Dataset Information

Effects of homologous phosphoenolpyruvate-carbohydrate phosphotransferase system proteins on carbohydrate uptake and poly(3-Hydroxybutyrate) accumulation in Ralstonia eutropha H16.

ABSTRACT: Seven gene loci encoding putative proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified in the genome of Ralstonia eutropha H16 by in silico analysis. Except the N-acetylglucosamine-specific PEP-PTS, an additional complete PEP-PTS is lacking in strain H16. Based on these findings, we generated single and multiple deletion mutants defective mainly in the PEP-PTS genes to investigate their influence on carbon source utilization, growth behavior, and poly(3-hydroxybutyrate) (PHB) accumulation. As supposed, the H16 ?frcACB and H16 ?nagFEC mutants exhibited no growth when cultivated on fructose and N-acetylglucosamine, respectively. Furthermore, a transposon mutant with a ptsM-ptsH insertion site did not grow on both carbon sources. The observed phenotype was not complemented, suggesting that it results from an interaction of genes or a polar effect caused by the Tn5::mob insertion. ptsM, ptsH, and ptsI single, double, and triple mutants stored much less PHB than the wild type (about 10 to 39% [wt/wt] of cell dry weight) and caused reduced PHB production in mutants lacking the H16_A2203, H16_A0384, frcACB, or nagFEC genes. In contrast, mutant H16 ?H16_A0384 accumulated 11.5% (wt/wt) more PHB than the wild type when grown on gluconate and suppressed partially the negative effect of the ptsMHI deletion on PHB synthesis. Based on our experimental data, we discussed whether the PEP-PTS homologous proteins in R. eutropha H16 are exclusively involved in the complex sugar transport system or whether they are also involved in cellular regulatory functions of carbon and PHB metabolism.

SUBMITTER: Kaddor C

PROVIDER: S-EPMC3127587 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Effects of homologous phosphoenolpyruvate-carbohydrate phosphotransferase system proteins on carbohydrate uptake and poly(3-Hydroxybutyrate) accumulation in Ralstonia eutropha H16.

Kaddor Chlud C Steinbüchel Alexander A

Applied and environmental microbiology 20110408 11

Seven gene loci encoding putative proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified in the genome of Ralstonia eutropha H16 by in silico analysis. Except the N-acetylglucosamine-specific PEP-PTS, an additional complete PEP-PTS is lacking in strain H16. Based on these findings, we generated single and multiple deletion mutants defective mainly in the PEP-PTS genes to investigate their influence on carbon source utilization, growth behavior, and p ...[more]

PMID: 21478317

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Project description: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. eutrophaIMPORTANCE 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 high-performance 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 massive overproduction of PHB. In contrast, in the absence of (p)ppGpp, mobilization of PHB was dramatically enhanced.

Project description:Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with "phasin 2 motifs." To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ?phaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins can have additional functions. A chromosomal deletion of phaP6 or phaP7 had no visible effect on formation of PHB granules.

Dataset Information

Effects of homologous phosphoenolpyruvate-carbohydrate phosphotransferase system proteins on carbohydrate uptake and poly(3-Hydroxybutyrate) accumulation in Ralstonia eutropha H16.

Publications

Effects of homologous phosphoenolpyruvate-carbohydrate phosphotransferase system proteins on carbohydrate uptake and poly(3-Hydroxybutyrate) accumulation in Ralstonia eutropha H16.

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