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.     

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.     

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.     

Non-disruptive release of Pseudomonas putida proteins by in situ electric breakdown of intact cells

Analysis of the native proteome of bacterial cells typically involves physical procedures (sonication, French press) and/or biochemical methods (treatment with lysozyme, osmotic shock etc.) to break open the bacteria to yield a soluble protein fraction. Such procedures are not only time consuming, but they change bacterial physiology during manipulation and affect labile post-translational modifications such as His-P bonds. In this work, we document the efficacy of the dielectric breakdown of live bacteria for releasing and delivering the protein contents of intact cells directly into a non-denaturing gel system. By means of such an in situ electrophoresis, the protein pool enters the separation medium without any manipulation of the cells other than being exposed to a moderate electric voltage. To validate the method we have followed the fate of the two forms of the PtsN (EIIA(Ntr)) protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) of Pseudomonas putida through the various stages of the procedure. Apart of detecting the corresponding polypeptides, we show that this procedure releases the bulk of the proteome while keeping unharmed the phosphorylation state of EIIA(Ntr) as it was present in the cells prior to applying the electric field. The method is applicable to other bacteria as well.     

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.     

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).     

Polar landmark protein HubP recruits flagella assembly protein FapA under glucose limitation in Vibrio vulnificus

How motile bacteria recognize their environment and decide whether to stay or navigate toward more favorable location is a fundamental issue in survival. The flagellum is an elaborate molecular device responsible for bacterial locomotion, and the flagellum‐driven motility allows bacteria to move themselves to the appropriate location at the right time. Here, we identify the polar landmark protein HubP as a modulator of polar flagellation that recruits the flagellar assembly protein FapA to the old cell pole, thereby controlling its activity for the early events of flagellar assembly in Vibrio vulnificus. We show that dephosphorylated EIIA^Glc of the PEP‐dependent sugar transporting phosphotransferase system sequesters FapA from HubP in response to glucose and hence inhibits FapA‐mediated flagellation. Thus, flagellar assembly and motility is governed by spatiotemporal control of FapA, which is orchestrated by the competition between dephosphorylated EIIA^Glc and HubP, in the human pathogen V. vulnificus.     

Glucose induces delocalization of a flagellar biosynthesis protein from the flagellated pole

To survive in a continuously changing environment, bacteria sense concentration gradients of attractants or repellents, and purposefully migrate until a more favourable habitat is encountered. While glucose is known as the most effective attractant, the flagellar biosynthesis and hence chemotactic motility has been known to be repressed by glucose in some bacteria. To date, the only known regulatory mechanism of the repression of flagellar synthesis by glucose is via downregulation of the cAMP level, as shown in a few members of the family Enterobacteriaceae. Here we show that, in Vibrio vulnificus, the glucose‐mediated inhibition of flagellar motility operates by a completely different mechanism. In the presence of glucose, EIIA^Glc is dephosphorylated and inhibits the polar localization of FapA (flagellar assembly protein A) by sequestering it from the flagellated pole. A loss or delocalization of FapA results in a complete failure of the flagellar biosynthesis and motility. However, when glucose is depleted, EIIA^Glc is phosphorylated and releases FapA such that free FapA can be localized back to the pole and trigger flagellation. Together, these data provide new insight into a bacterial strategy to reach and stay in the glucose‐rich area.     

Structure of the Enterococcus faecalis EIIA PTS component

In Eubacteria, the utilization of a number of extracellular carbohydrates is mediated by sugar specific phosphoenolepyruvate (PEP) dependent sugar phosphotransferase systems (PTSs), which simultaneously import und phosphorylate their target sugars. Here, we report the crystal structure of the EIIA^gnt component of the so far little investigated Enterococcus faecalis gluconate specific PTS. The crystal structure shows a tightly interacting dimer of EIIA^gnt which is structurally similar to the related EIIA^man from Escherichia coli. Homology modeling of E. faecalis HPr, EIIB^man and their complexes with EIIA^man suggests that despite moderate sequence identity between EIIA^man and EIIA^gnt, the active sites closely match the situation observed in the E. coli system with His-9 of EIIA^gnt being the likely phosphoryl group carrier. We therefore propose that the phosphoryl transfer reactions involving EIIA^gnt proceed according to a mechanism analog to the one described for E. coli EIIA^man.     

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.     

Comparative proteome profiling of host–pathogen interactions: insights into the adaptation mechanisms of Francisella tularensis in the host cell environment

The intracellular pathogens have the unique capacity to sense the host cell environment and to respond to it by alteration in gene expression and protein synthesis. Proteomic analysis of bacteria exposed directly to the host cell milieu might thus greatly contribute to the elucidation of processes leading to bacterial adaptation and proliferation inside the host cell. Here we have performed a global proteome analysis of a virulent Francisella tularensis subsp. holarctica strain during its intracellular cycle within the macrophage-like murine cell line J774.2 using the metabolic pulse-labeling of bacterial proteins with ³⁵S-methionine and ³⁵S-cysteine in various periods of infection. The two-dimensional gel analysis revealed macrophage-induced bacterial proteome changes in which 64 identified proteins were differentially expressed in comparison to controls grown in tissue culture medium. Nevertheless, activation of macrophages with interferon gamma before in vitro infection decreased the number of detected alterations in protein levels. Thus, these proteomic data indicate the F. tularensis ability to adapt to the intracellular hostile environment that is, however, diminished by prior interferon gamma treatment of host cells.     

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human ��-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors¹. This process leads to bacterial invasion of endothelial cells². More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells³. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as α-actinin. As no surface expressed α-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and α-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with α-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.     

Activity of the Enterococcus faecalis EIIA PTS component and its strong interaction with EIIB

Eubacteria can import and simultaneously phosphorylate a range of different carbohydrates by means of sugar specific phosphoenolpyruvate (PEP) dependent sugar phosphotransferase systems (PTSs). Here, we report the biochemical characterization of the gluconate specific PTS component EIIA^gnt from Enterococcus faecalis and its unexpectedly strong complex with EIIB^gnt. We analyze the activity of the complex regarding phosphoryl transfer using kinetic measurements and demonstrate by mutagenesis that His-9 of EIIA^gnt is essential for this process and represents most likely the phosphoryl group carrier of EIIA^gnt. With a combination of isothermal titration calorimetry (ITC), analytical ultracentrifugation (AUC), native gel electrophoresis and chemical crosslinking experiments we show that EIIA^gnt and EIIB^gnt form a strong 2:2 heterotetrameric complex, which seems to be destabilized upon phosphorylation of EIIB^gnt.     

Phosphatidylglycerol Directs Binding and Inhibitory Action of EIIAGlc Protein on the Maltose Transporter

The signal-transducing protein EIIA^Glc belongs to the phosphoenolpyruvate carbohydrate phosphotransferase system. In its dephosphorylated state, EIIA^Glc is a negative regulator for several permeases, including the maltose transporter MalFGK₂. How EIIA^Glc is targeted to the membrane, how it interacts with the transporter, and how it inhibits sugar uptake remain obscure. We show here that acidic phospholipids together with the N-terminal tail of EIIA^Glc are essential for the high affinity binding of the protein to the transporter. Using protein docking prediction and chemical cross-linking, we demonstrate that EIIA^Glc binds to the MalK dimer, interacting with both the nucleotide-binding and the C-terminal regulatory domains. Dissection of the ATPase cycle reveals that EIIA^Glc does not affect the binding of ATP but rather inhibits the capacity of MalK to cleave ATP. We propose a mechanism of maltose transport inhibition by this central amphitropic regulatory protein.     

Development of a microfluidic chip-based plasmid miniprep

Plasmids are the workhorse of contemporary molecular biology, serving as vectors in the multitude of molecular cloning approaches now available. Plasmid minipreps are a routine and essential means of extracting plasmid DNA from bacteria, such as Escherichia coli, for identification, characterization, and further manipulation. Although there have been many approaches described and miniprep kits are commercially available, traditional minipreps typically require more than 16 h, including the time needed for bacterial cell culture. Here we describe the development of a microfluidic chip (MFC)-based miniprep that uses on-chip lysis and trapping of large DNA in agarose to differentially separate plasmid DNA from the bacterial chromosome. Our approach greatly decreases both the time required for the miniprep itself and the time required for growth of the bacterial cultures because our on-chip miniprep uses 10⁵ times fewer E. coli cells. Because the quality of the isolated plasmid is comparable to that obtained using conventional miniprep protocols, this approach allows growth of E. coli and isolation of plasmid within hours, thereby making it ideal for rapid screening approaches. This MFC-based miniprep, coupled with recently demonstrated on-chip transfection capabilities, lays the groundwork for seamless manipulation of plasmids on MFC platforms.     

Crystal structure of nitrogen regulatory protein IIA<Superscript>Ntr</Superscript> from <Emphasis Type="Italic">Neisseria meningitidis</Emphasis>

Background  

The NMB0736 gene of Neisseria meningitidis serogroup B strain MC58 encodes the putative nitrogen regulatory protein, IIA^Ntr (abbreviated to NM-IIA^Ntr). The homologous protein present in Escherichia coli is implicated in the control of nitrogen assimilation. As part of a structural proteomics approach to the study of pathogenic Neisseria spp., we have selected this protein for structure determination by X-ray crystallography.