Biology subjects

Warren, M. J.

Publications and source records attributed to Warren, M. J..

11 recordsLinked to original sources

bioRxiv2026

From Magnetosomes to Vesicles: LemA Proteins as Drivers of Bacterial Membrane Reorganization

The cellular machineries that remodel bacterial membranes during organelle and vesicle formation remain poorly defined. Herein, we report MamQ as a key determinant of the lipid invaginations that enclose the magnetosome. MamQ belongs to the broadly conserved LemA protein family, whose members localize to either the inner or outer membrane. Heterologous overproduction of 13 different LemA proteins in Escherichia coli induced extensive membrane restructuring and vesiculation, indicating a conserved membrane-remodelling activity. In Pseudomonas aeruginosa, overproduction of the inner membrane protein LemA1 (PaLemA1) promoted cytoplasmic membrane vesicle (CMV) formation, whereas the periplasmic-located outer membrane-anchored LemA2 (PaLemA2) drove the formation of bacterial outer-membrane vesicles (OMVs). Conversely, deletion of the corresponding genes reduced extracellular vesicle production. Interactome analyses identified 256 protein partners of PaLemA1 and PaLemA2, including multiple components of the cellular divisome, such as CpoB, FtsE, FtsI, FtsK, FtsN, FtsW, MinE, ParB, ZapG, ZipA. Bacterial two-hybrid assays confirmed interactions between both LemA proteins and the cell division regulator CpoB, whose perturbation markedly altered vesicle formation. Additional interaction partners included PspA, an ESCRT-III-like protein implicated in membrane remodelling. Proteins involved in cell wall metabolism, membrane turnover, lipopolysaccharide (LPS) biogenesis, protein export and maturation, energy metabolism, and stress-responsive gene regulation were also enriched, suggesting a supporting role for PaLemA proteins in coordinating membrane-associated processes. Consistent with this, LemA deletion mutants displayed altered vesicle composition and global changes in gene expression. Furthermore, PalemA2 expression was induced in stationary phase and under envelope stress via the extracytoplasmic sigma factor AlgU, itself an interaction partner of the LemA proteins. Together, our findings indicate the LemA family act as a conserved membrane-remodelling system that, in conjunction with PspA, CpoB and divisome components, organises membrane-localized protein complexes required for bacterial organelle biogenesis and vesicle formation while executing a controlled, protein-machinery-driven mechanism that operates independently of passive chemical blebbing (such as hydrophobic PQS intercalation) or lytic membrane fragmentation.

microbiology↗

bioRxiv2026

Assembly and activation of the AAA+ motor of the multi-subunit cobaltochelatase

Cobalamin is a complex yet essential cobalt-containing cofactor synthesized exclusively by prokaryotes and archaea. Aerobic cobalamin biosynthesis requires the CobNST multi-subunit class I cobaltochelatase for cobalt incorporation. Cobalt chelation is dependent on the energy transfer from the CobST AAA+/VWA motor unit to the substrate-binding catalytic unit (CobN). However, the mechanism for assembly and function of CobNST cobaltochelatase remains poorly understood. Here, we used cryo-EM to solve the structures of the AAA+ motor CobS, in the absence and presence of ATP and its VWA-partner protein CobT. Our data reveal that the AAA+ CobS assembles into various conformations, including hexameric planar rings and different spiral left-handed assemblies: hexamers, double rings and filaments. In the absence of nucleotide, the CobT N-terminal domain interacts with CobS left-handed oligomers, remaining in a half-assembled, nonfunctional motor unit. In the CobST complex, ATP induces conformational rearrangement of CobS from left-handed helical oligomers to a right-handed closed hexamer. The simultaneous repositioning of the CobT VWA domain to the AAA+ central pore shifts the motor unit to a functional state, ready-to-drive CobN-catalyzed chelation reaction. These findings define the mechanism for assembly and activation of the class I cobaltochelatase CobST motor.

biochemistry↗

bioRxiv2025

Extracellular Vesicle-Linked Vitamin B12 Acquisition via Novel Binding Proteins in Bacteroides thetaiotaomicron

The acquisition and utilization of vitamin B12 (cobalamin) are essential for the metabolic functions of many gut bacteria, including Bacteroides thetaiotaomicron, which relies on external sources of cobalamin due to its inability to synthesize it de novo. To scavenge cobalamin efficiently, B. thetaiotaomicron employs a sophisticated cobamide uptake system comprising multiple operons encoding outer membrane binding proteins and transporters. This study identifies and characterizes several novel cobalamin-binding proteins and elucidates their roles in cobamide uptake and delivery via bacterial extracellular vesicles (BEVs), highlighting their siderophore-like function for competitive nutrient acquisition. We demonstrate that BtuJ1 and BtuJ2, members of the IPR027828 protein family, bind cobalamin with distinct structural and kinetic properties and are key components of BEVs responsible for cobamide capture and transfer to cells. Structural analyses reveal a conserved augmented {beta}-jellyroll architecture in these proteins, with tyrosine residues playing a central role in stabilizing cobalamin binding. Comparative proteomics of BEVs and cells under cobamide starvation underscore the selective enrichment of BtuJ proteins in BEVs, suggesting a specialized mechanism for nutrient acquisition. Additionally, we identify another novel B12-binding protein, BtuK1. We further establish BtuL as a critical player in early BEV release and propose a theoretical model in which BEVs function similarly to siderophores, scavenging cobalamin in the environment and delivering it to cells via specific receptors. This study provides new insights into the interplay between BEV-mediated transport, cobamide uptake, and the metabolic strategies employed by gut bacteria to thrive in nutrient-limited environments.

biochemistry↗

bioRxiv2025

Three-dimensional single-particle reconstruction by atomic force microscopy allows rapid structural-based validation of recombinant SARS-CoV-2 Spike protein from a single topology image

Atomic force microscopy (AFM) is a versatile multi-modal imaging method frequently used for structural characterisation of biological surfaces at the nanoscale. However, AFM-based three-dimensional single-particle reconstruction has hitherto not been possible due to the tip-sample convolution artifact that distorts AFM images of individual molecules, and the disconnect between two-dimensional AFM images of surface deposited molecules and their three-dimensional structures. Here, three-dimensional single-particle analysis was developed for rapid structure-based validation of protein structures using as few as a single AFM topology image, based on contact-point reconstruction AFM (CPR-AFM) in an integrative approach with cryo-electron microscopy maps available in the Electron Microscopy Data Bank by template matching. This approach was demonstrated on the structural validation of recombinant trimeric ectodomain of SARS-CoV-2 Spike glycoprotein to show its immediate utility as a rapid structure-based sample quality control method in the recombinant expression and purification of the Spike protein samples that can be used in vaccines and therapeutics research. These results show that three-dimensional single-particle reconstruction by AFM is possible, that high signal-to-noise AFM imaging offers a rapid and cost-effective way of validation or identification of three-dimensional protein structures at single particle level, and that AFM can be linked to structural data derived from methods such as cryo-electron microscopy, resulting in integrative methodologies with new capabilities for structural biology.

biophysics↗

bioRxiv2023

The conserved protein CBA1 is required for vitamin B12 uptake in different algal lineages

Microalgae play an essential role in global net primary productivity and global biogeochemical cycling, but despite their phototrophic lifestyle, over half of algal species depend on a supply of the corrinoid vitamin B12 (cobalamin) for growth. This essential organic micronutrient is produced only by a subset of prokaryotic organisms, which implies that for algal species to use this compound, they must first acquire it from external sources. Previous studies have identified protein components involved in vitamin B12 uptake in bacterial species and humans. However, little is known about how it is taken up in algae. Here, we demonstrate the essential role of a protein, CBA1 (for cobalamin acquisition protein 1), in B12 uptake in Phaeodactylum tricornutum, using CRISPR-Cas9 to generate targeted knockouts, and in Chlamydomonas reinhardtii, by insertional mutagenesis. In both cases, CBA1 knockout lines are no longer able to take up exogenous vitamin B12. Complementation of the C. reinhardtii mutants with the wildtype CBA1 gene restores B12 uptake, and regulation of CBA1 expression via a riboswitch element can be used to control the phenotype. When visualised by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 shows association with membranes. A bioinformatics analysis found that CBA1-like sequences are present in all the major eukaryotic phyla. Its presence is correlated with B12-dependent enzymes in many, although not all, taxa, suggesting CBA1 has a conserved role. Our results thus provide insight into the molecular basis of algal B12 acquisition, a process that likely underpins many interactions in aquatic microbial communities. One sentence summaryKnockout mutants and physiological studies demonstrate that the CBA1 protein is essential for uptake of vitamin B12 in both Chlamydomonas reinhardtii and the unrelated Phaeodactylum tricornutum.

biochemistry↗

bioRxiv2022

Enzyme encapsulation peptides bind to the groove between tessellating subunits of the bacterial microcompartment shell

Bacterial microcompartments are prokaryotic organelles consisting of encapsulated enzymes within a thin protein shell. They are involved in metabolic processing including propanediol, choline, glycerol and ethanolamine utilization, and carbon fixation. Enzymes targeted to the inside of the microcompartment frequently possess a cargo-encapsulation peptide, but the binding-site has not been revealed. We provide evidence that the encapsulation peptides bind to the hydrophobic groove formed between tessellating tiles of major shell proteins. In silico docking studies provide a compelling model of peptide binding to this hydrophobic groove. This result is consistent with the now widely accepted view that the convex side of the shell proteins faces the lumen of the microcompartment. Binding between shell protein tiles explains why the encapsulation peptide binding site has been elusive, how encapsulation-peptide bearing enzymes can promote shell assembly, and how the presence of cargo affects the size and shape of the bacterial microcompartment.

biochemistry↗

bioRxiv2022

Exploring the onset of B12-based mutualisms using a recently evolved Chlamydomonas auxotroph and B12-producing bacteria

Cobalamin (vitamin B12), is a cofactor for crucial metabolic reactions in multiple eukaryotic taxa, including major primary producers such as algae, and yet only prokaryotes can produce it. Many bacteria can colonise the algal phycosphere, forming stable communities that gain preferential access to exudates and in return provide compounds, such as B12. Extended coexistence can then drive gene loss, leading to greater algal-bacterial interdependence. In this study, we investigate how a recently evolved B12-dependent strain of Chlamydomonas reinhardtii, metE7, forms a mutualism with certain bacteria, including the rhizobium Mesorhizobium loti and even a strain of the gut bacterium E. coli engineered to produce cobalamin. Although metE7 was supported by B12 producers, its growth in co-culture was slower than the B12-independent wild-type, suggesting that high bacterial B12 provision may be necessary to favour B12 auxotrophs and their evolution. Moreover, we found that an E. coli strain that releases more B12 makes a better mutualistic partner, and although this trait may be more costly in isolation, greater B12 release provided an advantage in co-cultures. We hypothesise that, given the right conditions, bacteria that release more B12 may be selected for, particularly if they form close interactions with B12-dependent algae. Originality-Significance statementMicroalgae are fundamental to the global carbon cycle, and yet despite being photosynthetic they often rely on other organisms for micronutrients. One of these micronutrients is vitamin B12 (cobalamin), which they receive from bacteria. Many environmental studies support the widespread role of B12 in algal-bacterial mutualisms, so here we wished to investigate how these mutualisms may arise evolutionarily by using an experimentally evolved B12-dependent alga and various B12-producers. A B12-producing rhizobium, Mesorhizobium loti, could stably support the B12-dependent Chlamydomonas reinhardtii metE7 strain, and vice versa, but nutrient supplementation increased growth of both species further. metE7 could also be supported by E. coli strains engineered to produce B12, and engineering a strain to release a higher proportion of B12 led to better algal growth, which increased bacterial growth in turn. We suggest that as B12-based mutualisms develop, increased B12 release may be selected for and therefore lead to more productive symbioses.

microbiology↗

bioRxiv2021

Plasmodium falciparum hydroxymethylbilane synthase does not house any cosynthase activity within the haem biosynthetic pathway

The production of uroporphyrinogen III, the universal progenitor of macrocyclic, modified tetrapyrroles, is produced from aminolaevulinic acid (ALA) by a conserved pathway involving three enzymes: porphobilinogen synthase (PBGS), hydroxymethylbilane synthase (HmbS) and uroporphyrinogen III synthase (UroS). The gene encoding uroporphyrinogen III synthase has not yet been identified in Plasmodium falciparum but it has been suggested that this activity is housed inside a bifunctional hybroxymethylbilane synthase (HmbS). In this present study it is demonstrated that P. falciparum HmbS does not have uroporphyrinogen III synthase activity. This was demonstrated by the failure of a codon optimised P. falciparum hemC gene, encoding HmbS, to compliment a defined E. coli hemD- mutant (SASZ31) deficient in uroporphyrinogen III synthase activity. Furthermore, HPLC analysis of the oxidsed reaction product from recombinant, purified HmbS showed that only uroporphyrin I could be detected (corresponding to hydroxymethylbilane production). No uroporphyrin III was detected, thus showing that P. falciparum HmbS does not have UroS activity and can only catalyse the formation of hydroxymethylbilane from porphobilinogen.

biochemistry↗

bioRxiv2020

Calculating metalation in cells reveals CobW acquires CoII for vitamin B12 biosynthesis upon binding nucleotide

Protein metal-occupancy (metalation) in vivo has been elusive. Here we develop a metalation-calculator which accounts for inter-metal competition and changing metal-availabilities inside cells. The calculations are based on available free-energies of metals determined from the responses of metal sensors. We use the calculator to understand the function and mechanism of CobW, a predicted CoII-chaperone for vitamin B12. CobW is calculated to acquire negligible metal alone: But, upon binding nucleotide (GTP) and MgII, CobW assembles a high-affinity site that can obtain CoII or ZnII from the intracellular milieu. In idealised cells with sensors at the mid-points of their responses, competition within the cytosol enables CoII to outcompete ZnII for binding CobW. Thus, CoII is the cognate metal. However, after growth in different [CoII], CoII-occupancy ranges from 10 to 97% which matches CobW-dependent B12 synthesis. The calculator reveals how CobW acquires its metal and is made available for use with other proteins.

biochemistry↗

bioRxiv2017

Experimental warming reduces the diversity and functional potential of the Sphagnum microbiome

Climate change may reduce biodiversity leading to a reduction in ecosystem productivity. Despite numerous reports of a strong correlation of microbial diversity and ecosystem productivity, little is known about the warming effects on plant associated microbes. Here we explore the impact of experimental warming on the microbial and nitrogen-fixing (diazotroph) community associated with the widespread and ecologically relevant Sphagnum genus in a field warming experiment. To quantify changes in the abundance, diversity, and community composition of Sphagnum microbiomes with warming we utilized qPCR and Illumina sequencing of the 16S SSU rRNA and nifH gene. Microbial and diazotroph community richness and Shannon diversity decreased with warming (p<0.05). The diazotroph communities shifted from diverse communities to domination by primarily Nostocaceae (25% in control samples to 99% in elevated temperature samples). In addition, the nitrogen fixation activity measured with the acetylene reduction assay (ARA) decreased with warming treatment. This suggests the negative correlation of temperature and microbial diversity corresponds to a reduction in functional potential within the diazotroph community. The results indicate that climate warming may alter the community structure and function in peat moss microbiomes, with implications for impacts to host fitness and ecosystem productivity, and carbon uptake potential of peatlands.

ecology↗

bioRxiv2017

Alphaproteobacteria fix nitrogen in a Sphagnum-dominated peat bog using molybdenum-dependent nitrogenase

Microbial N2 fixation (diazotrophy) represents an important nitrogen source to oligotrophic peatland ecosystems, which are important sinks for atmospheric CO2 and susceptible to changing climate. The objectives of this study were: (i) to determine the active microbial group and type of nitrogenase mediating diazotrophy in a ombrotrophic Sphagnum-dominated peat bog (the S1 peat bog, Marcell Experimental Forest, Minnesota, USA); and (ii) to determine the effect of environmental parameters (light, O2, CO2, CH4) on potential rates of diazotrophy measured by acetylene (C2H2) reduction and 15N2 incorporation. Molecular analysis of metabolically active microbial communities suggested that diazotrophy in surface peat was primarily mediated by Alphaproteobacteria (Bradyrhizobiaceae and Beijerinckiaceae). Despite higher dissolved vanadium (V; 11 nM) than molybdenum (Mo; 3 nM) in surface peat, a combination of metagenomic, amplicon sequencing and activity measurements indicated that Mo-containing nitrogenases dominate over the V-containing form. Acetylene reduction was only detected in surface peat exposed to light, with the highest rates observed in peat collected from hollows with the highest water content. Incorporation of15N2 was suppressed 90% by O2 and 55% by C2H2, and was unaffected by CH4 and CO2 amendments. These results suggest that peatland diazotrophy is mediated by a combination of C2H2 - sensitive and C2H2 - insensitive microbes that are more active at low O2 and show similar activity at high and low CH4.\n\nImportancePrevious studies indicate that diazotrophy provides an important nitrogen source and is linked to methanotrophy in Sphagnum-dominated peatlands. However, the environmental controls and enzymatic pathways of peatland diazotrophy, as well as the metabolically active microbial populations that catalyze this process remain in question. Our findings indicate that oxygen levels and photosynthetic activity override low nutrient availability in limiting diazotrophy, and that members of the Alphaproteobacteria (Rhizobiales) catalyze this process at the bog surface using the molybdenum - based form of the nitrogenase enzyme.

microbiology↗