The transporter AatJQMP has a fundamental role in the utilization of L-glutamate and L-glutamine in P. aeruginosa PAO1
The genome of P. aeruginosa PAO1 encodes for three putative glutamate transporters: the ABC-transporter complex AatJQMP (PA1339 – PA13342), the sodium-glutamate symporter GltS (PA3176), and the proton-glutamate symporter GltP (PA5479). Deletion of the aatJ or aatQM genes in P. aeruginosa PAO1 significantly reduced the growth of this bacterium on L-glutamate as either the sole source of nitrogen or carbon (Figs. 2a and 3a); although as a sole carbon source, growth was more severely hindered. Similar growth deficiencies on L-glutamate have been observed for aatQMP-transposon mutants of P. aeruginosa PAK [26]. It would appear that the importance of AatJQMP in L-glutamate utilization is not restricted to PAO1, but is likely common among other various strains and isolates of P. aeruginosa. The roles of GltS, GltP and other dicarboxylate-transport proteins in L-glutamate utilization have yet to be resolved, but the presence of these transporters would explain the lingering or residual growth observed for the ΔaatJ and ΔaatQM mutants of P. aeruginosa PAO1 on L-glutamate. Lastly, it should be mentioned that transposon insertions of the aatJ, aatQ, and aatP genes were previously reported to abolish the growth of P. aeruginosa PAO1 on D-glutamate as the sole carbon and nitrogen source [27]. This suggest that the transporter AatJQMP is not stereospecific, and thus, plays a central role in the utilization of both L- and D-isomers of glutamate in P. aeruginosa PAO1.
The aatJ-aatQMP genes were required for optimal growth of P. aeruginosa PAO1 on L-glutamine as the sole source of carbon but not nitrogen (Figs. 2b and 3b). The PA5073 – PA5076 operon of P. aeruginosa PAO1 encodes for a putative glutamine ABC-transporter complex, but a previous analysis of transposon mutants indicates that this locus is not required for growth on any of the twenty common amino acids [26]. Nonetheless, it is still plausible that L-glutamine enters the cell through PA5073 – PA5076 and then undergoes transamination and/or deamidation to yield L-glutamate. Another route for assimilation of L-glutamine involves deamidation in the periplasm catalyzed by the glutaminase-asparaginase AnsB (PA1337) [28] with the subsequent transport of the L-glutamate product via AatJQMP, GltS, GltP and/or additional dicarboxylate-transport proteins. Data from the HPLC analysis (Fig. 5b) supports the existence of this extracellular metabolic conversion. Notably, because the periplasmic deamidation of L-glutamine liberates ammonia, a readily consumable nitrogen source, the bacterium does not have a strict requirement for the uptake of L-glutamine directly or the L-glutamate product to fulfill its nitrogen demands. This would account for the unperturbed growth observed for the ΔaatJ and ΔaatQM mutants of P. aeruginosa PAO1 on L-glutamine as a source of nitrogen but not carbon (Figs. 2b and 3b); the latter of which requires the L-glutamate product as a precursor for cellular biosynthesis. A similar result was observed for P. aeruginosa PAK in which a transposon mutation of the aatM gene eliminated its growth on L-glutamine as a source of carbon but not nitrogen [26].
The transporter AatJQMP affects the utilization of L-aspartate but not L-asparagine in P. aeruginosa PAO1
Growth was observed for both the ΔaatJ and ΔaatQM mutants on L-aspartate as a nitrogen or carbon source, but the final cell densities for these mutants were 2-fold lower than that of wild-type P. aeruginosa PAO1 (Figs. 2c and 3c). This indicates that the transporter AatJQMP is required for optimal utilization of L-aspartate as a preferred or sole nutrient. In an earlier study, data from LacZ assays and microarray analysis clearly demonstrated that exogenous L-aspartate induced expression of the dctA and dctPQM genes in P. aeruginosa PAO1 [29]. These previous results suggest that the C4-dicarboxylate transporters DctA and DctPQM might facilitate the uptake of L-aspartate, and therefore, would give reason as to why the ΔaatJ and ΔaatQM mutations had a less deleterious effect in the utilization of L-aspartate compared to that of L-glutamate, a C5-dicarboxylate. For comparison purposes, neither L-aspartate nor L-glutamate induced expression of the C5-dicarboxylate transporter gene PA5530 [13, 29].
The utilization of L-asparagine is not dependent on AatJQMP in P. aeruginosa PAO1. Analogous to that of L-glutamine, one might expect that exogenous L-asparagine is deaminated in the periplasm through the actions of AnsB, and the resulting L-aspartate transported intracellularly via AatJQMP, DctA and DctPQM. However, results from our HPLC analysis indicated that very little L-aspartate (< 1.0 mM) accumulated in the extracellular milieu of P. aeruginosa PAO1 when fed an initial concentration of 20 mM L-asparagine as the sole nitrogen source (Fig. 5d). This is in sharp contrast to the > 10 mM L-glutamate that amassed in the spent medium when cells were given a starting concentration of 20 mM L-glutamine as the sole nitrogen source (Fig. 5b). In a previous study, exogenous L-asparagine was shown to induce expression of two transporter-gene lacZ fusions, PA5530-lacZ and PA2252-lacZ [29]. The PA2252 gene encodes for a putative sodium-amino acid symporter and potentially forms an operon with the ansA gene, encoding for a cytoplasmic glutaminase-asparaginase. The direct transport of L-asparagine through PA5530 and/or PA2252 followed by AnsA-catalyzed deamidation would account for the non-essential nature of the aatJ-aatQMP genes in the utilization this amino acid in P. aeruginosa PAO1.
AauR and RpoN are essential for the induction of aatJ-aatQMP in response to acidic amino acids and their amide derivatives P. aeruginosa PAO1
The results from the LacZ assays for aatJ-lacZ and aatQ-lacZ revealed several key factors surrounding the expression and regulation of the aatJ-aatQMP genes in P. aeruginosa PAO1. First, expression of both aatJ-lacZ and aatQ-lacZ were induced or upregulated in the presence of exogenous L-glutamate (Figs. 7a and 8a), suggesting that the expression of the aatJ-aatQMP genes are coordinately upregulated in response to this amino acid. Such coordinated upregulation is not limited to L-glutamate, because the presence of L-aspartate, L-glutamine and L-asparagine also induced expression of aatJ-lacZ and aatQ-lacZ (Figs. 7a and 8a). Second, the upregulation of aatJ-lacZ and aatQ-lacZ in response to these four amino acids was completely dependent on the aauR and rpoN genes. This is consistent with the EBP AauR and the sigma factor RpoN being directly involved in the activation of the aatJ-aatQMP genes in P. aeruginosa.
Third, both aatJ-lacZ and aatQ-lacZ were still expressed in the absence of (i) the aauR and rpoN genes, and (ii) exogenous L-glutamate, L-aspartate, L-glutamine or L-asparagine. These expression levels, however, were less than that of their induced states. The same appears to be true for P. putida KT2440 in which deletion of the aauR gene did not eliminate expression of an aatJ-lacZ reporter, but instead, reduced its expression by ~ 50% in response to L-glutamate [16]. Fourth and last, the expression levels of aatJ-lacZ were significantly greater (> 5-fold) than that of aatQ-lacZ under both inducing and non-inducing conditions. Collectively, these findings indicate that although the aatJ-aatQMP genes in P. aeruginosa PAO1 are coordinately upregulated by AauR-RpoN in response to certain amino acids, they are also expressed independently of these two regulatory proteins, including a potential mechanism in which the aatJ gene is expressed separately or distinctly from that of aatQMP.
The multivariate expression surrounding the aatJ-aatQMP genes of P. aeruginosa PAO1 is evident in the response of aatJ-lacZ and aatQ-lacZ to various substrates (Figs. 7 and 8). The presence of L-arginine repressed expression of aatJ-lacZ but not aatQ-lacZ whereas exogenous L-histidine induced expression of the latter and not the former. An earlier study did report that the transcript levels of aatJ in P. aeruginosa PAO1 were ~ 2-fold higher for cells grown in L-glutamate compared to L-arginine as the sole nitrogen source [27]. However, the analysis of the ArgR regulon of P. aeruginosa PAO1 revealed that the transcript levels for aatJ were not significantly different between cells grown in L-glutamate versus a mixture of L-glutamate and L-arginine [24]. Therefore, it would appear that L-arginine represses the expression of aatJ when present as the sole nutrient, i.e., in the absence of L-glutamate. ArgR was reported to repress genes associated with glutamate metabolism [24], so it possible that aatJ is also a target of negative control by this regulator. In the presence of both L-arginine and L-glutamate, expression of aatJ might be a competition between repression and activation via ArgR and AauR, respectively. Curiously, the presence of dicarboxylates such as succinate and α-KG, which are preferred carbon sources in Pseudomonas [3, 4], caused ~ 2-fold increase in the expression levels of aatJ-lacZ and aatQ-lacZ. An earlier transcriptomic study did identify the aatJ gene as a potential regulatory target of catabolite repression in P. aeruginosa PAO1 [30], and results from ChIP seq showed that the sigma factor FecI binds to the aatJ locus in P. aeruginosa PA14 [18] . Further investigation is needed to determine how such control points and regulatory factors contribute to the expression of the aatJ-aatQMP genes in P. aeruginosa.
The EBP AauR plays a pivotal role in the utilization of L-glutamate and L-glutamine as sole or preferred nutrients
Two significant growth deficiencies were observed for the ΔaauR mutant of P. aeruginosa PAO1. The ΔaauR mutant did not grow on L-glutamate as the sole carbon source (Fig. 3a), and it displayed a ~ 2-fold growth reduction on L-glutamine as the sole carbon source (Fig. 3b). The results of the genetic-complementation experiments (Fig. 6) and LacZ assays (Figs. 7b and 8b) suggest that these growth deficiencies were due to inadequate expression of the aatQMP genes. Namely, plasmid-based expression of the aatQMP genes restored the growth of the ΔaauR mutant to wild-type levels, and expression of aatQ-lacZ was downregulated by > 2-fold in the absence of the aauR gene. In comparison, plasmid-based expression of aatJ did not significantly impact or improve the growth of the ΔaauR mutant, and even though expression of aatJ-lacZ was not induced in the ΔaauR mutant, the non-induced or basal expression levels were still substantial and greater than that of aatQ-lacZ. It is AauR-mediated upregulation of the aatQMP genes, and not aatJ, which is a decisive factor in the utilization of L-glutamate and L-glutamine as sole or preferred nutrients in P. aeruginosa PAO1. Unlike the ΔaatJ and ΔaatQM mutants, the growth of the ΔaauR mutant did not differ significantly from wild-type P. aeruginosa PAO1 when L-glutamate served as the only nitrogen source (Fig. 2a) or L-aspartate served as the sole nitrogen or carbon source (Figs. 2c and 3c). The upregulation of the aatJ-aatQMP genes via AauR-RpoN is non-essential under such conditions.
The sigma factor RpoN is necessary for the utilization of acidic amino acids and their amide derivatives
The rpoN::Ω-Km mutant of P. aeruginosa PAO1 exhibited growth deficiencies on both L-glutamate and L-glutamine when either amino acid served as the sole source of carbon or nitrogen (Figs. 2 and 3). Based on the results of the LacZ assays (Figs. 7c and 8c), the observed growth deficiencies of the rpoN::Ω-Km mutant were likely caused to some degree by the downregulation of the aatJ-aatQMP genes. In addition, the rpoN::Ω-Km mutant displayed growth deficiencies on L-aspartate as either a nitrogen or carbon source (Figs. 2c and 3c). As noted earlier, the dicarboxylate-transport proteins DctA and DctPQM have been implicated in the transport of L-aspartate [29], and the expression of the corresponding dctA and dctPQM genes are dependent on RpoN [11]. The downregulation or reduced expression of the aatJ-aatQMP, dctA and dctPQM genes is expected to have a negative effect in the utilization of L-aspartate as observed for the rpoN::Ω-Km mutant. The possible role or function of RpoN in the utilization of L-asparagine is less clear. While RpoN was required for growth on L-asparagine as the sole carbon source (Fig. 3d), it was unnecessary when this amino acid was the only available nitrogen source (Fig. 2d). One potential contributing factor is the transporter PA5530, whose expression is positively regulated by both RpoN and L-asparagine [13, 29]. In contrast, the PA2252-ansA genes, which have also been associated with L-asparagine utilization [29], are neither predicted nor experimentally-proven targets of RpoN regulation in P. aeruginosa.
The growth deficiencies observed for the rpoN::Ω-Km mutant can be attributed to some extent to the downregulation of specific target genes, but it is plausible that pleiotropic effects were also an underlying factor. The RpoN regulon of P. aeruginosa is extensive - second only to that of the housekeeping sigma factor RpoD [18]. The transcription of > 500 genes are affected by RpoN [18], which regulates a consortium of diverse cellular processes such as motility [31], quorum sensing [32, 33], transport of dicarboxylates [11, 13], catabolite repression and nitrogen assimilation [34, 35]. Consequently, the deregulation of core metabolic and assimilatory pathways is expected to hinder or limit the overall growth of the rpoN::Ω-Km mutant. The expression patterns of aatJ-lacZ (Fig. 7c) and aatQ-lacZ (Fig. 8c) in the rpoN::Ω-Km mutant is also suggestive of such deregulation. For example, exogenous L-arginine repressed the expression of aatJ-lacZ in the ΔaauR mutant and wild-type P. aeruginosa PAO1, whereas in the rpoN::Ω-Km mutant, it had no effect. Furthermore, both aatJ-lacZ and aatQ-lacZ were unresponsive in the rpoN::Ω-Km mutant.
The scope and magnitude of AauR regulation in P. aeruginosa PAO1
The last 38 amino acid residues of the C-terminus of AauR form a putative FIS-type helix-turn-helix (HTH) motif. This FIS-type HTH or DNA-binding domain is 79% identical between the AauR proteins of P. aeruginosa PAO1 and P. putida KT2440, suggesting that these two EBPs recognize similar if not identical DNA-binding sites. Located 131–144 and 233–278 bp upstream of the aatJ ORF in P. aeruginosa PAO1 are sequences resembling the unique − 12/− 24 promoter recognized by RpoN and the consensus DNA-binding site for AauR, respectively (Fig. 1). Apart from aatJ, no other genes in P. aeruginosa PAO1 possess sequences matching the consensus DNA-recognition site of AauR in their 5′-regulatory regions. Biochemical characterization of AauR from P. aeruginosa PAO1 should reveal the exact DNA-binding site for this regulator and will be invaluable in generating a more definitive answer as to the number and range of genes that are directly governed by this EBP.
While the aatJ-aatQMP genes were the focus of the current study, it is probable that AauR and RpoN regulate the expression of two other genes associated with this locus (Figs. 1 and 9). Namely, the aatJ-aatQMP genes might form an operon with genes encoding for the periplasmic glutaminase-asparaginase AnsB and a periplasmic gamma-glutamyltranspeptidase (Ggt) [19]. As mentioned earlier, the utilization of L-glutamine is thought to involve AnsB, which catalyzes the deamidation of this amino acid in the periplasm to liberate L-glutamate [28]. Deletion of the aauR did hinder the growth of P. aeruginosa PAO1 on L-glutamine as the sole carbon source, but the total disappearance or depletion of exogenously fed L-glutamine was only delayed and not abolished in the ΔaauR mutant, arguing that the deamidation of L-glutamine is not strictly dependent on AauR in P. aeruginosa PAO1. Interestingly, growth on L-asparagine but not L-glutamine was significantly reduced in an ΔansA ΔansB double mutant of P. aeruginosa PAO1 [29]. This suggests that additional glutaminases apart from the cytoplasmic AnsA and periplasmic AnsB are involved in the deamidation and metabolism of L-glutamine in P. aeruginosa. The enzyme Ggt catalyzes the transfer of γ-glutamyl groups from donor molecules, most notably glutathione, to target or acceptor substrates consisting of amino acids, peptides or even water. Although Ggt has been implicated in the metabolism of glutathione and cysteine in some bacteria [36], little is known on the biological importance of this enzyme in Pseudomonas, including any potential role it may have in glutamate-related metabolism.
The sensor histidine kinase AauS is hypothesized to be essential for optimal utilization of L-glutamate and L-glutamine in P. aeruginosa
The sensor histidine kinase AauS and the EBP AauR form a putative DctBD-type TCS. The transcriptional activity of AauR is, therefore, believed to be controlled through phosphorylation catalyzed by AauS. Because AauS controls the transcriptional activity of AauR, it is reasonable to predict that optimal utilization of L-glutamate and L-glutamine in P. aeruginosa will be dependent on AauS. Indeed, an earlier study reported that the growth phenotypes of aauS and aauR mutants of P. putida KT2440 were similar to one another [16]. Thus, comparable results are expected in P. aeruginosa.
The results of the LacZ assays strongly suggest that the AauSR TCS regulates the expression of the aatJ-aatQMP genes in response to L-glutamate, L-aspartate, L-glutamine and L-asparagine. The question then becomes: what is the substrate specificity of AauS? Does AauS recognize each amino acid, or are only the acidic amino acids capable of binding to and stimulating the histidine kinase activity of AauS? In the latter scenario, the periplasmic deamidation of L-glutamine and L-asparagine would generate the requisite acidic amino acid substrates for AauS. Another potential and unexpected substrate of AauS is that of L-histidine, which was found to induce expression of aatQ-lacZ in a manner dependent on AauR. Intracellular L-glutamate is an intermediate in the catabolism of L-histidine, and studies involving P. putida and Rhizobium leguminosarum have indicated that L-glutamate efflux may occur under certain conditions [17, 37, 38]. Perhaps the AauSR TCS of P. aeruginosa facilitates the recapture of effluxed or escaped L-glutamate during L-histidine catabolism. Lastly, given that the transporter AatJQMP is required for growth on both D- and L-glutamate in P. aeruginosa, it would suggest that AauS is also not stereospecific, i.e., either isomer of glutamate is sufficient for stimulating AauS activity. Current efforts are underway to determine the substrate specificity and biological significance of AauS in P. aeruginosa PAO1.