The Entner-Doudoroff and Nonoxidative Pentose Phosphate Pathways Bypass Glycolysis and the Oxidative Pentose Phosphate Pathway in Ralstonia solanacearum

Abstract

In <i>Ralstonia solanacearum</i>, a devastating phytopathogen whose metabolism is poorly understood, we observed that the Entner-Doudoroff (ED) pathway and nonoxidative pentose phosphate pathway (non-OxPPP) bypass glycolysis and OxPPP under glucose oxidation. Evidence derived from ¹³C stable isotope feeding and genome annotation-based comparative metabolic network analysis supported the observations. Comparative metabolic network analysis derived from the currently available 53 annotated <i>R. solanacearum</i> strains, including a recently reported strain (F1C1), representing the four phylotypes, confirmed the lack of key genes coding for phosphofructokinase (<i>pfk-1</i>) and phosphogluconate dehydrogenase (<i>gnd</i>) enzymes that are relevant for glycolysis and OxPPP, respectively. <i>R. solanacearum</i> F1C1 cells fed with [¹³C]glucose (99% [1-¹³C]glucose or 99% [1,2-¹³C]glucose or 40% [¹³C₆]glucose) followed by gas chromatography-mass spectrometry (GC-MS)-based labeling analysis of fragments from amino acids, glycerol, and ribose provided clear evidence that rather than glycolysis and the OxPPP, the ED pathway and non-OxPPP are the main routes sustaining metabolism in <i>R. solanacearum</i> The ¹³C incorporation in the mass ions of alanine (<i>m/z</i> 260 and <i>m/z</i> 232), valine (<i>m/z</i> 288 and <i>m/z</i> 260), glycine (<i>m/z</i> 218), serine (<i>m/z</i> 390 and <i>m/z</i> 362), histidine (<i>m/z</i> 440 and <i>m/z</i> 412), tyrosine (<i>m/z</i> 466 and <i>m/z</i> 438), phenylalanine (<i>m/z</i> 336 and <i>m/z</i> 308), glycerol (<i>m/z</i> 377), and ribose (<i>m/z</i> 160) mapped the pathways supporting the observations. The outcomes help better define the central carbon metabolic network of <i>R. solanacearum</i> that can be integrated with ¹³C metabolic flux analysis as well as flux balance analysis studies for defining the metabolic phenotypes.<b>IMPORTANCE</b> Understanding the metabolic versatility of <i>Ralstonia solanacearum</i> is important, as it regulates the trade-off between virulence and metabolism (1, 2) in a wide range of plant hosts. Due to a lack of clear evidence until this work, several published research papers reported on the potential roles of glycolysis and the oxidative pentose phosphate pathway (OxPPP) in <i>R. solanacearum</i> (3, 4). This work provided evidence from ¹³C stable isotope feeding and genome annotation-based comparative metabolic network analysis that the Entner-Doudoroff pathway and non-OxPPP bypass glycolysis and OxPPP during the oxidation of glucose, a component of the host xylem pool that serves as a potential carbon source (5). The outcomes help better define the central carbon metabolic network of <i>R. solanacearum</i> that can be integrated with ¹³C metabolic flux analysis as well as flux balance analysis studies for defining the metabolic phenotypes. The study highlights the need to critically examine phytopathogens whose metabolism is poorly understood.