Ribosomal Mutations Research Articles

Macrolide resistance through uL4 and uL22 ribosomal mutations in Pseudomonas aeruginosa

Macrolides are widely used antibiotics for the treatment of bacterial airway infections. Due to its elevated minimum inhibitory concentration in standardized culture media, Pseudomonas aeruginosa is considered intrinsically resistant and, therefore, antibiotic susceptibility testing against macrolides is not performed. Nevertheless, due to macrolides’ immunomodulatory effect and suppression of virulence factors, they are used for the treatment of persistent P. aeruginosa infections. Here, we demonstrate that macrolides are, instead, effective antibiotics against P. aeruginosa airway infections in an Air-Liquid Interface (ALI) infection model system resembling the human airways. Importantly, macrolide treatment in both people with cystic fibrosis and primary ciliary dyskinesia patients leads to the accumulation of uL4 and uL22 ribosomal protein mutations in P. aeruginosa which causes antibiotic resistance. Consequently, higher concentrations of antibiotics are needed to modulate the macrolide-dependent suppression of virulence. Surprisingly, even in the absence of antibiotics, these mutations also lead to a collateral reduction in growth rate, virulence and pathogenicity in airway ALI infections which are pivotal for the establishment of a persistent infection. Altogether, these results lend further support to the consideration of macrolides as de facto antibiotics against P. aeruginosa and the need for resistance monitoring upon prolonged macrolide treatment.

Fluorescent-protein co-expression to select CHO cells expressing high quantities of vaccine antigens.

Cell line development for production of vaccine antigens or therapeutic proteins typically involves transfection, selection, and enrichment for high-expressing cells. Enrichment methods include minipool enrichment, antibody-based enrichment, and enrichment based on co-expressed fluorescent biosensor proteins. However, these methods have limitations regarding labor and cost intensity, the generation of antibodies and assurance of their viral safety, and potential expression-interference or signal-saturation of the co-expressed fluorescent protein. To improve the method of fluorescent-protein co-expression, expression constructs were created that constitutively express a model vaccine antigen together with one of three fluorescent proteins having translation initiation controlled by a wildtype or mutant internal ribosome entry site (IRES), for a total of six constructs. The constructs were transfected into Chinese hamster ovary cells (CHO) cells, enriched for high fluorescence, cultured, and tested in a mini bioreactor to identify the most promising construct. The fluorescent protein, Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) with a mutant IRES performed best and was further tested with three additional vaccine antigens. Across the four vaccine antigens, the FUCCI fluorescent protein yielded productivity enhancements, without the need for generating an antibody and assuring its viral safety. Furthermore, FUCCI protein was present in negligible quantities in the cell supernatant, indicating a low risk for contaminating drug substances or vaccine antigen.

Ribosomal mutations enable a switch between high fitness and high stress resistance in Listeria monocytogenes.

Multiple stress resistant variants of Listeria monocytogenes with mutations in rpsU encoding ribosomal protein RpsU have previously been isolated after a single exposure to acid stress. These variants, including L. monocytogenes LO28 variant V14 with a complete deletion of the rpsU gene, showed upregulation of the general stress sigma factor Sigma B-mediated stress resistance genes and had a lower maximum specific growth rate than the LO28 WT, signifying a trade-off between stress resistance and fitness. In the current work V14 has been subjected to an experimental evolution regime, selecting for higher fitness in two parallel evolving cultures. This resulted in two evolved variants with WT-like fitness: 14EV1 and 14EV2. Comparative analysis of growth performance, acid and heat stress resistance, in combination with proteomics and RNA-sequencing, indicated that in both lines reversion to WT-like fitness also resulted in WT-like stress sensitivity, due to lack of Sigma B-activated stress defense. Notably, genotyping of 14EV1 and 14EV2 provided evidence for unique point-mutations in the ribosomal rpsB gene causing amino acid substitutions at the same position in RpsB, resulting in RpsB22Arg-His and RpsB22Arg-Ser, respectively. Combined with data obtained with constructed RpsB22Arg-His and RpsB22Arg-Ser mutants in the V14 background, we provide evidence that loss of function of RpsU resulting in the multiple stress resistant and reduced fitness phenotype, can be reversed by single point mutations in rpsB leading to arginine substitutions in RpsB at position 22 into histidine or serine, resulting in a WT-like high fitness and low stress resistance phenotype. This demonstrates the impact of genetic changes in L. monocytogenes' ribosomes on fitness and stress resistance.

Arabidopsis ribosomal RNA processing meerling mutants exhibit suspensor-derived polyembryony due to direct reprogramming of the suspensor.

Embryo development in Arabidopsis (Arabidopsis thaliana) starts off with an asymmetric division of the zygote to generate the precursors of the embryo proper and the supporting extraembryonic suspensor. The suspensor degenerates as the development of the embryo proper proceeds beyond the heart stage. Until the globular stage, the suspensor maintains embryonic potential and can form embryos in the absence of the developing embryo proper. We report a mutant called meerling-1 (mrl-1), which shows a high penetrance of suspensor-derived polyembryony due to delayed development of the embryo proper. Eventually, embryos from both apical and suspensor lineages successfully develop into normal plants and complete their life cycle. We identified the causal mutation as a genomic rearrangement altering the promoter of the Arabidopsis U3 SMALL NUCLEOLAR RNA-ASSOCIATED PROTEIN 18 (UTP18) homolog that encodes a nucleolar-localized WD40-repeat protein involved in processing 18S preribosomal RNA. Accordingly, root-specific knockout of UTP18 caused growth arrest and accumulation of unprocessed 18S pre-rRNA. We generated the mrl-2 loss-of-function mutant and observed asynchronous megagametophyte development causing embryo sac abortion. Together, our results indicate that promoter rearrangement decreased UTP18 protein abundance during early stage embryo proper development, triggering suspensor-derived embryogenesis. Our data support the existence of noncell autonomous signaling from the embryo proper to prevent direct reprogramming of the suspensor toward embryonic fate.

Establishment of a Diamond-Blackfan anemia like model in zebrafish.

Anemia is defined as a lack of erythrocytes, low hemoglobin levels, or abnormal erythrocyte morphology. Diamond-Blackfan anemia (DBA) is a rare and severe congenital hypoplastic anemia that occurs due to the dominant inheritance of a ribosomal protein gene mutation. Even rarer is a case described as Diamond-Blackfan anemia like (DBAL), which occurs due to a loss-of-function EPO mutation recessive inheritance. The effective cures for DBAL are bone marrow transfusion and treatment with erythropoiesis-stimulating agents (ESAs). To effectively manage the condition, construction of DBAL models to identify new medical methods or screen drugs are necessary. Here, an epoa-deficient mutant zebrafish called epoaszy8 was generated to model DBAL. The epoa-deficiency in zebrafish caused developmental defects in erythroid cells, leading to severe congenital anemia. Using the DBAL model, we validated a loss-of-function EPO mutation using an in vivo functional analysis and explored the ability of ESAs to alleviate congenital anemia. Together, our study demonstrated that epoa deficiency in zebrafish leads to a phenotype resembling DBAL. The DBAL zebrafish model was found to be beneficial for the in vivo assessment of patient-derived EPO variants with unclear implications and for devising potential therapeutic approaches for DBAL.

A connection between the ribosome and two S. pombe tRNA modification mutants subject to rapid tRNA decay.

tRNA modifications are crucial in all organisms to ensure tRNA folding and stability, and accurate translation. In both the yeast Saccharomyces cerevisiae and the evolutionarily distant yeast Schizosaccharomyces pombe, mutants lacking certain tRNA body modifications (outside the anticodon loop) are temperature sensitive due to rapid tRNA decay (RTD) of a subset of hypomodified tRNAs. Here we show that for each of two S. pombe mutants subject to RTD, mutations in ribosomal protein genes suppress the temperature sensitivity without altering tRNA levels. Prior work showed that S. pombe trm8Δ mutants, lacking 7-methylguanosine, were temperature sensitive due to RTD, and that one class of suppressors had mutations in the general amino acid control (GAAC) pathway, which was activated concomitant with RTD, resulting in further tRNA loss. We now find that another class of S. pombe trm8Δ suppressors have mutations in rpl genes, encoding 60S subunit proteins, and that suppression occurs with minimal restoration of tRNA levels and reduced GAAC activation. Furthermore, trm8Δ suppression extends to other mutations in the large or small ribosomal subunit. We also find that S. pombe tan1Δ mutants, lacking 4-acetylcytidine, are temperature sensitive due to RTD, that one class of suppressors have rpl mutations, associated with minimal restoration of tRNA levels, and that suppression extends to other rpl and rps mutations. However, although S. pombe tan1Δ temperature sensitivity is associated with some GAAC activation, suppression by an rpl mutation only modestly inhibits GAAC activation. We propose a model in which ribosomal protein mutations result in reduced ribosome concentrations, leading to both reduced ribosome collisions and a reduced requirement for tRNA, with these effects having different relative importance in trm8Δ and tan1Δ mutants. This model is consistent with our results in S. cerevisiae trm8Δ trm4Δ mutants, known to undergo RTD, fueling speculation that this model applies across eukaryotes.

Genomic Epidemiology of Treponema pallidum and Circulation of Strains With Diminished tprK Antigen Variation Capability in Seattle, 2021-2022.

The incidence of syphilis continues to increase in the United States, yet little is known about Treponema pallidum genomic epidemiology within American metropolitan areas. We performed whole-genome sequencing and tprK deep sequencing of 28 T. pallidum-containing specimens, collected mostly from remnant Aptima swab specimens from 24 individuals from Seattle Sexual Health Clinic during 2021-2022. All 12 individuals infected with Nichols-lineage strains were men who have sex with men, while a specific SS14 cluster (mean, 0.33 single-nucleotide variant) included 1 man who has sex with women and 5 women. All T. pallidum strains sequenced were azithromycin resistant via 23S ribosomal RNA A2058G mutation. Identical T. pallidum genomic sequences were found in pharyngeal and rectal swab specimens taken concurrently from the same individuals. The tprK sequences were less variable between patient-matched specimens and between epidemiologically linked clusters. We detected a 528-base pair deletion in the tprK donor site locus, eliminating 9 donor sites, in T. pallidum genomes of 3 individuals with secondary syphilis, associated with diminution of TprK diversity. We developed an end-to-end workflow for public health genomic surveillance of T. pallidum from remnant Aptima swab specimens. tprK sequencing may assist in linking cases beyond routine T. pallidum genome sequencing. T. pallidum strains with deletions in tprK donor sites currently circulate and are associated with diminished TprK antigenic diversity.

P19 Our experience diagnosing and treating deficiency of adenine deaminase 2 (DADA2) vasculitis

Abstract Introduction Deficiency of adenine deaminase 2 (DADA2) is caused by biallelic pathogenic variants in the gene on chromosome 22q11. It is autosomal recessive with incomplete penetrance, and a variable presentation. It typically manifests as a systemic vasculopathy with early onset strokes, cutaneous manifestations, immunodeficiency and cytopenia. Traditional immunosuppressants have limited efficacy, but vasculitic manifestations have been reported to respond to biologic TNF-inhibitors. We report a patient with confirmed ADA2 deficiency whose cutaneous manifestations were previously classified as Sneddon syndrome. After a period of stability on adalimumab she developed marked cutaneous ulceration that healed following a therapy switch to intravenous infliximab. Case description We report a 39-year-old female with a long clinical history that started in childhood when she developed Diamond-Blackfan anaemia that went into remission following corticosteroid treatment and transfusions. In 2006, she suddenly had two thalamic infarcts and was thoroughly investigated but was found to have unremarkable vascular imaging, a negative antiphospholipid syndrome and autoantibody testing, a modestly elevated CRP (5-15 mg/L) and a notably persistent low serum IgM. She continued to experience frequent migraines associated with transient sensorimotor symptoms. Subsequently, she developed recurrent botchy, polymorphic macular eruptions on the lower limbs. A deep cutaneous biopsy demonstrated lymphocytic predominant perivascular inflammation of deep dermal vessels associated with thrombus formation and endothelial proliferation. A putative diagnosis of Sneddon’s syndrome was made. However, after the subsequent identification of ADA2-deficiency vasculitis, the patient underwent genetic screening which identified a compound heterozygosity for two novel ADA2 mutations (c6634_636, delTTC (p.Phe212del) and c.1225C>T (p.Pro409Ser)). Her mother was as a carrier. She was started on adalimumab injections to which she had a good clinical response with improved cutaneous disease and no further neurological symptoms. However, after two years of disease stability, she then developed a significant ‘punched out’ ulcer on the mid-calf which was painful and increasing in size to a maximum of 2 x 3 cm. Dermatology colleagues were reluctant to re-biopsy due to a high likelihood of non-specific appearances and increased risk of poor healing leading to worsening ulceration. Drawing on experience from rheumatoid arthritis that a switch in anti-TNF biological will often restore efficacy following secondary non-response to adalimumab; we empirically switched treatment to intravenous infliximab, with a reducing course of prednisolone (40mg per day tapering to 5mg after 12 weeks). This was associated with a notable improvement within one month and a complete healing of the ulcer within 4 months. Discussion DADA2 remains a rare condition with a carrier frequency estimated at 1 in 236 persons worldwide and a predicted disease prevalence of approximately 1 in 222,000 persons. Patients with DADA2 who have haematological manifestations tend to present during early infancy, whereas delayed presentation is common in patients with vasculitis and systemic inflammation. It should be suspected in patients with early onset strokes, livedo and other haematological and immunological features. Additionally, many previously diagnosed cases of Sneddon syndrome may in fact have DADA2, with a low IgM level favouring the later. Diagnosis can be confirmed by genetic studies or by a biochemical assay that demonstrates near-absent levels of ADA2 activity in the plasma or serum. Interestingly, on screening of adult patients with polyarteritis nodosa (PAN) 3.4% possessed biallelic pathogenic ADA2 variants and similarly, in genomic survey of patients with Diamond-Blackfan anaemia 2% possessed biallelic pathogenic ADA2 variants in the absence of the ribosomal protein gene mutations associated with it. Treatment is phenotype based with a preference for lifelong TNF inhibitor and biosimilar use in those with vasculitis and/or systemic inflammation, though not currently commissioned for this indication. Glucocorticoids, disease-modifying antirheumatic drugs, and other biologic agents may partially ameliorate systemic inflammation. Unfortunately, they are all ineffective for severe hematologic manifestations and hematopoietic cell transplantation (HCT) is currently the only treatment option. Treating asymptomatic patients and mildly symptomatic patients picked up on screening remains senior clinician led on a risk benefit basis. Disease activity is clinically monitored with an overall mortality rate estimated at 8%. Key learning points The main learning points for me included an increased awareness of this syndrome, its variable presentation, association with other autoimmune illnesses, diagnosis, and treatment initiation. In addition, I learnt about screening of family members, weighing the benefits versus risks when considering treating mildly symptomatic and asymptomatic patients, clinical monitoring for response, and when to switch to other anti-TNFs and biosimilars. From this conference, I would like to explore the experience of our rheumatology colleagues with similar cases.

Parallel evolution of linezolid-resistant Staphylococcus aureus in patients with cystic fibrosis.

Linezolid is an antibiotic used to treat serious Staphylococcus aureus infections. Resistance to linezolid is considered rare but could emerge with repeated dosing. We recently reported widespread prescription of linezolid for a cohort of patients with cystic fibrosis (CF). The goals of this study were to determine the incidence of linezolid-resistant methicillin-resistant Staphylococcus aureus (MRSA) in CF and identify molecular mechanisms for linezolid resistance. We identified patients who cultured S. aureus resistant to linezolid with minimum inhibitory concentration (MIC) >4 at the University of Iowa CF Center between 2008 and 2018. We obtained isolates from these patients and retested susceptibility to linezolid using broth microdilution. We used whole genome sequencing to perform phylogenetic analysis of linezolid-resistant isolates and examine sequences for mutations or accessory genes that confer linezolid resistance. Between 2008 and 2018, 111 patients received linezolid, and 4 of these patients cultured linezolid-resistant S. aureus. We sequenced 11 resistant and 21 susceptible isolates from these 4 subjects. Phylogenetic analysis indicated that linezolid resistance developed in ST5 or ST105 backgrounds. Three individuals had linezolid-resistant S. aureus with a G2576T mutation in 23S rRNA. One of these subjects additionally had a mutS- mutL- hypermutating S. aureus that produced five resistant isolates with multiple ribosomal subunit mutations. In one subject, the genetic basis for linezolid resistance was unclear. We conclude that linezolid resistant S. aureus can occur through multiple genetic mechanisms in patients with repeated exposure to this antibiotic. IMPORTANCE Patients with cystic fibrosis have persistent lung infections with Staphylococcus aureus that require extensive antibiotic treatments. Linezolid, an antibiotic given by oral or intravenous route, is prescribed repeatedly for patients whose lung disease has progressed. After treatment with linezolid, S. aureus strains can evolve antibiotic resistance through multiple genetic mechanisms. In addition to a common mutation in the 23S ribosomal RNA known to confer linezolid resistance, S. aureus strains can evolve novel resistance based on a combination of mutations affecting the bacterial ribosome. This combination of mutations was observed in a strain that exhibited hypermutation owing to the loss of the DNA repair genes mutS and mutL. In this cohort of patients with cystic fibrosis, linezolid resistance was transient, possibly due to the growth disadvantage of resistant strains. However, ongoing chronic exposure to linezolid may create optimal conditions for the future emergence of resistance to this critical antibiotic.

Extensive breaking of genetic code degeneracy with non-canonical amino acids

Genetic code expansion (GCE) offers many exciting opportunities for the creation of synthetic organisms and for drug discovery methods that utilize in vitro translation. One type of GCE, sense codon reassignment (SCR), focuses on breaking the degeneracy of the 61 sense codons which encode for only 20 amino acids. SCR has great potential for genetic code expansion, but extensive SCR is limited by the post-transcriptional modifications on tRNAs and wobble reading of these tRNAs by the ribosome. To better understand codon-tRNA pairing, here we develop an assay to evaluate the ability of aminoacyl-tRNAs to compete with each other for a given codon. We then show that hyperaccurate ribosome mutants demonstrate reduced wobble reading, and when paired with unmodified tRNAs lead to extensive and predictable SCR. Together, we encode seven distinct amino acids across nine codons spanning just two codon boxes, thereby demonstrating that the genetic code hosts far more re-assignable space than previously expected, opening the door to extensive genetic code engineering.

Ribosomal Biogenesis and Heterogeneity in Development, Disease, and Aging.

Although reported in the literature, ribosome heterogeneity is a phenomenon whose extent and implications in cell and organismal biology is not fully appreciated. This has been the case due to the lack of the appropriate techniques and approaches. Heterogeneity can arise from alternative use and differential content of protein and RNA constituents, as well as from post-transcriptional and post-translational modifications. In the few examples we have, it is apparent that ribosomal heterogeneity offers an additional level and potential for gene expression regulation and might be a way towards tuning metabolism, stress, and growth programs to external and internal stimuli and needs. Here, we introduce ribosome biogenesis and discuss ribosomal heterogeneity in various reported occasions. We conclude that a systematic approach in multiple organisms will be needed to delineate this biological phenomenon and its contributions to growth, aging, and disease. Finally, we discuss ribosome mutations and their roles in disease.

Characterization of In Vitro Resistance to Linezolid in Mycobacterium abscessus.

Single-step selection of Mycobacterium abscessus mutants resistant to linezolid yielded high-level resistance at a low frequency that was associated with mutations in 23S rRNA or the ribosomal protein L3. Surprisingly, linezolid-resistant rRNA mutations conferred cross-resistance to several unrelated antibiotics. Low-level linezolid-resistant mutants were isolated at a higher frequency and were due to loss-of-function mutations in the transcriptional regulator MAB_4384, the repressor of the drug efflux pump MmpL5-MmpS5. IMPORTANCE The protein synthesis inhibitor linezolid is used for the treatment of lung disease caused by Mycobacterium abscessus. However, many strains of the bacterium show poor susceptibility to the antibiotic. For most clinical isolates, resistance is not due to mutations in the target of the drug, the ribosome. The mechanism responsible for non-target-related, indirect linezolid resistance is unknown. Here, we analyzed the development of linezolid resistance in the M. abscessus reference strain in vitro. We found, as expected, resistance mutations in the ribosome. In addition, we identified mutations in a system that involves a drug pump, suggesting drug efflux as a mechanism of resistance to linezolid. This finding may inform the analysis of clinical resistance to linezolid. Surprisingly, a subset of linezolid-resistant ribosome mutations conferred cross-resistance to several structurally and mechanistically unrelated drugs, uncovering a novel multidrug resistance mechanism.

Ribosomal protein mutations and cell competition: autonomous and nonautonomous effects on a stress response.

Ribosomal proteins (Rps) are essential for viability. Genetic mutations affecting Rp genes were first discovered in Drosophila, where they represent a major class of haploinsufficient mutations. One mutant copy gives rise to the dominant "Minute" phenotype, characterized by slow growth and small, thin bristles. Wild-type (WT) and Minute cells compete in mosaics, that is, Rp+/- are preferentially lost when their neighbors are of the wild-type genotype. Many features of Rp gene haploinsufficiency (i.e. Rp+/- phenotypes) are mediated by a transcriptional program. In Drosophila, reduced translation and slow growth are under the control of Xrp1, a bZip-domain transcription factor induced in Rp mutant cells that leads ultimately to the phosphorylation of eIF2α and consequently inhibition of most translation. Rp mutant phenotypes are also mediated transcriptionally in yeast and in mammals. In mammals, the Impaired Ribosome Biogenesis Checkpoint activates p53. Recent findings link Rp mutant phenotypes to other cellular stresses, including the DNA damage response and endoplasmic reticulum stress. We suggest that cell competition results from nonautonomous inputs to stress responses, bringing decisions between adaptive and apoptotic outcomes under the influence of nearby cells. In Drosophila, cell competition eliminates aneuploid cells in which loss of chromosome leads to Rp gene haploinsufficiency. The effects of Rp gene mutations on the whole organism, in Minute flies or in humans with Diamond-Blackfan Anemia, may be inevitable consequences of pathways that are useful in eliminating individual cells from mosaics. Alternatively, apparently deleterious whole organism phenotypes might be adaptive, preventing even more detrimental outcomes. In mammals, for example, p53 activation appears to suppress oncogenic effects of Rp gene haploinsufficiency.

DksA is a conserved master regulator of stress response in Acinetobacter baumannii.

Coordination of bacterial stress response mechanisms is critical for long-term survival in harsh environments for successful host infection. The general and specific stress responses of well-studied Gram-negative pathogens like Escherichia coli are controlled by alternative sigma factors, archetypically RpoS. The deadly hospital pathogen Acinetobacter baumannii is notoriously resistant to environmental stresses, yet it lacks RpoS, and the molecular mechanisms driving this incredible stress tolerance remain poorly defined. Here, using functional genomics, we identified the transcriptional regulator DksA as a master regulator for broad stress protection and virulence in A. baumannii. Transcriptomics, phenomics and in vivo animal studies revealed that DksA controls ribosomal protein expression, metabolism, mutation rates, desiccation, antibiotic resistance, and host colonization in a niche-specific manner. Phylogenetically, DksA was highly conserved and well-distributed across Gammaproteobacteria, with 96.6% containing DksA, spanning 88 families. This study lays the groundwork for understanding DksA as a major regulator of general stress response and virulence in this important pathogen.

Design and screening of tetracycline antibiotics: an in-silico approach

A prominent class of broad-spectrum antibiotics known as tetracycline works by inhibiting the synthesis of proteins, which prevents the development of bacteria. Tetracycline resistance is typically attributed to one or more of the following causes: ribosomal binding site mutations, acquisition of mobile genetic elements carrying tetracycline-specific resistance genes, and/or chromosomal mutations that increase the expression of intrinsic resistance mechanisms. In this research, our objective is to virtually plan and conduct in-silico experiments to find tetracycline derivatives with inhibitory capability against tetracycline resistance protein. The tetracycline derivatives were screened using the Data Warrior, Discovery Studio, PyRx, and Swiss ADME web tools. Initially, 19 tetracycline derivatives were primarily screened for ADME and toxicity study followed by docking study. Among the tetracycline derivatives, C1, C11, C12, C14, C16, and C17 were found to be the potential drug-like molecules with binding energies of -8.9 kcal/mol, -8.4 kcal/mol, -8.5 kcal/mol, -7.7 kcal/mol, -7.7 kcal/mol, -8.6 kcal/mol respectively. In particular, C1 was predicted to have a better binding affinity towards the target protein than the others.

Rare ribosomal RNA sequences from archaea stabilize the bacterial ribosome.

The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3'-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not affect cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation across archaeal ribosomes in the peptidyl transferase center that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

Computationally-guided design and selection of high performing ribosomal active site mutants.

Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.g. library size). Here, to address these limitations, we developed a computational rRNA design approach for screening guided libraries of mutant ribosomes. Our method includes in silico library design and selection using a Rosetta stepwise Monte Carlo method (SWM), library construction and in vitro testing of combined ribosomal assembly and translation activity, and functional characterization in vivo. As a model, we apply our method to making modified ribosomes with mutant PTCs. We engineer ribosomes with as many as 30 mutations in their PTCs, highlighting previously unidentified epistatic interactions, and show that SWM helps identify sequences with beneficial phenotypes as compared to random library sequences. We further demonstrate that some variants improve cell growth in vivo, relative to wild type ribosomes. We anticipate that SWM design and selectionmay serve as a powerful tool for rRNA engineering.

Longitudinal metatranscriptomic sequencing of Southern California wastewater representing 16 million people from August 2020–21 reveals widespread transcription of antibiotic resistance genes

Municipal wastewater provides a representative sample of human fecal waste across a catchment area and contains a wide diversity of microbes. Sequencing wastewater samples provides information about human-associated and medically important microbial populations, and may be useful to assay disease prevalence and antimicrobial resistance (AMR).Here, we present a study in which we used untargeted metatranscriptomic sequencing on RNA extracted from 275 sewage influent samples obtained from eight wastewater treatment plants (WTPs) representing approximately 16 million people in Southern California between August 2020 – August 2021. We characterized bacterial and viral transcripts, assessed metabolic pathway activity, and identified over 2,000 AMR genes/variants across all samples. Because we did not deplete ribosomal RNA, we have a unique window into AMR carried as ribosomal mutants. We show that AMR diversity varied between WTPs (as measured through PERMANOVA, P < 0.001) and that the relative abundance of many individual AMR genes/variants increased over time (as measured with MaAsLin2, Padj < 0.05). Similarly, we detected transcripts mapping to human pathogenic bacteria and viruses suggesting RNA sequencing is a powerful tool for wastewater-based epidemiology and that there are geographical signatures to microbial transcription. We captured the transcription of gene pathways common to bacterial cell processes, including central carbon metabolism, nucleotide synthesis/salvage, and amino acid biosynthesis. We also posit that due to the ubiquity of many viruses and bacteria in wastewater, new biological targets for microbial water quality assessment can be developed.To the best of our knowledge, our study provides the most complete longitudinal metatranscriptomic analysis of a large population's wastewater to date and demonstrates our ability to monitor the presence and activity of microbes in complex samples. By sequencing RNA, we can track the relative abundance of expressed AMR genes/variants and metabolic pathways, increasing our understanding of AMR activity across large human populations and sewer sheds.

The CRL4 E3 ligase Mahjong/DCAF1 controls cell competition through the transcription factor Xrp1, independently of polarity genes.

Cell competition, the elimination of cells surrounded by more fit neighbors, is proposed to suppress tumorigenesis. Mahjong (Mahj), a ubiquitin E3 ligase substrate receptor, has been thought to mediate competition of cells mutated for lethal giant larvae (lgl), a neoplastic tumor suppressor that defines apical-basal polarity of epithelial cells. Here, we show that Drosophila cells mutated for mahjong, but not for lgl [l(2)gl], are competed because they express the bZip-domain transcription factor Xrp1, already known to eliminate cells heterozygous for ribosomal protein gene mutations (Rp/+ cells). Xrp1 expression in mahj mutant cells results in activation of JNK signaling, autophagosome accumulation, eIF2α phosphorylation and lower translation, just as in Rp/+ cells. Cells mutated for damage DNA binding-protein 1 (ddb1; pic) or cullin 4 (cul4), which encode E3 ligase partners of Mahj, also display Xrp1-dependent phenotypes, as does knockdown of proteasome subunits. Our data suggest a new model of mahj-mediated cell competition that is independent of apical-basal polarity and couples Xrp1 to protein turnover.

Prevalence and risk factors of obesity in children with Diamond-Blackfan anemia

To investigate the distribution of body mass index (BMI) and risk factors for obesity in children with Diamond-Blackfan Anemia (DBA). The children with DBA who attended National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, from January 2003 to December 2020 were enrolled as subjects. The related clinical data and treatment regimens were recorded. The height and weight data measured within 1 week before or after follow-up time points were collected to calculate BMI. The risk factors for obesity were determined by multivariate regression analysis in children with DBA. A total of 129 children with DBA were enrolled, among whom there were 80 boys (62.0%) and 49 girls (38.0%), with a median age of 49 months (range 3-189 months). The prevalence rate of obesity was 14.7% (19/129). The multivariate logistic regression analysis showed that the absence of ribosomal protein gene mutation was closely associated with obesity in children with DBA (adjusted OR=3.63, 95%CI: 1.16-11.38, adjusted P=0.027). In children with glucocorticoid-dependent DBA, obesity was not associated with age of initiation of glucocorticoid therapy, duration of glucocorticoid therapy, and maintenance dose of glucocorticoids (P>0.05). There is a high prevalence rate of obesity in children with DBA, and the absence of ribosomal protein gene mutation is closely associated with obesity in children with DBA.