Biology subjects

Kropp, A.

Publications and source records attributed to Kropp, A..

8 recordsLinked to original sources

bioRxiv2026

Quinone-transporting filaments extend the respiratory chain of Gram positive bacteria

Cellular respiration depends on transferring electrons to hydrophobic quinones in membrane bilayers, constraining capacity to available surface area. Expanding this capacity is thought to have driven cellular complexity and eukaryogenesis, with Gram-negative bacteria evolving internal invaginations and eukaryotes using membrane-bound organelles. Whether Gram-positive bacteria, which lack such membranes, evolved alternatives was unknown. Here, we show that Bacillus subtilis forms a quinone-transporting pseudomembrane composed of filaments of the NADH dehydrogenase Ndh and the quinone-transporting protein Ncp. Cryo-EM, lipidomics, and molecular dynamics reveal that Ndh and Ncp co-assemble with phospholipids into a complex containing a solvent-excluded hydrophobic lumen that sequesters quinones. These complexes further assemble into filaments, linking chambers into a continuous conduit that amplifies quinone reduction while occupying minimal membrane space. Phylogenetic analysis suggests this recent innovation is widespread in Bacillota. Quinone-transporting filaments thus reveal a third strategy for overcoming surface-area limits and provide principles for engineering synthetic energy systems.

biochemistry↗

bioRxiv2025

Atmospheric hydrogen consumption is regulated by catabolite repression in mycobacteria

Consumption of atmospheric hydrogen (H2) enables diverse aerobic microorganisms to grow and persist in resource-deprived environments. In the aerobic saprophyte Mycobacterium smegmatis, hydrogen oxidation is catalyzed by two differentially expressed, high-affinity, oxygen-insensitive uptake hydrogenases, Huc and Hhy. Huc enables mixotrophic growth and facilitates the transition from growth to dormancy. Although the huc operon is known to be upregulated in response to organic carbon deprivation, the specific signals and regulators modulating its expression remain unresolved. Here, we show that GylR, a glycerol-3-phosphate-sensing regulator of glycerol metabolism, plays a role in repression of huc expression in response to the availability of glycerol but not other carbon sources. Based on proteomic analyses and activity assays, mutation or knockdown of gylR leads to enhanced Huc production and activity. GylR and other key catabolite repressor proteins (Crp1, Crp2) do not directly bind the huc operon, indicating repression is mediated by unidentified transcription factors, with GylR acting as an upstream sensor. Here, we present data that suggests atmospheric H2 oxidation is regulated in response to organic carbon source availability through the process of catabolite repression. By identifying a key signal that prompts atmospheric H2 oxidation, these findings advance understanding of how aerobic bacteria adapt to changing environmental conditions and suggest that organic carbon levels are a key factor regulating the main sink of atmospheric H2 in soils globally. ImportanceSoil microorganisms collectively consume 70 million tonnes of atmospheric H2 a year, regulating atmospheric composition and climate change. In turn, consuming this dependable trace gas enables these microorganisms to survive even when their preferred organic energy sources are exhausted. Despite the importance of H2 consumption for soil biodiversity and atmospheric regulation, the signals and sensors that regulate this process remain to be understood. Here, we demonstrate that a model soil bacterium turns on the machinery required for atmospheric H2 consumption in direct response to being limited by organic carbon availability, through the process of catabolite repression. Specifically, in the absence of a sensor of the organic carbon source glycerol, a H2-consuming hydrogenase is highly expressed and active. These findings suggest that organic carbon levels have a major role in regulating trace gas oxidation, with implications for predicting how trace gas consumption and soil biodiversity respond to environmental change.

microbiology↗

bioRxiv2025

A Protein Antibiotic Inhibits the BAM Complex to Kill Without Cell Entry

Many antibiotics are ineffective against Gram-negative pathogens such as Pseudomonas aeruginosa because they cannot penetrate the bacterial outer membrane. Here, we show that protein antibiotics called L-type pyocins kill P. aeruginosa by inhibiting the {beta}-barrel assembly machinery (BAM) complex at the cell surface, halting outer-membrane protein assembly. Using single-particle cryo-electron microscopy, we show that L-type pyocins bind a surface-exposed region of BamA and deploy a C-terminal peptide that competitively inhibits the BAM complex, demonstrating that cell entry is not required for antibiotic activity. We combine genetics, multi-omics and cryo-electron tomography to show that BAM complex inhibition by L-type pyocins or the peptide antibiotic darobactin triggers a multifaceted transcriptomic, proteomic and morphological response. Despite this, BAM inhibition ultimately leads to a catastrophic loss of membrane integrity and cell death. These results validate BAM as a target for antibiotics that do not enter the cell and define an engineerable system for their development.

microbiology↗

bioRxiv2025

Hydrogenase-driven ATP synthesis from air

All cells require a continuous supply of the universal energy currency, adenosine triphosphate (ATP), to drive countless cellular reactions. The universally conserved F1Fo-ATP synthase regenerates ATP from ADP and Pi by harnessing a transmembrane electrochemical proton gradient (pmf). Bacteria have evolved a wide diversity of pmf-forming proteins to make ATP using light, organic, and inorganic energy sources. Recently, we proposed that many bacteria survive using atmospheric trace gases to produce ATP when limited for other energy sources. However, there is no direct proof that atmospheric energy sources are sufficient to generate pmf or drive ATP synthesis. Here, we show that the membrane-associated hydrogen:quinone oxidoreductase Huc from Mycobacterium smegmatis enables ATP synthesis from air. Purified Huc couples H2 oxidation to the reduction of various ubiquinone and menaquinone analogues. We designed and optimised a minimal respiratory chain in which Huc is reconstituted into liposomes with a pmf-generating terminal oxidase and the ATP-generating F1Fo-ATP synthase. Our experiments show that passive hydrogen exchange from air to solution is sufficient for the electron transfer and pmf generation required to accumulate ATP. Finally, by combining continuous culture bioenergetics measurements with theoretical calculations, we show this process is sufficient for mycobacteria to sustain pmf and ATP synthesis (two ATP molecules per H2 oxidised) for maintenance energy requirements during nutrient starvation. These findings prove that atmospheric energy sources are dependable lifeline substrates that enable continuous energy conservation during nutrient starvation. In addition, this work provides a new tool for ATP production in synthetic applications, which unlike other approaches is traceless without by-product accumulation.

microbiology↗

bioRxiv2024

Inhibiting heme-piracy by pathogenic Escherichia coli using de novo-designed proteins

Iron is an essential nutrient for most bacteria and is often growth-limiting during infection, due to the host sequestering free iron as part of the innate immune response. To obtain the iron required for growth, many bacterial pathogens encode transporters capable of extracting the iron-containing cofactor heme directly from host proteins. Pathogenic E. coli and Shigella spp. produce the outer membrane transporter ChuA, which binds host hemoglobin and extracts its heme cofactor, before importing heme into the cell. Heme extraction by ChuA is a dynamic process, with the transporter capable of rapidly extracting heme from hemoglobin in the absence of an external energy source, without forming a stable ChuA-hemoglobin complex. In this work, we utilise a combination of structural modelling, Cryo-EM, X-ray crystallography, mutagenesis, and phenotypic analysis to understand the mechanistic detail of this process. Based on this understanding we utilise artificial intelligence-based protein design to create binders capable of inhibiting E. coli growth by blocking hemoglobin binding to ChuA. By screening a limited number of these designs, we identify several binders that inhibit E. coli growth at low nanomolar concentrations, without experimental optimisation. We determine the structure of a subset of these binders, alone and in complex with ChuA, demonstrating that they closely match the computational design. This work demonstrates the utility of de novo-designed proteins for inhibiting bacterial nutrient uptake and uses a workflow that could be applied to integral membrane proteins in other organisms.

biochemistry↗

bioRxiv2024

Quinone extraction drives atmospheric carbon monoxide oxidation in bacteria

Diverse bacteria and archaea use atmospheric carbon monoxide (CO) as an energy source during long-term survival. This process enhances the biodiversity of soil and marine ecosystems globally and removes 250 million tonnes of a toxic, climate-relevant pollutant from the atmosphere each year. Bacteria use [MoCu]-carbon monoxide dehydrogenases (Mo-CODH) to convert CO to carbon dioxide, then transfer the liberated high-energy electrons to the aerobic respiratory chain. However, given no high-affinity Mo-CODH has been purified, it is unknown how these enzymes oxidise CO at low concentrations and interact with the respiratory chain. Here we resolve these knowledge gaps by analysing Mo-CODH (CoxSML) and its hypothetical partner CoxG from Mycobacterium smegmatis. Kinetic and electrochemical analyses show purified Mo-CODH is a highly active high-affinity enzyme (Km = 139 nM, kcat = 54.2 s-1). Based on its 1.85 [A] resolution cryoEM structure, Mo-CODH forms a CoxSML homodimer similar to characterised low-affinity homologs, but has distinct active site coordination and narrower gas channels that may modulate affinity. We provide structural, biochemical, and genetic evidence that Mo-CODH transfers CO-derived electrons to the aerobic respiratory chain via the membrane-bound menaquinone-binding protein CoxG. Consistently, CoxG is required for CO-driven respiration, extracts menaquinone from mycobacterial membranes, and binds quinones in a hydrophobic pocket. Finally, we show that Mo-CODH and CoxG genetically and structurally associate in diverse bacteria and archaea. These findings reveal the basis of a biogeochemically and ecologically important process, while demonstrating that the newly discovered process of long-range quinone transport is a general mechanism of energy conservation, which convergently evolved on multiple occasions.

biochemistry↗

bioRxiv2022

Energy extraction from air: structural basis of atmospheric hydrogen oxidation

Diverse aerobic bacteria use atmospheric H2 as an energy source for growth and survival. This recently discovered yet globally significant process regulates the composition of the atmosphere, enhances soil biodiversity, and drives primary production in certain extreme environments. Atmospheric H2 oxidation has been attributed to still uncharacterised members of the [NiFe]-hydrogenase superfamily. However, it is unresolved how these enzymes overcome the extraordinary catalytic challenge of selectively oxidizing picomolar levels of H2 amid ambient levels of the catalytic poison O2, and how the derived electrons are transferred to the respiratory chain. Here we determined the 1.52 [A] resolution CryoEM structure of the mycobacterial hydrogenase Huc and investigated its mechanism by integrating kinetics, electrochemistry, spectroscopy, mass spectrometry, and molecular dynamics simulations. Purified Huc is an oxygen-insensitive enzyme that couples the oxidation of atmospheric H2 at its large subunit to the hydrogenation of the respiratory electron carrier menaquinone at its small subunit. The enzyme uses a narrow hydrophobic gas channel to selectively bind atmospheric H2 at the expense of O2, while three [3Fe-4S] clusters and their unusual ligation by a D-histidine modulate the electrochemical properties of the enzyme such that atmospheric H2 oxidation is energetically feasible. Huc forms an 833 kDa complex composed of an octamer of catalytic subunits around a membrane-associated central stalk, which extracts and transports menaquinone a remarkable 94 [A] from the membrane, enabling its reduction. These findings provide a mechanistic basis for the biogeochemically and ecologically critical process of atmospheric H2 oxidation. Through the first characterisation of a group 2 [NiFe]-hydrogenase, we also uncover a novel mode of energy coupling dependent on long-range quinone transport and pave way for the development of biocatalysts that oxidize H2 in ambient air.

biochemistry↗

bioRxiv2022

When two is a crowd - Structural mapping of PEAK pseudokinase interactions identifies 14 3 3 as a molecular switch for PEAK3/Crk signaling.

PEAK pseudokinases regulate cell migration, invasion and proliferation by recruiting key signaling proteins to the cytoskeleton. Despite lacking catalytic activity, alteration in their expression level is associated with several aggressive cancers. Here, we elucidate new molecular details of key PEAK signaling interactions with the adapter proteins CrkII and Grb2 and the scaffold protein 14-3-3. Our findings rationalize why the dimerization of PEAK proteins has a crucial function in signal transduction and provide biophysical and structural data to unravel binding specificity within the PEAK interactome. We identify a conserved high affinity 14-3-3 motif on PEAK3 and demonstrate its role as a molecular switch to regulate CrkII binding. Together, our studies provide a detailed structural snapshot of PEAK interaction networks and further elucidate how PEAK proteins, especially PEAK3, act as dynamic scaffolds that exploit adapter proteins to control signal transduction in cell growth/motility and cancer.

biochemistry↗