Ribosomal Protein L1 Research Articles

Molecular characterization of Cry1F resistance in fall armyworm, Spodoptera frugiperda from Brazil

Fall armyworm, Spodoptera frugiperda (J.E. Smith) is a major lepidopteran pest of maize in Brazil and its control particularly relies on the use of genetically engineered crops expressing Bacillus thuringiensis (Bt) toxins such as Cry1F. However, control failures compromising the efficacy of this technology have been reported in many regions in Brazil, but the mechanism of Cry1F resistance in Brazilian fall armyworm populations remained elusive. Here we investigated the molecular mechanism of Cry1F resistance in two field-collected strains of S. frugiperda from Brazil exhibiting high levels of Cry1F resistance. We first rigorously evaluated several candidate reference genes for normalization of gene expression data across strains, larval instars and gut tissues, and identified ribosomal proteins L10, L17 and RPS3A to be most suitable. We then investigated the expression pattern of ten potential Bt toxin receptors/enzymes in both neonates and 2nd instar gut tissue of Cry1F resistant fall armyworm strains compared to a susceptible strain. Next we sequenced the ATP-dependent Binding Cassette subfamily C2 gene (ABCC2) and identified three mutated sites present in ABCC2 of both Cry1F resistant strains: two of them, a GY deletion (positions 788–789) and a P799 K/R amino acid substitution, located in a conserved region of ABCC2 extracellular loop 4 (EC4) and another amino acid substitution, G1088D, but in a less conserved region. We further characterized the role of the novel mutations present in EC4 by functionally expressing both wild type and mutated ABCC2 transporters in insect cell lines, and confirmed a critical role of both sites for Cry1F binding by cell viability assays. Finally, we assessed the frequency of the mutant alleles by pooled population sequencing and pyrosequencing in 40 fall armyworm populations collected from maize fields in different regions in Brazil. We found that the GY deletion being present at high frequency. However we also observed many rare alleles which disrupt residues between sites 783–799, and their diversity and abundance in field collected populations lends further support to the importance of the EC4 domain for Cry1F toxicity.

Slowing ribosome velocity restores folding and function of mutant CFTR.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), with approximately 90% of patients harboring at least one copy of the disease-associated variant F508del. We utilized a yeast phenomic system to identify genetic modifiers of F508del-CFTR biogenesis, from which ribosomal protein L12 (RPL12/uL11) emerged as a molecular target. In the present study, we investigated mechanism(s) by which suppression of RPL12 rescues F508del protein synthesis and activity. Using ribosome profiling, we found that rates of translation initiation and elongation were markedly slowed by RPL12 silencing. However, proteolytic stability and patch-clamp assays revealed RPL12 depletion significantly increased F508del-CFTR steady-state expression, interdomain assembly, and baseline open-channel probability. We next evaluated whether Rpl12-corrected F508del-CFTR could be further enhanced with concomitant pharmacologic repair (e.g., using clinically approved modulators lumacaftor and tezacaftor) and demonstrated additivity of these treatments. Rpl12 knockdown also partially restored maturation of specific CFTR variants in addition to F508del, and WT Cftr biogenesis was enhanced in the pancreas, colon, and ileum of Rpl12 haplosufficient mice. Modulation of ribosome velocity therefore represents a robust method for understanding both CF pathogenesis and therapeutic response.

Light-inducible expression of translation factor EF-Tu during acclimation to strong light enhances the repair of photosystem II

In photosynthetic organisms, the repair of photosystem II (PSII) is enhanced after acclimation to strong light, with the resultant mitigation of photoinhibition of PSII. We previously reported that oxidation of translation elongation factor EF-Tu, which delivers aminoacyl-tRNA to the ribosome, depresses the repair of PSII in the cyanobacterium Synechocystis sp. PCC 6803. In the present study, we investigated the role of EF-Tu in the repair of PSII after acclimation of Synechocystis to strong light. In cells that had been grown under strong light, both the repair of PSII and the synthesis of proteins de novo were enhanced under strong light, with the resultant mitigation of photoinhibition of PSII. Moreover, levels of EF-Tu were elevated, whereas levels of other components of the translation machinery, such as translation factor EF-G and ribosomal proteins L2 and S12, did not change significantly. The expression of the gene for EF-Tu was induced by light, as monitored at the transcriptional level. Elevation of the level of EF-Tu was strongly correlated with the subsequent enhancement of PSII repair in cells that had been grown under light at various intensities. Furthermore, overexpression of EF-Tu in Synechocystis enhanced protein synthesis and PSII repair under strong light, even after cell culture under nonacclimating conditions. These observations suggest that elevation of the level of EF-Tu might be a critical factor in enhancing the capacity for repair of PSII that develops during acclimation to strong light.

Oncogenic MYC Induces the Impaired Ribosome Biogenesis Checkpoint and Stabilizes p53 Independent of Increased Ribosome Content.

The role of MYC in regulating p53 stability as a function of increased ribosome biogenesis is controversial. On the one hand, it was suggested that MYC drives the overexpression of ribosomal proteins (RP)L5 and RPL11, which bind and inhibit HDM2, stabilizing p53. On the other, it has been proposed that increased ribosome biogenesis leads the consumption of RPL5/RPL11 into nascent ribosomes, reducing p53 levels and enhancing tumorigenesis. Here, we show that the components that make up the recently described impaired ribosome biogenesis checkpoint (IRBC) complex, RPL5, RPL11, and 5S rRNA, are reduced following MYC silencing. This leads to a rapid reduction in p53 protein half-life in an HDM2-dependent manner. In contrast, MYC induction leads to increased ribosome biogenesis and p53 protein stabilization. Unexpectedly, there is no change in free RPL5/RPL11 levels, but there is a striking increase in IRBC complex bound to HDM2. Our data support a cell-intrinsic tumor-suppressor response to MYC expression, which is presently being exploited to treat cancer. SIGNIFICANCE: Oncogenic MYC induces the impaired ribosome biogenesis checkpoint, which could be potentially targeted for cancer treatment.

Comparative genomic analyses of two segregating mutants reveal seven genes likely involved in resistance to Fusarium equiseti in soybean via whole genome re-sequencing.

The candidate genes involved in resistance to Fusarium equiseti in soybean PI 437654 were identified through comparative genomic analyses of mutants via whole genome re-sequencing. The fungus Fusarium infects each stage of the growth and development of soybean and causes soybean (Glycine max (L.)) seed and root rot and seedling damping-off and wilt with a large quantity of annual yield loss worldwide. It is very important to identify the resistant genes in soybean to prevent and control this pathogen. One Fusarium equiseti isolate was previously identified to be incompatible with 'PI 437654' but compatible with a Chinese soybean cultivar 'Zhonghuang 13'. In this study, with the infection of this isolate on the seedling roots of developed PI 437654 mutants, 6 mutants were identified from 500 mutants to significantly alter their phenotypes to F. equiseti compared to wild-type PI 437654. Then, two identified segregating mutants were selected to directly perform whole genome re-sequencing. Finally, through comparative genomic analyses 7 genes including one cluster of 4 nucleotide binding site-leucine-rich repeat genes on one genomic region of chromosome 7, a 60S ribosomal protein L12 gene and 2 uncharacterized genes were identified to be likely involved in the resistance to F. equiseti. These genes will facilitate the breeding of resistant germplasm resources and the identification of resistance of soybean to Fusarium spp.

A DEAD-box protein regulates ribosome assembly through control of ribosomal protein synthesis.

DEAD-box proteins (DBPs) comprise a large family of proteins that most commonly have been identified as regulators of ribosome assembly. The Escherichia coli DBP, SrmB, represents a model bacterial DBP whose absence impairs formation of the large ribosomal subunit (LSU). To define the basis for SrmB function, suppressors of the ribosomal defect of ΔsrmB strains were isolated. The major class of suppressors was found to map to the 5′ untranslated region (UTR) of the rplM-rpsI operon, which encodes the ribosomal proteins (r-proteins) L13 and S9. An analysis of protein abundance indicated that both r-proteins are under-produced in the ΔsrmB strain, but are increased in these suppressors, implicating r-protein underproduction as the molecular basis for the observed ribosomal defects. Reduced r-protein synthesis was determined to be caused by intrinsic transcription termination within the rplM 5′ UTR that is abrogated by SrmB. These results reveal a specific mechanism for DBP regulation of ribosomal assembly, indirectly mediated through its effects on r-protein expression.

Interactions of protein translocation machinery components of Streptococcus mutans

BackgroundPivotal virulence properties of the cariogenic bacterium Streptococcus mutans depend on secreted and integral membrane proteins. Bacterial protein trafficking involves the signal recognition particle (SRP) pathway components Ffh, scRNA and FtsY, the SecYEG translocon, the SecA motor protein, and membrane‐localized YidC chaperone/insertases. Unlike Escherichia coli, S. mutans survives loss of the SRP pathway. In addition, S. mutans has two yidC paralogs. The phenotype of a DyidC2 mutant largely parallels that of Dffh, while that of DyidC1 is far less severe. Also in S. mutans, YlxM acts as an accessory factor of the SRP that modulates GTP hydrolysis activities of Ffh and FtsY. The goal of this study is to identify interactions among S. mutans transport machinery components to better understand the respective functions of YidC1 and YidC2 alone and in concert with the SRP, ribosome, and/or Sec translocon that can affect the destiny of translocated proteins.MethodsA chemical cross‐linking approach was employed, whereby whole cell lysates (wcl) were treated with formaldehyde followed by Western blotting using anti‐Ffh, FtsY, YidC1 and YidC2 antibodies and mass spectrometry (MS) analysis of corresponding gel‐shifted bands. Wcl from WT and DyidC2 strains were also reacted with a‐YidC2 antibodies coupled to magnetic Dynabeads™ and co‐captured proteins identified by MS. In addition, biolayer interferometry (BLI) was used to evaluate specific interactions of the C‐terminal tails of YidC1 and YidC2, engineered as glutathione‐S‐transferase fusion proteins (GST‐C1 and GST‐C2), with the SRP components Ffh and YlxM. The same GST‐constructs were also subjected to 2D Difference Gel Electrophoresis (DIGE), in which each was reacted with wcl, captured with glutathione affinity resin, and labelled individually with fluorescent dyes. Spots unique to the GST‐C1 and/or GST‐C2 samples were analysed by MS.Results and ConclusionBand‐shift patterns detected with anti‐Ffh, FtsY and YidC2 antisera were similar to one another and distinct from that of anti‐YidC1. Cross‐linked proteins included elongation factor Tu, DNA‐directed RNA polymerase subunit b, and HSP 70. Immunocaptured proteins from anti‐YidC2 Dynabeads™ included two small, transmembrane proteins, LemA and SMU_1719c, which may represent translocation substrates. Both GST‐C1 and GST‐C2 interacted with Ffh by BLI with binding influenced by YlxM. Over one hundred spots were identified by DIGE. Among differentially identified proteins in the GST‐C1 sample were DnaK, RNA polymerase subunit d, and cell division protein FtsH. Ribosomal proteins S7 and L13 were prevalent in spots of the GST‐C2 sample, whereas ribosomal protein L2, DNA polymerase III g/t subunit, and the Cas5 homolog were all more abundant in spots from GST‐C1 and GST‐C2 samples compared to GST. This information suggests a coupled cellular machinery that integrates transcription and translation with protein transport and folding.Support or Funding InformationNIH R01DE008007This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of mitochondrial DNA transcription by protein post‐translational modifications

Mammalian cells contain genetic information in two compartments, the nucleus and the mitochondria. Mitochondrial DNA (mtDNA) encodes thirteen protein subunits required for oxidative phosphorylation. The remaining mitochondrial proteome, including additional oxidative phosphorylation subunits, and the proteins necessary for mtDNA replication, expression, and stability, is encoded by the nuclear genome. Therefore, to respond to metabolic changes, mitochondrial gene expression must be coordinated with nuclear gene expression. While the control of nuclear gene expression is widely studied, there is a need to understand the regulation of mtDNA transcription. Our central hypothesis is that reversible post‐translational modification of the mtDNA transcriptional machinery is a means to tune mtDNA transcription in response to changes in cellular metabolism. Here, using in vitro biochemical and cell biology approaches, we describe the effects of lysine acetylation and serine/threonine/tyrosine phosphorylation on the mtDNA binding ability and transcriptional activity of three proteins, mitochondrial transcription factor B2 (TFB2M), the mitochondrial polymerase (POLRMT), and mitochondrial ribosomal protein L12 (MRPL12). Our in vitro biochemical results indicate that phosphorylation of TFB2M may impede mtDNA binding and subsequently alter transcription initiation. Modification of MRPL12 may alter the ability of MRPL12 to bind and stabilize POLRMT. We also report a temporal response in the transcript level of mitochondrial and nuclear genes in response to cell growth conditions used to drive a dependence on mitochondrial metabolism. Importantly, mtDNA content is unchanged in these conditions, providing evidence for a regulated transcriptional response of the mitochondrial genome. It is expected that these findings will lead to a more complete understanding of the dynamics and coordination of mitochondrial and nuclear gene transcription, as well as adding to the understanding of the importance of protein post‐translational modifications in regulating aspects of mitochondrial function.Support or Funding InformationThis material is based upon work supported by the National Science Foundation Division of Molecular and Cellular Biosciences under Grant No. 1814845.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Post‐Translational Modifications of Mitochondrial Proteins MRPL12 and POLRMT as a Potential Regulatory Mechanism for Mitochondrial DNA Transcription

Mitochondrial DNA (mtDNA) encodes 13 of the protein subunits of the oxidative phosphorylation pathway, the pathway that produces the majority of ATP in eukaryotic cells. Mitochondrial dysfunction is associated with neurodegenerative disorders, cancer, inherited mitochondrial diseases, and other pathologies, making mitochondrial biochemistry an important area of investigation. However, there lacks an understanding of key elements of this biochemistry, including the mechanisms that regulate mtDNA transcription. mtDNA is known to be complexed to various proteins in structures known as nucleoids. Some nucleoid proteins are key members of the mtDNA transcription machinery; these include the mitochondrial RNA polymerase (POLRMT) and mitochondrial ribosomal protein L12 (MRPL12), which maintains POLRMT stability and promotes mtDNA transcription. We hypothesize that reversible post‐translational modifications (PTMs), such as phosphorylation of threonine, serine, or tyrosine residues or acetylation of lysine residues within these and other nucleoid proteins, may play a role in regulating mtDNA transcription. The objective of our study is to determine the effects of MRPL12 and POLRMT post‐translational modifications on the activities of these proteins in the context of mtDNA transcription in order to assess the roles of such modifications in regulating this process. To test this, we performed mutagenesis PCR on MRPL12 and POLRMT genes within bacterial expression vectors to replace the sequence encoding an amino acid at a PTM site with a sequence encoding a modified or unmodified amino acid mimic (T, S, Y → E/A, phosphorylated/dephosphorylated mimic; K → Q/R, acetylated/deacetylated mimic). We optimized bacterial expression and protein purification conditions and purified wild‐type and mutant MRPL12 proteins, as well as wild‐type POLRMT. We performed preliminary in vitro transcription assays to assess POLRMT functionality and found that our protein successfully transcribed short mtDNA templates. Going forward, we will perform protein‐protein binding assays to determine the effects of MRPL12 modifications on MRPL12's ability to bind POLRMT, as well as POLRMT mutagenesis and more in vitro transcription assays to measure the effects of MRPL12 and POLRMT modifications on mtDNA transcription.Support or Funding InformationThis research is based upon work supported by the National Science Foundation Division of Molecular and Cellular Biosciences under Grant No. 1814845, with additional funding from the Arnold and Mabel Beckman Foundation Beckman Scholars Award and the ASBMB Undergraduate Research award.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genome-Wide Identification and Evaluation of New Reference Genes for Gene Expression Analysis Under Temperature and Salinity Stresses in Ciona savignyi.

Rapid adaptation/accommodation to changing environments largely contributes to maximal survival of invaders during biological invasions, usually leading to success in crossing multiple barriers and finally in varied environments in recipient habitats. Gene expression is one of the most important and rapid ways during responses to environmental stresses. Selection of proper reference genes is the crucial prerequisite for gene expression analysis using the common approach, real-time quantitative PCR (RT-qPCR). Here we identified eight candidate novel reference genes from the RNA-Seq data in an invasive model ascidian Ciona savignyi under temperature and salinity stresses. Subsequently, the expression stability of these eight novel reference genes, as well as other six traditionally used reference genes, was evaluated using RT-qPCR and comprehensive tool RefFinder. Under the temperature stress, two traditional reference genes, ribosomal proteins S15 and L17 (RPS15, RPL17), and one novel gene Ras homolog A (RhoA), were recommended as the top three stable genes, which can be used to normalize target genes with a high and moderate expression level, respectively. Under the salinity stress, transmembrane 9 superfamily member (TMN), MOB kinase activator 1A-like gene (MOB) and ubiquitin-conjugating enzyme (UBQ2) were suggested as the top three stable genes. On the other hand, several commonly used reference genes such as α-tubulin (TubA), β-tubulin (TubB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) showed unstable expressions, thus these genes should not be used as internal controls for gene expression analysis. We also tested the expression level of an important stress response gene, large proline-rich protein bag6-like gene (BAG) using different reference genes. As expected, we observed different results and conclusions when using different normalization methods, thus suggesting the importance of selection of proper reference genes and associated normalization methods. Our results provide a valuable reference gene resource for the normalization of gene expression in the study of environmental adaptation/accommodation during biological invasions using C. savignyi as a model.

Ribosomal Proteins Regulate MHC Class I Peptide Generation for Immunosurveillance

The MHC class I antigen presentation system enables Tcell immunosurveillance of cancers and viruses. A substantial fraction of the immunopeptidome derives from rapidly degraded nascent polypeptides (DRiPs). By knocking down each of the 80 ribosomal proteins, we identified proteins that modulate peptide generation without altering source protein expression. We show that 60S ribosomal proteins L6 (RPL6) and RPL28, which are adjacent on theribosome, play opposite roles in generating an influenza A virus-encoded peptide. Depleting RPL6 decreases ubiquitin-dependent peptide presentation, whereas depleting RPL28 increases ubiquitin-dependent and -independent peptide presentation. 40S ribosomal protein S28 (RPS28) knockdown increases total peptide supply in uninfected cells by increasing DRiP synthesis from non-canonical translation of "untranslated" regions and non-AUG start codons and sensitizes tumor cells for Tcell targeting. Our findings raise the possibility of modulating immunosurveillance by pharmaceutical targeting ribosomes.

Antimicrobial susceptibility and genotyping of Mycoplasma pneumoniae isolates in Beijing, China, from 2014 to 2016

BackgroundThe presence of macrolide-resistant Myocplasma pneumoniae has been frequently reported in recent years, especially in China. In this study, we investigated the antimicrobial susceptibility and genotype against M. pneumoniae isolates from 2014 to 2016, Beijing.MethodsWe investigated the activities of four antibiotics against 81 M. pneumoniae isolates in vitro. All isolates were amplification of domains II and V of the 23S rRNA gene and the L4 and L22 ribosomal protein fragments. All isolates were genotyped with duplex real-time PCR, MLVA and VNTR detection in p1 gene.ResultsThe macrolide resistance rate was 65.4% (53/81). Each of the macrolide-resistant M. pneumoniae isolates was resistant to erythromycin (Minimum Inhibitory Concentration, MIC, ≥256 μg/ml) and azithromycin (MIC, 2–64 μg/ml), but susceptible to tetracycline and levofloxacin in vitro. Fifty two macrolide-resistant isolates harbored the A2063G mutation, and only 1 macrolide-resistant isolates harbored the A2064G mutation in domain V of the 23S ribosomal RNA gene. The C162A, A430G, and T279C mutations in the L4 and L22 ribosomal protein genes were not responsible for macrolide resistance, but they were related to the particular genotype of M. pneumoniae. 95.7% of type 1 isolates (45/47) were macrolide-resistance, and 23.5% of the type 2 isolates (8/34) were macrolide-resistance. Type 2 M. pneumoniae macrolide-resistance rate was 50.6% higher than that of the previous reports in China. The eight macrolide-resistant type 2 M. pneumoniae isolates were belong to 3/5/6/2 and 3/5/7/2 MLVA genotypes.ConclusionTo our knowledge, this phenomenon likely resulted from a combination of genotype shifting from type1 to type 2 and antibiotic selection pressure in M. pneumoniae in China in recent years. The increase of resistance in type 2 is not due to the spread of same clone. However, the relationship between genotype shifts and macrolide resistance in M. pneumoniae needs to be further verified with more extensive surveillance data.

Identification of key genes and pathways involved in microsatellite instability in colorectal cancer.

Microsatellite instability (MSI) has emerged as one of the key biological features of colorectal cancer (CRC). However, controversies remain regarding the association between the MSI status and clinicopathological characteristics of CRC. Therefore, it is crucial to identify potential key genes and pathways associated with MSI in CRC. In the present study, the GSE25071 gene expression profile was retrieved, with thirty-eight cases of microsatellite stable (MSS), five of MSI-High (MSI-H) and three of MSI-Low (MSI-L) CRC patients. The differentially expressed genes (DEGs) were analyzed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes pathway enrichment, gene set enrichment analysis (GSEA) and gene co-expression network analysis. Weighted gene correlation network analysis (WGCNA) was used for the gene modules and correlation of clinical traits. A total of forty-nine DEGs were identified between MSI-H and MSS, including six upregulated and forty-three downregulated DEGs. Only the DEGs of MSI-H and MSS were subjected to subsequent analysis (limited number of DEGs of MSI-L and MSS, MSI-H and MSI-L). RNA metabolic process, endoplasmic reticulum and chemokine receptor binding were the top ranked terms in GO enrichment. The hub genes of co-expression network of DEGs included zinc finger protein (ZNF) 813, ZNF426, ZNF611, ZNF320 and ZNF573. The GSEA of MSI-H and MSS indicated that the mammalian target of rapamycin complex 1 signaling was significantly enriched with a nominal P-value of 0.038 and normalized enrichment score of 0.446. The WGCNA results showed that the pink module was the top in correlation with MSI status (R2=0.5, P=0.0004). The genes in the pink module were significantly enriched in proteins targeting to endoplasmic reticulum, cytosolic part, structural constituent of ribosome and ribosome pathway. The hub genes identified in the pink module were ribosomal protein L12 (RPL12), RPS3A, RPS9, RPL27A, RPL7, RPL28, RPL14, RPS17, mitochondrial ribosomal protein L16, and G elongation factor, mitochondrial 2. The present study identified key genes and pathways associated with MSI, providing insightful mechanisms.

First Report of Prunus domestica as the Host of a Phytoplasma Belonging to Group 16SrI, Subgroup B/L

HomePlant DiseaseVol. 103, No. 1First Report of Prunus domestica as the Host of a Phytoplasma Belonging to Group 16SrI, Subgroup B/L PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Prunus domestica as the Host of a Phytoplasma Belonging to Group 16SrI, Subgroup B/LA. Zwolińska, N. Borodynko-Filas, D. Nowaczyk, and B. Hasiów-JaroszewskaA. Zwolińska†Corresponding author: A. Zwolińska; E-mail: E-mail Address: a.zwolinska@iorpib.poznan.plhttp://orcid.org/0000-0003-2681-2915Search for more papers by this author, N. Borodynko-FilasSearch for more papers by this author, D. NowaczykSearch for more papers by this author, and B. Hasiów-JaroszewskaSearch for more papers by this authorAffiliationsAuthors and Affiliations A. Zwolińska † N. Borodynko-Filas D. Nowaczyk B. Hasiów-Jaroszewska , Institute of Plant Protection – National Research Institute, Virology and Bacteriology Department, Poznań, Poland. Published Online:31 Oct 2018https://doi.org/10.1094/PDIS-06-18-0963-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In July 2016 and 2017, diseased plum trees (Prunus domestica L.) were observed in the orchard localized in the Wielkopolska region of Poland. The symptoms of witches’ brooms with internode shortening, axillary bud growth, and reduced leaf size indicated a potential phytoplasma infection. Samples of shortened shoots were collected from five symptomatic and five asymptomatic plants, respectively. DNA samples were extracted from 2 g of leaf tissues using a modified cetyltrimethylammonium bromide procedure (Maixner et al. 1995) and subsequently used as templates in nested polymerase chain reaction (PCR) for the amplification of genes encoding 16S rRNA; ribosomal proteins (rp) S19, L22, and S3 and elongation factor Tu (tuf) with primers P1/P7 followed by R16F2n/R16R2; rpF1/rpR1 followed by rp(I)F1A/rp(I)R1A and fTuf1/rTuf1 followed by fTufAY/rTufAY, respectively (Duduk et al. 2013; Martini et al. 2007; Schneider et al. 1997). Amplicons of correct sizes (1.2, 1.2, and 0.9 kb) were obtained from all the diseased plants tested (samples Plum1 to Plum5), respectively. No PCR products were obtained from the asymptomatic samples. All PCR products were ligated into pGEM-T Easy Vector Systems (Promega), and plasmid DNA was sequenced by an external company (Genomed, Warsaw, Poland). The 16S rDNA, tuf, and rp genes coding sequences from Plum1 to Plum5 were identical, and therefore one representative sequence of each region was deposited in GenBank under accession numbers MH061193, MH061368, and MH061366, respectively. The R16F2n/R16R2 (MH061193) primed fragment was subjected to in silico restriction digestion using iPhyClassifier, the online tool for phytoplasma classification (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi). The collective restriction fragment length polymorphism patterns indicated that diseased plants were infected by ‘Candidatus Phytoplasma asteris’ of subgroup 16SrI-L. Next, the 385-bp barcode fragments of the EF-Tu gene from representatives of different phytoplasma groups were retrieved from the GenBank database. The phylogenetic analysis was performed based on those sequences and the tuf sequence obtained in this study using the neighbor joining algorithm of MEGA 7 software (https://www.megasoftware.net/). The 1.2-kbp DNA segment containing rp genes was also analyzed. In the phylogenetic trees, the Polish isolate of plum witches’ broom phytoplasma clustered together with ‘Ca. P. asteris’ representatives of ribosomal subgroup 16SrI-B and 16SrI-B/L. Based on all the performed analyses, the plum witches’ broom phytoplasma (PlumWB) was classified as a member of group 16SrI, subgroup B/L, which consists of two heterogeneous 16Sr RNA genes (Jomantiene et al. 2010). To our knowledge, this is the first report of plums infected with ‘Ca. P. asteris’ representing subgroup I-B/L in Poland, suggesting that fruit trees can be perennial reservoirs of the phytoplasma, which has great significance in the pathogen’s epidemiology.

In silico study on the apicoplast L4 ribosomal protein and three domains from 23S rRNA from „Plasmodium falciparum“ and comparison with the existing cocrystal structures

We performed preliminary computational studies on the construction of a segment of ribosomal protein L4 from the apicoplast ribosome of Plasmodium falciparum. With a Z-score of -3.404, it is arguably the best constructed model of this drug target so far. Three domains from 23S rRNA were made from scratch using the software RNA2D3D: domain II, IV and V. They were not validated but show reasonable similarity with bacterial 23S rRNA. This model has technical limitations but is a starting point; refined models are expected to find use in antimalarial drug design.

Novel mechanism responsible for high-level macrolide resistance in Moraxella catarrhalis.

BackgroundHigh-level macrolide-resistant Moraxella catarrhalis strains have been isolated; however, the underlying mechanism has not been well elucidated. We investigated the role of mutations in the 23S rRNA gene and the L4 and L22 ribosomal proteins using spontaneous erythromycin-resistant mutants and transformants.Materials and methodsThe erythromycin-susceptible M. catarrhalis ATCC25238 and clinical isolate Mc19 were used as parental strains. To obtain spontaneous erythromycin-resistant mutants, in vitro stepwise selection was performed using brain–heart infusion agar plates containing various concentrations of erythromycin. The role of the mutations identified in the spontaneous mutants was validated using transformation experiments.ResultsWe obtained two spontaneous mutants with high-level resistance to erythromycin, S25-32-af10 and S19-256-af10, from ATCC25238 and Mc19, respectively. S25-32-af10 exhibited mutations of Q61R in L4 and Insertion98SRADRIS in L22. S19-256-af10 exhibited three C2611T-mutated alleles in the 23S rRNA gene and G65A in L4. Transformants with single mutations identified in S25-32-af10 or S19-256-af10 showed higher erythromycin and azithromycin minimum inhibitory concentrations (MICs) than those of each parental strain. However, transformants with multiple mutations identified in S25-32-af10 or S19-256-af10 showed macrolide MICs similar to those of each parental strain.ConclusionOur results provide the first evidence suggesting that Q61R in L4 and Insertion98SRADRIS in L22 are involved in the synergistic acquisition of high-level resistance to both 14- and 15-member macrolides, and that C2611T in the 23S rRNA gene and G65A in L4 also synergistically contribute toward conferring high-level 14-member macrolide resistance to M. catarrhalis.

Molecular Epidemiology and Mechanisms of 43 Low-Level Linezolid-Resistant Enterococcus faecalis Strains in Chongqing, China

BackgroundEnterococcus faecalis strains with low-level resistance to linezolid (an oxazolidinone antibiotic) have become common. No large-scale study has examined the underlying mechanisms in linezolid-resistant E. faecalis (LRE) strains. We investigated these mechanisms and molecular characteristics in Chongqing, China.MethodsA total of 1,120 non-duplicated E. faecalis strains collected from August 2014 to June 2017 underwent drug susceptibility testing. LRE strains were screened for optrA, cfr, and mutations in the 23S rRNA and ribosomal proteins L3 and L4 by PCR amplification and sequencing. Multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used for epidemiological analysis.ResultsAll 43 low-level LRE strains (minimum inhibitory concentration: 8–16 mg/L) harbored optrA; cfr and 23S rRNA mutations were not detected. Novel mutations in the ribosomal proteins L3 and L4—one deletion (Q103del) and four substitutions (S113L, T35A, I98V, and N79D)—were identified. Novel amino acid substitutions at positions E60K, G197D, and T285P of the OptrA protein were observed. MLST revealed 20 types of LRE strains; the most common type was ST16 (32.6%). PFGE showed 14 strains of ST16 with unique banding patterns. Eight novel sequence types (ST823 to ST830) and one allele (gki95) were identified for the first time in China.ConclusionsoptrA plays an important role in linezolid resistance and may serve as a marker for resistance screening. Since the L3 and L4 mutations did not simultaneously occur in the same strain, they play a negligible role in linezolid resistance. Epidemiological investigation suggested that the LRE cases were sporadic.

Detection of the Novel optrA Gene Among Linezolid-Resistant Enterococci in Barcelona, Spain.

The purpose of this study was to describe the presence of the novel optrA gene among clinical isolates of enterococci in a Spanish teaching hospital (May 2016-April 2017). optrA and cfr genes were screened by PCR in all isolates showing linezolid minimal inhibitory concentration (MIC) ≥4 mg/L. The genetic relatedness of the isolates, the presence of resistance and virulence genes, and the genetic environment of optrA were assessed by whole-genome sequencing (WGS). Six of 1,640 enterococci had linezolid MIC ≥4 mg/L. Among them, the optrA gene was detected in five Enterococcus faecalis isolated from unrelated patients. Although none of them had received linezolid or chloramphenicol, all had antecedents of recent quinolone consumption. WGS analysis revealed the existence of two different genotypes: ST585 and ST474. cfr was not detected in any of the isolates. No mutations were detected among the 23S ribosomal RNA and the ribosomal proteins L3, L4, and L22. Both genotypes also carried genes related to aminoglycoside, lincosamide, macrolide, phenicol, and tetracycline resistance. Detection of optrA in a setting with low linezolid consumption and among patients without antecedents of oxazolidinone therapy is of concern.

Identification of anti-Gram-negative bacteria agents targeting the interaction between ribosomal proteins L12 and L10

Gram-negative bacteria have become the main pathogens and cause serious clinical problems with increased morbidity and mortality. However, the slow discovery of new antimicrobial agents is unable to meet the need for the treatment of bacterial infections caused by drug-resistant strains. The interaction of L12 and L10 is essential for ribosomal function and protein synthesis. In this study, a yeast two-hybrid system was established to successfully detect the interaction between L12 and L10 proteins from gram-negative bacteria Escherichia coli, which allows us to screen compounds that specifically disrupt this interaction. With this system, we identified two compounds IMB-84 and IMB-87 that block L12−L10 interaction and show bactericidal activity against E. coli. We used glutathione-S-transferase (GST) pull-down and surface plasmon resonance (SPR) assays to demonstrate that these compounds disrupt L12−L10 interaction in vitro and the target of compounds was further confirmed by the overexpression of target proteins. Moreover, protein synthesis and elongation factor G-dependent GTPase activities are inhibited by two compounds. Therefore, we have identified two antibacterial agents that disrupt L12−L10 interaction by using yeast two-hybrid system.

Fitness advantages conferred by the L20-interacting RNAcis-regulator of ribosomal protein synthesis inBacillus subtilis

In many bacteria, ribosomal proteins autogenously repress their own expression by interacting with RNA structures typically located in the 5′-UTRs of their mRNA transcripts. This regulation is necessary to maintain a balance between ribosomal proteins and rRNA to ensure proper ribosome production. Despite advances in noncoding RNA discovery and validation of RNA-protein regulatory interactions, the selective pressures that govern the formation and maintenance of such RNA cis-regulators in the context of an organism remain largely undetermined. To examine the impact disruptions to this regulation have on bacterial fitness, we introduced point mutations that abolish ribosomal protein binding and regulation into the RNA structure that controls expression of ribosomal proteins L20 and L35 within the Bacillus subtilis genome. Our studies indicate that removing this regulation results in reduced log phase growth, improper rRNA maturation, and the accumulation of a kinetically trapped or misassembled ribosomal particle at low temperatures, suggesting defects in ribosome synthesis. Such work emphasizes the important role regulatory RNAs play in the stoichiometric production of ribosomal components for proper ribosome composition and overall organism viability and reinforces the potential of targeting ribosomal protein production and ribosome assembly with novel antimicrobials.