Biology subjects

van Niftrik, L.

Publications and source records attributed to van Niftrik, L..

10 recordsLinked to original sources

bioRxiv2026

Streamlining large-scale high-resolution electron tomography with VolWeaver

Researchers using electron microscopy must often balance a trade-off between obtaining high-resolution structural information and preserving sufficient cellular context. At one end of this spectrum, single particle cryo-electron microscopy and cryo-electron tomography provide near-molecular detail but are typically limited to relatively small fields of view. At the other, volume electron microscopy approaches, such as scanning electron microscopy of resin-embedded specimens, capture large cellular volumes but generally at lower resolution. Consequently, linking nanoscale structural information to larger cellular architecture remains a significant challenge. To address this gap, we optimised a transmission electron tomography workflow for resin-embedded malaria parasites that allows us to visualise targeted regions of interest at nanometre-scale resolution while retaining several micrometres of surrounding cellular context. Here, we present our current best-practice pipeline for sample preparation, tomogram acquisition, and reconstruction. In addition, we introduce VolWeaver, a data-processing framework, that integrates high-resolution tomographic datasets into serial section volume reconstructions, enabling the visualisation and interpretation of ultrastructural features within their broader cellular environment.

cell biology↗

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↗

bioRxiv2026

The molecular mechanism and activity of Kuenenia stuttgartiensis hydrazine synthase

Anaerobic ammonium-oxidizing (anammox) bacteria convert ammonium and nitrite into dinitrogen gas via the intermediates nitric oxide and hydrazine. To produce hydrazine, anammox bacteria harbor a biochemically unique enzyme: hydrazine synthase. Based on the hydrazine synthase crystal structure it was hypothesized that hydrazine is produced in a two-step mechanism. In this hypothesis, nitric oxide is first reduced to hydroxylamine (first half-reaction), followed by condensation of hydroxylamine with ammonium to hydrazine (second half-reaction). Here, we experimentally investigated the proposed molecular mechanism of hydrazine synthase and characterized and optimized the in vitro activity. First, we optimized the activity of isolated hydrazine synthase from anammox bacterium Kuenenia stuttgartiensis strain MBR1 via an anaerobic isolation method. We further compared hydrazine synthase activity measured via a coupled assay versus that of a newly established direct LC-MS assay. Next, the hypothesized second half-reaction was investigated via the direct LC-MS assay, quantifying biologically produced hydrazine from hydroxylamine and ammonium. Despite variation in hydrazine synthase activity across assays, we determined ammonium and hydroxylamine affinity and investigated product inhibition. Finally, we found that hydrazine synthesis from ammonium and hydroxylamine by isolated hydrazine synthase is oxygen-tolerant, strongly suggesting that the second half-reaction is initiated on an oxidized heme within hydrazine synthase. Taken together, the results corroborate that condensation of ammonium with hydroxylamine to form hydrazine is the second half-reaction of the proposed two-step mechanism for hydrazine synthesis by hydrazine synthase.

microbiology↗

bioRxiv2026

Quantification of hydrazine in biochemical assays and anammox bacteria using LC-MS

Hydrazine is an industrially valuable product. Strikingly, hydrazine is also a key intermediate in the energy metabolism of anaerobic ammonium-oxidizing (anammox) bacteria, where it is formed by hydrazine synthase. To study the molecular mechanism and activity of isolated hydrazine synthase, a sensitive, relatively fast and easy method to quantify hydrazine is needed. However, reported methods such as colorimetric assays, MALDI-TOF MS, and enzymatic conversion of hydrazine to dinitrogen gas, are either insensitive or laborious. In this study, we describe the validation and application of a fast and simple liquid chromatography-mass spectrometry (LC-MS) method to reproducibly quantify hydrazine produced by anammox hydrazine synthase. Hydrazine was derivatized with benzaldehyde, and directly injected onto a C18 column coupled to a Q-TOF MS. To increase assay performance, 15N2-hydrazine was included as internal standard. The response ratio of hydrazine was linearly proportional to the hydrazine concentration from 0.05-1 {micro}M with an average correlation coefficient of 0.9925. Intra- and inter-day accuracy lay between 88-113% and 95-105%, respectively. Intra- and inter-day precision (RSD, %) [≤] 11%. Hydrazine and derivatized hydrazine were stable when stored at -70{degrees}C or in the autosampler. We successfully applied the LC-MS method to determine hydrazine production by isolated hydrazine synthase and within cell lysate of anammox bacteria.

biochemistry↗

bioRxiv2025

Building without the architect: the dispensable role of MICOS in de novo cristae formation in malaria parasites

The malaria parasite mitochondria represent the only tractable, naturally occurring system in which cristae, the characteristic invaginations of the inner mitochondrial membrane, are known to be formed de novo. In traditional model organisms, the central complex involved in cristae organization is the mitochondrial contact site and cristae organizing system (MICOS). However, whether MICOS has an active role in the initial formation of cristae remains unclear. We identified two putative Plasmodium falciparum MICOS components, PfMIC19 and PfMIC60 and show that both genes are dispensable in asexual blood stages, gametocytes, and mosquito stages, albeit with a mild reduction in oocyst numbers. Immunofluorescence microscopy and electron tomography of gametocytes lacking either or both MICOS orthologues showed aberrant mitochondrial morphology and abnormal cristae, with a marked reduction of crista junctions. Thus, by utilizing the unique properties of P. falciparum, we confirmed the involvement of PfMIC19 and PfMIC60 in the organization of crista junctions, while providing evidence that MICOS is not required for the initial formation of cristae.

microbiology↗

bioRxiv2025

Nitric oxide-forming nitrite reductases in the anaerobic ammonium oxidizer Kuenenia stuttgartiensis

Anaerobic ammonium-oxidizing (anammox) bacteria contribute to the global nitrogen cycle by removing fixed nitrogen from the environment. They do so via the anaerobic oxidation of ammonium to dinitrogen gas, with nitrite as terminal electron acceptor. The first step in this so-called anammox reaction is the conversion of nitrite to nitric oxide by nitrite reductase. Next, nitric oxide is combined with ammonium to form hydrazine by hydrazine synthase, after which hydrazine is oxidized to dinitrogen gas by hydrazine dehydrogenase. In contrast to the other catabolic anammox enzymes, different anammox species encode different potential nitrite reductase enzymes. On top of that, there is a redundancy in genes encoding for nitrite reductase in single anammox species. The unusual diversity and redundancy in anammox nitrite reductases is unexplained. The genome of the model anammox species "Candidatus Kuenenia stuttgartiensis" encodes for three putative nitrite reductases. Here, we investigated which of these nitrite reductases is or are active in K. stuttgartiensis. Active nitric oxide-producing nitrite reductases were enriched from K. stuttgartiensis cells via fast protein liquid chromatography. Nitric oxide production by the enriched nitrite reductases was followed with membrane inlet mass spectrometry. Combining the activity assays with proteomics analysis indicated that the soluble nitrite reductases NirS and HAOr most strongly correlated with enzyme activity. This indicates that K. stuttgartiensis employs two distinct nitrite reductases to keep its nitric oxide pool replenished.

microbiology↗

bioRxiv2025

Immune responses to human pathogens exposed to simulated Mars conditions

The identification of health risks associated with long-term crewed missions to Mars is critical for mission planning and crew safety. Human-associated pathogens can be part of the microbiome and are likely to be transported during these missions. This study examines the immunological responses of human immune cells stimulated with non-fastidious bacterial species that cause opportunistic infections, i.e. Klebsiella pneumoniae and Serratia marcescens, after exposure to simulated Martian conditions, including UV radiation, desiccation and atmospheric pressure. We observed that exposure of the bacteria to these conditions altered cytokine secretion, reactive oxygen species (ROS) production, and phagocytic activity in human peripheral blood mononuclear cells (PBMCs). Specifically, exposure to desiccation reduced cytokines and ROS production, indicating impaired innate immune recognition and stimulation. Notably, the altered immune response was partially restored when desiccated bacteria were regrown in standard media. Flow cytometry revealed decreased bacterial size and complexity of both species post-exposure. These findings indicate that Martian conditions induce bacterial morphological and physiological changes, which could impair immune recognition and response. Expanding these studies to in vivo models and a broader range of potentially pathogenic microorganisms is essential to estimate infection risks during Mars missions, which is vital for developing strategies to mitigate infection risks and maintain astronaut health during long-term space travel. ImportanceSince Yuri Gagarins 1961 flight, human space exploration has expanded, unintentionally transporting microorganisms, including pathogens, into space environments. Our previous studies demonstrated that opportunistic pathogens like Klebsiella pneumoniae and Serratia marcescens can survive simulated Mars conditions. With upcoming Mars missions, it is crucial to understand how such conditions influence these pathogens and their interaction with the human immune system. This research evaluates immune responses to bacteria pathogens exposed to Martian stressors such as UV radiation and desiccation, revealing significant changes of the immune responses to the exposed bacteria. These findings provide essential insights into the health risks that astronauts may face if infected with Mars-adapted pathogens. Understanding these interactions will help to develop preventive strategies and therapeutic measures, ensuring the safety and health of crew members during long-term missions. Ultimately, this work contributes to the broader objective of safe human exploration and colonization of Mars.

immunology↗

bioRxiv2023

Spectroscopic insights into the mechanism of anammox hydrazine synthase

Anaerobic ammonium oxidizing bacteria make a living oxidizing ammonium with nitrite as electron acceptor, intermediates nitric oxide and hydrazine, and end product dinitrogen gas. Hydrazine is a biologically unique free intermediate in this metabolism, and is produced by the enzyme hydrazine synthase. Crystallization of Candidatus Kuenenia stuttgartiensis hydrazine synthase allowed for an initial hypothesis of its reaction mechanism. In this hypothesis, nitric oxide is first reduced to hydroxylamine after which hydroxylamine is condensed with ammonium to form hydrazine. Hydrazine synthase is a tetraheme cytochrome c, containing two proposed active site hemes ({gamma}I & I) in the {gamma}- and -subunit, respectively, connected by an intra-enzymatic tunnel. Here we combined the data from electrochemistry-induced Fourier transform infrared (FTIR) spectroscopy, EPR and optical spectroscopy to shed light on the redox properties and protein dynamics of hydrazine synthase in the context of its reaction mechanism. Redox titrations revealed two low potential low spin hemes with midpoint potentials of [~]-360 mV and [~]-310 mV for heme II and {gamma}II, respectively. Heme {gamma}I showed redox transitions in the range of 0 mV, consisting of both low spin and high spin characteristics in optical and EPR spectroscopy. Electrochemistry-induced FTIR spectroscopy indicated an aspartic acid ligating a OH-/H2O at the heme {gamma}I axial site as a possible candidate for involvement in this mixed spin characteristic. Furthermore, EPR spectroscopy confirmed the ability of heme {gamma}I to bind NO in the reduced state. Heme I exhibited a rhombic high spin signal, in line with its ligation by a proximal tyrosine observed in the crystal structure. Redox titrations down to -610 mV nor addition of dithionite resulted in the reduction of heme I, indicating a very low midpoint potential for this heme. In vivo chemistry at this heme I, the candidate for the comproportionation of hydroxylamine and ammonium, is thus likely to be initiated solely on the oxidized heme, in contrast to previously reported DFT calculations. The reduction potentials of the {gamma}-subunit hemes were in line with the proposed electron transfer of heme {gamma}II to heme {gamma}I for the reduction of NO to hydroxylamine (E0 = - 30 mV).

biochemistry↗

bioRxiv2019

On anammox activity at low temperature: effect of ladderane composition, process conditions and dominant anammox population

The application of partial nitritation-anammox (PN/A) under mainstream conditions can enable substantial cost savings at wastewater treatment plants (WWTPs), but how process conditions and cell physiology affect anammox performance at psychrophilic temperatures below 15 {degrees}C remains poorly understood. We tested 14 anammox communities, including 8 from globally-installed PN/A processes, for (i) specific activity at 10-30 {degrees}C (batch assays), (ii) composition of membrane lipids (U-HPLC-HRMS/MS), and (iii) microbial community structure (16S rRNA gene amplicon sequencing). Crucially, the key parameters impacting anammox activity were the membrane lipid composition and cultivation temperature. The size of ladderane lipids and the content of bacteriohopanoids were key physiological drivers of anammox performance at low temperatures. Higher contents of (i) short C18 [3]-ladderane alkyl and (ii) large phosphatidylcholine headgroup were determined in anammox more active at 15-30 {degrees}C and 10-15 {degrees}C, respectively. At below 15 {degrees}C, the activation energies of most mesophilic cultures severely increased while those of the psychrophilic cultures remained stable; this indicates that the adaptation of mesophilic cultures to psychrophilic regime necessitates months, but in some cases can take up to 5 years. Interestingly, biomass enriched in the marine genus "Candidatus Scalindua" displayed exceptionally highest activity at 10-20 {degrees}C (0.50 kg-N.kg-VSS-1.d-1 at 10 {degrees}C, Ea10-30 {degrees}C = 51{+/-}16 kJ.mol-1), indicating outstanding potential for nitrogen removal from cold streams. Collectively, our comprehensive study provides essential knowledge of cold adaptation mechanism, will enable more accurate modelling and suggests highly promising target anammox genera for inoculation and set-up of anammox reactors, in particular for mainstream WWTPs. HighlightsO_LILadderane size and cold exposure affected anammox activation energy (Ea). C_LIO_LIEa improved with more C18 [3]-ladderanes over C20 and larger polar headgroup. C_LIO_LILong-term cold exposure reduced Ea at 10-15 {degrees}C, not activity per se. C_LIO_LIMarine "Ca. Scalindua" was exceptionally suitable for cold streams. C_LIO_LIAnammox Ea at 15-30 {degrees}C was 79{+/-}18 kJ.mol-1. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/873869v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1c5c3aborg.highwire.dtl.DTLVardef@b11ed2org.highwire.dtl.DTLVardef@5e7ec9org.highwire.dtl.DTLVardef@1d5cba5_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2019

Extracellular electron transfer-dependent anaerobic oxidation of ammonium by anammox bacteria

Anaerobic ammonium oxidation (anammox) by anammox bacteria contributes significantly to the global nitrogen cycle, and plays a major role in sustainable wastewater treatment. Anammox bacteria convert ammonium (NH4+) to dinitrogen gas (N2) using nitrite (NO2-) or nitric oxide (NO) as the electron acceptor. In the absence of NO2- or NO, anammox bacteria can couple formate oxidation to the reduction of metal oxides such as Fe(III) or Mn(IV). Their genomes contain homologs of Geobacter and Shewanella cytochromes involved in extracellular electron transfer (EET). However, it is still unknown whether anammox bacteria have EET capability and can couple the oxidation of NH4+ with transfer of electrons to carbon-based insoluble extracellular electron acceptors. Here we show using complementary approaches that in the absence of NO2-, freshwater and marine anammox bacteria couple the oxidation of NH4+ with transfer of electrons to carbon-based insoluble extracellular electron acceptors such as graphene oxide (GO) or electrodes poised at a certain potential in microbial electrolysis cells (MECs). Metagenomics, fluorescence in-situ hybridization and electrochemical analyses coupled with MEC performance confirmed that anammox electrode biofilms were responsible for current generation through EET-dependent oxidation of NH4+. 15N-labelling experiments revealed the molecular mechanism of the EET-dependent anammox process. NH4+ was oxidized to N2 via hydroxylamine (NH2OH) as intermediate when electrode was the terminal electron acceptor. Comparative transcriptomics analysis supported isotope labelling experiments and revealed an alternative pathway for NH4+ oxidation coupled to EET when electrode is used as electron acceptor compared to NO2-as electron acceptor. To our knowledge, our results provide the first experimental evidence that marine and freshwater anammox bacteria can couple NH4+ oxidation with EET, which is a significant finding, and challenges our perception of a key player of anaerobic oxidation of NH4+ in natural environments and engineered systems.

microbiology↗