The evolution of microbial parasites involves the counterplay between natural selection forcing parasites to improve and genetic drifts forcing parasites to lose genes and accumulate deleterious mutations. Here, to understand how this counterplay occurs at the scale of individual macromolecules, we describe cryo-EM structure of ribosomes from Encephalitozoon cuniculi, a eukaryote with one of the smallest genomes in nature. The extreme rRNA reduction in E. cuniculi ribosomes is accompanied with unparalleled structural changes, such as the evolution of previously unknown molten rRNA linkers and bulgeless rRNA. Furthermore, E. cuniculi ribosomes withstand the loss of rRNA and protein segments by evolving an ability to use small molecules as structural mimics of degenerated rRNA and protein segments. Overall, we show that the molecular structures long viewed as reduced, degenerated, and suffering from debilitating mutations possess an array of compensatory mechanisms that allow them to remain active despite the extreme molecular reduction.

Despite the rapidly increasing number of organisms with sequenced genomes, there is no existing resource that simultaneously contains information about genome sequences and the optimal growth conditions for a given species. In the absence of such a resource, we cannot immediately sort genomic sequences by growth conditions, making it difficult to study how organisms and biological molecules adapt to distinct environments. To address this problem, we have created a database called GSHC (Genome Sequences: Hot, Cold, and everything in between). This database, available at http://melnikovlab.com/gshc, brings together information about the genomic sequences and optimal growth temperatures for 25,324 species, including ~89% of the bacterial species with known genome sequences. Using this database, it is now possible to readily compare genomic sequences from thousands of species and correlate variations in genes and genomes with optimal growth temperatures, at the scale of the entire tree of life. The database interface allows users to retrieve protein sequences sorted by optimal growth temperature for their corresponding species, providing a tool to explore how organisms, genomes, and individual proteins and nucleic acids adapt to certain temperatures. We hope that this database will contribute to medicine and biotechnology by helping to create a better understanding of molecular adaptations to heat and cold, leading to new ways to preserve biological samples, engineer useful enzymes, and develop new biological materials and organisms with the desired tolerance to heat and cold.

Ribosomal genes are widely used as ‘molecular clocks’ to infer evolutionary relationships between species. However, their utility as ‘molecular thermometers’ for estimating optimal growth temperature of microorganisms remains uncertain. Previously, some estimations were made using the nucleotide composition of ribosomal RNA (rRNA), but the universal application of this approach was hindered by numerous outliers. In this study, we aimed to address this problem by identifying additional indicators of thermal adaptation within the sequences of ribosomal proteins. By comparing sequences from 2021 bacteria with known optimal growth temperature, we identified novel indicators among the metal-binding residues of ribosomal proteins. We found that these residues serve as conserved adaptive features for bacteria thriving above 40°C, but not at lower temperatures. Furthermore, the presence of these metal-binding residues exhibited a stronger correlation with the optimal growth temperature of bacteria compared to the commonly used correlation with the 16S rRNA GC content. And an even more accurate correlation was observed between the optimal growth temperature and the YVIWREL amino acid content within ribosomal proteins. Overall, our work suggests that ribosomal proteins contain a more accurate record of bacterial thermal adaptation compared to rRNA. This finding may simplify the analysis of unculturable and extinct species.

During starvation and stress, virtually all organisms arrest their protein synthesis and convert their ribosomes into a state of dormancy in which a special class of proteins, known as hibernation factors, protect the ribosome from damage. In bacteria, two major families of hibernation factors have been characterized, but the low conservation of these proteins and the huge diversity of species, habitats and environmental stressors has made the discovery of new dormancy factors challenging. In this study, we identify a new bacterial protein involved in ribosome dormancy. We first characterize dormant ribosomes following cold shock in the psychrophilic bacterium Psychrobacter urativorans using proteomics and cryo-EM. We show that during cold shock, ribosomes in this organism associate with a previously uncharacterized protein that occupies ribosomal active centres and multiple drug-binding sites. Our structure reveals that this new factor, which we term Balon, bears no resemblance to known bacterial hibernation factors. Instead, it belongs to a family of bacterial homologs of the eukaryotic translation factor aeRF1. We identify unique motifs present only in Balon but not its archaeal or eukaryotic counterparts that allow it to engage with the peptidyl transferase center and decoding center of dormant bacterial ribosomes. We then show that, across the bacterial kingdom, Balon-encoding genes are found in nearly 20% of known species and are located in operons involved in different types of stress responses, including osmotic and thermal shock, hypoxia, acid, tRNA damage and antibiotic stress. Taken together, our work shows that bacterial cells possess a functional homolog of eukaryotic translation termination factor, which is likely repurposed into a stress-response protein. This finding illuminates a long-sought evolutionary connection between the bacterial and eukaryotic protein synthesis machinery.

The evolution of microbial parasites involves the interplay of two opposing forces. On the one hand, the pressure to survive drives parasites to improve through Darwinian natural selection. On the other, frequent genetic drifts result in genome decay, an evolutionary process in which an ever-increasing burden of deleterious mutations leads to gene loss and gradual genome reduction. Here, seeking to understand how this interplay occurs at the scale of individual macromolecules, we describe cryo-EM and evolutionary analyses of ribosomes from Encephalitozoon cuniculi, a eukaryote with one of the most reduced genomes in nature. We show that E. cuniculi ribosomes, the smallest eukaryotic cytoplasmic ribosomes to be structurally characterized, employ unparalleled structural innovations that allow extreme rRNA reduction without loss of ribosome integrity. These innovations include the evolution of previously unknown rRNA features such as molten rRNA linkers and bulgeless rRNA. Furthermore, we show that E. cuniculi ribosomes withstand the loss of rRNA and protein segments by evolving a unique ability to effectively trap small molecules and use them as ribosomal building-blocks and structural mimics of degenerated rRNA and protein segments. Overall, our work reveals a recurrent evolutionary pattern, which we term 'lose-to-gain' evolution, where it is only through the loss of rRNA and protein segments that E. cuniculi ribosomes evolve their major innovations. Our study shows that the molecular structures of intracellular parasites long viewed as reduced, degenerated, and suffering from various debilitating mutations instead possess an array of systematically overlooked and extraordinary structural features. These features allow them to not only adapt to molecular reduction but evolve new activities that parasites can possibly use to their advantage.