An Atlas of the base inter-RNA stacks involved in bacterial translation.

Nucleobase-specific noncovalent interactions play a crucial role in translation. Herein, we provide a comprehensive analysis of the stacks between different RNA components in the crystal structures of the bacterial ribosome caught at different translation stages. Analysis of tRNA||rRNA stacks reveals distinct behaviour; both the A-and E-site tRNAs exhibit unique stacking patterns with 23S rRNA bases, while P-site tRNAs stack with 16S rRNA bases. Furthermore, E-site stacks exhibit diverse face orientations and ring topologies-rare for inter-chain RNA interactions-with higher average interaction energies than A or P-site stacks. This suggests that stacking may be essential for stabilizing tRNA progression through the E-site. Additionally, mRNA||rRNA stacks reveal other geometries, which depend on the tRNA binding site, whereas 16S rRNA||23S rRNA stacks highlight the importance of specific bases in maintaining the integrity of the translational complex by linking the two rRNAs. Furthermore, tRNA||mRNA stacks exhibit distinct geometries and energetics at the E-site, indicating their significance during tRNA translocation and elimination. Overall, both A and E-sites display a more diverse distribution of inter-RNA stacks compared to the P-site. Stacking interactions in the active ribosome are not simply accidental byproducts of biochemistry but are likely invoked to compensate and support the integrity and dynamics of translation.

Ribosomal proteins can hold a more accurate record of bacterial thermal adaptation compared to rRNA.

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.

Acetic acid bacteria in agro-wastes: from cheese whey and olive mill wastewater to cellulose.

In this study, cheese whey and olive mill wastewater were investigated as potential feedstocks for producing bacterial cellulose by using acetic acid bacteria strains. Organic acids and phenolic compounds composition were assayed by high-pressure liquid chromatography. Fourier-transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction were used to investigate modifications in bacterial cellulose chemical and morphological structure. Cheese whey was the most efficient feedstock in terms of bacterial cellulose yield (0.300 g of bacterial cellulose/gram of carbon source consumed). Bacterial cellulose produced in olive mill wastewater presented a more well-defined network compared to pellicles produced in cheese whey, resulting in a smaller fiber diameter in most cases. The analysis of bacterial cellulose chemical structure highlighted the presence of different chemical bonds likely to be caused by the adsorption of olive mill wastewater and cheese whey components. The crystallinity ranged from 45.72 to 80.82%. The acetic acid bacteria strains used in this study were characterized by 16S rRNA gene sequencing, allowing to assign them to Komagataeibacter xylinus and Komagataeibacter rhaeticus species. This study proves the suitability to perform sustainable bioprocesses for producing bacterial cellulose, combining the valorisation of agro-wastes with microbial conversions carried out by acetic acid bacteria. The high versatility in terms of yield, morphology, and fiber diameters obtained in cheese whey and olive mill wastewater contribute to set up fundamental criteria for developing customized bioprocesses depending on the final use of the bacterial cellulose. KEY POINTS: • Cheese whey and olive mill wastewater can be used for bacterial cellulose production. • Bacterial cellulose structure is dependent on the culture medium. • Komagataeibacter strains support the agro-waste conversion in bacterial cellulose.

DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms.

Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13C, 18O, or 15N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labelled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labelled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labelling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, or even metagenomes and metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labelled microorganisms. Analysis of labelled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allowed using labelled substrates at environmentally relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies, leading to further breakthroughs in our understanding of novel microbial populations and elucidation of the metabolic function of complex microbial communities. In this chapter, we provide protocols for obtaining labelled DNA, RNA, and proteins that can be used for downstream omics-based analyses.

Dominance of coniferous and broadleaved trees drives bacterial associations with boreal feather mosses.

The composition of ecologically important moss-associated bacterial communities seems to be mainly driven by host species but may also be shaped by environmental conditions related with tree dominance. The moss phyllosphere has been studied in coniferous forests while broadleaf forests remain understudied. To determine if host species or environmental conditions defined by tree dominance drives the bacterial diversity in the moss phyllosphere, we used 16S rRNA gene amplicon sequencing to quantify changes in bacterial communities as a function of host species (Pleurozium schreberi and Ptilium crista-castrensis) and forest type (coniferous black spruce versus deciduous broadleaf trembling aspen) in eastern Canada. The overall composition of moss phyllosphere was defined by the interaction of both factors, though most of the bacterial phyla were determined by a strong effect of forest type. Bacterial α-diversity was highest in spruce forests, while there was greater turnover (ß-diversity) and higher γ-diversity in aspen forests. Unexpectedly, Cyanobacteria were much more relatively abundant in aspen than in spruce forests, with the cyanobacteria family Nostocaceae differing the most between forest types. Our results advance the understanding of moss-associated microbial communities among coniferous and broadleaf deciduous forests, which are important with the increasing changes in tree dominance in the boreal system.

The breast tissue microbiome, stroma, immune cells and breast cancer.

BACKGROUND: Stromal and immune cell composition alterations in benign breast tissue associate with future cancer risk. Pilot data suggest the innate microbiome of normal breast tissue differs between women with and without breast cancer. Microbiome alterations might explain tissue microenvironment variations associated with disease status. METHODS: Prospectively-collected sterile normal breast tissues from women with benign (n=16) or malignant (n=17) disease underwent 16SrRNA sequencing with Illumina MiSeq and Hybrid-denovo pipeline processing. Breast tissue was scored for fibrosis and fat percentages and immune cell infiltrates (lobulitis) classified as absent/mild/moderate/severe. Alpha and beta diversity were calculated on rarefied OTU data and associations analyzed with multiple linear regression and PERMANOVA. RESULTS: Breast tissue stromal fat% was lower and fibrosis% higher in benign disease versus cancer (median 30% versus 60%, p=0.01, 70% versus 30%, p=0.002, respectively). The microbiome varied with stromal composition. Alpha diversity (Chao1) correlated with fat% (r=0.38, p=0.02) and fibrosis% (r=-0.32, p=0.05) and associated with different microbial populations as indicated by beta diversity metrics (weighted UniFrac, p=0.08, fat%, p=0.07, fibrosis%). Permutation testing with FDR control revealed taxa differences for fat% in Firmicutes, Bacilli, Bacillales, Staphylococcaceae and genus Staphylococcus, and fibrosis% in Firmicutes, Spirochaetes, Bacilli, Bacillales, Spirochaetales, Proteobacteria RF32, Sphingomonadales, Staphylococcaceae, and genera Clostridium, Staphylococcus, Spirochaetes, Actinobacteria Adlercreutzia. Moderate/severe lobulitis was more common in cancer (73%) than benign disease (13%), p=0.003, but no significant microbial associations were seen. CONCLUSION: These data suggest a link between breast tissue stromal alterations and its microbiome, further supporting a connection between the breast tissue microenvironment and breast cancer.

50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA.

Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2'-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a postcatalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144, which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes, even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues.

Decoding the Mechanism of Specific RNA Targeting by Ribosomal Methyltransferases.

Methylation of specific nucleotides is integral for ribosomal biogenesis and also serves as a common mechanism to confer antibiotic resistance by pathogenic bacteria. Here, by determining the high-resolution structure of the 30S-KsgA complex by cryo-electron microscopy, a state was captured, where KsgA juxtaposes between helices h44 and h45 of the 30S ribosome, separating them, thereby enabling remodeling of the surrounded rRNA and allowing the cognate site to enter the methylation pocket. With the structure as a guide, several mutant versions of the ribosomes, where interacting bases in the catalytic helix h45 and surrounding helices h44, h24, and h27, were mutated and evaluated for their methylation efficiency revealing factors that direct the enzyme to its cognate site with high fidelity. The biochemical studies show that the three-dimensional environment of the ribosome enables the interaction of select loop regions in KsgA with the ribosome helices paramount to maintain selectivity.

Molecular phylogeography reveals multiple Pleistocene divergence events in estuarine crabs from the tropical West Pacific.

Due to the lack of visible barriers to gene flow, it was a long-standing assumption that marine coastal species are widely distributed, until molecular studies revealed geographically structured intraspecific genetic differentiation in many taxa. Historical events of sea level changes during glacial periods are known to have triggered sequential disjunctions and genetic divergences among populations, especially of coastal organisms. The Parasesarma bidens species complex so far includes three named plus potentially cryptic species of estuarine brachyuran crabs, distributed along East to Southeast Asia. The aim of the present study is to address phylogeography and uncover real and hidden biological diversity within this complex, by revealing the underlying genetic structure of populations and species throughout their distribution ranges from Japan to West Papua, with a comparison of mitochondrial COX1 and 16S rRNA gene sequences. Our results reveal that the P. bidens species complex consists of at least five distinct clades, resulting from four main cladogenesis events during the mid to late Pleistocene. Among those clades, P. cricotum and P. sanguimanus are recovered as monophyletic taxa. Geographically restricted endemic clades are encountered in southeastern Indonesia, Japan and China respectively, whereas the Philippines and Taiwan share two clades. As individuals of the Japanese clade can also be found in Taiwan, we provide evidence of a third lineage and the occurrence of a potential cryptic species on this island. Ocean level retreats during Pleistocene ice ages and present oceanic currents appear to be the main triggers for the divergences of the five clades that are here addressed as the P. bidens complex. Secondary range expansions converted Taiwan into the point of maximal overlap, sharing populations with Japan and the Philippines, but not with mainland China.

A novel ribosome-dimerization protein found in the hyperthermophilic archaeon Pyrococcus furiosus using ribosome-associated proteomics.

Ribosome dimerization is one of the bacterial events that suppresses protein synthesis in the stationary phase. Protein factors responsible for ribosome dimerization in bacteria are well characterized, whereas no information is available for the corresponding factors in archaeal and eukaryotic cells. Here we describe a protein found among the ribosome-associated proteins which dimerizes the 30S ribosomal subunit of the archaeon Pyrococcus furiosus. The ribosome-associated proteins were prepared by high-salt wash of crude ribosomes, and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). Of the detected proteins we focused on a protein (PF0560) whose Protein Score was the highest of all of the function-unknown proteins. PF0560 protein had a pronounced effect on the sedimentation pattern of the 30S ribosomal subunit; addition of this protein to isolated 30S subunit reduced the 30S fraction and increased the amount of the 50S fraction. This increase presumably corresponds to the dimer of the 30S subunit. The PF0560-dependent 30S-dimerization, was also observed by gel electrophoretic analysis. This effect was not observed in EDTA-treated 30S subunit, with protein-free 16S rRNA or with bacterial/eukaryotic ribosomal small subunits. Furthermore, PF0560 protein suppressed the formation of functional 70S ribosomes. These results suggest that PF0560 is a novel 30S dimerization factor, which might participate in regulation of archaeal translation.

The Clinical Utility of Two High-Throughput 16S rRNA Gene Sequencing Workflows for Taxonomic Assignment of Unidentifiable Bacterial Pathogens in Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Bacterial pathogens that cannot be identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) are occasionally encountered in clinical laboratories. The 16S rRNA gene is often used for sequence-based analysis to identify these bacterial species. Nevertheless, traditional Sanger sequencing is laborious, time-consuming, and low throughput. Here, we compared two commercially available 16S rRNA gene sequencing tests that are based on Illumina and Nanopore sequencing technologies, respectively, in their ability to identify the species of 172 clinical isolates that failed to be identified by MALDI-TOF MS. Sequencing data were analyzed by the respective built-in programs (MiSeq Reporter software of Illumina and Epi2me of Nanopore) and BLAST+ (v2.11.0). Their agreement with Sanger sequencing on species-level identification was determined. Discrepancies were resolved by whole-genome sequencing. The diagnostic accuracy of each workflow was determined using the composite sequencing result as the reference standard. Despite the high base-calling accuracy of Illumina sequencing, we demonstrated that the Nanopore workflow had a higher taxonomic resolution at the species level. Using built-in analysis algorithms, the concordance of Sanger 16S with the Illumina and Nanopore workflows was 33.14% and 87.79%, respectively. The agreement was 65.70% and 83.14%, respectively, when BLAST+ was used for analysis. Compared with the reference standard, the diagnostic accuracy of Nanopore 16S was 96.36%, which was identical to that of Sanger 16S and better than that of Illumina 16S (69.07%). The turnaround time of the Illumina workflow and the Nanopore workflow was 78 h and 8.25 h, respectively. The per-sample cost of the Illumina and Nanopore workflows was US$28.5 and US$17.7, respectively.

Protective Effects of Lactobacillus plantarum CCFM8610 against Acute Toxicity Caused by Different Food-Derived Forms of Cadmium in Mice.

Cadmium (Cd) is an environmental pollutant that is toxic to almost every human organ. Oral supplementation with lactic acid bacteria (LAB) has been reported to alleviate cadmium toxicity. However, research on the mitigation of cadmium toxicity by LAB is still limited to inorganic cadmium, which is not representative of the varied forms of cadmium ingested daily. In this study, different foodborne forms of cadmium were adopted to establish an in vivo toxicity model, including cadmium-glutathione, cadmium-citrate, and cadmium-metallothionein. The ability of Lactobacillus plantarum CCFM8610 to reduce the toxic effects of these forms of cadmium was further investigated. The 16S rRNA gene sequencing and metabolomics technologies based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) were adopted for the exploration of relevant protective mechanisms. The results demonstrated that the consumption of CCFM8610 can reduce the content of cadmium in mice and relieve the oxidative stress caused by different food-derived forms of cadmium, indicating that CCFM8610 has a promising effect on the remediation of the toxic effects of cadmium food poisoning. Meanwhile, protective effects on gut microflora and serum metabolites might be an important mechanism for probiotics to alleviate cadmium toxicity. This study provides a theoretical basis for the application of L. plantarum CCFM8610 to alleviate human cadmium poisoning.

Deciphering the low abundance microbiota of presumed aseptic hip and knee implants.

16S rRNA gene sequencing of DNA extracted from clinically uninfected hip and knee implant samples has revealed polymicrobial populations. However, previous studies assessed 16S rRNA gene sequencing as a technique for the diagnosis of periprosthetic joint infections, leaving the microbiota of presumed aseptic hip and knee implants largely unstudied. These communities of microorganisms might play important roles in aspects of host health, such as aseptic loosening. Therefore, this study sought to characterize the bacterial composition of presumed aseptic joint implant microbiota using next generation 16S rRNA gene sequencing, and it evaluated this method for future investigations. 248 samples were collected from implants of 41 patients undergoing total hip or knee arthroplasty revision for presumed aseptic failure. DNA was extracted using two methodologies-one optimized for high throughput and the other for human samples-and amplicons of the V4 region of the 16S rRNA gene were sequenced. Sequencing data were analyzed and compared with ancillary specific PCR and microbiological culture. Computational tools (SourceTracker and decontam) were used to detect and compensate for environmental and processing contaminants. Microbial diversity of patient samples was higher than that of open-air controls and differentially abundant taxa were detected between these conditions, possibly reflecting a true microbiota that is present in clinically uninfected joint implants. However, positive control-associated artifacts and DNA extraction methodology significantly affected sequencing results. As well, sequencing failed to identify Cutibacterium acnes in most culture- and PCR-positive samples. These challenges limited characterization of bacteria in presumed aseptic implants, but genera were identified for further investigation. In all, we provide further support for the hypothesis that there is likely a microbiota present in clinically uninfected joint implants, and we show that methods other than 16S rRNA gene sequencing may be ideal for its characterization. This work has illuminated the importance of further study of microbiota of clinically uninfected joint implants with novel molecular and computational tools to further eliminate contaminants and artifacts that arise in low bacterial abundance samples.

Phylogeny of Lithobiidae Newport, 1844, with emphasis on the megadiverse genus Lithobius Leach, 1814 (Myriapoda, Chilopoda).

Phylogenetic analyses based on molecular and morphological data were conducted to shed light on relationships within the mostly Palaearctic/Oriental centipede family Lithobiidae, with a particular focus on the Palaearctic genus Lithobius Leach, 1814 (Lithobiidae, Lithobiomorpha), which contains >500 species and subspecies. Previous studies based on morphological data resolved Lithobius as nonmonophyletic, but molecular-based phylogenetic analyses have until now sampled few species. To elucidate species inter-relationships of the genus, test the validity of its classification into subgenera, and infer its relationships with other Lithobiidae, we obtained molecular data (nuclear markers: 18S rRNA, 28S rRNA; mitochondrial markers: 16S rRNA, COI) and 61 morphological characters for 44 species of Lithobius representing four of its eight subgenera and nine other representatives of Lithobiidae. The data were analyzed phylogenetically using maximum-likelihood, parsimony and Bayesian inference. This study suggests that (i) a close relationship between L. giganteus and the pterygotergine Disphaerobius loricatus highlighted in recent morphological analyses is also strongly supported by molecular data, and Pterygoterginae is formally synonymized with Lithobiinae; (ii) the Oriental/Australian genus Australobius is consistently resolved as sister group to all other sampled Lithobiidae by the molecular and combined data; (iii) the subfamily Ethopolyinae may be paraphyletic; (iv) the genus Lithobius is nonmonophyletic; (v) the subgenera Lithobius, Sigibius and Monotarsobius are nonmonophyletic and should not be used in future taxonomic studies; and (vi) there are instances of cryptic species and cases in which subspecies should be elevated to full species status, as identified for some European taxa within Lithobius.

Directed evolution of rRNA improves translation kinetics and recombinant protein yield.

In bacteria, ribosome kinetics are considered rate-limiting for protein synthesis and cell growth. Enhanced ribosome kinetics may augment bacterial growth and biomanufacturing through improvements to overall protein yield, but whether this can be achieved by ribosome-specific modifications remains unknown. Here, we evolve 16S ribosomal RNAs (rRNAs) from Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae towards enhanced protein synthesis rates. We find that rRNA sequence origin significantly impacted evolutionary trajectory and generated rRNA mutants with augmented protein synthesis rates in both natural and engineered contexts, including the incorporation of noncanonical amino acids. Moreover, discovered consensus mutations can be ported onto phylogenetically divergent rRNAs, imparting improved translational activities. Finally, we show that increased translation rates in vivo coincide with only moderately reduced translational fidelity, but do not enhance bacterial population growth. Together, these findings provide a versatile platform for development of unnatural ribosomal functions in vivo.

Elucidate microbial characteristics in a full-scale treatment plant for offshore oil produced wastewater.

Oil-produced wastewater treatment plants, especially those involving biological treatment processes, harbor rich and diverse microbes. However, knowledge of microbial ecology and microbial interactions determining the efficiency of plants for oil-produced wastewater is limited. Here, we performed 16S rDNA amplicon sequencing to elucidate the microbial composition and potential microbial functions in a full-scale well-worked offshore oil-produced wastewater treatment plant. Results showed that microbes that inhabited the plant were diverse and originated from oil and marine associated environments. The upstream physical and chemical treatments resulted in low microbial diversity. Organic pollutants were digested in the anaerobic baffled reactor (ABR) dominantly through fermentation combined with sulfur compounds respiration. Three aerobic parallel reactors (APRs) harbored different microbial groups that performed similar potential functions, such as hydrocarbon degradation, acidogenesis, photosynthetic assimilation, and nitrogen removal. Microbial characteristics were important to the performance of oil-produced wastewater treatment plants with biological processes.

Structural basis for +1 ribosomal frameshifting during EF-G-catalyzed translocation.

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.

A distinct assembly pathway of the human 39S late pre-mitoribosome.

Assembly of the mitoribosome is largely enigmatic and involves numerous assembly factors. Little is known about their function and the architectural transitions of the pre-ribosomal intermediates. Here, we solve cryo-EM structures of the human 39S large subunit pre-ribosomes, representing five distinct late states. Besides the MALSU1 complex used as bait for affinity purification, we identify several assembly factors, including the DDX28 helicase, MRM3, GTPBP10 and the NSUN4-mTERF4 complex, all of which keep the 16S rRNA in immature conformations. The late transitions mainly involve rRNA domains IV and V, which form the central protuberance, the intersubunit side and the peptidyltransferase center of the 39S subunit. Unexpectedly, we find deacylated tRNA in the ribosomal E-site, suggesting a role in 39S assembly. Taken together, our study provides an architectural inventory of the distinct late assembly phase of the human 39S mitoribosome.

Whole-genome characterization and comparative genomics of a novel freshwater cyanobacteria species: Pseudanabaena punensis.

Cyanobacteria are emerging as a potential source of novel, beneficial bioactive compounds. However, some cyanobacteria species can harm water quality and public health through the production of toxins. Therefore, surveying the occurrence and generating genomic resources of cyanobacteria producing harmful compounds could help develop the control methods necessary to manage their growth and limit the release contaminants into the water bodies. Here, we describe a novel strain, Pseudanabaena punensis isolated from the open ends of pipelines supplying freshwater. This isolate was characterized morphologically, biochemically and by whole-genome sequence analysis. We also provide genomic information for P. punensis to help understand and highlight the features unique to this isolate. Morphological and genetic (analysis using 16S rRNA and rbcL genes) data were used to assign this novel strain to phylogenetic and taxonomic groups. The isolate was identified as a filamentous and non-heterocystous cyanobacteria. Based on morphological and 16S rRNA phylogeny, this isolate shares characteristics with the Pseudanabaenaceae family, but remains distinct from well-characterized species suggesting its polyphyletic assemblage. The whole-genome sequence analysis suggests greater genomic and phenotypic plasticity. Genome-wide sequence and comparative genomic analyses, comparing against several closely related species, revealed diverse and important genes associated with synthesizing bioactive compounds, multi-drug resistance pathway, heavy metal resistance, and virulence factors. This isolate also produces several important fatty acids with potential industrial applications. The observations described in this study emphasize both industrial applications and risks associated with the freshwater contamination, and therefore genomic resources provided in this study offer an opportunity for further investigations.

Structural basis for late maturation steps of the human mitoribosomal large subunit.

Mitochondrial ribosomes (mitoribosomes) synthesize a critical set of proteins essential for oxidative phosphorylation. Therefore, mitoribosomal function is vital to the cellular energy supply. Mitoribosome biogenesis follows distinct molecular pathways that remain poorly understood. Here, we determine the cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factor GTPBP5, allowing us to capture consecutive steps during mitoribosomal large subunit (mt-LSU) biogenesis. Our structures provide essential insights into the last steps of 16S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, which require the coordinated action of nine assembly factors. We show that mammalian-specific MTERF4 contributes to the folding of 16S rRNA, allowing 16 S rRNA methylation by MRM2, while GTPBP5 and NSUN4 promote fine-tuning rRNA rearrangements leading to PTC formation. Moreover, our data reveal an unexpected involvement of the elongation factor mtEF-Tu in mt-LSU assembly, where mtEF-Tu interacts with GTPBP5, similar to its interaction with tRNA during translational elongation.