Genetic Analysis Reveals the Essential Role of Nitrogen Phosphotransferase System Components in Sinorhizobium fredii CCBAU 45436 Symbioses with Soybean and Pigeonpea Plants

The nitrogen phosphotransferase system (PTS^Ntr) consists of EI^Ntr, NPr, and EIIA^Ntr. The active phosphate moiety derived from phosphoenolpyruvate is transferred through EI^Ntr and NPr to EIIA^Ntr. Sinorhizobium fredii can establish a nitrogen-fixing symbiosis with the legume crops soybean (as determinate nodules) and pigeonpea (as indeterminate nodules). In this study, S. fredii strains with mutations in ptsP and ptsO (encoding EI^Ntr and NPr, respectively) formed ineffective nodules on soybeans, while a strain with a ptsN mutation (encoding EIIA^Ntr) was not defective in symbiosis with soybeans. Notable reductions in the numbers of bacteroids within each symbiosome and of poly-β-hydroxybutyrate granules in bacteroids were observed in nodules infected by the ptsP or ptsO mutant strains but not in those infected with the ptsN mutant strain. However, these defects of the ptsP and ptsO mutant strains were recovered in ptsP ptsN and ptsO ptsN double-mutant strains, implying a negative role of unphosphorylated EIIA^Ntr in symbiosis. Moreover, the symbiotic defect of the ptsP mutant was also recovered by expressing EI^Ntr with or without the GAF domain, indicating that the putative glutamine-sensing domain GAF is dispensable in symbiotic interactions. The critical role of PTS^Ntr in symbiosis was also observed when related PTS^Ntr mutant strains of S. fredii were inoculated on pigeonpea plants. Furthermore, nodule occupancy and carbon utilization tests suggested that multiple outputs could be derived from components of PTS^Ntr in addition to the negative role of unphosphorylated EIIA^Ntr.     

A role for EIIANtr in controlling fluxes in the central metabolism of E. coli K12

To investigate a possible role of the nitrogen-PTS (PTS^Ntr) in controlling carbon metabolism, we determined the growth of Escherichia coli LJ110 and of isogenic derivatives, mutated in components of the PTS^Ntr, on different carbon sources. The PTS^Ntr is a set of proteins homologous to the PEP-dependent phosphotransferase system (C-PTS) that transfers a phosphate group from PEP over EI^Ntr (encoded by ptsP) and NPr (encoded by ptsO) to EIIA^Ntr (encoded by ptsN). Strains deleted in ptsN were characterized by a high acetate production coupled to slow growth on glycolytic substrates. The ΔptsP and the ΔptsO strain showed the same behavior as the parent strain. As the phosphorylation level of EIIA^Ntr in these mutants differed significantly from that of the parent strain, phosphorylation of EIIA^Ntr obviously is not important for its function. During growth in minimal medium with defined carbon sources, EIIA^Ntr was always completely phosphorylated in LJ110. Significant amounts of dephosphorylated EIIA^Ntr were only visible in strains lacking EI^Ntr or NPr. mRNA expression studies on glucose revealed a downregulation of genes encoding TCA cycle enzymes when EIIA^Ntr was absent. ¹³C-flux analyses confirmed higher fluxes towards acetate and lower fluxes in the TCA cycle in the ptsN mutants but additionally hinted to a slightly but significantly increased flux through the pyruvate dehydrogenase complex (PDH). During growth on succinate the ΔptsN strain accumulated mutations in rpoS, while no rpoS mutants were observed for the ΔptsN-O strain. This hints to an additional function of NPr during growth with succinate.     

The phosphotransferase protein EIIA(Ntr) modulates the phosphate starvation response through interaction with histidine kinase PhoR in Escherichia coli

Many Proteobacteria possess the paralogous PTS^Ntr, in addition to the sugar transport phosphotransferase system (PTS). In the PTS^Ntr phosphoryl‐groups are transferred from phosphoenolpyruvate to protein EIIA^Ntr via the phosphotransferases EI^Ntr and NPr. The PTS^Ntr has been implicated in regulation of diverse physiological processes. In Escherichia coli, the PTS^Ntr plays a role in potassium homeostasis. In particular, EIIA^Ntr binds to and stimulates activity of a two‐component histidine kinase (KdpD) resulting in increased expression of the genes encoding the high‐affinity K⁺ transporter KdpFABC. Here, we show that the phosphate (pho) regulon is likewise modulated by PTS^Ntr. The pho regulon, which comprises more than 30 genes, is activated by the two‐component system PhoR/PhoB under conditions of phosphate starvation. Mutants lacking EIIA^Ntr are unable to fully activate the pho genes and exhibit a growth delay upon adaptation to phosphate limitation. In contrast, pho expression is increased above the wild‐type level in mutants deficient for EIIA^Ntr phosphorylation suggesting that non‐phosphorylated EIIA^Ntr modulates pho. Protein interaction analyses reveal binding of EIIA^Ntr to histidine kinase PhoR. This interaction increases the amount of phosphorylated response regulator PhoB. Thus, EIIA^Ntr is an accessory protein that modulates the activities of two distinct sensor kinases, KdpD and PhoR, in E. coli.     

Potential Regulatory Role of Competitive Encounter Complexes in Paralogous Phosphotransferase Systems

There are two paralogous Escherichia coli phosphotransferase systems, one for sugar import (PTS^sugar) and one for nitrogen regulation (PTS^Ntr), that utilize proteins enzyme I^sugar (EI^sugar) and HPr, and enzyme I^Ntr (EI^Ntr) and NPr, respectively. The enzyme I proteins have similar folds, as do their substrates HPr and NPr, yet they show strict specificity for their cognate partner both in stereospecific protein–protein complex formation and in reversible phosphotransfer. Here, we investigate the mechanism of specific EI^Ntr:NPr complex formation by the study of transient encounter complexes. NMR paramagnetic relaxation enhancement experiments demonstrated transient encounter complexes of EI^Ntr not only with the expected partner, NPr, but also with the unexpected partner, HPr. HPr occupies transient sites on EI^Ntr but is unable to complete stereospecific complex formation. By occupying the non-productive transient sites, HPr promotes NPr transient interaction to productive sites closer to the stereospecific binding site and actually enhances specific complex formation between NPr and EI^Ntr. The cellular level of HPr is approximately 150 times higher than that of NPr. Thus, our finding suggests a potential mechanism for cross-regulation of enzyme activity through formation of competitive encounter complexes.     

Biochemical Characterization of a Nitrogen-Type Phosphotransferase System Reveals that Enzyme EINtr Integrates Carbon and Nitrogen Signaling in Sinorhizobium meliloti

In Sinorhizobium meliloti, catabolite repression is influenced by a noncanonical nitrogen-type phosphotransferase system (PTS^Ntr). In this PTS^Ntr, the protein HPr is phosphorylated on histidine-22 by the enzyme EI^Ntr and the flux of phosphate through this residue onto downstream proteins leads to an increase in succinate-mediated catabolite repression (SMCR). In order to explore the molecular determinants of HPr phosphorylation by EI^Ntr, both proteins were purified and the activity of EI^Ntr was measured. Experimentally determined kinetic parameters of EI^Ntr activity were significantly slower than those determined for the carbohydrate-type EI in Escherichia coli. Enzymatic assays showed that glutamine, a signal of nitrogen availability in many Gram-negative bacteria, strongly inhibits EI^Ntr. Binding experiments using the isolated GAF domain of EI^Ntr (EI_GAF) showed that it is the domain responsible for detection of glutamine. EI^Ntr activity was not affected by α-ketoglutarate, and no binding between the EI_GAF and α-ketoglutarate could be detected. These data suggest that in S. melilloti, EI^Ntr phosphorylation of HPr is regulated by signals from both carbon metabolism (phosphoenolpyruvate) and nitrogen metabolism (glutamine).     

New molecular interaction of IIANtr and HPr from Burkholderia pseudomallei identified by X‐ray crystallography and docking studies

The nitrogen‐related phosphoenolpyruvate phosphotransferase system (PTS^Ntr) is involved in controlling ammonia assimilation and nitrogen fixation. The additional role of PTS^Ntr as a regulatory link between nitrogen and carbon utilization in Escherichia coli is assumed to be closely related to molecular functions of IIA^Ntr in potassium homeostasis. We have determined the crystal structure of IIA^Ntr from Burkholderia pseudomallei (BpIIA^Ntr), which is a causative agent of melioidosis. The crystal structure of dimeric BpIIA^Ntr determined at 3.0 Å revealed that its active sites are mutually blocked. This dimeric state is stabilized by charge and weak hydrophobic interactions. Overall monomeric structure and the active site residues, Arg51 and His67, of BpIIA^Ntr are well conserved with those of IIA^Ntr enzymes from E. coli and Neisseria meningitides. Interestingly, His113 of BpIIA^Ntr, which corresponds to a key residue in another phosphoryl group relay in the mannitol‐specific enzyme EIIA family (EIIA^Mtl), is located away from the active site due to the loop connecting β5 and α3. Combined with other differences in molecular surface properties, these structural signatures distinguish the IIA^Ntr family from the EIIA^Mtl family. Since, there is no gene for NPr in the chromosome of B. pseudomallei, modeling and docking studies of the BpIIA^Ntr–BpHPr complex has been performed to support the proposal on the NPr‐like activity of BpHPr. A potential dual role of BpHPr as a nonspecific phosphocarrier protein interacting with both sugar EIIAs and IIA^Ntr in B. pseudomallei has been discussed. Proteins 2013. © 2013 Wiley Periodicals, Inc.     

The interplay of the EIIA component of the nitrogen-related phosphotransferase system (PTS) of Pseudomonas putida with pyruvate dehydrogenase

Background

Pseudomonas putida KT2440 is endowed with a variant of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS^Ntr), which is not related to sugar transport but believed to rule the metabolic balance of carbon vs. nitrogen. The metabolic targets of such a system are largely unknown.

Methods

Dielectric breakdown of P. putida cells grown in rich medium revealed the presence of forms of the EIIA^Ntr (PtsN) component of PTS^Ntr, which were strongly associated to other cytoplasmic proteins. To investigate such intracellular partners of EIIA^Ntr, a soluble protein extract of bacteria bearing an E epitope tagged version of PtsN was immunoprecipitated with a monoclonal anti-E antibody and the pulled-down proteins identified by mass spectrometry.

Results

The E1 subunit of the pyruvate dehydrogenase (PDH) complex, the product of the aceE gene, was identified as a major interaction partner of EIIA^Ntr. To examine the effect of EIIA^Ntr on PDH, the enzyme activity was measured in extracts of isogenic ptsN⁺/ptsN⁻P. putida strains and the role of phosphorylation was determined. Expression of PtsN and AceE proteins fused to different fluorescent moieties and confocal laser microscopy indicated a significant co-localization of the two proteins in the bacterial cytoplasm.

Conclusion

EIIA^Ntr down-regulates PDH activity. Both genetic and biochemical evidence revealed that the non-phosphorylated form of PtsN is the protein species that inhibits PDH.

General significance

EIIA^Ntr takes part in the node of C metabolism that checks the flux of carbon from carbohydrates into the Krebs cycle by means of direct protein–protein interactions with AceE. This type of control might connect metabolism to many other cellular functions. This article is part of a Special Issue entitled: Systems Biology of Microorganisms.     

Colony‐morphology screening uncovers a role for the Pseudomonas aeruginosa nitrogen‐related phosphotransferase system in biofilm formation

Pseudomonas aeruginosa is an opportunistic human pathogen whose survival is aided by forming communities known as biofilms, in which cells are encased in a self‐produced matrix. We devised a mutant screen based on colony morphology to identify additional genes with previously unappreciated roles in biofilm formation. Our screen, which identified most known biofilm‐related genes, also uncovered PA14_16550 and PA14_69700, deletions of which abrogated and augmented biofilm formation respectively. We also identified ptsP, which encodes enzyme I of the nitrogen‐regulated phosphotransferase (PTS^Ntr) system, as being important for cyclic‐di‐GMP production and for biofilm formation. Further experiments showed that biofilm formation is hindered in the absence of phosphotransfer through the PTS^Ntr, but only in the presence of enzyme II (PtsN), the putative regulatory module of the PTS^Ntr. These results implicate unphosphorylated PtsN as a negative regulator of biofilm formation and establish one of the first known roles of the PTS^Ntr in P. aeruginosa.     

Reciprocal regulation of the autophosphorylation of enzyme INtr by glutamine and α‐ketoglutarate in Escherichia coli

In addition to the phosphoenolpyruvate:sugar phosphotransferase system (sugar PTS), most proteobacteria possess a paralogous system (nitrogen phosphotransferase system, PTS^Ntr). The first proteins in both pathways are enzymes (enzyme I^sugar and enzyme I^Ntr) that can be autophosphorylated by phosphoenolpyruvate. The most striking difference between enzyme I^sugar and enzyme I^Ntr is the presence of a GAF domain at the N‐terminus of enzyme I^Ntr. Since the PTS^Ntr was identified in 1995, it has been implicated in a variety of cellular processes in many proteobacteria and many of these regulations have been shown to be dependent on the phosphorylation state of PTS^Ntr components. However, there has been little evidence that any component of this so‐called PTS^Ntr is directly involved in nitrogen metabolism. Moreover, a signal regulating the phosphorylation state of the PTS^Ntr had not been uncovered. Here, we demonstrate that glutamine and α‐ketoglutarate, the canonical signals of nitrogen availability, reciprocally regulate the phosphorylation state of the PTS^Ntr by direct effects on enzyme I^Ntr autophosphorylation and the GAF signal transduction domain is necessary for the regulation of enzyme I^Ntr activity by the two signal molecules. Taken together, our results suggest that the PTS^Ntr senses nitrogen availability.     

Deuteration of Escherichia coli Enzyme I alters its stability

Enzyme I^Ntr is the first protein in the nitrogen phosphotransferase pathway. Using an array of biochemical and biophysical tools, we characterized the protein, compared its properties to that of EI of the carbohydrate PTS and, in addition, examined the effect of substitution of all nonexchangeable protons by deuterium (perdeuteration) on the properties of EI^Ntr. Notably, we find that the catalytic function (autophosphorylation and phosphotransfer to NPr) remains unperturbed while its stability is modulated by deuteration. In particular, the deuterated form exhibits a reduction of approximately 4 °C in thermal stability, enhanced oligomerization propensity, as well as increased sensitivity to proteolysis in vitro. We investigated tertiary, secondary, and local structural changes, both in the absence and presence of PEP, using near- and far-UV circular dichroism and Trp fluorescence spectroscopy. Our data demonstrate that the aromatic residues are particularly sensitive probes for detecting effects of deuteration with an enhanced quantum yield upon PEP binding and apparent decreases in tertiary contacts for Tyr and Trp side chains. Trp mutagenesis studies showed that the region around Trp522 responds to binding of both PEP and NPr. The significance of these results in the context of structural analysis of EI^Ntr are evaluated.     

Dephosphorylated NPr of the nitrogen PTS regulates lipid A biosynthesis by direct interaction with LpxD

Bacterial phosphoenolpyruvate-dependent phosphotransferase systems (PTS) play multiple roles in addition to sugar transport. Recent studies revealed that enzyme IIA^Ntr of the nitrogen PTS regulates the intracellular concentration of K⁺ by direct interaction with TrkA and KdpD. In this study, we show that dephosphorylated NPr of the nitrogen PTS interacts with Escherichia coli LpxD which catalyzes biosynthesis of lipid A of the lipopolysaccharide (LPS) layer. Mutations in lipid A biosynthetic genes such as lpxD are known to confer hypersensitivity to hydrophobic antibiotics such as rifampin; a ptsO (encoding NPr) deletion mutant showed increased resistance to rifampin and increased LPS biosynthesis. Taken together, our data suggest that unphosphorylated NPr decreases lipid A biosynthesis by inhibiting LpxD activity.     

Biosynthesis of poly-β-hydroxybutyrate (PHB) with a high molecular mass by a mutant strain of Azotobacter vinelandii (OPN)

The aim of this study was to characterize the influence of the aeration conditions on the production of PHB and its molecular mass in a mutant strain of Azotobacter vinelandii (OPN), which carries a mutation on ptsN, the gene encoding enzyme IIA^Ntr, previously shown to increase the accumulation of PHB. Cultures of A. vinelandii wild-type strain OP and its mutant derivative strain OPN were grown in 500-mL flasks, containing 100 and 200 mL of PY sucrose medium. PHB production and its molecular mass were analyzed at the end of the culture. The molecular mass (MM) was significantly influenced by the aeration conditions and strain used. A polymer with a higher molecular weight was produced under low aeration conditions for both strains. A maximal molecular mass of 2,026 kDa (equivalent to 3,670 kDa measured by GPC) was obtained with strain OPN cultured under low-aeration conditions, reaching a value two-fold higher than that obtained from the parental strain OP (MM?=?1,013 kDa) grown under the same conditions. Aeration conditions and the ptsN mutation influence the molecular mass of the PHB produced by A. vinelandii affecting in turn its physico-chemical properties.