Co-transcriptional gene regulation in eukaryotes and prokaryotes.

Many steps of RNA processing occur during transcription by RNA polymerases. Co-transcriptional activities are deemed commonplace in prokaryotes, in which the lack of membrane barriers allows mixing of all gene expression steps, from transcription to translation. In the past decade, an extraordinary level of coordination between transcription and RNA processing has emerged in eukaryotes. In this Review, we discuss recent developments in our understanding of co-transcriptional gene regulation in both eukaryotes and prokaryotes, comparing methodologies and mechanisms, and highlight striking parallels in how RNA polymerases interact with the machineries that act on nascent RNA. The development of RNA sequencing and imaging techniques that detect transient transcription and RNA processing intermediates has facilitated discoveries of transcription coordination with splicing, 3'-end cleavage and dynamic RNA folding and revealed physical contacts between processing machineries and RNA polymerases. Such studies indicate that intron retention in a given nascent transcript can prevent 3'-end cleavage and cause transcriptional readthrough, which is a hallmark of eukaryotic cellular stress responses. We also discuss how coordination between nascent RNA biogenesis and transcription drives fundamental aspects of gene expression in both prokaryotes and eukaryotes.

Structural basis for plasmid restriction by SMC JET nuclease.

DNA loop-extruding SMC complexes play crucial roles in chromosome folding and DNA immunity. Prokaryotic SMC Wadjet (JET) complexes limit the spread of plasmids through DNA cleavage, yet the mechanisms for plasmid recognition are unresolved. We show that artificial DNA circularization renders linear DNA susceptible to JET nuclease cleavage. Unlike free DNA, JET cleaves immobilized plasmid DNA at a specific site, the plasmid-anchoring point, showing that the anchor hinders DNA extrusion but not DNA cleavage. Structures of plasmid-bound JetABC reveal two presumably stalled SMC motor units that are drastically rearranged from the resting state, together entrapping a U-shaped DNA segment, which is further converted to kinked V-shaped cleavage substrate by JetD nuclease binding. Our findings uncover mechanical bending of residual unextruded DNA as molecular signature for plasmid recognition and non-self DNA elimination. We moreover elucidate key elements of SMC loop extrusion, including the motor direction and the structure of a DNA-holding state.

The immune modules conserved across the tree of life: Towards a definition of ancestral immunity.

Immune defence mechanisms exist across the tree of life in such diversity that prokaryotic antiviral responses have historically been considered unrelated to eukaryotic immunity. Mechanisms of defence in divergent eukaryotes were similarly believed to be largely clade specific. However, recent data indicate that a subset of modules (domains and proteins) from prokaryote defence systems are conserved in eukaryotes and populate many stages of innate immune pathways. In this Essay, we propose the notion of ancestral immunity, which corresponds to the set of immune modules conserved between prokaryotes and eukaryotes. After offering a typology of ancestral immunity, we speculate on the selective pressures that could have led to the differential conservation of specific immune modules across domains of life. The exploration of ancestral immunity is in its infancy and appears full of promises to illuminate immune evolution, and also to identify and decipher immune mechanisms of economic, ecological, and therapeutic importance.

Contingency, repeatability, and predictability in the evolution of a prokaryotic pangenome.

Pangenomes exhibit remarkable variability in many prokaryotic species, much of which is maintained through the processes of horizontal gene transfer and gene loss. Repeated acquisitions of near-identical homologs can easily be observed across pangenomes, leading to the question of whether these parallel events potentiate similar evolutionary trajectories, or whether the remarkably different genetic backgrounds of the recipients mean that postacquisition evolutionary trajectories end up being quite different. In this study, we present a machine learning method that predicts the presence or absence of genes in the Escherichia coli pangenome based on complex patterns of the presence or absence of other accessory genes within a genome. Our analysis leverages the repeated transfer of genes through the E. coli pangenome to observe patterns of repeated evolution following similar events. We find that the presence or absence of a substantial set of genes is highly predictable from other genes alone, indicating that selection potentiates and maintains gene-gene co-occurrence and avoidance relationships deterministically over long-term bacterial evolution and is robust to differences in host evolutionary history. We propose that at least part of the pangenome can be understood as a set of genes with relationships that govern their likely cohabitants, analogous to an ecosystem's set of interacting organisms. Our findings indicate that intragenomic gene fitness effects may be key drivers of prokaryotic evolution, influencing the repeated emergence of complex gene-gene relationships across the pangenome.

Chromosome structure modeling tools and their evaluation in bacteria.

The three-dimensional (3D) structure of bacterial chromosomes is crucial for understanding chromosome function. With the growing availability of high-throughput chromosome conformation capture (3C/Hi-C) data, the 3D structure reconstruction algorithms have become powerful tools to study bacterial chromosome structure and function. It is highly desired to have a recommendation on the chromosome structure reconstruction tools to facilitate the prokaryotic 3D genomics. In this work, we review existing chromosome 3D structure reconstruction algorithms and classify them based on their underlying computational models into two categories: constraint-based modeling and thermodynamics-based modeling. We briefly compare these algorithms utilizing 3C/Hi-C datasets and fluorescence microscopy data obtained from Escherichia coli and Caulobacter crescentus, as well as simulated datasets. We discuss current challenges in the 3D reconstruction algorithms for bacterial chromosomes, primarily focusing on software usability. Finally, we briefly prospect future research directions for bacterial chromosome structure reconstruction algorithms.

Target DNA-dependent activation mechanism of the prokaryotic immune system SPARTA.

In both prokaryotic and eukaryotic innate immune systems, TIR domains function as NADases that degrade the key metabolite NAD+ or generate signaling molecules. Catalytic activation of TIR domains requires oligomerization, but how this is achieved varies in distinct immune systems. In the Short prokaryotic Argonaute (pAgo)/TIR-APAZ (SPARTA) immune system, TIR NADase activity is triggered upon guide RNA-mediated recognition of invading DNA by an unknown mechanism. Here, we describe cryo-EM structures of SPARTA in the inactive monomeric and target DNA-activated tetrameric states. The monomeric SPARTA structure reveals that in the absence of target DNA, a C-terminal tail of TIR-APAZ occupies the nucleic acid binding cleft formed by the pAgo and TIR-APAZ subunits, inhibiting SPARTA activation. In the active tetrameric SPARTA complex, guide RNA-mediated target DNA binding displaces the C-terminal tail and induces conformational changes in pAgo that facilitate SPARTA-SPARTA dimerization. Concurrent release and rotation of one TIR domain allow it to form a composite NADase catalytic site with the other TIR domain within the dimer, and generate a self-complementary interface that mediates cooperative tetramerization. Combined, this study provides critical insights into the structural architecture of SPARTA and the molecular mechanism underlying target DNA-dependent oligomerization and catalytic activation.

The missing part: the Archaeoglobus fulgidus Argonaute forms a functional heterodimer with an N-L1-L2 domain protein.

Argonaute (Ago) proteins are present in all three domains of life (bacteria, archaea and eukaryotes). They use small (15-30 nucleotides) oligonucleotide guides to bind complementary nucleic acid targets and are responsible for gene expression regulation, mobile genome element silencing, and defence against viruses or plasmids. According to their domain organization, Agos are divided into long and short Agos. Long Agos found in prokaryotes (long-A and long-B pAgos) and eukaryotes (eAgos) comprise four major functional domains (N, PAZ, MID and PIWI) and two structural linker domains L1 and L2. The majority (∼60%) of pAgos are short pAgos, containing only the MID and inactive PIWI domains. Here we focus on the prokaryotic Argonaute AfAgo from Archaeoglobus fulgidus DSM4304. Although phylogenetically classified as a long-B pAgo, AfAgo contains only MID and catalytically inactive PIWI domains, akin to short pAgos. We show that AfAgo forms a heterodimeric complex with a protein encoded upstream in the same operon, which is a structural equivalent of the N-L1-L2 domains of long pAgos. This complex, structurally equivalent to a long PAZ-less pAgo, outperforms standalone AfAgo in guide RNA-mediated target DNA binding. Our findings provide a missing piece to one of the first and the most studied pAgos.

Adaptation of a eukaryote-like ProRS to a prokaryote-like tRNAPro.

Prolyl-tRNA synthetases (ProRSs) are unique among aminoacyl-tRNA synthetases (aaRSs) in having two distinct structural architectures across different organisms: prokaryote-like (P-type) and eukaryote/archaeon-like (E-type). Interestingly, Bacillus thuringiensis harbors both types, with P-type (BtProRS1) and E-type ProRS (BtProRS2) coexisting. Despite their differences, both enzymes are constitutively expressed and functional in vivo. Similar to BtProRS1, BtProRS2 selectively charges the P-type tRNAPro and displays higher halofuginone tolerance than canonical E-type ProRS. However, these two isozymes recognize the primary identity elements of the P-type tRNAPro-G72 and A73 in the acceptor stem-through distinct mechanisms. Moreover, BtProRS2 exhibits significantly higher tolerance to stresses (such as heat, hydrogen peroxide, and dithiothreitol) than BtProRS1 does. This study underscores how an E-type ProRS adapts to a P-type tRNAPro and how it may contribute to the bacterium's survival under stress conditions.

POCP-nf: an automatic Nextflow pipeline for calculating the percentage of conserved proteins in bacterial taxonomy.

SUMMARY: Sequence technology advancements have led to an exponential increase in bacterial genomes, necessitating robust taxonomic classification methods. The Percentage Of Conserved Proteins (POCP), proposed initially by Qin et al. (2014), is a valuable metric for assessing prokaryote genus boundaries. Here, I introduce a computational pipeline for automated POCP calculation, aiming to enhance reproducibility and ease of use in taxonomic studies. AVAILABILITY AND IMPLEMENTATION: The POCP-nf pipeline uses DIAMOND for faster protein alignments, achieving similar sensitivity to BLASTP. The pipeline is implemented in Nextflow with Conda and Docker support and is freely available on GitHub under https://github.com/hoelzer/pocp. The open-source code can be easily adapted for various prokaryotic genome and protein datasets. Detailed documentation and usage instructions are provided in the repository.

Intracellular signaling in proto-eukaryotes evolves to alleviate regulatory conflicts of endosymbiosis.

The complex eukaryotic cell resulted from a merger between simpler prokaryotic cells, yet the role of the mitochondrial endosymbiosis with respect to other eukaryotic innovations has remained under dispute. To investigate how the regulatory challenges associated with the endosymbiotic state impacted genome and network evolution during eukaryogenesis, we study a constructive computational model where two simple cells are forced into an obligate endosymbiosis. Across multiple in silico evolutionary replicates, we observe the emergence of different mechanisms for the coordination of host and symbiont cell cycles, stabilizing the endosymbiotic relationship. In most cases, coordination is implicit, without signaling between host and symbiont. Signaling only evolves when there is leakage of regulatory products between host and symbiont. In the fittest evolutionary replicate, the host has taken full control of the symbiont cell cycle through signaling, mimicking the regulatory dominance of the nucleus over the mitochondrion that evolved during eukaryogenesis.

Challenges in computational discovery of bioactive peptides in 'omics data.

Peptides have a plethora of activities in biological systems that can potentially be exploited biotechnologically. Several peptides are used clinically, as well as in industry and agriculture. The increase in available 'omics data has recently provided a large opportunity for mining novel enzymes, biosynthetic gene clusters, and molecules. While these data primarily consist of DNA sequences, other types of data provide important complementary information. Due to their size, the approaches proven successful at discovering novel proteins of canonical size cannot be naïvely applied to the discovery of peptides. Peptides can be encoded directly in the genome as short open reading frames (smORFs), or they can be derived from larger proteins by proteolysis. Both of these peptide classes pose challenges as simple methods for their prediction result in large numbers of false positives. Similarly, functional annotation of larger proteins, traditionally based on sequence similarity to infer orthology and then transferring functions between characterized proteins and uncharacterized ones, cannot be applied for short sequences. The use of these techniques is much more limited and alternative approaches based on machine learning are used instead. Here, we review the limitations of traditional methods as well as the alternative methods that have recently been developed for discovering novel bioactive peptides with a focus on prokaryotic genomes and metagenomes.

ProGlycProt V3.0: updated insights into prokaryotic glycoproteins and their glycosyltransferases.

ProGlycProt is a comprehensive database of experimentally validated information about protein glycosylation in prokaryotes, including the glycoproteins, glycosyltransferases, and their accessory enzymes. The first release of ProGlycProt featured experimentally validated information on glycoproteins only. For the second release in 2019, the size and scope of the database were expanded twofold, and experimental data on cognate glycosyltransferases and their accessory proteins was incorporated. The growing research and technology interest in microbial glycoproteins and their enzymes is evident from the steady rise in academic publications and patents in this area. Accordingly, the third update comprises a new section on patents related to glycosylation methods, novel glycosyltransferases, and technologies developed therefrom. The structure gallery is reorganized, wherein the number and quality of the models are upgraded with the help of AlphaFold2. Over the years, the influx of experimental proteomics data into public repositories like PRIDE has surged. Harnessing this legacy data for in-silico glycoprotein identification is a smart approach. Version 3.0 adds 45 N-glycoprotein entries annotated from MS datasets available on PRIDE and reviewed by independent research groups. With a 67% rise in entries corresponding to 119 genera of prokaryotes, the ProGlycProt continues to be the exclusive database of experimentally validated comprehensive information about protein glycosylation in prokaryotes.

Distinct bacterial succession and functional response to alginate in the South, Equatorial, and North Pacific Ocean.

The availability of alginate, an abundant macroalgal polysaccharide, induces compositional and functional responses among marine microbes, but these dynamics have not been characterized across the Pacific Ocean. We investigated alginate-induced compositional and functional shifts (e.g., heterotrophic production, glucose turnover, hydrolytic enzyme activities) of microbial communities in the South Subtropical, Equatorial, and Polar Frontal North Pacific in mesocosms. We observed that shifts in response to alginate were site-specific. In the South Subtropical Pacific, prokaryotic cell counts, glucose turnover, and peptidase activities changed the most with alginate addition, along with the enrichment of the widest range of particle-associated taxa (161 amplicon sequence variants; ASVs) belonging to Alteromonadaceae, Rhodobacteraceae, Phormidiaceae, and Pseudoalteromonadaceae. Some of these taxa were detected at other sites but only enriched in the South Pacific. In the Equatorial Pacific, glucose turnover and heterotrophic prokaryotic production increased most rapidly; a single Alteromonas taxon dominated (60% of the community) but remained low (<2%) elsewhere. In the North Pacific, the particle-associated community response to alginate was gradual, with a more limited range of alginate-enriched taxa (82 ASVs). Thus, alginate-related ecological and biogeochemical shifts depend on a combination of factors that include the ability to utilize alginate, environmental conditions, and microbial interactions.

Dinucleotide biases in the genomes of prokaryotic and eukaryotic dsDNA viruses and their hosts.

The genomes of cellular organisms display CpG and TpA dinucleotide composition biases. Such biases have been poorly investigated in dsDNA viruses. Here, we show that in dsDNA virus, bacterial, and eukaryotic genomes, the representation of TpA and CpG dinucleotides is strongly dependent on genomic G + C content. Thus, the classical observed/expected ratios do not fully capture dinucleotide biases across genomes. Because a larger portion of the variance in TpA frequency was explained by G + C content, we explored which additional factors drive the distribution of CpG dinucleotides. Using the residuals of the linear regressions as a measure of dinucleotide abundance and ancestral state reconstruction across eukaryotic and prokaryotic virus trees, we identified an important role for phylogeny in driving CpG representation. Nonetheless, phylogenetic ANOVA analyses showed that few host associations also account for significant variations. Among eukaryotic viruses, most significant differences were observed between arthropod-infecting viruses and viruses that infect vertebrates or unicellular organisms. However, an effect of viral DNA methylation status (either driven by the host or by viral-encoded methyltransferases) is also likely. Among prokaryotic viruses, cyanobacteria-infecting phages resulted to be significantly CpG-depleted, whereas phages that infect bacteria in the genera Burkolderia and Staphylococcus were CpG-rich. Comparison with bacterial genomes indicated that this effect is largely driven by the general tendency for phages to resemble the host's genomic CpG content. Notably, such tendency is stronger for temperate than for lytic phages. Our data shed light into the processes that shape virus genome composition and inform manipulation strategies for biotechnological applications.

Nitrate reduction to ammonium: a phylogenetic, physiological, and genetic aspects in Prokaryotes and eukaryotes.

The microbe-mediated conversion of nitrate (NO3-) to ammonium (NH4+) in the nitrogen cycle has strong implications for soil health and crop productivity. The role of prokaryotes, eukaryotes and their phylogeny, physiology, and genetic regulations are essential for understanding the ecological significance of this empirical process. Several prokaryotes (bacteria and archaea), and a few eukaryotes (fungi and algae) are reported as NO3- reducers under certain conditions. This process involves enzymatic reactions which has been catalysed by nitrate reductases, nitrite reductases, and NH4+-assimilating enzymes. Earlier reports emphasised that single-cell prokaryotic or eukaryotic organisms are responsible for this process, which portrayed a prominent gap. Therefore, this study revisits the similarities and uniqueness of mechanism behind NO3- -reduction to NH4+ in both prokaryotes and eukaryotes. Moreover, phylogenetic, physiological, and genetic regulation also shed light on the evolutionary connections between two systems which could help us to better explain the NO3--reduction mechanisms over time. Reports also revealed that certain transcription factors like NtrC/NtrB and Nit2 have shown a major role in coordinating the expression of NO3- assimilation genes in response to NO3- availability. Overall, this review provides a comprehensive information about the complex fermentative and respiratory dissimilatory nitrate reduction to ammonium (DNRA) processes. Uncovering the complexity of this process across various organisms may further give insight into sustainable nitrogen management practices and might contribute to addressing global environmental challenges.

Light Control in Microbial Systems.

Light is a key environmental component influencing many biological processes, particularly in prokaryotes such as archaea and bacteria. Light control techniques have revolutionized precise manipulation at molecular and cellular levels in recent years. Bacteria, with adaptability and genetic tractability, are promising candidates for light control studies. This review investigates the mechanisms underlying light activation in bacteria and discusses recent advancements focusing on light control methods and techniques for controlling bacteria. We delve into the mechanisms by which bacteria sense and transduce light signals, including engineered photoreceptors and light-sensitive actuators, and various strategies employed to modulate gene expression, protein function, and bacterial motility. Furthermore, we highlight recent developments in light-integrated methods of controlling microbial responses, such as upconversion nanoparticles and optical tweezers, which can enhance the spatial and temporal control of bacteria and open new horizons for biomedical applications.

The Constructive Black Queen hypothesis: new functions can evolve under conditions favouring gene loss.

Duplication is a major route for the emergence of new gene functions. However, the emergence of new gene functions via this route may be reduced in prokaryotes, as redundant genes are often rapidly purged. In lineages with compact, streamlined genomes, it thus appears challenging for novel function to emerge via duplication and divergence. A further pressure contributing to gene loss occurs under Black Queen dynamics, as cheaters that lose the capacity to produce a public good can instead acquire it from neighbouring producers. We propose that Black Queen dynamics can favour the emergence of new function because, under an emerging Black Queen dynamic, there is high gene redundancy spread across a community of interacting cells. Using computational modelling, we demonstrate that new gene functions can emerge under Black Queen dynamics. This result holds even if there is deletion bias due to low duplication rates and selection against redundant gene copies resulting from the high cost associated with carrying a locus. However, when the public good production costs are high, Black Queen dynamics impede the fixation of new functions. Our results expand the mechanisms by which new gene functions can emerge in prokaryotic systems.

On the ever-growing functional versatility of the CRISPR-Cas13 system.

CRISPR-Cas systems evolved in prokaryotes to implement a powerful antiviral immune response as a result of sequence-specific targeting by ribonucleoproteins. One of such systems consists of an RNA-guided RNA endonuclease, known as CRISPR-Cas13. In very recent years, this system is being repurposed in different ways in order to decipher and engineer gene expression programmes. Here, we discuss the functional versatility of the CRISPR-Cas13 system, which includes the ability for RNA silencing, RNA editing, RNA tracking, nucleic acid detection and translation regulation. This functional palette makes the CRISPR-Cas13 system a relevant tool in the broad field of systems and synthetic biology.

ARCTIC-3D: automatic retrieval and clustering of interfaces in complexes from 3D structural information.

The formation of a stable complex between proteins lies at the core of a wide variety of biological processes and has been the focus of countless experiments. The huge amount of information contained in the protein structural interactome in the Protein Data Bank can now be used to characterise and classify the existing biological interfaces. We here introduce ARCTIC-3D, a fast and user-friendly data mining and clustering software to retrieve data and rationalise the interface information associated with the protein input data. We demonstrate its use by various examples ranging from showing the increased interaction complexity of eukaryotic proteins, 20% of which on average have more than 3 different interfaces compared to only 10% for prokaryotes, to associating different functions to different interfaces. In the context of modelling biomolecular assemblies, we introduce the concept of "recognition entropy", related to the number of possible interfaces of the components of a protein-protein complex, which we demonstrate to correlate with the modelling difficulty in classical docking approaches. The identified interface clusters can also be used to generate various combinations of interface-specific restraints for integrative modelling. The ARCTIC-3D software is freely available at github.com/haddocking/arctic3d and can be accessed as a web-service at wenmr.science.uu.nl/arctic3d.

Oxygen-dependent biofilm dynamics in leaf decay: an in vitro analysis.

Biofilms are important in the natural process of plant tissue degradation. However, fundamental knowledge of biofilm community structure and succession on decaying leaves under different oxygen conditions is limited. Here, we used 16S rRNA and ITS gene amplicon sequencing to investigate the composition, temporal dynamics, and community assembly processes of bacterial and fungal biofilms on decaying leaves in vitro. Leaves harvested from three plant species were immersed in lake water under aerobic and anaerobic conditions in vitro for three weeks. Biofilm-covered leaf samples were collected weekly and investigated by scanning electron microscopy. The results showed that community composition differed significantly between biofilm samples under aerobic and anaerobic conditions, though not among plant species. Over three weeks, a clear compositional shift of the bacterial and fungal biofilm communities was observed. The alpha diversity of prokaryotes increased over time in aerobic assays and decreased under anaerobic conditions. Oxygen availability and incubation time were found to be primary factors influencing the microbial diversity of biofilms on different decaying plant species in vitro. Null models suggest that stochastic processes governed the assembly of biofilm communities of decaying leaves in vitro in the early stages of biofilm formation and were further shaped by niche-associated factors.