From Enigma to Revelation: Unravelling Biological Functions of Ubiquitous Small Ribozymes.

RNA, widely recognized as an information-carrier molecule, is capable of catalyzing essential biological processes through ribozymes. Despite their ubiquity, specific functions in a biological context and phenotypes based on the ribozymes' activity are often unknown. Here, we present the discovery of a subgroup of minimal HDV-like ribozymes, which reside 3' to viral tRNAs and appear to cleave the 3'-trailers of viral premature tRNA transcripts. This proposed tRNA-processing function is unprecedented for any ribozymes, thus, we designate this subgroup as theta ribozymes. Most theta ribozymes were identified in Caudoviricetes bacteriophages, the main constituent (>90%) of the mammalian gut virome. Intriguingly, our findings further suggest the involvement of theta ribozymes in the transition of certain bacteriophages between distinct genetic codes, thus possibly contributing to the phage lysis trigger. Our discovery expands the limited repertoire of biological functions attributed to HDV-like ribozymes and provides insights into the fascinating world of RNA catalysis.

Identification of HDV-like theta ribozymes involved in tRNA-based recoding of gut bacteriophages.

Trillions of microorganisms, collectively known as the microbiome, inhabit our bodies with the gut microbiome being of particular interest in biomedical research. Bacteriophages, the dominant virome constituents, can utilize suppressor tRNAs to switch to alternative genetic codes (e.g., the UAG stop-codon is reassigned to glutamine) while infecting hosts with the standard bacterial code. However, what triggers this switch and how the bacteriophage manipulates its host is poorly understood. Here, we report the discovery of a subgroup of minimal hepatitis delta virus (HDV)-like ribozymes - theta ribozymes - potentially involved in the code switch leading to the expression of recoded lysis and structural phage genes. We demonstrate their HDV-like self-scission behavior in vitro and find them in an unreported context often located with their cleavage site adjacent to tRNAs, indicating a role in viral tRNA maturation and/or regulation. Every fifth associated tRNA is a suppressor tRNA, further strengthening our hypothesis. The vast abundance of tRNA-associated theta ribozymes - we provide 1753 unique examples - highlights the importance of small ribozymes as an alternative to large enzymes that usually process tRNA 3'-ends. Our discovery expands the short list of biological functions of small HDV-like ribozymes and introduces a previously unknown player likely involved in the code switch of certain recoded gut bacteriophages.

From microbiome composition to functional engineering, one step at a time.

SUMMARYCommunities of microorganisms (microbiota) are present in all habitats on Earth and are relevant for agriculture, health, and climate. Deciphering the mechanisms that determine microbiota dynamics and functioning within the context of their respective environments or hosts (the microbiomes) is crucially important. However, the sheer taxonomic, metabolic, functional, and spatial complexity of most microbiomes poses substantial challenges to advancing our knowledge of these mechanisms. While nucleic acid sequencing technologies can chart microbiota composition with high precision, we mostly lack information about the functional roles and interactions of each strain present in a given microbiome. This limits our ability to predict microbiome function in natural habitats and, in the case of dysfunction or dysbiosis, to redirect microbiomes onto stable paths. Here, we will discuss a systematic approach (dubbed the N+1/N-1 concept) to enable step-by-step dissection of microbiome assembly and functioning, as well as intervention procedures to introduce or eliminate one particular microbial strain at a time. The N+1/N-1 concept is informed by natural invasion events and selects culturable, genetically accessible microbes with well-annotated genomes to chart their proliferation or decline within defined synthetic and/or complex natural microbiota. This approach enables harnessing classical microbiological and diversity approaches, as well as omics tools and mathematical modeling to decipher the mechanisms underlying N+1/N-1 microbiota outcomes. Application of this concept further provides stepping stones and benchmarks for microbiome structure and function analyses and more complex microbiome intervention strategies.

Chemotaxis and autoinducer-2 signalling mediate colonization and contribute to co-existence of Escherichia coli strains in the murine gut.

Bacteria communicate and coordinate their behaviour at the intra- and interspecies levels by producing and sensing diverse extracellular small molecules called autoinducers. Autoinducer 2 (AI-2) is produced and detected by a variety of bacteria and thus plays an important role in interspecies communication and chemotaxis. Although AI-2 is a major autoinducer molecule present in the mammalian gut and can influence the composition of the murine gut microbiota, its role in bacteria-bacteria and bacteria-host interactions during gut colonization remains unclear. Combining competitive infections in C57BL/6 mice with microscopy and bioinformatic approaches, we show that chemotaxis (cheY) and AI-2 signalling (via lsrB) promote gut colonization by Escherichia coli, which is in turn connected to the ability of the bacteria to utilize fructoselysine (frl operon). We further show that the genomic diversity of E. coli strains with respect to AI-2 signalling allows ecological niche segregation and stable co-existence of different E. coli strains in the mammalian gut.

proGenomes3: approaching one million accurately and consistently annotated high-quality prokaryotic genomes.

The interpretation of genomic, transcriptomic and other microbial 'omics data is highly dependent on the availability of well-annotated genomes. As the number of publicly available microbial genomes continues to increase exponentially, the need for quality control and consistent annotation is becoming critical. We present proGenomes3, a database of 907 388 high-quality genomes containing 4 billion genes that passed stringent criteria and have been consistently annotated using multiple functional and taxonomic databases including mobile genetic elements and biosynthetic gene clusters. proGenomes3 encompasses 41 171 species-level clusters, defined based on universal single copy marker genes, for which pan-genomes and contextual habitat annotations are provided. The database is available at http://progenomes.embl.de/.

Phylogeny and Metabolic Potential of the Candidate Phylum SAR324.

The bacterial SAR324 cluster is ubiquitous and abundant in the ocean, especially around hydrothermal vents and in the deep sea, where it can account for up to 30% of the whole bacterial community. According to a new taxonomy generated using multiple universal protein-coding genes (instead of the previously used 16S rRNA single gene marker), the former Deltaproteobacteria cluster SAR324 has been classified since 2018 as its own phylum. Yet, very little is known about its phylogeny and metabolic potential. We downloaded all publicly available SAR324 genomes (65) from all natural environments and reconstructed 18 new genomes using publicly available oceanic metagenomic data and unpublished data from the waters underneath the Ross Ice Shelf. We calculated a global SAR324 phylogenetic tree and identified six clusters (namely 1A, 1B, 2A, 2B, 2C and 2D) within this clade. Genome annotation and metatranscriptome read mapping showed that SAR324 clades possess a flexible array of genes suited for survival in various environments. Clades 2A and 2C are mostly present in the surface mesopelagic layers of global oceans, while clade 2D dominates in deeper regions. Our results show that SAR324 has a very versatile and broad metabolic potential, including many heterotrophic, but also autotrophic pathways. While one surface water associated clade (2A) seems to use proteorhodopsin to gain energy from solar radiation, some deep-sea genomes from clade 2D contain the complete Calvin-Benson-Bassham cycle gene repertoire to fix carbon. This, in addition to a variety of other genes and pathways for both oxic (e.g., dimethylsulfoniopropionate degradation) and anoxic (e.g., dissimilatory sulfate reduction, anaerobic benzoate degradation) conditions, can help explain the ubiquitous presence of SAR324 in aquatic habitats.