Biology subjects

Meheust, R.

Publications and source records attributed to Meheust, R..

12 recordsLinked to original sources

bioRxiv2024

Versatile roles of protein flavinylation in bacterial extracyotosolic electron transfer

Bacteria perform diverse redox chemistries in the periplasm, cell wall, and extracellular space. Electron transfer for these extracytosolic activities is frequently mediated by proteins with covalently bound flavins, which are attached through post-translational flavinylation by the enzyme ApbE. Despite the significance of protein flavinylation to bacterial physiology, the basis and function of this modification remains unresolved. Here we apply genomic context analyses, computational structural biology, and biochemical studies to address the role of ApbE flavinylation throughout bacterial life. We find that ApbE flavinylation sites exhibit substantial structural heterogeneity. We identify two novel classes of flavinylation substrates that are related to characterized proteins with non-covalently bound flavins, providing evidence that protein flavinylation can evolve from a non-covalent flavoprotein precursor. We further find a group of structurally related flavinylation-associated cytochromes, including those with the domain of unknown function DUF4405, that presumably mediate electron transfer in the cytoplasmic membrane. DUF4405 homologs are widespread in bacteria and related to ferrosome iron storage organelle proteins that may facilitate iron redox cycling within ferrosomes. These studies reveal a complex basis for flavinylated electron transfer and highlight the discovery power of coupling comparative genomic analyses with high-quality structural models.

microbiology↗

bioRxiv2022

Exceptionally versatile respiratory metabolisms drive metabolite production by diverse gut bacteria

Respiratory reductases enable microbes to utilize molecules present in anaerobic ecosystems as energy-generating respiratory electron acceptors. Here we identify three taxonomically distinct families of human gut bacteria (Burkholderiaceae, Eggerthellaceae, Erysipelotrichaceae) that encode large arsenals of tens-to-hundreds of respiratory-like reductases per genome. Screening species from each family (Sutterella wadsworthensis, Eggerthella lenta, and Holdemania filiformis), we discover 22 metabolites used as respiratory electron acceptors in a species-specific manner. Identified reactions transform multiple classes of dietary- and host-derived metabolites, including bioactive molecules resveratrol and itaconate. Products of identified respiratory metabolisms highlight poorly characterized compounds, such as the itaconate-derived 2-methylsuccinate. Reductase substrate-profiling defines enzyme-substrate pairs and reveals a complex picture of reductase evolution, providing evidence that reductases with specificities for related cinnamate substrates independently emerged at least four times. These studies thus establish an exceptionally versatile form of anaerobic respiration that directly links microbial energy metabolism to the gut metabolome.

microbiology↗

bioRxiv2022

Distinct energy-coupling factor transporter subunits enable flavin acquisition and extracytosolic trafficking for extracellular electron transfer in Listeria monocytogenes

A variety of electron transfer mechanisms link bacterial cytosolic electron pools with functionally diverse redox activities in the cell envelope and extracellular space. In Listeria monocytogenes, the ApbE-like enzyme FmnB catalyzes extracytosolic protein flavinylation, covalently linking a flavin cofactor to proteins that transfer electrons to extracellular acceptors. L. monocytogenes uses an energy-coupling factor (ECF) transporter complex that contains distinct substrate-binding, transmembrane, ATPase A, and ATPase A subunits (RibU, EcfT, EcfA, and EcfA) to import environmental flavins, but the basis of extracytosolic flavin trafficking for FmnB flavinylation remains poorly defined. In this study, we show that the proteins EetB and FmnA are related to ECF transporter substrate-binding and transmembrane subunits, respectively, and are essential for exporting flavins from the cytosol for flavinylation. Comparisons of the flavin import versus export capabilities of L. monocytogenes strains lacking different ECF transporter subunits demonstrates a strict directionality of substrate-binding subunit transport but partial functional redundancy of transmembrane and ATPase subunits. Based on these results, we propose that ECF transporter complexes with different subunit compositions execute directional flavin import/export through a broadly conserved mechanism. Finally, we present genomic context analyses which show that related ECF exporter genes are distributed across the Firmicutes phylum and frequently co-localize with genes encoding flavinylated extracytosolic proteins. These findings clarify the basis of ECF transporter export and extracytosolic flavin cofactor trafficking in Firmicutes.

microbiology↗

bioRxiv2021

Protein family content uncovers lineage relationships and bacterial pathway maintenance mechanisms in DPANN archaea

DPANN are small-celled archaea that are generally predicted to be symbionts, and in some cases are known episymbionts of other archaea. As the monophyly of the DPANN remains uncertain, we hypothesized that proteome content could reveal relationships among DPANN lineages, constrain genetic overlap with bacteria, and illustrate how organisms with hybrid bacterial and archaeal protein sets might function. We tested this hypothesis using protein family content that was defined in part using 569 newly reconstructed genomes. Protein family content clearly separates DPANN from other archaea, paralleling the separation of Candidate Phyla Radiation (CPR) bacteria from all other bacteria. This separation is partly driven by hypothetical proteins, some of which may be symbiosis-related. Pacearchaeota with the most limited predicted metabolic capacities have Form II/III and III-like Rubisco, suggesting metabolisms based on scavenged nucleotides. Intriguingly, the Pacearchaeota and Woesearchaeota with the smallest genomes also tend to encode large extracellular murein-like lytic transglycosylase domain proteins that may bind and degrade components of bacterial cell walls, indicating that some might be episymbionts of bacteria. The pathway for biosynthesis of bacterial isoprenoids is widespread in Woesearchaeota genomes and is encoded in proximity to genes involved in bacterial fatty acids synthesis. Surprisingly, in some DPANN genomes we identified a pathway for synthesis of queuosine, an unusual nucleotide in tRNAs of bacteria. Other bacterial systems are predicted to be involved in protein refolding. For example, many DPANN have the complete bacterial DnaK-DnaJ-GrpE system and many Woesearchaeota and Pacearchaeota possess bacterial group I chaperones. Thus, many DPANN appear to have mechanisms to ensure efficient protein folding of both archaeal and laterally acquired bacterial proteins.

microbiology↗

bioRxiv2020

Early acquisition of conserved, lineage-specific proteins currently lacking functional predictions were central to the rise and diversification of archaea

Recent genomic analyses of Archaea have profoundly reshaped our understanding of their distribution, functionalities and roles in eukaryotic evolution. Within the domain, major supergroups are Euryarchaeota, which includes many methanogens, the TACK, which includes Thaumarchaeaota that impact ammonia oxidation in soils and the ocean, the Asgard, which includes lineages inferred to be ancestral to eukaryotes, and the DPANN, a group of mostly symbiotic small-celled archaea. Here, we investigated the extent to which clustering based on protein family content recapitulates archaeal phylogeny and identified the proteins that distinguish the major subdivisions. We also defined 10,866 archaeal protein families that will serve as a community resource. Clustering based on these families broadly recovers the archaeal phylogenetic tree. Interestingly, all major groups are distinguished primarily by the presence of families of conserved hypothetical proteins that are either novel or so highly diverged that their functions are obscured. Given that these hypothetical proteins are near ubiquitous within phyla, we conclude that they were important in the origin of most of the major archaeal lineages.

microbiology↗

bioRxiv2020

Structure of the Bacterial Ribosome at 2 A Resolution

Continuing advances in cryo-electron microscopy (cryo-EM) demonstrate the promise it holds for revealing biological structures at chemical resolution, in which noncovalent interactions, RNA and protein modifications, and solvation can be modeled accurately. At present, the best cryo-EM-derived models of the bacterial ribosome are of the large (50S) ribosomal subunit with effective global resolutions of 2.4-2.5 [A], based on map-to-model Fourier shell correlation (FSC). Here we present a model of the E. coli 70S ribosome with an effective global resolution of 2.0 [A], based on maps showcasing unambiguous positioning of residues, their detailed chemical interactions, and chemical modifications. These modifications include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former of which is likely conserved in all domains of life. The model also defines extensive solvation of the small (30S) ribosomal subunit for the first time, as well as interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The high quality of the maps now allows a deeper phylogenetic analysis of ribosomal components, and identification of structural conservation to the level of solvation. The maps and models of the bacterial ribosome presented here should enable future structural analysis of the chemical basis for translation, and the development of robust tools for cryo-EM structure modeling and refinement.

biophysics↗

bioRxiv2020

Large Freshwater Phages with the Potential to Augment Aerobic Methane Oxidation

There is growing evidence that phages with unusually large genomes are common across various natural and human microbiomes, but little is known about their genetic inventories or potential ecosystem impacts. Here, we reconstructed large phage genomes from freshwater lakes known to contain bacteria that oxidize methane. Twenty-two manually curated genomes (18 are complete) ranging from 159 to 527 kbp in length were found to encode the pmoC gene, an enzymatically critical subunit of the particulate methane monooxygenase, the predominant methane oxidation catalyst in nature. The phage-associated PmoC show high similarity (> 90%) and affiliate phylogenetically with those of coexisting bacterial methanotrophs, and their abundance patterns correlate with the abundances of these bacteria, supporting host-phage relationships. We suggest that phage PmoC has similar functions to additional copies of PmoC encoded in bacterial genomes, thus contribute to growth on methane. Transcriptomics data from one system showed that the phage-associated pmoC genes are actively expressed in situ. Augmentation of bacterial methane oxidation by pmoC-phages during infection could modulate the efflux of this powerful greenhouse gas into the environment.

microbiology↗

bioRxiv2019

Bacterial secondary metabolite biosynthetic potential in soil varies with phylum, depth, and vegetation type

Bacteria isolated from soils are major sources of specialized metabolites, including antibiotics and other compounds with clinical value that likely shape interactions among microbial community members and impact biogeochemical cycles. Yet, isolated lineages represent a small fraction of all soil bacterial diversity. It remains unclear how the production of specialized metabolites varies across the phylogenetic diversity of bacterial species in soils, and whether the genetic potential for production of these metabolites differs with soil type. We sampled soils and saprolite from three sites in a northern California Critical Zone Observatory with varying vegetation and bedrock characteristics and used metagenomic sequencing and assembly to reconstruct 1,334 microbial genomes containing diverse biosynthetic gene clusters (BGCs) for secondary metabolite production. We obtained genomes for prolific producers of secondary metabolites, including novel groups within the Actinobacteria, Chloroflexi and candidate phylum Dormibactereota. Surprisingly, one genome of a Candidate Phyla Radiation bacterium encoded for a ribosomally synthesized linear azole/azoline-containing peptide, a capacity we found in other publicly available CPR bacterial genomes. Overall, bacteria with higher biosynthetic potential were enriched in shallow soils and grassland soils, with patterns of abundance of BGC type varying by taxonomy.

microbiology↗

bioRxiv2019

Aquatic Elusimicrobia are metabolically diverse compared to gut microbiome Elusimicrobia and some have novel nitrogenase-like gene clusters

Currently described members of Elusimicrobia, a relatively recently defined phylum, are animal-associated and rely on fermentation. However, free-living Elusimicrobia have been detected in sediments, soils and groundwater, raising questions regarding their metabolic capacities and evolutionary relationship to animal-associated species. Here, we analyzed 94 draft-quality, non-redundant genomes, including 30 newly reconstructed genomes, from diverse animal-associated and natural environments. Genomes group into 12 clades, 10 of which previously lacked reference genomes. Groundwater-associated Elusimicrobia are predicted to be capable of heterotrophic or autotrophic lifestyles, reliant on oxygen or nitrate/nitrite-dependent respiration, or a variety of organic compounds and Rhodobacter nitrogen fixation-dependent (Rnf-dependent) acetogenesis with hydrogen and carbon dioxide as the substrates. Genomes from two clades of groundwater-associated Elusimicrobia often encode a new group of nitrogenase paralogs that co-occur with an extensive suite of radical S-Adenosylmethionine (SAM) proteins. We identified similar genomic loci in genomes of bacteria from the Gracilibacteria phylum and the Myxococcales order and predict that the gene clusters reduce a tetrapyrrole, possibly to form a novel cofactor. The animal-associated Elusimicrobia clades nest phylogenetically within two free-living-associated clades. Thus, we propose an evolutionary trajectory in which some Elusimicrobia adapted to animal-associated lifestyles from free-living species via genome reduction.

microbiology↗

bioRxiv2019

Clades of huge phage from across Earth’s ecosystems

Phage typically have small genomes and depend on their bacterial hosts for replication. DNA sequenced from many diverse ecosystems revealed hundreds of huge phage genomes, between 200 kbp and 716 kbp in length. Thirty-four genomes were manually curated to completion, including the largest phage genomes yet reported. Expanded genetic repertoires include diverse and new CRISPR-Cas systems, tRNAs, tRNA synthetases, tRNA modification enzymes, translation initiation and elongation factors, and ribosomal proteins. Phage CRISPR-Cas systems have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phage may repurpose bacterial CRISPR-Cas systems to eliminate competing phage. We phylogenetically define major clades of huge phage from human and other animal microbiomes, oceans, lakes, sediments, soils and the built environment. We conclude that their large gene inventories reflect a conserved biological strategy, observed over a broad bacterial host range and across Earths ecosystems.

microbiology↗

bioRxiv2018

Candidate Phyla Radiation Roizmanbacteria from hot springs have novel, unexpectedly abundant, and potentially alternatively functioning CRISPR-Cas systems

The Candidate Phyla Radiation (CPR) comprises a huge group of bacteria that have small genomes that rarely encode CRISPR-Cas systems for phage defense. Consequently, questions remain about their mechanisms of phage resistance and the nature of phage that infect them. The compact CRISPR-CasY system (Cas12d) with potential value in genome editing was first discovered in these organisms. Relatively few CasY sequences have been reported to date, and little is known about the function and activity of these systems in the natural environment. Here, we conducted a genome-resolved metagenomic investigation of hot spring microbiomes and recovered CRISPR systems mostly from Roizmanbacteria that involve CasY proteins that are divergent from published sequences. Within population diversity in the spacer set indicates current in situ diversification of most of the loci. In addition to CasY, some Roizmanbacteria genomes also encode large type I-B and/or III-A systems that, based on spacer targeting, are used in phage defense. CRISPR targeting identified three phage represented by complete genomes and a prophage, which are the first reported for bacteria of the Microgenomates superphylum. Interestingly, one phage encodes a Cas4-like protein, a scenario that has been suggested to drive acquisition of self-targeting spacers. Consistent with this, the Roizmanbacteria population that it infects has a CRISPR locus that includes self-targeting spacers and a fragmented CasY gene (fCasY). Despite gene fragmentation, the PAM sequence is the same as that of other CasY reported in this study. Fragmentation of CasY may avoid the lethality of self-targeting spacers. However, the spacers may still have some biological role, possibly in genome regulation. The findings expand our understanding of CasY diversity, and more broadly, CRISPR-Cas systems and phage of CPR bacteria.

microbiology↗

bioRxiv2018

Biological capacities clearly define a major subdivision in Domain Bacteria

ResumePhylogenetic analyses separate candidate phyla radiation (CPR) bacteria from other bacteria, but the degree to which their proteomes are distinct remains unclear. Here, we leveraged a proteome database that includes sequences from thousands of uncultivated organisms to identify protein families and examine their organismal distributions. We focused on widely distributed protein families that co-occur in genomes, as they likely foundational for metabolism. Clustering of genomes using the protein family presence/absence patterns broadly recapitulates the phylogenetic structure of the tree, suggesting persistence of core sets of protein families after lineage divergence. CPR bacteria group together and away from all other bacteria and archaea, in part due to novel proteins, some of which may be involved in cell-cell interactions. The diversity of combinations of protein families in CPR may exceed that of all other bacteria. Overall, the results extend the phylogeny-based suggestion that the CPR represent a major subdivision within Bacteria.

microbiology↗