Biology subjects

Garg, S. G.

Publications and source records attributed to Garg, S. G..

9 recordsLinked to original sources

bioRxiv2024

A general substitution matrix for structural phylogenetics.

Sequence-based maximum likelihood (ML) phylogenetics is a widely used method for inferring evolutionary relationships, which has illuminated the evolutionary histories of proteins and the organisms that harbour them. But modern implementations with sophisticated models of sequence evolution struggle to resolve deep evolutionary relationships, which can be obscured by excessive sequence divergence and substitution saturation. Structural phylogenetics has emerged as a promising alternative, because protein structure evolves much more slowly than protein sequences. Recent developments protein structure prediction using AI have made it possible to predict protein structures for entire protein families, and then to translate these structures into a sequence representation - the 3Di structural alphabet - that can in theory be directly fed into existing sequence based phylogenetic software. To unlock the full potential of this idea, however, requires the inference of a general substitution matrix for structural phylogenetics, which has so far been missing. Here we infer this matrix from large datasets of protein structures and show that it results in a better fit to empirical datasets that previous approaches. We then use this matrix to re-visit the question of the root of the tree of life. Using structural phylogenies of universal paralogs, we provide the first unambiguous evidence for a root between and archaea and bacteria. Finally, we discuss some practical and conceptual limitations of structural phylogenetics. Our 3Di substitution matrix provides a starting point for revisiting many deep phylogenetic problems that have so far been extremely difficult to solve.

bioinformatics↗

bioRxiv2023

Molecular model of a bacterial flagellar motor in situ reveals a "parts-list" of protein adaptations to increase torque

One hurdle to understanding how molecular machines work, and how they evolve, is our inability to see their structures in situ. Here we describe a minicell system that enables in situ cryogenic electron microscopy imaging and single particle analysis to investigate the structure of an iconic molecular machine, the bacterial flagellar motor, which spins a helical propeller for propulsion. We determine the structure of the high-torque Campylobacter jejuni motor in situ, including the subnanometre-resolution structure of the periplasmic scaffold, an adaptation essential to high torque. Our structure enables identification of new proteins, and interpretation with molecular models highlights origins of new components, reveals modifications of the conserved motor core, and explain how these structures both template a wider ring of motor proteins, and buttress the motor during swimming reversals. We also acquire insights into universal principles of flagellar torque generation. This approach is broadly applicable to other membrane-residing bacterial molecular machines complexes.

molecular biology↗

bioRxiv2021

The ER transmembrane complex (EMC) can functionally replace the Oxa1 insertase in mitochondria

Two multisubunit protein complexes for membrane protein insertion were recently identified in the endoplasmic reticulum (ER): The guided entry of tail anchor proteins (GET) complex and ER membrane complex (EMC). The structures of both of their hydrophobic core subunits, that are required for the insertion reaction, revealed an overall similarity to the YidC/Oxa1/Alb3 family members found in bacteria, mitochondria and chloroplasts. This suggests that these membrane insertion machineries all share a common ancestry. To test whether these ER proteins can functionally replace Oxa1 in yeast mitochondria, we generated strains that express mitochondria-targeted Get2-Get1 and Emc6-Emc3 fusion proteins in Oxa1 deletion mutants. Interestingly, the Emc6-Emc3 fusion was able to complement an{Delta} oxa1 mutant and restored its respiratory competence. The Emc6-Emc3 fusion promoted the insertion of the mitochondrially encoded protein Cox2 as well as of nuclear encoded inner membrane proteins though was not able to facilitate the assembly of the Atp9 ring. Our observations indicate that protein insertion into the ER is functionally conserved to the insertion mechanism in bacteria and mitochondria and adheres to similar topological principles.

biochemistry↗

bioRxiv2021

The asgardarchaeal-unique contribution to protein families of the eukaryotic common ancestor was 0.3%

Significance StatementEver since the first report of a new archaeal lineage, the asgardarchaea, their metagenome analyses have encouraged continued speculations on a type of cell biology ranging between that of prokaryotes and eukaryotes. While it appears a tempting notion, recent microscopic images of an asgardarchaeon suggest otherwise. We inspected the origin of eukaryotic protein families with respect to their distribution across bacteria and archaea. This reveals that the protein families shared exclusively between asgardarchaea and eukaryotes amounts to only 0.3% of the protein families conserved across all eukaryotes. Asgardarchaeal diversity is likely unrivaled across archaea, but their cell biology remains prokaryotic in nature and lends support for the importance of endosymbiosis in evolving eukaryotic traits. SummaryThe difference between pro- and eukaryotic biology is evident in their genomes, cell biology, and evolution of complex and macroscopic body plans. The lack of intermediates between the two types of cells places the endosymbiotic acquisition of the mitochondrion through an archaeal host at the event horizon of eukaryote origin. The identification of eukaryote specific proteins in a new archaeal phylum, the asgardarchaea, has fueled speculations about their cellular complexity, suggesting they could be eukaryote-like. Here we analyzed the coding capacity of 150 eukaryotes, 1000 bacteria, and 226 archaea, including the only cultured member of the asgardarchaea, Candidatus Prometheoarchaeon syntrophicum MK-D1. Established clustering methods that recover endosymbiotic contributions to eukaryotic genomes, recover an asgardarchaeal-unique contribution of a mere 0.3% to protein families present in the last eukaryotic common ancestor, while simultaneously suggesting that asgardarchaeal diversity rivals that of all other archaea combined. Furthermore, we show that the number of homologs shared exclusively between asgardarchaea and eukaryotes is only 27 on average. Genomic and in particular cellular complexity remains a eukaryote-specific feature and, we conclude, is best understood as the archaeal hosts solution to housing an endosymbiont and not as a preparation for obtaining one.

evolutionary biology↗

bioRxiv2019

Major changes in plastid protein import and the origin of the Chloroplastida

While core components of plastid protein import (Toc and Tic) and the principle of using N-terminal targeting sequences (NTS) are conserved, lineage-specific differences are known. Rhodophytes and glaucophytes carry a conserved NTS motif, which was lost in the green lineage that also added novel proteins to Toc and Tic. Here we compare the components of plastid protein import and generated RNA-Seq, pigment profile and trans-electron microscopy data based on high-light stress from representatives of the three archaeplastidal groups. In light of plastid protein targeting, we compare the response to high-light stress of archaeplastidal representatives based on RNA-Seq, pigment profile and trans-electron microscopy data. Like land plants, the chlorophyte Chlamydomonas reinhardtii displays a broad respond to high-light stress, not observed to the same degree in the glaucophyte Cyanophora paradoxa or the rhodophyte Porphyridium purpureum. We find that only the green lineage encodes a conserved duplicate of the outer plastid membrane protein channel Oep80, namely Toc75 and suggest that the ability to respond to high-light stress entailed evolutionary changes in protein import, including the departure from phenylalanine-based targeting and the introduction of a green-specific Toc75 next to other import components unique to Chloroplastida. One consequence of relaxed NTS specificity was the origin of dual-targeting of plastid derived proteins to mitochondria and vice versa, using a single ambiguous NTS. Changes in the plastid protein import enabled the green lineage to import proteins at a more efficient rate, including those required for high-light stress response, a prerequisite for the colonization of land.\n\nHigh-lightsO_LILoss of Phe-based N-terminal targeting sequences (NTS) triggered the origin of dual-targeting using a single ambiguous NTS\nC_LIO_LIThe Chloroplastida evolved a green-specific Toc75 for high throughput import, next to a universal and ancient Omp85 present in all Archaeplastida\nC_LIO_LIA broad response to high-light stress appears unique to Chloroplastida\nC_LIO_LIRelaxation of functional constraints allowed a broader modification of the green Toc/Tic machinery\nC_LIO_LICritical changes in plastid targeting enabled the origin and success of the Chloroplastida and their later conquer of land\nC_LI

evolutionary biology↗

bioRxiv2019

Gene duplications trace mitochondria to the onset of eukaryote complexity

The last eukaryote common ancestor (LECA) lived 1.6 billion years ago1,2. It possessed nuclei, sex, an endomembrane system, mitochondria, and all key traits that make eukaryotic cells more complex than their prokaryotic ancestors2-6. The closest known relatives of the host lineage that acquired the mitochondrion are, however, small obligately symbiotic archaea that lack any semblance of eukaryotic cell complexity7. Although the steep evolutionary grade separating prokaryotes from eukaryotes increasingly implicates mitochondrial symbiosis at eukaryote origin4,7, the timing and evolutionary significance of mitochondrial origin remains debated. Gradualist theories contend that eukaryotes arose from archaea by slow accumulation of eukaryotic traits8-10 with mitochondria arriving late11, while symbiotic theories have it that mitochondria initiated the onset of eukaryote complexity in a non-nucleated archaeal host7 by gene transfers from the organelle4,12-14. The evolutionary process leading to LECA should be recorded in its gene duplications. Among 163,545 duplications in 24,571 gene trees spanning 150 sequenced eukaryotic genomes we identified 713 gene duplication events that occurred in LECA. LECAs bacterially derived genes were duplicated more frequently than archaeal derived or eukaryote specific genes, reflecting the serial copying15,16 of genes from the mitochondrial endosymbiont to the archaeal hosts chromosomes prior to the onset of eukaryote genome complexity. Bacterial derived genes for mitochondrial functions, lipid synthesis, biosynthesis, as well as core carbon and energy metabolism in LECA were duplicated more often than archaeal derived genes and even more often than eukaryote-specific inventions for endomembrane, cytoskeletal or cell cycle functions. Gene duplications record the sequence of events at LECAs origin and indicate that recurrent gene transfer from a resident mitochondrial endosymbiont preceded the onset of eukaryotic cellular complexity.

evolutionary biology↗

bioRxiv2019

Anomalous phylogenetic behavior of ribosomal proteins in metagenome assembled genomes

Metagenomic studies have claimed the existence of novel lineages with unprecedented properties never before observed in prokaryotes. Such lineages include Asgard archaea1-3, which are purported to represent archaea with eukaryotic cell complexity, and the Candidate Phyla Radiation (CPR), a novel domain level taxon erected solely on the basis of metagenomic data4. However, it has escaped the attention of most biologists that these metagenomic sequences are not assembled into genomes by sequence overlap, as for cultured archaea and bacteria. Instead, short contigs are sorted into computer files by a process called binning in which they receive taxonomic assignment on the basis of sequence properties like GC content, dinucleotide frequencies, and stoichiometric co-occurrence across samples. Consequently, they are not genome sequences as we know them, reflecting the gene content of real organisms. Rather they are metagenome assembled genomes (MAGs). Debates that Asgard data are contaminated with individual eukaryotic sequences5-7 are overshadowed by the more pressing issue that no evidence exists to indicate that any sequences in binned Asgard MAGs actually stem from the same chromosome, as opposed to simply stemming from the same environment. Here we show that Asgard and CPR MAGs fail spectacularly to meet the most basic phylogenetic criterion8 fulfilled by genome sequences of all cultured prokaryotes investigated to date: the ribosomal proteins of Asgard and CPR MAGs do not share common evolutionary histories. Their phylogenetic behavior is anomalous to a degree never observed with genomes of real organisms. CPR and Asgard MAGs are binning artefacts, assembled from environments where up to 90% of the DNA is from dead cells9-12. Asgard and CPR MAGs are unnatural constructs, genome-like patchworks of genes that have been stitched together into computer files by binning.

evolutionary biology↗

bioRxiv2019

Adaptation to life on land at 21% O2 via transition from ferredoxin- to NADH-dependent redox balance

Pyruvate:ferredoxin oxidoreductase (PFO) and iron only hydrogenase ([Fe]-HYD) are common enzymes among eukaryotic microbes that inhabit anaerobic niches. Their function is to maintain redox balance by donating electrons from food oxidation via ferredoxin (Fd) to protons, generating H2 as a waste product. Operating in series, they constitute a soluble electron transport chain of one-electron transfers between FeS clusters. They fulfill the same function -- redox balance -- served by two electron-transfers in the NADH- and O2-dependent respiratory chains of mitochondria. Although they possess O2-sensitive FeS clusters, PFO, Fd and [Fe]-HYD are also present among numerous algae that produce O2. The evolutionary persistence of these enzymes among eukaryotic aerobes is traditionally explained as enabling facultative anaerobic growth. Here we show that algae express enzymes of anaerobic energy metabolism at ambient O2 levels (21% v/v), Chlamydomonas reinhardtii expresses them with diurnal regulation. High O2 environments arose on Earth only some [~]450 million years ago. Gene presence absence and gene expression data indicate that during the transition to high O2 environments and terrestrialization, diverse algal lineages retained enzymes of Fd-dependent one-electron based redox balance, while the land plant and land animal lineages underwent irreversible specialization to redox balance involving the O2-insensitive two-electron carrier NADH.\n\nHighlights- Algae express enzymes of anaerobic metabolism in 21% [v/v] O2 atmosphere, independent of anaerobiosis\n- Retention of a plastid-encoded NADH dehydrogenase-like (NDH) was likely a prerequisite for the transition to life on land\n- Terrestrialization and adaption to high O2 is accompanied by a shift to redox balance at higher midpoint potentials\n- Eukaryotes adapted to high O2 life on land via specialization to two-electron based redox balance

evolutionary biology↗

bioRxiv2018

BspA and Pmp proteins of Trichomonas vaginalis mediate adherence to host cells

Trichomonas vaginalis is one of the most widespread, sexually transmitted pathogens. The infection involves a morphological switch from a free-swimming pyriform trophozoite to an amoeboid cell upon adhesion to host epithelial cells. While details on how the switch is induced and to what proteins of the host surface the parasite adheres remain poorly characterized, several surface proteins of the parasite itself have been identified as potential candidates. Among those are two expanded protein families that harbor domains that share similarity to functionally investigated surface proteins of prokaryotic oral pathogens; these are the BspA proteins of Bacteroidales and Spirochaetales, and the Pmp proteins of Chlamydiales. We sequenced the transcriptomes of five Trichomonads and screened for the presence of BspA and Pmp domain-containing proteins and tested the ability of individual T. vaginalis candidates to mediate adhesion. Here we demonstrate that (i) BspA and Pmp domain-containing proteins are specifically expanded in T. vaginalis in comparison to other Trichomonads, and that (ii) individual proteins of both families have the ability to increase adhesion performance in a non-virulent T. vaginalis strain and Tetratrichomonas gallinarum, a parasite usually known to infect birds but not humans. Our results initiate the functional characterization of these two broadly distributed protein families, whose origin we trace back to the origin of Trichomonads themselves.

cell biology↗