Ribosomal Protein L1 Research Articles

Single mutations introduced in the essential ribosomal proteins L3 and S10 cause a sporulation defect in Bacillus subtilis

We introduced single mutations into the rplC and rpsJ genes, which encode the essential ribosomal proteins L3 (RplC) and S10 (RpsJ), respectively, and are located in the S10 gene cluster of the gram-positive, endospore-forming bacterium Bacillus subtilis, and examined whether these mutations affected their growth rate, sporulation, competence development and 70S ribosome formation. Mutant cells harboring the G52D mutation in the L3 ribosomal protein, which is located at the peptidyl transferase center of 50S, accumulated 30S subunit at 45°C, probably due to a defect in 50S formation, and exhibited a reduction in the sporulation frequency at high temperature. On the other hand, mutant cells harboring the H56R mutation in the S10 protein, which is located near the aminoacyl-tRNA site of 30S, showed severe growth defect and deficiency in spore formation, and also exhibited significant delay in competence development.

Microbiological characterization of Streptococcus pneumoniae and non-typeable Haemophilus influenzae isolates as primary causes of acute otitis media in Bulgarian children before the introduction of conjugate vaccines

BackgroundPneumococcal and Haemophilus influenzae type b (Hib) vaccines were introduced in our national immunisation program in April 2010. The aims of this retrospective, laboratory-based study were to determine the serotypes and antibiotic resistance of Streptococcus pneumoniae and H. influenzae isolates from middle ear fluid (MEF) collected before the introduction of immunization.MethodsS. pneumoniae (n = 128) and H. influenzae (n = 40) strains isolated from MEF of children with AOM between 1994 and 2011 were studied. MICs were determined by a microdilution assay. Serotyping of S. pneumoniae was done by Quellung method and PCR capsular typing was used for H. influenzae. Macrolide resistance genes were detected by PCR for erythromycin resistant S. pneumoniae (ERSP). DNA sequencing of ftsI gene was performed for ampicillin nonsusceptible H. influenzae.ResultsThe most common serotypes found among children with pneumococcal AOM were 19 F (20.3%), 6B (15.6%), and 19A (10.9%). The potential coverage rates by the PCV7, PCV10 and PCV13 of children aged < 5 years were 63.6%, 66.4% and 85.5%, respectively. Reduced susceptibility to oral penicillin was seen in 68.1%; resistance to erythromycin was 46.9%. We found erm(B) gene in 56.7% of the ERSP, mef(E) gene in 25%; 15% harbored both genes erm(B) + mef(E) and 3.3% had mutations of L4 ribosomal protein. Of the 40 H. influenzae isolates 97.5% were nontypeable. Nonsusceptibility to ampicillin occurred in 25%. Ampicillin resistance groups were: β-lactamase-positive ampicillin resistant (BLPAR) strains (10%), β-lactamase-negative ampicillin resistant (BLNAR) strains (12.5%) and β-lactamase-positive amoxicillin-clavulanate resistant (BLPACR) strains (2.5%). Among BLNAR and BLPACR most of the isolates (5/6) belonged to group II, defined by the Asn526Lys substitution.ConclusionsThe levels of antibiotic resistance among S. pneumoniae and H. influenzae causing severe AOM in children are high in our settings. The existence of multidrug-resistant S. pneumoniae serotype 19A is of particular concern. The rate of BLNAR and BLPACR strains among H. influenzae isolates was 15%.

Mutational and Transcriptomic Changes Involved in the Development of Macrolide Resistance in Campylobacter jejuni

Macrolide antibiotics are important for clinical treatment of infections caused by Campylobacter jejuni. Development of resistance to this class of antibiotics in Campylobacter is a complex process, and the dynamic molecular changes involved in this process remain poorly defined. Multiple lineages of macrolide-resistant mutants were selected by stepwise exposure of C. jejuni to escalating doses of erythromycin or tylosin. Mutations in target genes were determined by DNA sequencing, and the dynamic changes in the expression of antibiotic efflux transporters and the transcriptome of C. jejuni were examined by real-time reverse transcription-PCR, immunoblotting, and DNA microarray analysis. Multiple types of mutations in ribosomal proteins L4 and L22 occurred early during stepwise selection. On the contrary, the mutations in the 23S rRNA gene, mediating high resistance to macrolides, were observed only in the late-stage mutants. Upregulation of antibiotic efflux genes was observed in the intermediately resistant mutants, and the magnitude of upregulation declined with the occurrence of mutations in the 23S rRNA gene. DNA microarray analysis revealed the differential expression of 265 genes, most of which occurred in the intermediate mutant, including the upregulation of genes encoding ribosomal proteins and the downregulation of genes involved in energy metabolism and motility. These results indicate (i) that mutations in L4 and L22 along with temporal overexpression of antibiotic efflux genes precede and may facilitate the development of high-level macrolide resistance and (ii) that the development of macrolide resistance affects the pathways important for physiology and metabolism in C. jejuni, providing an explanation for the reduced fitness of macrolide-resistant Campylobacter.

Yeast polypeptide exit tunnel ribosomal proteins L17, L35 and L37 are necessary to recruit late-assembling factors required for 27SB pre-rRNA processing

Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2.

The Genetic Environment of the cfr Gene and the Presence of Other Mechanisms Account for the Very High Linezolid Resistance of Staphylococcus epidermidis Isolate 426-3147L

The clinical Staphylococcus epidermidis isolate 426-3147L exhibits an unusually high resistance to linezolid that exceeds 256 μg/ml. The presence of the cfr gene, encoding the RNA methyltransferase targeting an rRNA nucleotide located in the linezolid binding site, accounts for a significant fraction of resistance. The association of cfr with a multicopy plasmid is one of the factors that contribute to its elevated expression. Mapping of the cfr transcription start sites identified the native cfr promoter. Furthermore, analysis of the cfr transcripts in Staphylococcus epidermidis 426-3147L showed that some of them originate from the upstream plasmid-derived promoters whose activity contributes to efficient cfr transcription. The genetic environment of the cfr gene and its idiosyncratic transcription pattern result in increased activity of Cfr methyltransferase, leading to a high fraction of the ribosomes being methylated at A2503 of the 23S rRNA. Curing of the Staphylococcus epidermidis 426-3147L isolate from the cfr-containing plasmid reduced the linezolid MIC to 64 μg/ml, indicating that other determinants contribute to resistance. Nucleotide sequence analysis revealed the presence of the C2534T mutation in two of the six 23S rRNA gene alleles as well as the presence of mutations in the genes of ribosomal proteins L3 and L4, which were previously implicated in linezolid resistance. Thus, the combination of resistance mechanisms operating through alteration of the drug target site appears to cause an unusually high level of linezolid resistance in the isolate.

Mutual protection of ribosomal proteins L5 and L11 from degradation is essential for p53 activation upon ribosomal biogenesis stress

Impairment of ribosomal biogenesis can activate the p53 protein independently of DNA damage. The ability of ribosomal proteins L5, L11, L23, L26, or S7 to bind Mdm2 and inhibit its ubiquitin ligase activity has been suggested as a critical step in p53 activation under these conditions. Here, we report that L5 and L11 are particularly important for this response. Whereas several other newly synthesized ribosomal proteins are degraded by proteasomes upon inhibition of Pol I activity by actinomycin D, L5 and L11 accumulate in the ribosome-free fraction where they bind to Mdm2. This selective accumulation of free L5 and L11 is due to their mutual protection from proteasomal degradation. Furthermore, the endogenous, newly synthesized L5 and L11 continue to be imported into nucleoli even after nucleolar disruption and colocalize with Mdm2, p53, and promyelocytic leukemia protein. This suggests that the disrupted nucleoli may provide a platform for L5- and L11-dependent p53 activation, implying a role for the nucleolus in p53 activation by ribosomal biogenesis stress. These findings may have important implications with respect to understanding the pathogenesis of diseases caused by impaired ribosome biogenesis.

New partners in regulation of gene expression: the enhancer of Trithorax and Polycomb Corto interacts with methylated ribosomal protein l12 via its chromodomain.

Chromodomains are found in many regulators of chromatin structure, and most of them recognize methylated lysines on histones. Here, we investigate the role of the Drosophila melanogaster protein Corto's chromodomain. The Enhancer of Trithorax and Polycomb Corto is involved in both silencing and activation of gene expression. Over-expression of the Corto chromodomain (CortoCD) in transgenic flies shows that it is a chromatin-targeting module, critical for Corto function. Unexpectedly, mass spectrometry analysis reveals that polypeptides pulled down by CortoCD from nuclear extracts correspond to ribosomal proteins. Furthermore, real-time interaction analyses demonstrate that CortoCD binds with high affinity RPL12 tri-methylated on lysine 3. Corto and RPL12 co-localize with active epigenetic marks on polytene chromosomes, suggesting that both are involved in fine-tuning transcription of genes in open chromatin. RNA–seq based transcriptomes of wing imaginal discs over-expressing either CortoCD or RPL12 reveal that both factors deregulate large sets of common genes, which are enriched in heat-response and ribosomal protein genes, suggesting that they could be implicated in dynamic coordination of ribosome biogenesis. Chromatin immunoprecipitation experiments show that Corto and RPL12 bind hsp70 and are similarly recruited on gene body after heat shock. Hence, Corto and RPL12 could be involved together in regulation of gene transcription. We discuss whether pseudo-ribosomal complexes composed of various ribosomal proteins might participate in regulation of gene expression in connection with chromatin regulators.

Identification of antituberculosis agents that target ribosomal protein interactions using a yeast two-hybrid system

Mycobacterium tuberculosis kills about 2 million people annually and antibiotic resistance is a cause of increased mortality. Therefore, development of new antituberculosis drugs is urgent for the control of widespread tuberculosis infections. For this purpose, we performed an innovative screen to identify new agents that disrupt the function of ribosomes in M. tuberculosis. Two bacterial ribosomal proteins L12 and L10 interact with each other and constitute the stalk of the 50S ribosomal subunit, which recruits initiation and elongation factors (EFs) during translation. Therefore, the L12-L10 interaction should be essential for ribosomal function and protein synthesis. We established a yeast two-hybrid system to identify small molecules that block the interaction between L12 and L10 proteins from M. tuberculosis. Using this system, we identified two compounds T766 and T054 that show strong bactericidal activity against tuberculosis but with low toxicity to mice and other bacterial strains. Moreover, using surface plasmon resonance (SPR) assay, we have demonstrated that these compounds bind specifically to L12 to disrupt L12-L10 interaction. Overproduction of L12 protein, but not L10, lowers the antibacterial activity of T766 and T054, indicating that the ribosome is likely the cellular target. Therefore, our data demonstrate that this yeast two-hybrid system is a useful tool to identify unique antituberculosis agents targeting the ribosomal protein L12-L10 interaction.

Caracterización de cepas de Staphylococcus epidermidis y S. haemolyticus resistentes a meticilina y linezolid en un hospital español

IntroductionLinezolid resistance is mainly due to mutations in the 23S rRNA target. The aim of this study was to characterize linezolid and methicillin resistant Staphylococcus epidermidis (SE-LMR) and S. haemolyticus (SH-LMR) strains detected in a Spanish hospital. MethodsSE-LMR and SH-LMR strains obtained in the period June 2009-August 2011 in a second level hospital were recorded along with the epidemiological characteristics of the patients. These strains were typed, and their resistance, phenotype, genotype and the factors determining their virulence were analysed. ResultsLinezolid resistance was explained by the presence of G2603T mutation (23S rRNA) and aminoacid changes in L3 and L4 ribosomal proteins. The 25 SE-LMR strains belonged to sequence type ST2, presented SCCmec typeIII, and two different PFGE patterns. The two SH-LMR strains showed non-typeable SCCmec. SE-LMR strains harboured the resistance genes aac(6’)-aph(2”), and dfrS1. SH-LMR strains contained these genes and the gene erm(C). No lincomycin resistance mechanism was identified in SE-LMR strains regardless of showing lincomycin resistance and diminished susceptibility to clindamycin. ConclusionsLinezolid resistance is of concern in hospitals, and requires continued vigilance. Several linezolid resistance mechanisms (mutation in 23S RNAr and amino acid changes in L3 and L4) were identified in this study.

Antimicrobial HPA3NT3 peptide analogs: Placement of aromatic rings and positive charges are key determinants for cell selectivity and mechanism of action

In an earlier study, we determined that HP(2‐20) (residues 2‐20 of parental HP derived from the N-terminus of the Helicobacter pylori ribosomal protein L1) and its analog, HPA3NT3, had potent antimicrobial effects. However, HPA3NT3 also showed undesirable cytotoxicity against HaCaT cells. In the present study, we designed peptide analogs including HPA3NT3-F1A (‐F1A), HPA3NT3-F8A (‐F8A), HPA3NT3-F1AF8A (‐F1AF8A), HPA3NT3-A1 (‐A1) and HPA3NT3-A2 (‐A2) in an effort to investigate the effects of amino acid substitutions in reducing their hydrophobicity or increasing their cationicity, and any resulting effects on their selectivity in their interactions with human cells and pathogens, as well as their mechanism of antimicrobial action. With the exception of HPA3NT3-A1, all of these peptides showed potent antimicrobial activity. Moreover, substitution of Ala for Phe at positions 1 and/or 8 of the HPA3NT3 peptides (‐F1A, -F8A and -F1AF8A) dramatically reduced their cytotoxicity. Thus the cytotoxicity of HPA3NT3 appears to be related to its Phe residues (positions 1 and 8), which strongly interact with sphingomyelin in the mammalian cell membrane. HPA3NT3 exerted its bactericidal effects through membrane permeabilization mediated by pore formation. In contrast, fluorescent dye leakage and nucleic acid gel retardation assays showed that ‐A2 acted by penetrating into the cytoplasm, where it bound to nucleic acids and inhibited protein synthesis. Notably, Staphylococcus aureus did not develop resistance to -A2 as it did with rifampin. These results suggest that the -A2 peptide could potentially serve as an effective antibiotic agent against multidrug-resistant bacterial strains.

The effect of zinc limitation on the transcriptome ofPseudomonas protegens Pf‐5

Zinc is an important nutrient but can be lacking in some soil environments, influencing the physiology of soil-dwelling bacteria. Hence, we studied the global effect of zinc limitation on the transcriptome of the rhizosphere biocontrol strain Pseudomonas protegens Pf-5 (formerly Pseudomonas fluorescens). We observed that the expression of the putative zinc uptake regulator (Zur) gene was upregulated, and we mapped putative Zur binding sites in the Pf-5 genome using bioinformatic approaches. In line with the need to regulate intracellular zinc concentrations, an array of potential zinc transporter genes was found to be zinc-regulated. To adapt to low-zinc conditions, a gene cluster encoding non-zinc-requiring paralogues of zinc-dependent proteins was also significantly upregulated. Similarly, transcription of genes encoding non-zinc-requiring paralogues of ribosomal proteins L31 and L36 was increased by zinc limitation. A strong transcriptional downregulation of the putative copper chaperone gene (copZ) was also observed, suggesting interplay between zinc and copper homeostasis. Importantly, zinc also affected biocontrol attributes in Pf-5, most notably reducing the expression of the gene cluster responsible for biosynthesis of the antibiotic 2,4-diacetylphloroglucinol (DAPG) under zinc limitation. This study clearly defines changes to the molecular physiology of Pf-5 that enable it to survive under zinc limitation.

Calcium-Dependent Interaction of Calmodulin with Human 80S Ribosomes and Polyribosomes

Ribosomes are the protein factories of every living cell. The process of protein translation is highly complex and tightly regulated by a large number of diverse RNAs and proteins. Earlier studies indicate that Ca(2+) plays a role in protein translation. Calmodulin (CaM), a ubiquitous Ca(2+)-binding protein, regulates a large number of proteins participating in many signaling pathways. Several 40S and 60S ribosomal proteins have been identified to interact with CaM, and here, we report that CaM binds with high affinity to 80S ribosomes and polyribosomes in a Ca(2+)-dependent manner. No binding is observed in buffer with 6 mM Mg(2+) and 1 mM EGTA that chelates Ca(2+), suggesting high specificity of the CaM-ribosome interaction dependent on the Ca(2+) induced conformational change of CaM. The interactions between CaM and ribosomes are inhibited by synthetic peptides comprising putative CaM-binding sites in ribosomal proteins S2 and L14. Using a cell-free in vitro translation system, we further found that these synthetic peptides are potent inhibitors of protein synthesis. Our results identify an involvement of CaM in the translational activity of ribosomes.

Ribosomal proteins L7 and L8 function in concert with six A₃ assembly factors to propagate assembly of domains I and II of 25S rRNA in yeast 60S ribosomal subunits.

Ribosome biogenesis is a complex multistep process that involves alternating steps of folding and processing of pre-rRNAs in concert with assembly of ribosomal proteins. Recently, there has been increased interest in the roles of ribosomal proteins in eukaryotic ribosome biogenesis in vivo, focusing primarily on their function in pre-rRNA processing. However, much less is known about participation of ribosomal proteins in the formation and rearrangement of preribosomal particles as they mature to functional subunits. We have studied ribosomal proteins L7 and L8, which are required for the same early steps in pre-rRNA processing during assembly of 60S subunits but are located in different domains within ribosomes. Depletion of either leads to defects in processing of 27SA(3) to 27SB pre-rRNA and turnover of pre-rRNAs destined for large ribosomal subunits. A specific subset of proteins is diminished from these residual assembly intermediates: six assembly factors required for processing of 27SA(3) pre-rRNA and four ribosomal proteins bound to domain I of 25S and 5.8S rRNAs surrounding the polypeptide exit tunnel. In addition, specific sets of ribosomal proteins are affected in each mutant: In the absence of L7, proteins bound to domain II, L6, L14, L20, and L33 are greatly diminished, while proteins L13, L15, and L36 that bind to domain I are affected in the absence of L8. Thus, L7 and L8 might establish RNP structures within assembling ribosomes necessary for the stable association and function of the A(3) assembly factors and for proper assembly of the neighborhoods containing domains I and II.

Antibiotic susceptibilities and resistance genes of Ureaplasma parvum isolated in South Africa

There is only limited information on the antimicrobial susceptibilities and resistance genes of Ureaplasma parvum in South Africa. This study was designed to detect and characterize resistance genes in U. parvum. Fifteen U. parvum isolates were investigated employing the broth microdilution method (tetracycline, doxycycline, ofloxacin, erythromycin, azithromycin and josamycin). Gene analyses were performed on target regions of: tet(M); gyrA, gyrB, parC and parE; erm(A), erm(B), erm(C) and erm(E); msr(A), msr(B), msr(C) and msr(D); 23S rRNA operons; and L4 and L22 ribosomal proteins. Seven of the U. parvum isolates were fully susceptible to the antibiotics tested. Five strains exhibited resistance to tetracycline (MICs 16-256 mg/L), one strain was resistant to ofloxacin (MIC 128 mg/L) and four strains were resistant to macrolides (MICs 128 mg/L); two strains showed dual resistance to tetracycline and erythromycin. The five tetracycline-resistant strains were found to have mosaic tet(M) genes, with one strain containing different specific regions to those previously described. Mutations in the L22 ribosomal protein were seen in three strains that were resistant to erythromycin (two strains) and erythromycin + azithromycin (one strain). For a further strain that was resistant to erythromycin and azithromycin, possible mechanisms of resistance remained elusive. This is the first report of quinolone, erythromycin and azithromycin resistance development in U. parvum from South Africa. A point mutation in parC (Pro-57 → Leu) and two novel mutations in parE (Ile-73 → Thr and a methionine insertion at codon 86) were found in an ofloxacin-resistant strain. The study reinforces the adaptability of U. parvum to develop resistance and acquire, modify and maintain transposon-located resistance genes.

Silencing expression of ribosomal protein L26 and L29 by RNA interfering inhibits proliferation of human pancreatic cancer PANC-1 cells

Oncogenic KRAS, an important target for antitumor therapy, contributes to pancreatic cancer tumorigenesis, progression and maintenance. However, intracellular compensation regulation can help cells to resist the targeted therapy. Gene knockdown method such as RNAi may help to understand this intracellular regulatory system and discover novel therapeutic approach. In this study, two stable transfected cell lines were constructed through lentivirus-mediated shRNA targeting KRAS of PANC-1 cells, to investigate cell response to the knockdown of KRAS. Human whole genome microarray was then used to compare the gene expression profile. As a result, ribosomal proteins L26 and L29 (RPL26 and RPL29) were dramatically upregulated by KRAS-shRNA specifically. To identify whether RPL26 or RPL29 was critical for PANC-1 cells, siRNAs against RPL26 and RPL29 were designed and transfected in vitro. The results showed that knockdown of RPL26 or RPL29 expression significantly suppressed cell proliferation, induced cell arrest at G0/G1 phase and enhanced cell apoptosis. Reactive oxygen species (ROS) assay indicated that silencing of RPL26 or RPL29 markedly decreased the intracellular ROS generation. Our findings imply that siRNA interference against RPL26 and RPL29 is of potential value for intervention of pancreatic cancer.

Reduced Expression of the rplU-rpmA Ribosomal Protein Operon in mexXY -Expressing Pan-Aminoglycoside-Resistant Mutants of Pseudomonas aeruginosa

Pan-aminoglycoside-resistant Pseudomonas aeruginosa mutants expressing the mexXY components of the aminoglycoside-accommodating MexXY-OprM multidrug efflux system but lacking mutations in the mexZ gene encoding a repressor of this efflux system and in the mexXY promoter have been reported (S. Fraud and K. Poole, Antimicrob. Agents Chemother. 55:1068-1074, 2011). Genome sequencing of one of these mutants, K2966, revealed the presence of a mutation within the predicted promoter region of the rplU-rpmA operon encoding ribosomal proteins L21 and L27, consistent with an observed 2-fold decrease in expression of this operon in the mutant relative to wild-type P. aeruginosa PAO1. Moreover, correction of the mutation restored rplU-rpmA expression and, significantly, reversed the elevated mexXY expression and pan-aminoglycoside resistance of the mutant. Reduced rplU-rpmA expression was also observed in a second mexXY-expressing pan-aminoglycoside-resistant mutant, K2968, which, however, lacked a mutation in the rplU-rpmA promoter region. Restoration of rplU-rpmA expression in the K2968 mutant following chromosomal integration of the rplU-rpmA operon derived from wild-type P. aeruginosa failed, however, to reverse the elevated mexXY expression and pan-aminoglycoside resistance of this mutant, although it did so for K2966, suggesting that the mutation impacting rplU-rpmA expression in K2968 also impacts other mexXY-related genes. Increased mexXY expression owing to reduced rplU-rpmA expression in K2966 and K2968 was dependent on PA5471, whose expression was also elevated in these mutants. Thus, mutational disruption of ribosome function, by limiting expression of ribosomal constituents, promotes recruitment of mexXY and does so via PA5471, reminiscent of mexXY induction by ribosome-disrupting antimicrobial agents. Interestingly, reduced rplU-rpmA expression was also observed in a mexXY-expressing pan-aminoglycoside-resistant clinical isolate, suggesting that ribosome-perturbing mutations have clinical relevance in the recruitment of the MexXY-OprM aminoglycoside resistance determinant.

Protein L5 is crucial for in vivo assembly of the bacterial 50S ribosomal subunit central protuberance

In the present work, ribosomes assembled in bacterial cells in the absence of essential ribosomal protein L5 were obtained. After arresting L5 synthesis, Escherichia coli cells divide a limited number of times. During this time, accumulation of defective large ribosomal subunits occurs. These 45S particles lack most of the central protuberance (CP) components (5S rRNA and proteins L5, L16, L18, L25, L27, L31, L33 and L35) and are not able to associate with the small ribosomal subunit. At the same time, 5S rRNA is found in the cytoplasm in complex with ribosomal proteins L18 and L25 at quantities equal to the amount of ribosomes. Thus, it is the first demonstration that protein L5 plays a key role in formation of the CP during assembly of the large ribosomal subunit in the bacterial cell. A possible model for the CP assembly in vivo is discussed in view of the data obtained.

Molecular mechanism of macrolide–lincosamide resistance in Moraxella catarrhalis

We identified a Moraxella catarrhalis strain with high-level resistance to azithromycin (MIC>256 mg l(-1)), NSH1, isolated from nasopharyngeal swab samples from an inpatient with acute bronchitis in a Japanese hospital in 2011 and determined its mechanism of macrolide-lincosamide resistance. Antimicrobial susceptibility of M. catarrhalis strains was determined using the Etest and agar dilution methods. Mutations in the four 23S rRNA alleles, the ribosomal proteins L4 and L22, and methylase genes erm(B) and erm(F) were tested by PCR and/or sequencing. The efflux system was examined using appropriate inhibitors. Transformation experiments were performed using DNA amplicons of the 23S rRNA gene of M. catarrhalis strain NSH1. This strain showed high-level resistance to erythromycin, clarithromycin, azithromycin, clindamycin (MICs>256 mg l(-1)) and josamycin (MIC = 128 mg l(-1)), and contained the A2058T mutation (Escherichia coli numbering) in four of the 23S rRNA alleles. Mutation of the ribosomal proteins and overproduction of the efflux system were not observed, and methylase genes were not detected. When amplified DNA containing the single A2058T mutation was transformed into M. catarrhalis strains, transformants with three A2058T-mutated 23S rRNA alleles showed high-level resistance to macrolide-lincosamide, similar to strain NSH1. In contrast, transformants with two A2058T-mutated 23S rRNA alleles showed low-level MICs (azithromycin: 0.38-0.5 mg l(-1)). Thus, a single A2058T mutation occurring in at least three 23S rRNA alleles confers high-level resistance to 14-, 15- and 16-membered macrolides and lincosamides in M. catarrhalis possessing four 23S rRNA alleles. This study represents the first evidence, to our knowledge, of this effect in M. catarrhalis.

High-resolution crystal structure of the isolated ribosomal L1 stalk

The crystal structure of the isolated full-length ribosomal L1 stalk, consisting of Thermus thermophilus ribosomal protein L1 in complex with a specific 80-nucleotide fragment of 23S rRNA, has been solved for the first time at high resolution. The structure revealed details of protein-RNA interactions in the L1 stalk. Analysis of the crystal packing enabled the identification of sticky sites on the protein and the 23S rRNA which may be important for ribosome assembly and function. The structure was used to model different conformational states of the ribosome. This approach provides an insight into the roles of domain II of L1 and helix 78 of rRNA in ribosome function.

A study of the molecular basis of quinolone and macrolide resistance in a selection of Campylobacter isolates from intensive poultry flocks

The aim of this study was to investigate the molecular basis for observed high-level quinolone and macrolide resistance in poultry Campylobacter isolates. Seventeen Campylobacter isolates displaying high-level resistance to nalidixic acid, ciprofloxacin and/or erythromycin were investigated. Minimum inhibitory concentrations were initially determined using both the broth microdilution and E-test methods. The contribution of target gene mutations and active efflux to the observed resistances were then investigated using PCR and sequencing methods. High-level resistance to nalidixic acid was attributed to amino acid substitutions Thr-86-Ile and Asn-203-Ser in GyrA in some but not all isolates. Contrary to previous reports, the Thr-86-Ile substitution did not confer universal resistance to all quinolones. Strains displaying a high level of resistance to erythromycin carried the 23S rRNA transition mutation A2075G and/or carried mutations in the L4 and/or L22 ribosomal-encoding proteins. Interestingly and in contrast to previous studies, not all of the isolates carrying substitutions within the β-hairpin region of the L22 ribosomal protein displayed erythromycin resistance. With the exception of a single isolate, efflux did not contribute to either quinolone or macrolide resistance.This study further expands our understanding of the molecular basis of quinolone and macrolide resistance in Campylobacter spp. and suggests that other factors, remaining to be elucidated, may also contribute to the resistant phenotypes observed.