Mutations in 23S rRNA in Helicobacter pylori conferring resistance to erythromycin do not always confer resistance to clarithromycin

Mutations conferring resistance to erythromycin or clarithromycin in Helicobacter pylori were studied. Mutation A2142G was consistently associated with clarithromycin MIC of > 256 micrograms/ml, whereas mutants carrying A2143G had MICs ranging from < or = 0.016 to > 256 micrograms/ml, suggesting that additional factors account for the observed multiple levels of resistance to clarithromycin.     

Genotypic characterization of clarithromycin-resistant and -susceptible Helicobacter pylori strains from the same patient demonstrates existence of two unrelated isolates

Clarithromycin-susceptible and clarithromycin-resistant Helicobacter pylori isolates from the same patient were investigated for the mode of development and mechanism of clarithromycin resistance. The clarithromycin-resistant strain UA1182 harbors homozygous A-to-G mutations at position 2143 in both copies of the 23S rRNA gene and has a phenotype of resistance to clarithromycin and clindamycin but no significant resistance to streptogramin B. Pulsed-field gel electrophoresis patterns of NruI- and NotI-digested genomic DNA from the Clas and Clar isolates demonstrated that they are genetically distinct, suggesting that the development of clarithromycin resistance is not from the mutation of the existing Clas strain but from a completely new strain.     

Cloning and characterization of cDNA for human adenylate kinase 2A

Considerable genomic microdiversity has been reported previously among Helicobacter pylori isolates. We have constructed genome maps of four unrelated H. pylori strains (NCTC11637, NCTC11639, UA802 and UA861) using pulsed-field gel electrophoresis (PFGE) with NotI and NruI, hybridization with extracted PFGE DNA fragments and probing with 17 gene probes. These strains of H. pylori were compared with a fifth unrelated H. pylori strain NCTC11638 mapped previously. Considerable diversity in gene arrangement was evident among the five H. pylori maps, and no consistent gene clustering was found. The association of only four genes, katA (catalase gene), vacA (vacuolating cytotoxin gene), hpaA (a putative adhesin gene), and pfr (bacterial ferritin gene) were generally conserved within approximately the same 25% of the genome; however, the order of these genes also varied. Our study demonstrates that macrodiversity, i.e. variability in gene order, in addition to microdiversity, is a characteristic of the H. pylori genome.     

Ribosomal protein L9 interactions with 23 S rRNA: the use of a translational bypass assay to study the effect of amino acid substitutions

During translation of bacteriophage T4 gene 60 mRNA, ribosomes bypass 50 nucleotides with high efficiency. One of the mRNA signals for bypass is a stem-loop in the first part of the coding gap. When the length of this stem-loop is extended by 36 nucleotides, bypass is reduced to 0.35% of the wild-type level. Bypass is partially restored by a mutation in the C-terminal domain of Escherichia coli large ribosomal subunit protein L9. Previous work has shown that L9 is an elongated protein with an alpha-helix that connects and orients the N and C-terminal domains that both contain a predicted RNA binding site. We have determined two binding sites of L9 on 23 S rRNA. A 778 nucleotide RNA fragment encompassing domain V (nucleotides 1999 to 2776) of the 23 S rRNA is retained on filters by L9 and contains both sites. The N and C-terminal domains of L9 were shown to interact with nucleotides just 5' to nucleotide 2231 and 2179 of the 23 S rRNA, respectively, using the toeprint assay. These L9 binding sites on 23 S rRNA suggest that L9 functions as a brace across helix 76 to position helices 77 and 78 relative to the peptidyl transferase center. In this study, bypass on a mutant gene 60 mRNA has been used as an assay to probe the importance of particular L9 amino acids for function. Amino acid substitutions in the C-terminal domain are shown to partially restore bypass. These mutant L9 proteins have reduced binding to a 23 S rRNA fragment (nucleotides 1999 to 2274) containing domain V, to which L9 binds. They partially retain both the N and C-terminal domain interactions. On the other hand, substitutions of amino acids in the N-terminal domain, which greatly reduce RNA binding, do not restore bypass. The latter mutants have completely lost the N-terminal domain interaction. Addition of an amino acid to the alpha-helix also restores gene 60 bypass. RNA binding by this mutant is similar to that observed for the C-terminal domain mutants that partially restore bypass.     

Strategy for the detection of Helicobacter species by amplification of 16S rRNA genes and identification of H. felis in a human gastric biopsy

The aim of the present work was to develop polymerase chain reactions (PCRs) based on the conserved nucleotide sequence of the 16S rRNA gene for detection of bacteria of the Helicobacter genus in human antral biopsy samples. The assay for Helicobacter spp was developed by amplifying a 399-bp 16S rRNA gene sequence specific to the genus Helicobacter. The identity of the amplicon was confirmed by hybridization with an internal probe and by restriction by endonuclease VspI showing two expected fragments of 295 and 104 base pairs. A total of 65 dyspeptic patients from France and New Caledonia were screened for Helicobacter spp infection through the use of the following diagnostic assays on biopsy specimens collected through endoscopy: direct detection of bacteria in histological sections by Giemsa and Warthin Starry staining, urease test and bacterial isolation, PCR for Helicobacter pylori ureC/glmM gene, and PCR targeted to 16S rRNA genes. The 16S rRNA gene PCR assay was able to detect down to 680 bacterial cells, as assessed by agarose gel electrophoresis, and down to 4 bacterial cells by hybridization of amplicon with the internal probe. The 16S rRNA PCR test was 100% specific and sensitive; results obtained with this test were in agreement with the visualization of bacteria by histology. Urease test and culture were 86.4% and 22.7% sensitive, and 96.5 and 100% specific, respectively. The H. pylori ureC/glmM gene-based PCR was 100% specific and only 95.4% sensitive, since one biopsy from a Melanesian patient contained a Helicobacter strain other than H. pylori. For this Melanesian patient, a branch-specific PCR targeting the epsilon branch of Proteobacteria was used to amplify a 967-bp amplicon. This amplicon was sequenced and matched with the H. felis sequence. This was confirmed using an H. felis-specific urease PCR test.     

Detection of point mutations associated with resistance of Helicobacter pylori to clarithromycin by hybridization in liquid phase

When the standard procedure for determining antibiotic susceptibility of bacteria is used, the results are delayed, especially for bacteria that grow slowly, such as Helicobacter pylori. Treatment for this bacterium may involve clarithromycin, a compound for which resistance has been associated with point mutations on the 23S rRNA gene. This resistance is currently found in organisms isolated from 0 to 15% of patients and jeopardizes the success of the treatment. We have designed a test involving amplification and colorimetric hybridization in the liquid phase to detect the mutation at the molecular level. First, four reference strains, including the wild type and three strains with the mutations A2143C, A2143G, and A2144G, were used to optimize the method. Amplification was carried out with primers previously published. The amplified products were added to probe-coated microtiter wells. A DNA enzyme immunoassay was used to detect the hybrids. The optimal conditions of the hybridization were defined for each probe. Nineteen H. pylori strains resistant to clarithromycin and 22 susceptible according to phenotypic data were submitted to restriction with BsaI and BbsI, and part of the 23S rRNA gene was sequenced in order to determine the mutation involved for the resistant strains. The new assay showed a complete correlation with the reference methods, except for one strain. Cross-hybridizations as well as application of the reaction to other bacteria did not lead to optical densities higher than the cutoff values chosen with the receiving operating characteristic curve. This method can be easily standardized and gives a result within a day. Its application directly to the biopsy specimens or infected gastric juice is planned in the future.     

A molecular scheme based on 23S rRNA gene polymorphisms for rapid identification of Campylobacter and Arcobacter species

23S rRNA gene PCR amplicons from 118 strains representing 15 species of Campylobacter and four species of Arcobacter were consecutively digested with HpaII, CfoI, and HinfI. A reproducible and discriminatory identification scheme based on combined restriction patterns was developed by using reference strains and was applied to the identification of a variety of isolates.     

In vitro protein folding by ribosomes from Escherichia coli, wheat germ and rat liver: the role of the 50S particle and its 23S rRNA

Ribosomes from a number of prokaryotic and eukaryotic sources (e.g. Escherichia coli, wheat germ and rat liver) can refold a number of enzymes which are denatured with guanidine/HC1 prior to incubation with ribosomes. In this report, we present our observations on the refolding of denatured lactate dehydrogenase from rabbit muscle and glucose-6-phosphate dehydrogenase from baker's yeast by ribosomes from E. coli, wheat germ and rat liver. The protein-folding activity of E. coli ribosomes was found to be present in 50S particles and in 23S rRNA. The 30S particle or 16S rRNA did not show any protein-folding activity. The protein-folding activity of 23S rRNA may depend on its tertiary conformation. Loss of tertiary structure, by incubation with low concentrations of EDTA, inhibited the protein-folding activity of 23S rRNA. This low concentration of EDTA had no effect on folding of the denatured enzymes by themselves.     

Detection of kanamycin-resistant Mycobacterium tuberculosis by identifying mutations in the 16S rRNA gene

In Mycobacterium smegmatis and a limited number of Mycobacterium tuberculosis strains, the involvement of alterations of the 16S rRNA gene (rrs) in resistance to kanamycin has been shown. To investigate the extent to which mutations in a specific region of the rrs gene and the kanamycin-resistant phenotype in clinically isolated M. tuberculosis strains were correlated, 43 kanamycin-resistant strains (MICs, > or =200 microg/ml), 71 kanamycin-susceptible strains, and 4 type strains were examined. The 300-bp DNA fragments carrying the rrs gene and the intervening sequence between the rrs gene and 23S rRNA (rrl) gene fragments were amplified by PCR and were subjected to PCR-based direct sequencing. By comparing the nucleotide sequences, substitutions were found in 29 of 43 (67.4%) kanamycin-resistant clinical isolates at positions 1400, 1401, and 1483 but in none of the 71 sensitive isolates or the 4 type strains. The most frequent substitution, from A to G, occurred at position 1400. A substitution from C to T at position 1401 was found once. Two clinical isolates carried the double mutation from C to A at position 1401 and from G to T at position 1483. In addition, we found that these mutants can be distinguished from wild-type strains by digestion with the restriction endonucleases TaiI and Tsp45I. Furthermore, we found that the genotypes of kanamycin-resistant strains can be discriminated from each other by digestion with a restriction endonuclease, BstUI or DdeI.     

23S rRNA positions essential for tRNA binding in ribosomal functional sites

rRNA plays an important role in function of peptidyl transferase, the catalytic center of the ribosome responsible for the peptide bond formation. Proper placement of the peptidyl transferase substrates, peptidyl-tRNA and aminoacyl-tRNA, is essential for catalysis of the transpeptidation reaction and protein synthesis. In this report, we define a small set of rRNA nucleotides that are most likely directly involved in binding of tRNA in the functional sites of the large ribosomal subunit. By binding biotinylated tRNA substrates to randomly modified large ribosomal subunits from Escherichia coli and capturing resulting complexes on the avidin resin, we identified four nucleotides in the large ribosomal subunit rRNA (positions G2252, A2451, U2506, and U2585) whose modifications prevent binding of a peptidyl-tRNA analog in the P site and one residue (U2555) whose modification interferes with transfer of peptidyl moiety to puromycin. These nucleotides represent a subset of positions protected by tRNA analogs from chemical modification and significantly narrow the number of 23S rRNA nucleotides that may be directly involved in tRNA binding in the ribosomal functional sites.     

Ribosomal proteins neighboring 23 S rRNA nucleotides 803-811 within the 50 S subunit

We report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe and its exploitation in identifying 50 S ribosomal subunit components neighboring its target site, nucleotides 803-811 in 23 S rRNA. Photolysis of the complex formed between the probe and 50 S subunits leads to site-specific probe photoincorporation into proteins L15, L17, and L20, labeled to greater extents, and L13 and L21, labeled to lesser extents. Portions of each of these proteins thus lie within 23 A of nucleotide U803. These results lead us to conclude that nucleotides 803-811 fall on the side of the L13-L17-L20-L21 protein cluster [Walleczek et al. (1988) EMBO J. 7, 3571-3576] that points from the back of the 50 S particle toward the peptidyl transferase center within the 50 S subunit. Such placement is consistent with the observation that an oligoDNA probe directed to nucleotides 803-811 decreases P-site binding of tRNA [Hill et al. (1990) Biochim. Biophys. Acta 1050, 45-50].     

Overexpression of the thiostrepton-resistance gene from Streptomyces azureus in Escherichia coli and characterization of recognition sites of the 23S rRNA A1067 2'-methyltransferase in the guanosine triphosphatase center of 23S ribosomal RNA

The thiostrepton-resistance gene encoding the 23S rRNA A1067 methyltransferase from Streptomyces azureus has been overexpressed in Escherichia coli using a T7-RNA-polymerase-dependent expression vector. The protein was efficiently expressed at levels up to 20% of total soluble protein and purified to near homogeneity. Kinetic parameters for S-adenosyl-L-methionine (Km = 0.1 mM) and an RNA fragment containing nucleotides 1029-1122 of the 23S ribosomal RNA from E. coli (Km = 0.001 mM) were determined. S-Adenosyl-L-homocysteine showed competitive product inhibition (Ki = 0.013 mM). Binding of either thiostrepton or protein L11 inhibited methylation. RNA sequence variants of the RNA fragment with mutations in nucleotides 1051-1108 were tested as substrates for the methylase. The experimental data indicate that methylation is dependent on the secondary structure of the hairpin including nucleotide A1067 and the exact sequence U(1066)-A(1067)-G(1068)-A(1069)-A(1070) of the single strand.     

Clarithromycin resistance in Helicobacter pylori: prevalence in untreated dyspeptic patients and stability in vitro

Susceptibilities to clarithromycin and metronidazole of 444 Helicobacter pylori isolates cultured from antral biopsies of 444 dyspeptic patients were determined by disc diffusion tests (15 mu g disc for clarithromycin, 5 mu g disc for metronidazole). Susceptibility of 46 of these isolates to erythromycin (5 mu g disc) was also tested. Minimal inhibitory concentrations (MICs) of clarithromycin for 42 selected isolates were determined by a plate dilution method. A zone diameter of 30 mm was defined as a 'cut-off' size differentiating susceptibility and resistance of the organism to clarithromycin, by comparing results obtained with the two methods. Of the 444 isolates, 424 (95.5%) were highly sensitive to clarithromycin, with zone diameters ranging from 30 to 98 mm. Twenty isolates (4.5%) were defined as resistant to clarithromycin, with zone diameters ranging between 6 and 28 mm. The incidence of clarithromycin resistance was similar in men and women and in different age groups, and was not significantly different between patients with peptic ulcer and non-ulcer dyspepsia. Among the 444 isolates, 168 (37.8%) were metronidazole resistant. There was cross resistance between clarithromycin and erythromycin, but not between clarithromycin and metronidazole. Stability of clarithromycin resistance was evaluated by the disc diffusion test and confirmed by the plate dilution method. Among the 20 clarithromycin-resistant isolates, nine (45%) reverted to be sensitive after 25 subcultures on drug-free agar. The findings in this study indicate that the incidence of clarithromycin-resistant H. pylori in untreated dyspeptic patients is low. Cross-resistance occurs between macrolides and resistance to clarithromycin in some strains is reversible.     

Detection of Helicobacter-like bacteria in porcine gastric biopsy samples by amplification of 16S rRNA, ureB, vacA and cagA genes by PCR

Preliminary evidence for the presence of Helicobacter-like bacteria was sought in 395 porcine gastric samples by a urease test. Of the samples, 37% (146/395) were urease-positive and 82% (82/100) of the Gram-stained urease-positive samples showed large, tightly spiralled organisms. Several methods were applied to culture the organisms but isolation was unsuccessful, contaminant organisms being considered to be one of the major problems. PCR with Helicobacter genus-specific primers for 16S rRNA and ureB genes, and primers for H. pylori vacA and cagA genes were tested with 102 ureasepositive biopsy samples. The PCR results showed some evidence for the presence of the urease and the vacA genes in porcine Helicobacter-like bacteria and raises the possibility of pathogenicity by these organisms.     

Sites of interaction of streptogramin A and B antibiotics in the peptidyl transferase loop of 23 S rRNA and the synergism of their inhibitory mechanisms

Streptogramin antibiotics contain two active A and B components that inhibit peptide elongation synergistically. Mutants resistant to the A component (virginiamycin M1 and pristinamycin IIA) were selected for the archaeon Halobacterium halobium. The mutations mapped to the universally conserved nucleotides A2059 and A2503 within the peptidyl transferase loop of 23 S rRNA (Escherichia coli numbering). When bound to wild-type and mutant haloarchaeal ribosomes, the A and B components (pristinamycins IIA and IA, respectively) produced partially overlapping rRNA footprints, involving six to eight nucleotides in the peptidyl transferase loop of 23 S rRNA, including the two mutated nucleotides. An rRNA footprinting study, performed both in vivo and in vitro, on the A and B components complexed to Bacillus megaterium ribosomes, indicated that similar drug-induced effects occur on free ribosomes and within the bacterial cells. It is inferred that position 2058 and the sites of mutation, A2059 and A2503, are involved in the synergistic inhibition by the two antibiotics. A structural model is presented which links A2059 and A2503 and provides a structural rationale for the rRNA footprints.     

New features of 23S ribosomal RNA folding: the long helix 41-42 makes a "U-turn" inside the ribosome

23S rRNA from Escherichia coli was cleaved at single internucleotide bonds using ribonuclease H in the presence of appropriate chimeric oligonucleotides; the individual cleavage sites were between residues 384 and 385, 867 and 868, 1045 and 1046, and 2510 and 2511, with an additional fortuitous cleavage at positions 1117 and 1118. In each case, the 3' terminus of the 5' fragment was ligated to radioactively labeled 4-thiouridine 5'-,3'-biphosphate ("psUp"), and the cleaved 23S rRNA carrying this label was reconstituted into 50S subunits. The 50S subunits were able to associate normally with 30S subunits to form 70S ribosomes. Intra-RNA crosslinks from the 4-thiouridine residues were induced by irradiation at 350 nm, and the crosslink sites within the 23S rRNA were analyzed. The rRNA molecules carrying psUp at positions 867 and 1117 showed crosslinks to nearby positions on the opposite strand of the same double helix where the cleavage was located, and no crosslinking was detected from position 2510. In contrast, the rRNA carrying psUp at position 384 showed crosslinking to nt 420 (and sometimes also to 416 and 425) in the neighboring helix in 23S rRNA, and the rRNA with psUp at position 1045 gave a crosslink to residue 993. The latter crosslink demonstrates that the long helix 41-42 of the 23S rRNA (which carries the region associated with GTPase activity) must double back on itself, forming a "U-turn" in the ribosome. This result is discussed in terms of the topography of the GTPase region in the 50S subunit, and its relation to the locations of the 5S rRNA and the peptidyl transferase center.