Proteomic and Transcriptomic Elucidation of the Mutant Ralstonia eutropha G + 1 with Regard to Glucose Utilization

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.Ralstonia eutropha H16 is a Gram-negative betaproteobacterium that serves as a model organism to investigate the hydrogen-based chemolithoautotrophy and the metabolism of poly(3-hydroxybutyrate) (PHB) and other polyesters. When cells are cultivated under imbalanced growth conditions, PHB is accumulated and occurs as insoluble granules in the cytoplasm to serve as a storage compound for energy and carbon (31, 32). Our investigations of strain H16 focused on, among other things, its polyester metabolism and its ability to produce these polyesters from cheap and abundant carbon sources.R. eutropha H16 utilizes fructose, N-acetylglucosamine, gluconate, and other organic acids as carbon and energy sources for heterotrophic growth (4). Despite the detailed knowledge of its central carbon metabolism, including the Entner-Doudoroff (ED) pathway and the citric acid cycle, little is known about the carbohydrate uptake and transport in this bioplasticproducing bacterium. It is currently assumed that fructose is imported by a CUT2 family ATP binding cassette (ABC)-type transporter whose genes frcACB are located on chromosome 2 of R. eutropha H16 (10, 23). Gluconate is transported via a gluconate-H ϩ symporter catalyzed by a gluconate permease and transporter (gntP and gntT, respectively) (23).R. eutropha possesses a functional phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) which is specifi...

Section: Discussionmentioning

confidence: 99%

Effects of Homologous Phosphoenolpyruvate-Carbohydrate Phosphotransferase System Proteins on Carbohydrate Uptake and Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha H16

Kaddor

Steinbüchel

2011

“…In these reports, many genes and their products previously annotated as hypothetical or putative could be assigned predicted functions. In addition, this type of approach allows the elucidation of complex regulatory networks involving posttranscription control (9)(10)(11)(12).…”

mentioning

confidence: 99%

Profile of Secreted Hydrolases, Associated Proteins, and SlpA in Thermoanaerobacterium saccharolyticum during the Degradation of Hemicellulose

Currie

Guss

Herring³

et al. 2014

Thermoanaerobacterium saccharolyticum, a Gram-positive thermophilic anaerobic bacterium, grows robustly on insoluble hemicellulose, which requires a specialized suite of secreted and transmembrane proteins. We report here the characterization of proteins secreted by this organism. Cultures were grown on hemicellulose, glucose, xylose, starch, and xylan in pH-controlled bioreactors, and samples were analyzed via spotted microarrays and liquid chromatography-mass spectrometry. Key hydrolases and transporters employed by T. saccharolyticum for growth on hemicellulose were, for the most part, hitherto uncharacterized and existed in two clusters (Tsac_1445 through Tsac_1464 for xylan/xylose and Tsac_1344 through Tsac_1349 for starch). A phosphotransferase system subunit, Tsac_0032, also appeared to be exclusive to growth on glucose. Previously identified hydrolases that showed strong conditional expression changes included XynA (Tsac_1459), XynC (Tsac_0897), and a pullulanase, Apu (Tsac_1342). An omnipresent transcript and protein making up a large percentage of the overall secretome, Tsac_0361, was tentatively identified as the primary S-layer component in T. saccharolyticum, and deletion of the Tsac_0361 gene resulted in gross morphological changes to the cells. The view of hemicellulose degradation revealed here will be enabling for metabolic engineering efforts in biofuel-producing organisms that degrade cellulose well but lack the ability to catabolize C 5 sugars.

“…We also identified pyruvate phosphate dikinase (Y88_0878) (Fig. 3C, spot 30), which regenerates pyruvate into phosphoenolpyruvate (PEP) to reinitiate the transport signal transduction chain (39).…”

Section: Resultsmentioning

confidence: 99%

Proteomic Phenotyping of Novosphingobium nitrogenifigens Reveals a Robust Capacity for Simultaneous Nitrogen Fixation, Polyhydroxyalkanoate Production, and Resistance to Reactive Oxygen Species

Smit

Strabala

Peng

et al. 2012

ABSTRACTNovosphingobium nitrogenifigensY88T(Y88) is a free-living, diazotrophicAlphaproteobacterium, capable of producing 80% of its biomass as the biopolymer polyhydroxybutyrate (PHB). We explored the potential utility of this species as a polyhydroxybutyrate production strain, correlating the effects of glucose, nitrogen availability, dissolved oxygen concentration, and extracellular pH with polyhydroxybutyrate production and changes in the Y88 proteomic profile. Using two-dimensional differential in-gel electrophoresis and tandem mass spectrometry, we identified 217 unique proteins from six growth conditions. We observed reproducible, characteristic proteomic signatures for each of the physiological states we examined. We identified proteins that changed in abundance in correlation with either nitrogen fixation, dissolved oxygen concentration, or acidification of the growth medium. The proteins that correlated with nitrogen fixation were identified either as known nitrogen fixation proteins or as novel proteins that we predict play roles in aspects of nitrogen fixation based on their proteomic profiles. In contrast, the proteins involved in central carbon and polyhydroxybutyrate metabolism were constitutively abundant, consistent with the constitutive polyhydroxybutyrate production that we observed in this species. Three proteins with roles in detoxification of reactive oxygen species were identified in this obligate aerobe. The most abundant protein in all experiments was a polyhydroxyalkanoate granule-associated protein, phasin. The full-length isoform of this protein has a long, intrinsically disordered Ala/Pro/Lys-rich N-terminal segment, a feature that appears to be unique to sphingomonad phasins. The data suggest that Y88 has potential as a PHB production strain due to its aerobic tolerance and metabolic orientation toward polyhydroxybutyrate accumulation, even in low-nitrogen growth medium.