Biology subjects

de Graaf, R. M.

Publications and source records attributed to de Graaf, R. M..

3 recordsLinked to original sources

bioRxiv2026

A new stress-response pathway in Mycobacterium tuberculosis

Metabolic adaptations are key in the virulence of the pathogen Mycobacterium tuberculosis (Mtb). However, our current understanding of these adaptations is limited to common pathways in central carbon metabolism. Here we apply an untargeted bottom-up approach to discover unknown stress-responsive metabolites and their biosynthetic enzyme. We show that upon exposure to hypoxia, nitric oxide and activated macrophages, Mtb rapidly produces high levels of an unknown metabolite that we identify as 6-{gamma}-amino-butyric acid-trehalose (GABA-trehalose). Formation of millimolar GABA-trehalose levels under these stresses is driven by a rapid rise in GABA, which is also excreted. We demonstrate that GABA-trehalose is produced from GABA and trehalose by the uncharacterized ATP-grasp enzyme Rv1722, involving a carboxylate-hydroxyl ligation that is non-canonical for ATP-grasp enzymes. Phylogenetic analyses demonstrate that the gene rv1722 is present in most slow-growing mycobacteria but absent in most rapid-growing mycobacteria. While the role of GABA-trehalose in Mtb metabolism remains unclear, we postulate that the increased NADH/NAD+ ratio under hypoxia and nitric oxide exposure promotes GABA formation and inhibits its breakdown, leading to GABA accumulation and excretion. Rv1722-driven coupling of GABA and trehalose constitutes an alternative to excretion that conserves carbon and nitrogen. Taken together, our bottom-up approach reveals a new stress-response pathway in Mtb that rapidly produces large quantities of GABA-trehalose. These findings extend our knowledge of the metabolic adaptations that a major human pathogen utilizes in response to immune system-imposed stresses.

biochemistry↗

bioRxiv2025

Sediments from a seasonally euxinic coastal ecosystem show high nitrogen cycling potential

Coastal ecosystems are susceptible to eutrophication and deoxygenation, which may alter their nitrogen cycle dynamics. Here, we investigated the microbial nitrogen cycling potential in the sediment of a seasonally euxinic coastal ecosystem (Lake Grevelingen, NL), in winter and summer. Porewater profiles showed ammonium (NH4+) concentrations exceeding 10 mM and rapid depletion of electron acceptors with depth. Activity tests revealed NH4+ oxidation potential up to 53 {micro}mol g-1 day-1, even in anoxic sediment layers. A nitrifying microbial community was present in both oxic and anoxic sediment sections (up to 1.4% relative abundance). NO-, nitrite (NO2-) and nitrous oxide (N2O) reduction potential were prominent across all sediment sections, with the highest rates (167 {micro}mol NO3- g-1 day-1) in the surface sediment in summer. Denitrification (79.3-98.4%) and dissimilatory nitrate reduction to ammonium (DNRA; 1.6-20.7%) were the major NO3- removal pathways, as supported by the detection of the narG/napA, nirK/nirS, norB, nosZ and nrfA/otr genes in all sediment sections. The DNRA contribution increased with depth and with the addition of electron donors, such as monomethylamine. Anaerobic ammonium oxidation (anammox) was not detected in these eutrophic sediments. Combined, our results show that there is high potential for nitrogen removal in eutrophic coastal ecosystems which may help further restoration measures.

microbiology↗

bioRxiv2019

Autotrophic and mixotrophic metabolic network fluxes suggest versatile lifestyle for the anammox bacterium Candidatus Kuenenia stuttgartiensis

Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and mixotrophy beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus Kuenenia stuttgartiensis using time-series 13C and 2H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis (INST-MFA). Our findings confirm predicted metabolic pathways used for CO2 fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of an oxidative tricarboxylic acid cycle, despite the genome not encoding a known citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood-Ljungdahl pathway instead of oxidizing it completely to CO2 followed by reassimilation. In contrast, our data suggests that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactors side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.

microbiology↗