Abstract
Bacillus megaterium is a bacterium of great importance as a plant-beneficial bacterium in agricultural applications and in industrial bioproduction of proteins. Understanding intracellular processing of carbohydrates in this species is crucial to predicting natural carbon utilization as well as informing strategies in metabolic engineering. Here, we applied stable isotope-assisted metabolomics profiling and metabolic flux analysis to elucidate, at high resolution, the connections of the different catabolic routes for carbohydrate metabolism immediately following substrate uptake in B. megaterium QM B1551. We performed multiple 13C tracer experiments to obtain both kinetic and long-term 13C profiling of intracellular metabolites. In addition to the direct phosphorylation of glucose to glucose-6-phosphate (G6P) prior to oxidation to 6-phosphogluconate (6P-gluconate), the labeling data also captured glucose catabolism through the gluconate pathway involving glucose oxidation to gluconate followed by phosphorylation to 6P-gluconate. Our data further confirmed the absence of the Entner–Doudoroff pathway in B. megaterium and showed that subsequent catabolism of 6P-gluconate was instead through the oxidative pentose–phosphate (PP) pathway. Quantitative flux analysis of glucose-grown cells showed equal partition of consumed glucose from G6P to the Embden–Meyerhof–Parnas (EMP) pathway and from G6P to the PP pathway through 6P-gluconate. Growth on fructose alone or xylose alone was consistent with the ability of B. megaterium to use each substrate as a sole source of carbon. However, a detailed 13C mapping during simultaneous feeding of B. megaterium on glucose, fructose, and xylose indicated non-uniform intracellular investment of the different carbohydrate substrates. Flux of glucose-derived carbons dominated the gluconate pathway and the PP pathway, whereas carbon flux from both glucose and fructose populated the EMP pathway; there was no assimilatory flux of xylose-derived carbons. Collectively, our findings provide new quantitative insights on the contribution of the different catabolic routes involved in initiating carbohydrate catabolism in B. megaterium and related Bacillus species.
Highlights
Bacillus megaterium, an aerobic bacterium ubiquitous in a diverse range of environments, has been of special investigative interest for its applications in promoting plant health (Eppinger et al, 2011; Santos et al, 2014), bioremediation of contaminants (Quinn et al, 1989), and industrial bioproduction (Vary, 1994; Vary et al, 2007; Biedendieck et al, 2010)
The 13C labeling of gluconate was indicative of glucose oxidation to gluconate, which was reported in spores of B. megaterium QM B1551 (Otani et al, 1986; Sano et al, 1988)
We employed 13C-carbon mapping of intracellular metabolism to address knowledge gaps regarding the routes for carbohydrate catabolism in B. megaterium QM B1551
Summary
An aerobic bacterium ubiquitous in a diverse range of environments, has been of special investigative interest for its applications in promoting plant health (Eppinger et al, 2011; Santos et al, 2014), bioremediation of contaminants (Quinn et al, 1989), and industrial bioproduction (Vary, 1994; Vary et al, 2007; Biedendieck et al, 2010). Much of what is known about carbon metabolism in Bacillus species is derived from Bacillus subtilis, the primary model organism for Gram-positive bacteria (Sauer et al, 1997; Dauner and Sauer, 2001; Dauner et al, 2001; Fuhrer et al, 2005). Previous metabolic studies highlighted incongruent metabolic fluxes between B. subtilis and B. megaterium species (Sauer et al, 1997; Dauner and Sauer, 2001; Dauner et al, 2001; Fuhrer et al, 2005; Furch et al, 2007a,b; Tannler et al, 2008; Youngster et al, 2017). Metabolic flux modeling has been performed on mutant strains of B. megaterium that lacked specific metabolic functions (Furch et al, 2007b). Of particular interest is an investigation of the different catabolic routes that initiate carbohydrate metabolism in B. megaterium due to the ubiquitous presence of carbohydrate-containing feedstocks
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