes that had been previously described in other bacteria were tested in P. putida KT2440ΔapaH. Although the inactivation of the apaH gene did not cause a significant change in the cellular morphology of P. putida KT2440, this mutant strain showed a reduced motility, decreased production of the siderophore pyoverdine, increased biofilm formation, and increased sensitivity to oxidative stress. However, the most remarkable and novel property of the P. putida KT2440ΔapaH strain was its increased lag phase when growing in different carbon sources, especially when using aromatic compounds such as AFA.
To explore for the first time at the molecular level the global effect of the lack of a functional apaH gene in bacteria, we compare the total proteome of the wild-type P. putida KT2440 strain with that of the P. putida KT2440ΔapaH mutant strain growing in AFA. Remarkably, the apaH mutant strain showed a significant number of proteins whose expression was induced or repressed, and that are potentially involved in all three major cellular programs, i.e., in the morphological, metabolic and stress programs. Therefore, these results highlight a previously unnoticed essential role of the Ap4A molecule controlling many diverse functions in the bacterial cell. Among the most induced proteins in the P. putida KT2440ΔapaH mutant, there is a group of proteins, such as catalases and alkyllhydroperoxydases, usually present in bacteria under oxidative stress conditions, strongly suggesting that inactivation of the apaH gene and, consequently, an increase in the Ap4A levels, triggers an oxidative stress-like cellular response. Some c-diGMP synthases and cell envelope proteins, such as adhesins, are also induced in the apaH mutant, which is in agreement with the observed increase in biofilm formation and may reflect the low fitness state of the mutant. However, the most abundant set of proteins whose expression changes in the apaH mutant are those related to the metabolic program. Thus, some TCA cycle isoenzymes become induced in the apaH mutant, revealing an unprecedented role of Ap4A remodeling the central metabolism in bacteria. Oxidative phosphorylation, iron uptake systems, amino acids metabolism, PHA and protein synthesis, and several signal transduction systems and RNA regulators, as the Hfq protein, are also likely altered in the mutant strain. An interesting observation is that a proposed trealose (and glycogen) metabolism was induced in apaH mutant, which suggests the activation of an osmotic stress response. Accordingly, the apaH mutant strain showed a higher resistance to a NaCl-dependent osmotic stress than the wild-type strain, which represents an apaH related phenotype that had not been described before in any bacteria. By using the iJN1411 model and the proteomic data obtained from the P. putida wild-type and apaH mutant strains growing in AFA, we constructed the cognate condition specific genome scale metabolic models that were then used to predict carbon fluxes throughout the different metabolic modules. This in silico analysis predicted that the flux throughout the TCA cycle was significantly reduced in the apaH mutant strain, especially regarding the enzymatic steps catalyzed by aconitase, citrate synthase succinate dehydrogenase and fumarase, which is in an agreement with the observed induction of unusual TCA isoenzymes in the proteome. Flux through the electron transport chain and oxidative phosphorylation was also significantly reduced in the apaH mutant strain, a prediction that matches the observed repression of the NADH dehydrogenase I complex in the proteome. All these analyses of the distribution of carbon fluxes pointed to a compromised energetic state in the P. putida KT2440ΔapaH strain. This hypothesis was confirmed experimentally by showing that the mutant strain had a lower oxygen consumption and ATP levels than the wild-type strain. Moreover, by using a respiratory uncoupling agent (FCCP) we could conclude that the respiratory flux was limited by reduced NAD(P)H levels in the P. putida KT2440ΔapaH strain, which in turn would explain the extended lag phase of the mutant in most carbon sources. This was confirmed later on by showing that the long lag phase of P. putida KT2440ΔapaH grown in AFA was significantly reduced by adding to the medium some minor amounts of compounds, e.g., octanoate, glucose, that do not need the TCA cycle for generating some reducing power.
By using the iJN1411 metabolic model and the measured lag phase of the apaH mutant in different carbon sources, we could predict in silico that fumarase was the limiting step that caused blockage of the TCA cycle. By measuring the FumA and FumC activity in crude extacts of the wild-type and apaH mutant strains, we were able to confirm the proteomic data that revealed the replacement of the standard FumA activity by the stress-induced FumC activity in P. putida KT2440ΔapaH. Gene expression studies also confirmed the induction of fumC2 and the repression of fumA in the apaH mutant strain. Taken together, all these data suggest that the increased levels of Ap4A in P. putida KT2440ΔapaH caused an inhibition of the standard fumarase activity which led to a certain blockage of the TCA cycle and, in turn, to a compromised energetic state that explains the extended lag phase observed as well as the relevant changes detected at the morphological and stress programs. To gain some insights on the regulatory circuit(s) that was altered in P. putida KT2440ΔapaH, we analyzed the expression of some RNA polymerase sigma factors that usually control the stress response in bacteria. These studies revealed that rpoS expression was significantly increased in the apaH mutant strain. Moreover, the overexpression of rpoS in P. putida KT2440 led to the characteristic extended lag phase of this strain. Therefore, it was concluded that RpoS was a central player in the regulatory cascade that senses and respond to the Ap4A cellular levels. To study further this regulatory circuit, we monitored the expression of different regulatory elements, i.e., psrA, crc, and rsm genes, that have been reported to control the expression of rpoS in bacteria. Interestingly, in the apaH mutant we only found a significant change in the expression levels of the rsmX and rsmY sRNAs, which have been shown to activate the expression of rpoS by sequestering the RsmA repressor through a post-transcriptional regulation mechanism (Kojic y Venturi, 2001; Dong et al., 2013; Heeb et al., 2005; Whistler et al., 1998; Sonnleitner y Haas, 2011). Since the rsm sRNAs are controlled by the global GacS/GacA two-component regulatory system in some Pseudomonas strains, we checked whether this regulatory cascade was also controlling the P. putida KT2440ΔapaH behaviour. To this end, we constructed a double gacA/apaH P. putida KT2440 strain, and we could confirm that this strain lacked the extended lag growth phase that characterizes the apaH mutant. In summary, all these results suggest a new mechanism of action of Ap4A acting as a signal of the pleiotropic GacS/GacA transduction system.
Descripción235 p.-73 fig.-10 tab.-1 tab suppl.
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