Tobias Warnecke

Nucleosomes organize genomes and regulate DNA access, yet accumulating evidence suggests that their constituent histones may have functions beyond canonical chromatin regulation, but the breadth of such regulatory diversity remains unclear. Here, we used the six-residue loop 2 (L2) of H2A and H2A.Z to map, at single-residue resolution, how nucleosome-core variation reshapes cellular function. Genome-scale interaction mapping identified hundreds of regulatory connections spanning chromatin, as well as actin organization, endocytosis, and membrane trafficking. Interactions were residue-specific and differed between H2A and H2A.Z, revealing a regulatory landscape encoded by single residues. Transcriptome profiling showed limited expression changes and little overlap between differentially expressed genes and regulatory partners, indicating that non-chromatin connections are not readily explained by altered transcription. L2 substitutions also preferentially conferred benefits under cell wall and membrane stress. Thus, the nucleosome is linked to cellular-periphery functions beyond classical chromatin regulation, identifying histone variation as a source of phenotypic innovation.

Inside cells, DNA is intimately associated with proteins, forming chromatin. The protein constituents of chromatin vary across the tree of life: histones are the principal building blocks of chromatin in eukaryotes and many archaea, whereas bacteria typically encode a collection of nucleoid-associated proteins (NAPs) that wrap, bend, bridge or coat the DNA. Although chromatin proteins appear to be a universal feature of cellular life, DNA-templated processes such as transcription, replication, and DNA repair can take place in vitro in the absence of chromatin, raising the possibility that cellular systems might exist — or could be built — that lack chromatin proteins. To explore this possibility, the molecular consequences and potential systemic adjustments required for life without chromatin, we serially deleted the nine most abundant NAPs from E. coli ( hupA , hupB , ihfA , ihfB , hns , stpA , fis , dps , lrp ), resulting in a strain (ΔNAP9) that lacks its native chromatin. Using an array of different techniques, we document change — and sometimes surprising lack thereof — in compaction, composition and 3D architecture of the nucleoid, supercoiling, prophage activity, growth, viability, and genetic make-up of ΔNAP9. Most notably, we find that ΔNAP9 exhibits global dysregulation of gene expression, marked by a striking homogenization of transcriptional output across the genome that is reminiscent of the effects of histone depletion in eukaryotic cells. Our results reinforce the notion that chromatin plays a key role in compartmentalizing the use of genomic information, enabling both the localized suppression of selfish elements and dynamic reprogramming of genome activity in response to environmental change. At the same time, the successful construction of ΔNAP9 demonstrates that bacterial cells can carry out basic cellular functions in the absence of co-evolved chromatin proteins, highlighting the potential for radical (re-)engineering of prokaryotic chromatin and systems of gene expression.

Archaea and bacteria routinely live side by side in microbial communities and must interact at least on occasion. Whether such cross-Domain interactions are dominated by mutual disregard, co-operation, or conflict remains fundamentally unknown. One potential window into archaeal-bacterial conflict is to ask whether some of the molecular weapons bacteria wield to kill other bacteria are present in archaea, and vice versa. Here, to start to address this question, we carry out a phylogenomic survey of bacteriocins in archaeal genomes and archaeocins in bacterial genomes. We find that more than 20% of known bacteriocins – proteins deployed by bacteria against other bacteria - have at least one homolog in archaea. Typically, these archaeal homologs are related to bacteriocins targeting (and encoded by) monoderm bacteria. Based on conservation of functionally critical residues, protein structure, and accessory genes critical for bacteriocin biosynthesis, we highlight homologs of subtilosin A, encoded in some Thermococcus archaea, as promising candidates for experimental follow- up work. We also show that halocin C8, originally described in Natrinema archaea, is comparatively common in bacterial genomes, including a number of skin-resident Staphylococcus species. Our results suggest that bacteriocins/archaeocins are shared across Domain boundaries with some regularity. While many instances are phylogenetically isolated – raising doubts about their functional importance and integration into host physiology – some bacteriocins are present in multiple related genomes and embedded in broader biosynthetic gene clusters that are also found in the original producers, suggesting that archaea and bacteria periodically use the same weapon systems in conflicts with other microbes. Further study of these systems might elucidate cross-Domain conflict and the nature of archaeal-bacterial interactions in different environments.

Archaea kill bacteria, at least on occasion. The molecular underpinnings of these lethal interactions are barely understood. Here, we describe cinquedea, an α/β hydrolase secreted by the halophilic archaeon Haloferax larsenii s5a-1. Cinquedea exhibits bactericidal activity in the nanomolar range, killing halophilic Pontibacillus bacteria. Bacterial death is accompanied by gross morphological abnormalities, indicative of severe damage to the cell envelope. We predict, and confirm in vitro, that cinquedea is a phospholipase, with structural similarities to a phospholipase A1 enzyme isolated from hornet venom. Exposing lipids extracted from a cinquedea-sensitive Pontibacillus strain to the enzyme leads to accumulation of lysophosphatidylglycerol, a cleavage product of phospholipase A activity, supporting direct activity of cinquedea against the Pontibacillus membrane, which is chiefly composed of phosphatidylglycerol. Following the recent discovery that some archaea encode bactericidal peptidoglycan hydrolases, these findings suggest that archaea can kill bacteria in mechanistically diverse ways. Our work provides a template for future experimental discovery and characterization of bactericidal proteins of archaeal origin and reinforces an emerging view that archaea represent a substantial reservoir for the discovery of new antibacterial compounds.

For more than a century, scientists have worked to characterize, understand, and predict the consequences of mutations. For almost as long, scientists—always on the lookout for general principles—have categorized these mutations, hoping that putting them into labeled boxes might help reveal the molecular logic that governs mutational effects. Here, I will dive into one of these boxes, labeled “gain-of-function”, a term that will ring familiar to undergraduates, (clinical) geneticists, and virologists alike. I will emerge from the box with a profound sense of bewilderment and the conclusion that its contents appear to have very little in common. What is a gain-of-function mutation? What do we know (or can reasonably assume) about a mutation once it has attracted this label? Do gain-of-function mutations share anything in common in terms of their molecular features or the consequences they cause? I will argue that the answers to these three questions are “I don’t know,” “not much,” and “not really,” and that the term gain-of-function tells us rather little. Worse, it often misleads our intuition regarding what a given mutation is or does. I will suggest that this is because the gain-of-function label has historically been applied, with liberal abandon, across different levels of biological complexity, from the behavior of individual proteins, to protein complexes, to cells, to whole-organism physiology. I will discuss the implications (all bad…) of this heterogeneous labeling history for recent efforts to train machine learning algorithms to discriminate different types of mutations. Above all, I hope to highlight that the myriad ways in which mutational effects can percolate through biological systems often defy easy categorization and that, while classifying things is often useful, it is best not to forget that molecular biology is a glorious mess.

DNA topoisomerases are essential enzymes found in all cells, where they regulate DNA supercoiling. Reverse gyrase (RG) is a unique type of topoisomerase that introduces positive supercoils into DNA and appears exclusively in hyperthermophiles where it was proposed to play a key, yet still elusive, role. Here, we investigate RG activity in the hyperthermophilic archaeon Thermococcus kodakarensis at 85C, its optimal growth temperature, using genetics and functional genomics assays. Deletion of RG led to a loss of positive supercoiling in plasmid DNA and the reduced dynamic range of transcription, without affecting histone occupancy. To investigate the effects of RG loss on the topology of chromosomal DNA, we established a psoralen photobinding assay (TMP-seq) in T. kodakarensis under native growth conditions. TMP enrichment patterns were consistent with the twin-domain model of transcription and further revealed that promoters of expressed transcription units are, on average, underwound. TMP-seq profiles in an RG deletion strain revealed that promoters are hotspots for RG activity, consistent with RG acting on hyper-negatively supercoiled substrates. We propose that RG acts not as a global modulator of supercoiling, but as a local genome guardian, selectively stabilising vulnerable regulatory regions to ensure a delicate balance between DNA accessibility and integrity under extremely high temperatures.

The social life of archaea is poorly understood. In particular, even though competition and conflict are common themes in microbial communities, there is scant evidence documenting antagonistic interactions between archaea and their abundant prokaryotic brethren: bacteria. Do archaea specifically target bacteria for destruction? If so, what molecular weaponry do they use? Here, we present an approach to infer antagonistic interactions between archaea and bacteria from genome sequence. We show that a large and diverse set of archaea encode peptidoglycan hydrolases, enzymes that recognize and cleave a structure-peptidoglycan-that is a ubiquitous component of bacterial cell walls but absent from archaea. We predict the bacterial targets of archaeal peptidoglycan hydrolases using a structural homology approach and demonstrate that the predicted target bacteria tend to inhabit a similar niche to the archaeal producer, indicative of ecologically relevant interactions. Using a heterologous expression system, we demonstrate that two peptidoglycan hydrolases from the halophilic archaeaon Halogranum salarium B-1 kill the halophilic bacterium Halalkalibacterium halodurans, a predicted target, and do so in a manner consistent with peptidoglycan hydrolase activity. Our results suggest that, even though the tools and rules of engagement remain largely unknown, archaeal-bacterial conflicts are likely common, and we present a roadmap for the discovery of additional antagonistic interactions between these two domains of life. Our work has implications for understanding mixed microbial communities that include archaea and suggests that archaea might represent a large untapped reservoir of novel antibacterials.

The social life of archaea is poorly understood. In particular, even though competition and conflict are common themes in microbial communities, there is scant evidence documenting antagonistic interactions between archaea and their abundant prokaryotic brethren: bacteria. Do archaea specifically target bacteria for destruction? If so, what molecular weaponry do they use? Here, we present an approach to infer antagonistic interactions between archaea and bacteria from genome sequence. We show that a large and diverse set of archaea encode peptidoglycan hydrolases, enzymes that recognize and cleave a structure – peptidoglycan – that is a ubiquitous component of bacterial cell walls but absent from archaea. We predict the bacterial targets of archaeal peptidoglycan hydrolases using a structural homology approach and demonstrate that the predicted target bacteria tend to inhabit a similar niche to the archaeal producer, indicative of ecologically relevant interactions. Using a heterologous expression system, we demonstrate that two peptidoglycan hydrolases from the halophilic archaeaon Halogranum salarium B-1 kill the halophilic bacterium Halalkalibacterium halodurans, a predicted target, and do so in a manner consistent with peptidoglycan hydrolase activity. Our results suggest that, even though the tools and rules of engagement remain largely unknown, archaeal-bacterial conflicts are likely common, and we present a roadmap for the discovery of additional antagonistic interactions between these two domains of life. Our work has implications for understanding mixed microbial communities that include archaea and suggests that archaea might represent a large untapped reservoir of novel antibacterials.

Histones are among the most conserved proteins in the eukaryotic genome, and their function is thought to be largely invariant across species. Here, we tested this assumption, examining over a billion years of the essential histone H2A.Z’s evolution in a single synthetic host. We identify single residue substitutions within the H2A.Z core domain that led to its neofunctionalization. Such H2A.Z neomorphs are distinct by their ability to directly interact with the transcription apparatus, rewiring gene expression genome-wide by tuning transcription processivity. Our results reveal that even changes of single residues within the histones core domain can transform their function, catalysing the rapid emergence of phenotypic diversity by directly imposing both fitness opportunities and costs. We propose that the entire histone sequence has the potential to evolve new regulatory relationships, providing a framework to understand the mechanistic underpinnings of disease-associated histone mutations.