Phosphotransferase protein EIIANtr interacts with SpoT, a key enzyme of the stringent response, in Ralstonia eutropha H16

Karstens, Katja; Zschiedrich, Christopher P.; Bowien, Botho; Stülke, Jörg; Görke, Boris

doi:10.1099/mic.0.075226-0

Cited by 44 publications

(54 citation statements)

References 51 publications

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“…The assumption of a covalent modification such as phosphorylation would be in line with previous data. Karstens and coworkers previously showed that in R. eutropha, SpoT1 but not SpoT2 interacted with the nonphosphorylated form of the EIIA Ntr component of the PTS system (33). This indicates a possible phosphorylation cascade from the carbon source uptake system to SpoT1, which could, in turn, influence the activity of the PHB mobilization system.…”

Section: Discussionmentioning

confidence: 99%

“…Support for a connection between PHB metabolism and the stringent response in R. eutropha was recently published in two reports in which the authors identified an interaction of the PTS protein EIIA Ntr with the ppGpp synthase/hydrolase SpoT1 (33) and showed that an EIIA Ntr knockout mutant accumulated substantially more PHB than the wild type (34). Furthermore, the North American authors of this study claimed in a previous study that a mutant (Re2411) with a deletion in the H16_A1337 gene (ΔspoT2) had completely lost the ability to accumulate PHB, pointing to an essential function of SpoT2 for PHB metabolism (24).…”

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confidence: 99%

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Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha H16

Juengert

Borisova

Mayer

et al. 2017

Appl Environ Microbiol

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In this study, we constructed a set of Ralstonia eutropha H16 strains with single, double, or triple deletions of the (p)ppGpp synthase/hydrolase (spoT1), (p)ppGpp synthase (spoT2), and/or polyhydroxybutyrate (PHB) depolymerase (phaZa1 or phaZa3) gene, and we determined the impact on the levels of (p)ppGpp and on accumulated PHB. Mutants with deletions of both the spoT1 and spoT2 genes were unable to synthesize detectable amounts of (p)ppGpp and accumulated only minor amounts of PHB, due to PhaZa1-mediated depolymerization of PHB. In contrast, unusually high levels of PHB were found in strains in which the (p)ppGpp concentration was increased by the overexpression of (p)ppGpp synthase (SpoT2) and the absence of (p)ppGpp hydrolase. Determination of (p)ppGpp levels in wild-type R. eutropha under different growth conditions and induction of the stringent response by amino acid analogs showed that the concentrations of (p)ppGpp during the growth phase determine the amount of PHB remaining in later growth phases by influencing the efficiency of the PHB mobilization system in stationary growth. The data reported for a previously constructed ΔspoT2 strain (C. J. Brigham, D. R. Speth, C. Rha, and A. J. Sinskey, Appl Environ Microbiol 78:8033-8044, 2012, https://doi.org/ 10.1128/AEM.01693-12) were identified as due to an experimental error in strain construction, and our results are in contrast to the previous indication that the spoT2 gene product is essential for PHB accumulation in R. eutropha. IMPORTANCE Polyhydroxybutyrate (PHB) is an important intracellular carbon and energy storage compound in many prokaryotes and helps cells survive periods of starvation and other stress conditions. Research activities in several laboratories over the past 3 decades have shown that both PHB synthase and PHB depolymerase are constitutively expressed in most PHB-accumulating bacteria, such as Ralstonia eutropha. This implies that PHB synthase and depolymerase activities must be well regulated in order to avoid a futile cycle of simultaneous PHB synthesis and PHB degradation (mobilization). Previous reports suggested that the stringent response in Rhizobium etli and R. eutropha is involved in the regulation of PHB metabolism. However, the levels of (p)ppGpp and the influence of those levels on PHB accumulation and PHB mobilization have not yet been determined for any PHB-accumulating species. In this study, we optimized a (p)ppGpp extraction procedure and a highperformance liquid chromatography-mass spectrometry (HPLC-MS)-based detection method for the quantification of (p)ppGpp in R. eutropha. This enabled us to study the relationship between the concentrations of (p)ppGpp and the accumulated levels of PHB in the wild type and in several constructed mutant strains. We show that overproduction of the alarmone (p)ppGpp correlated with reduced growth and mas-

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha H16

Juengert

Borisova

Mayer

et al. 2017

Appl Environ Microbiol

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“…Bacterial two-hybrid assays indicated that this interaction occurs only with the unphosphorylated form of EIIA Ntr (133). Interaction with EIIB components.…”

Section: Fig 5 Pts-catalyzed Glucose Uptake and The Eiiamentioning

confidence: 99%

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

Deutscher

Aké

Derkaoui

et al. 2014

Microbiol Mol Biol Rev

341

374

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SUMMARY The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.

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“…However, it is noteworthy that regulation of K + transport by PTS Ntr as observed in E. coli is not necessarily conserved in other bacteria. For instance, interaction of EIIA Ntr and KdpD is also operative in Rhizobium leguminosarum but absent in Ralstonia eutropha , suggesting that PTS Ntr serves different functions in different species [Karstens et al, 2014;Prell et al, 2012].…”

Section: Eiiamentioning

confidence: 99%

“…According to current evidence, only nonphosphorylated EIIA Ntr is engaged in protein-protein interactions [Karstens et al, 2014;Lee et al, 2007;Lüttmann et al, 2009Lüttmann et al, , 2012Pflüger-Grau et al, 2011]. In contrast to the canonical EI, EI Ntr carries an additional GAF domain fused to its N-terminus [Reizer et al, 1996].…”

Section: Eiiamentioning

confidence: 99%

Cross-Talk between the Canonical and the Nitrogen-Related Phosphotransferase Systems Modulates Synthesis of the KdpFABC Potassium Transporter in <b><i>Escherichia coli</i></b>

2015

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Many Proteobacteria possess the regulatory nitrogen-related phosphotransferase system (PTS^Ntr), which operates in parallel to the transport PTS. PTS^Ntr is composed of the proteins EI^Ntr and NPr and the final phosphate acceptor EIIA^Ntr. Both PTSs can exchange phosphoryl groups among each other. Proteins governing K⁺ uptake represent a major target of PTS^Ntr in Escherichia coli. Nonphosphorylated EIIA^Ntr binds and stimulates the K⁺ sensor KdpD, which activates expression of the kdpFABC operon encoding a K⁺ transporter. Here we show that this regulation also operates in an ilvG⁺ strain ruling out previous concern about interference with a nonfunctional ilvG allele present in many strains. Furthermore, we analyzed phosphorylation of EIIA^Ntr. In wild-type cells EIIA^Ntr is predominantly phosphorylated, regardless of the growth stage and the utilized carbon source. However, cross-phosphorylation of EIIA^Ntr by the transport PTS becomes apparent in the absence of EI^Ntr: EIIA^Ntr is predominantly nonphosphorylated when cells grow on a PTS sugar and phosphorylated when a non-PTS carbohydrate is utilized. These differences in phosphorylation are transduced into corresponding kdpFABC transcription levels. Thus, the transport PTS may affect phosphorylation of EIIA^Ntr and accordingly modulate processes controlled by EIIA^Ntr. Our data suggest that this cross-talk becomes most relevant under conditions that would inhibit activity of EI^Ntr.

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Phosphotransferase protein EIIANtr interacts with SpoT, a key enzyme of the stringent response, in Ralstonia eutropha H16

Abstract: EIIANtr is a member of a truncated phosphotransferase (PTS) system that serves regulatory functions and exists in many Proteobacteria in addition to the sugar transport PTS.

Cited by 44 publications

References 51 publications

Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha H16

Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha H16

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

Cross-Talk between the Canonical and the Nitrogen-Related Phosphotransferase Systems Modulates Synthesis of the KdpFABC Potassium Transporter in <b><i>Escherichia coli</i></b>

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