Involvement of Escherichia coli YbeX/CorC in ribosomal metabolism.

YbeX of Escherichia coli, a member of the CorC protein family, is encoded in the same operon with ribosome-associated proteins YbeY and YbeZ. Here, we report the involvement of YbeX in ribosomal metabolism. The ΔybeX cells accumulate distinct 16S rRNA degradation intermediates in the 30S particles and the 70S ribosomes. E. coli lacking ybeX has a lengthened lag phase upon outgrowth from the stationary phase. This growth phenotype is heterogeneous at the individual cell level and especially prominent under low extracellular magnesium levels. The ΔybeX strain is sensitive to elevated growth temperatures and to several ribosome-targeting antibiotics that have in common the ability to induce the cold shock response in E. coli. Although generally milder, the phenotypes of the ΔybeX mutant overlap with those caused by ybeY deletion. A genetic screen revealed partial compensation of the ΔybeX growth phenotype by the overexpression of YbeY. These findings indicate an interconnectedness among the ybeZYX operon genes, highlighting their roles in ribosomal assembly and/or degradation.

A field-wide assessment of differential expression profiling by high-throughput sequencing reveals widespread bias.

We assess inferential quality in the field of differential expression profiling by high-throughput sequencing (HT-seq) based on analysis of datasets submitted from 2008 to 2020 to the NCBI GEO data repository. We take advantage of the parallel differential expression testing over thousands of genes, whereby each experiment leads to a large set of p-values, the distribution of which can indicate the validity of assumptions behind the test. From a well-behaved p-value set π0, the fraction of genes that are not differentially expressed can be estimated. We found that only 25% of experiments resulted in theoretically expected p-value histogram shapes, although there is a marked improvement over time. Uniform p-value histogram shapes, indicative of <100 actual effects, were extremely few. Furthermore, although many HT-seq workflows assume that most genes are not differentially expressed, 37% of experiments have π0-s of less than 0.5, as if most genes changed their expression level. Most HT-seq experiments have very small sample sizes and are expected to be underpowered. Nevertheless, the estimated π0-s do not have the expected association with N, suggesting widespread problems of experiments with controlling false discovery rate (FDR). Both the fractions of different p-value histogram types and the π0 values are strongly associated with the differential expression analysis program used by the original authors. While we could double the proportion of theoretically expected p-value distributions by removing low-count features from the analysis, this treatment did not remove the association with the analysis program. Taken together, our results indicate widespread bias in the differential expression profiling field and the unreliability of statistical methods used to analyze HT-seq data.

Causal variation modelling identifies large inter- and intra-regional disparities in physical therapy offered to hip fracture patients in Estonia.

PURPOSE: An essential measure of hip fracture (HF) rehabilitation, the amount of physical therapy (PT) used per patient, has been severely understudied. This study (1) evaluates post-acute PT use after HF in Estonia, (2) presents causal variation modelling for examining inter- and intra-regional disparities, and (3) analyses its temporal trends. MATERIALS AND METHODS: This retrospective cohort study used validated population-wide health data, including patients aged ≥50 years, with an index HF diagnosed between January 2009 and September 2017. Patients' 6-month PT use was analysed and reported separately for acute and post-acute phases. RESULTS: While most of the included 11,461 patients received acute rehabilitation, only 40% of them received post-acute PT by a median of 6 h. Analyses based on measures of central tendency revealed 2.5 to 2.6-fold inter-regional differences in HF post-acute rehabilitation. Variation modelling additionally detected intra-regional disparities, showing imbalances in the fairness of allocating local rehabilitation resources between a county's patients. CONCLUSIONS: This study demonstrates the advantages of causal variation modelling for identifying inter- and intra-regional disparities in rehabilitation. The analyses revealed persisting large multi-level disparities and accompanying overall inaccessibility of PT in HF rehabilitation in Estonia, showing an urgent need for system-wide improvements.Implications for rehabilitationThis study demonstrates the advantages of causal variation modelling for identifying inter- and intra-regional disparities in rehabilitation, using an essential outcome measure - used physical therapy hours.The study revealed large multi-level disparities and overall inaccessibility of physical therapy in hip fracture rehabilitation in Estonia, showing an urgent need for system-wide improvements.This study expands our knowledge on unstudied topics - hip fracture post-acute care and long-term physical therapy use.This regional analysis provides the first evidence-based regional-level basis for improving the rehabilitation system in Estonia.

Microbiome of root vegetables-a source of gluten-degrading bacteria.

Gluten is a cereal protein that is incompletely digested by human proteolytic enzymes that create immunogenic peptides that accumulate in the gastrointestinal tract (GIT). Although both environmental and human bacteria have been shown to expedite gluten hydrolysis, gluten intolerance is a growing concern. Here we hypothesize that together with food, we acquire environmental bacteria that could impact our GIT with gluten-degrading bacteria. Using in vitro gastrointestinal simulation conditions, we evaluated the capacity of endophytic bacteria that inhabit root vegetables, potato (Solanum tuberosum), carrot (Daucus sativus), beet (Beta vulgaris), and topinambur (Jerusalem artichoke) (Helianthus tuberosus), to resist these conditions and degrade gluten. By 16S rDNA sequencing, we discovered that bacteria from the families Enterobacteriaceae, Bacillaceae, and Clostridiaceae most effectively multiply in conditions similar to the human GIT (microoxic conditions, 37 °C) while utilizing vegetable material and gluten as nutrients. Additionally, we used stomach simulation (1 h, pH 3) and intestinal simulation (1 h, bile salts 0.4%) treatments. The bacteria that survived this treatment retained the ability to degrade gluten epitopes but at lower levels. Four bacterial strains belonging to species Bacillus pumilus, Clostridium subterminale, and Clostridium sporogenes isolated from vegetable roots produced proteases with postproline cleaving activity that successfully neutralized the toxic immunogenic epitopes. KEY POINTS: • Bacteria from root vegetables can degrade gluten. • Some of these bacteria can resist conditions mimicking gastrointestinal tract.

High variability in hip fracture post-acute care and dementia patients having worse chances of receiving rehabilitation: an analysis of population-based data from Estonia.

PURPOSE: Rehabilitation is a fundamental part of hip fracture (HF) care; however, the best strategies are unclear. This study maps index HF patients' post-acute care (PAC) in Estonia and compares the PAC of patients with and without a diagnosis of dementia. METHODS: A retrospective cohort study was conducted using validated population-based data from the Estonian Health Insurance Fund using inclusion criteria: age ≥ 50 years, International Classification of Diseases code (S72.0-2) indicating HF between 1 January 2009 and 30 September 2017, and the survival of PAC. The presence of dementia diagnosis was based on the 10th revision of the International Classification of Diseases codes. RESULTS: A total of 8729 patients were included in the study, 11% of whom had a dementia diagnosis. The PAC of HF patients varied from extensive to no care: 8.7% received combined inpatient and outpatient care; 59% received hospital care (13% had a length of stay (LOS) > 6 weeks; 33% had LOS between 2-4 weeks, 14% had LOS < 2 weeks); 4% received only community-based care; 28% received no PAC. Physical therapy (PT) was received by 56% of patients and by 35% of those with dementia diagnosis. Dementia patients had 1.5-fold higher odds of not receiving PT. CONCLUSION: In Estonia, the PAC after index HF varies from extensive to no care, and the provision of PT is limited and unequal, affecting dementia patients in particular. Thus, there is an urgent need to standardise index HF PAC by reviewing the resources of PT and developing effective rehabilitation practices.

Endophytic bacterial communities in peels and pulp of five root vegetables.

Plants contain endophytic bacteria, whose communities both influence plant growth and can be an important source of probiotics. Here we used deep sequencing of a 16S rRNA gene fragment and bacterial cultivation to independently characterize the microbiomes of five plant species from divergent taxonomic orders-potato (Solanum tuberosum), carrot (Daucus sativus), beet (Beta vulgaris), neep (Brassica napus spp. napobrassica), and topinambur (Helianthus tuberosus). We found that both species richness and diversity tend to be higher in the peel, where Alphaproteobacteria and Actinobacteria dominate, while Gammaproteobacteria and Firmicutes dominate in the pulp. A statistical analysis revealed that the main characteristic features of the microbiomes of plant species originate from the peel microbiomes. Topinambur pulp displayed an interesting characteristic feature: it contained up to 108 CFUs of lactic acid bacteria, suggesting its use as a source of probiotic bacteria. We also detected Listeria sp., in topinambur pulps, however, the 16S rRNA gene fragment is unable to distinguish between pathogenic versus non-pathogenic species, so the evaluation of this potential health risk is left to a future study.

Fragmentation of Escherichia coli mRNA by MazF and MqsR.

MazEF and MqsRA are toxin-antitoxin systems, where the toxins MazF and MqsR sequence-specifically cleave single-stranded RNA, thereby shutting down protein synthesis and cell growth. However, it has been proposed that MazF functions in a highly specific pathway, where it truncates the 5' ends of a set of E. coli transcripts (the MazF regulon), which are then translated under stress conditions by specialized ribosomes. We mapped the cleavage sites of MazF and MqsR throughout the E. coli transcriptome. Our results show that both toxins cleave mRNA independently of the recognition site position and MazF freely cleaves transcripts of the proposed MazF regulon within coding sequences. Proteome analysis indicated that MazF expression leads to overall inhibition of protein synthesis and the putative MazF regulon proteins are not selectively synthesized in response to the toxin. Our results support a simpler role for endoribonuclease TA systems as indifferent destroyers of unstructured RNA.

Antibody response against cancer-testis antigens MAGEA4 and MAGEA10 in patients with melanoma.

Melanoma-associated antigen A (MAGEA) represent a class of tumor antigens that are expressed in a variety of malignant tumors, however, their expression in healthy normal tissues is restricted to germ cells of testis, fetal ovary and placenta. The restricted expression and immunogenicity of these antigens make them ideal targets for immunotherapy in human cancer. In the present study the presence of naturally occurring antibodies against two MAGEA subfamily proteins, MAGEA4 and MAGEA10, was analyzed in patients with melanoma at different stages of disease. Results indicated that the anti-MAGEA4/MAGEA10 immune response in melanoma patients was heterogeneous, with only ~8% of patients having a strong response. Comparing the number of strongly responding patients between different stages of disease revealed that the highest number of strong responses was detected among stage II melanoma patients. These findings support the model that the immune system is involved in the control of melanoma in the early stages of disease.

Toxins MazF and MqsR cleave Escherichia coli rRNA precursors at multiple sites.

The endoribonuclease toxins of the E. coli toxin-antitoxin systems arrest bacterial growth and protein synthesis by targeting cellular mRNAs. As an exception, E. coli MazF was reported to cleave also 16S rRNA at a single site and separate an anti-Shine-Dalgarno sequence-containing RNA fragment from the ribosome. We noticed extensive rRNA fragmentation in response to induction of the toxins MazF and MqsR, which suggested that these toxins can cleave rRNA at multiple sites. We adapted differential RNA-sequencing to map the toxin-cleaved 5'- and 3'-ends. Our results show that the MazF and MqsR cleavage sites are located within structured rRNA regions and, therefore, are not accessible in assembled ribosomes. Most of the rRNA fragments are located in the aberrant ribosomal subunits that accumulate in response to toxin induction and contain unprocessed rRNA precursors. We did not detect MazF- or MqsR-cleaved rRNA in stationary phase bacteria and in assembled ribosomes. Thus, we conclude that MazF and MqsR cleave rRNA precursors before the ribosomes are assembled and potentially facilitate the decay of surplus rRNA transcripts during stress.

The effects of disruptions in ribosomal active sites and in intersubunit contacts on ribosomal degradation in Escherichia coli.

Although ribosomes are very stable under most conditions, ribosomal degradation does occur in diverse groups of organisms in response to specific stresses or environmental conditions. While non-functional ribosome decay (NRD) in yeast is well characterized, very little is known of the mechanisms that initiate ribosomal degradation in bacteria. Here we test ribosome degradation in growing Escherichia coli expressing mutant ribosomes. We found that mutations in the 16S rRNA decoding centre (G530U and A1492C) and 23S rRNA active site (A2451G) do not lead to ribosomal degradation. In contrast, 23S rRNA mutation U2585A causes degradation of both the large and small ribosomal subunits in E. coli. We further tested mutations in 23S rRNA, which disrupt ribosomal intersubunit bridges B2a and B3. Deletion of helix 69 of 23S rRNA and the point mutation A1912G in the same helix did not destabilize ribosomes, while expression of mutations A1919G in H69 and A1960G in H71 led to degradation of both mutant and wild-type ribosomes. Our results suggest an actively induced mechanism requiring de novo protein synthesis for ribosomal degradation in E. coli, which degrades both structurally inactive and active ribosomes.

When stable RNA becomes unstable: the degradation of ribosomes in bacteria and beyond.

This review takes a comparative look at the various scenarios where ribosomes are degraded in bacteria and eukaryotes with emphasis on studies involving Escherichia coli and Saccharomyces cerevisiae. While the molecular mechanisms of degradation in bacteria and yeast appear somewhat different, we argue that the underlying causes of ribosome degradation are remarkably similar. In both model organisms during ribosomal assembly, partially formed pre-ribosomal particles can be degraded by at least two different sequentially-acting quality control pathways and fully assembled but functionally faulty ribosomes can be degraded in a separate quality control pathway. In addition, ribosomes that are both structurally- and functionally-sound can be degraded as an adaptive measure to stress.

Ribosome degradation in growing bacteria.

Ribosomes are large ribozymes that synthesize all cellular proteins. As protein synthesis is rate-limiting for bacterial growth and ribosomes can comprise a large portion of the cellular mass, elucidation of ribosomal turnover is important to the understanding of cellular physiology. Although ribosomes are widely believed to be stable in growing cells, this has never been rigorously tested, owing to the lack of a suitable experimental system in commonly used bacterial model organisms. Here, we develop an experimental system to directly measure ribosomal stability in Escherichia coli. We show that (i) ribosomes are stable when cells are grown at a constant rate in the exponential phase; (ii) more than half of the ribosomes made during exponential growth are degraded during slowing of culture growth preceding the entry into stationary phase; and (iii) ribosomes are stable for many hours in the stationary phase. Ribosome degradation occurs in growing cultures that contain almost no dead cells and coincides with a reduction of comparable magnitude in the cellular RNA concentration.

Ribosome reactivation by replacement of damaged proteins.

Ribosomal functions are vital for all organisms. Bacterial ribosomes are stable 2.4 MDa particles composed of three RNAs and over 50 different proteins. Accumulating damage to ribosomal RNA or proteins can disturb ribosome functioning. Organisms could benefit from degrading or possibly repairing inactive or partially active ribosomes. Reactivation of chemically damaged ribosomes by a process of protein replacement was studied in vitro. Ribosomes were inactivated by chemical modification of Cys residues. Incubation of modified ribosomes with total ribosomal proteins led to reactivation of translational activity. Intriguingly, ribosomal proteins extracted by LiCl are equally active in the restoration of ribosome function. Incubation of 70S ribosomes with isotopically labelled r-proteins followed by separation of ribosomes was used to identify exchangeable proteins. A similar set of proteins was found to be exchanged in vivo under stress conditions in the stationary phase. We propose that repair of damaged ribosomes might be an important mechanism for maintaining protein synthesis activity following chemical damage.

Identification of nucleotides in E. coli 16S rRNA essential for ribosome subunit association.

The ribosome consists of two unequal subunits, which associate via numerous intersubunit contacts. Medium-resolution structural studies have led to grouping of the intersubunit contacts into 12 directly visualizable intersubunit bridges. Most of the intersubunit interactions involve RNA. We have used an RNA modification interference approach to determine Escherichia coli 16S rRNA positions that are essential for the association of functionally active 70S ribosomes. Modification of the N1 position of A702, A1418, and A1483 with DMS, and of the N3 position of U793, U1414, and U1495 with CMCT in 30S subunits strongly interferes with 70S ribosome formation. Five of these positions localize into previously recognized intersubunit bridges, namely, B2a (U1495), B2b (U793), B3 (A1483), B5 (A1418), and B7a (A702). The remaining position displaying interference, U1414, forms a base pair with G1486, which is a part of bridge B3. We contend that these five intersubunit bridges are essential for reassociation of the 70S ribosome, thus forming the functional core of the intersubunit contacts.

Analysis of the function of E. coli 23S rRNA helix-loop 69 by mutagenesis.

BACKGROUND: The ribosome is a two-subunit enzyme known to exhibit structural dynamism during protein synthesis. The intersubunit bridges have been proposed to play important roles in decoding, translocation, and the peptidyl transferase reaction; yet the physical nature of their contributions is ill understood. An intriguing intersubunit bridge, B2a, which contains 23S rRNA helix 69 as a major component, has been implicated by proximity in a number of catalytically important regions. In addition to contacting the small ribosomal subunit, helix 69 contacts both the A and P site tRNAs and several translation factors. RESULTS: We scanned the loop of helix 69 by mutagenesis and analyzed the mutant ribosomes using a plasmid-borne IPTG-inducible expression system. We assayed the effects of 23S rRNA mutations on cell growth, contribution of mutant ribosomes to cellular polysome pools and the ability of mutant ribosomes to function in cell-free translation. Mutations A1912G, and A1919G have very strong growth phenotypes, are inactive during in vitro protein synthesis, and under-represented in the polysomes. Mutation Psi1917C has a very strong growth phenotype and leads to a general depletion of the cellular polysome pool. Mutation A1916G, having a modest growth phenotype, is apparently defective in the assembly of the 70S ribosome. CONCLUSION: Mutations A1912G, A1919G, and Psi1917C of 23S rRNA strongly inhibit translation. Mutation A1916G causes a defect in the 50S subunit or 70S formation. Mutations Psi1911C, A1913G, C1914A, Psi1915C, and A1918G lack clear phenotypes.

Definition of bases in 23S rRNA essential for ribosomal subunit association.

The ribosome is a two-subunit molecular machine, sporting a working cycle that involves coordinated movements of the subunits. Recent structural studies of the 70S ribosome describe a rather large number of intersubunit contacts, some of which are dynamic during translocation. We set out to determine which intersubunit contacts are functionally indispensable for the association of ribosome subunits by using a modification interference approach. Modification of the N-1 position of A715, A1912, or A1918 in Escherichia coli 50S subunits is strongly detrimental to 70S ribosome formation. This result points to 23S rRNA helices 34 and 69, and thus bridges B2a and B4, as essential for ensuring stability of the 70S ribosome.

Accessibility of phosphates in domain I of 23 S rRNA in the ribosomal 50 S subunit as detected by R(P) phosphorothioates.

Recent atomic models of ribosomal structure emphasize the need for new biochemical methods, suitable for fine-scale studies of ribosomal structure and function. We have used the phosphorothioate approach to probe iodine accessibility of 23 S rRNA domain I phosphates inside functional 50 S ribosomal subunits. Five percent of R(P) isomers of nucleoside phosphorothioate were incorporated into Thermus aquaticus 23 S rRNA during in vitro transcription. Ribosomal large subunits were reconstituted from 23 S rRNA and 5 S rRNA transcripts and ribosomal large subunit proteins. The resulting particles sedimented as 50 S and were active in a peptide bond formation assay. Iodine-induced cleavage sites were determined for domain I of 23 S rRNA by reverse transcriptase-directed primer extension. Specific signals were detected at 360 positions, 80 of which were protected in reconstituted 50 S subunits. We argue that most observed protections are caused by shielding of phosphates by ribosomal proteins. The phosphorothioate approach can be extended to analyze dynamic structural changes during translation and the functional roles of individual chemical groups in rRNA.