Ribosomes from spiramycin resistant mutants of Escherichia coli Q13

Summary Mutants from Escherichia coli Q13 were selected for resistance to leucomycin, tylosin or spiramycin. Most of the mutants so selected exhibited cross resistance to all the macrolide antibiotics tested including erythromycin. A few mutants however seem to be less resistant to erythromycin. One mutant, QSP008, was highly resistant to tylosin, leucomycin and spiramycin but relatively sensitive to erythromycin. Another mutant, QSP006, was highly resistant to spiramycin but less resistant to erythromycin, tylosin and leucomycin. This selective resistance of cells to specific antibiotics could be due to the extent of conformational alteration of their ribosomes, which may be demonstrated by the extent of ¹⁴C-erythromycin binding to these ribosomes. The ribosomes from QSP008 cells were found to contain an altered 50-8 protein of the 50s ribosomal subunit, while in the ribosomes from QSP006 no such protein change could be detected by the methods used.A preliminary data of part of this work has been published (Tanaka, Teraoka, Tamaki, Watanabe, Osawa, Otaka, and Takata, 1971).     

Peptide analyses of a protein component, 50-8, of 50s ribosomal subunit from erythromycin resistant mutants of Escherichia coli and Escherichia freudii

Summary Chromatographic analyses on a Dowex 50x8 column of tryptic digests of the mutationally altered 50-8 protein component from several erythromycin resistant (ery ^r) mutants of Escherichia coli and Escherichia freundii have been performed. It was found that (1) the difference in the elution profile of the altered components detected with carboxymethyl cellulose column chromatography reflects the difference in their amino acid sequence, (2) the structural change(s) of the 50-8 protein from three E. coli ery ^r mutants examined seems to exist only in the same single peptide fragment and (3) the primary structure of the 50-8(R) protein of E. freundii (ery ^s: wild type) differs from that of E. coli Q13 (ery ^s) and the structural change in 50-8(R) component of E. freundii caused by the ery ^r mutation was found to take place in different peptide fragments from that in which the mutational change of the E. coli 50-8 component occurred.     

Phenotypes represented by a mutational change in a 50s ribosomal protein component, 50-8, in Escherichia coli

Summary An erythromycin resistant (ery ^r) mutant of Escherichia coli Q13, QE107, was characterized by (1) the cross resistance of the cells to several macrolide antibiotics such as erythromycin, tylosin, spiramycin, oleandomycin and leucomycin, (2) the reduced affinity of its ribosomes to erythromycin and probably to the other macrolides mentioned above, (3) a low peptidyl transferase activity of its ribosomes and (4) an altered 50-8 protein of the 50s ribosomal subunits. These characters were always transferred together with the ery marker in the transduction experiments.Preliminary data of part of this work has been published (Tanaka, Teraoka, Tamaki, Watanabe, Osawa, Otaka and Takata, 1971).     

Chloramphenicol resistant mutants of Bacillus subtilis

Summary Telve chloramphenicol resistant (CM ^r)-mutants were isolated from B. subtilis ATCC 6633 and were classified into the following six groups. Group I. No 50s ribosomal protein change was detectable. Ribosomes did not show alteration of the binding ability to CM or to erythromycin in vitro. Group II. A 50s protein, 50a, was altered. Ribosomes did not show alteration of the binding ability to CM or to erythromycin in vitro. The genes specifying the 50a protein was in the cysA-str region on B. subtilis chromosome. Group III. A 50s protein, 50b, was altered. Biological properties of the ribosomes were the same as Group I or II so fas as examined. The genes for 50b protein was in the cysA-str region. Group IV. A 50s protein, 50c, was altered. Ribosomes showed a definite decrease in ability to bind to CM in vitro. The binding of erythromycin to the ribosomes was not impaired. The chromosomal locus of the CM ^r (and for 50c protein) was in the cysA-str region. Group V. A 50s protein, 50e, was changed. The ability of the ribosomes to bind in vitro both to CM and to erythromycin was greatly reduced. The genetic locus of the CM ^r (and for 50e protein) was in the cysA-str region. Group VI. A 50s protein, 50f, was altered. Ribosomes showed a decrease in ability to bind in vitro both to CM and to erythromycin. The genes for 50f protein was in the cysA-str region.The results suggest that the ribosomal resistance to CM may be caused by an independent change of at least several 50s ribosomal protein species. The genetic data shown here and those reported previously show that at least two 30s and seven 50s ribosomal protein genes are situated in the cysA-str region on B. subtilis chromosome.     

The Erythromycin Resistance Gene of the Corynebacterium xerosis R-plasmid pTP10 Also Carrying Chloramphenicol, Kanamycin, and Tetracycline Resistances Is Capable of Transposition in Corynebacterium glutamicum

The clinical isolate Corynebacterium xerosis M82B carries the 50-kb R-plasmid pTP10 that confers resistance to the antibiotics chloramphenicol, kanamycin, erythromycin, and tetracycline. A detailed restriction map of pTP10 was constructed by cloning and analyzing restriction fragments of pTP10 in Escherichia coli . The resistance determinants of pTP10 were located by studying the phenotype of the recombinant plasmids in E. coli and Corynebacterium glutamicum . Restriction patterns of fragments encoding the kanamycin and erythromycin resistances revealed striking similarity to the kanamycin resistance of transposon Tn903 and the erythromycin resistance on plasmid pNG2 from Corynebacterium diphtheriae, respectively. Expression of the resistance determinants in E. coli and C. glutamicum ATCC 13032 led to high resistance levels in both strains, with the exception of the tetracycline resistance gene, which could be expressed only in C. glutamicum. Furthermore, the erythromycin resistance gene was found to be located on a transposable element which is functional in C. glutamicum strains.     

Interactions between viomycin resistance and streptomycin resistance on ribosomes of Mycobacterium smegmatis.

We have investigated the mechanism of the expression of resistance to high levels of viomycin and coresistance to streptomycin in a mutant strain of Mycobacterium smegmatis ATCC 14468 (AC-13) which was obtained by serial transfers of parental cells to media containing increasing concentrations of viomycin. It was shown previously that resistance to viomycin by strain AC-13 was due to an alteration in the 50 S ribosomal subunit (20). However, genetic analysis has shown that mutation in 50 S subunits alone gave only low level resistance to viomycin. When a streptomycin resistant mutation (caused by an alteration in the 30 S subunit) was introduced into the low level viomycin resistant recombinant strains, most of them were highly resistant to viomycin. Some recombinants were resistant to intermediate levels of viomycin, and the remainder were not affected by the introduction of the str^r allele. Studies with in vitro cell-free systems have shown that streptomycin resistant 30 S ribosomal subunits obtained from a high level viomycin resistant recombinant were able to modify the levels of resistance to viomycin expressed by the 50 S ribosomal subunit. These findings provide additional evidence concerning the functional relationship between 30 S and 50 S ribosomal components in ribosomes.     

MsrA Efflux Pump Inhibitory Activity of Piper cubeba L.f. and its Phytoconstituents against Staphylococcus aureus RN4220

MsrA, an efflux pump belonging to ATP‐binding cassette (ABC) transporter family that conferred resistance to macrolides, was detected in Staphylococcus aureus strains. Herein, we report the isolation of phytoconstituents from Piper cubeba fruit methanol extract and investigated their efflux pump inhibitory potential against S. aureus MsrA pump. Four isolated compounds, viz. pellitorine, sesamin, piperic acid and tetrahydropiperine studied in combination with erythromycin in S. aureus RN4220, exhibited 2–8‐fold reduction in minimum inhibitory concentration (MIC) of erythromycin. Pellitorine and sesamin decreased MIC of erythromycin by 8‐fold. The real‐time fluorometry‐based efflux and accumulation studies of ethidium bromide (EtBr) on S. aureus RN4220 in the presence of these compounds showed reduced efflux and enhanced uptake, thus indicating inhibition of the efflux pump. Pellitorine showed significant post‐antibiotic effect of erythromycin. The results revealed that the primary mechanism of action of these compounds involves steady ATP production impairment.     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.     

Genetic studies of the ribosomal proteins in Escherichia coli. 7. Mapping of several ribosomal protein components by transduction experiments between different strains of Escherichia coli

Summary Two 50s (50-10 and 50-12) and two 30s (30-4 and 30-7) ribosomal proteins could be distinguished between Shigella dysenteriae Sh/s and Escherichia coli K-12 JC411 with CMC column chromatography. On the other hand, E. coli K-12 AT2472 was shown to have a 30s ribosomal protein, 30-6(AT), which is specific to this strain and distinguishable from 30-6 of other E. coli K-12 strains. Transduction experiments by phage Plkc between Sh. dysenteriae Sh/s and E. coli ATSPCO1, a spectinomycin resistant mutant derived from AT2472 in which the 30-4 protein is altered, indicated that the genes specifying the above five ribosomal protein components are located in the streptomycin region on the E. coli chromosome.The gene order for three 50s (50-8, 50-10 and 50-12) and three 30s [str (30-?), 30-4 and 30-6] ribosomal proteins on the chromosome was determined by transduction technique between Sh. dysenteriae Sh/s and E. coli ATSPC01, between E. coli ATSPC01 and E. coli ER05 (an erythromycin resistant strain in which the 50-8 protein is altered), and between Sh. dysenteriae Sh/s and E. coli ERSPC14 (str ^s spc ^r ery ^r), respectively. It was found that these protein genes are arranged on the chromosome in the order of str (30-?)-30-4-30-6-50-8-50-10-50-12.     

Erythromycin inhibits the assembly of the large ribosomal subunit in growing Escherichia coli cells

Erythromycin and other macrolide antibiotics have been examined for their effects on ribosome assembly in growing Escherichia coli cells. Formation of the 50S ribosomal subunit was specifically inhibited by erythromycin and azithromycin. Other related compounds tested, including oleandomycin, clarithromycin, spiramycin, and virginiamycin M₁, did not influence assembly. Erythromycin did not promote the breakdown of ribosomes formed in the absence of the drug. Two erythromycin-resistant mutants with alterations in ribosomal proteins L4 and L22 were also examined for an effect on assembly. Subunit assembly was affected in the mutant containing the L22 alteration only at erythromycin concentrations fourfold greater than those needed to stop assembly in wild-type cells. Ribosomal subunit assembly was only marginally affected at the highest drug concentration tested in the cells that contained the altered L4 protein. These novel results indicate that erythromycin has two effects on translation, preventing elongation of the polypeptide chain and also inhibiting the formation of the large ribosomal subunit.     

Studies on the synthesis of plasmid-coded proteins and their control in Bacillus subtilis minicells.

The minicell system of Bacillus subtilis has been used to study the expression of plasmid genes using several R plasmids derived from Staphylococcus aureus. pE194, pC194, and pUB110 as well as several mutant and in vitro recombinant derivatives of these plasmids segregate into minicells. A copy control mutant of pE194 was used to show that the extent of segregation is proportional to the copy number. The polypeptides specified by these plasmids were examined by SDS-polyacrylamide gel electrophoresis. Six proteins specified by pE194, an erythromycin resistance plasmid, were identified using cop mutants. These comprise about 90% of the potential coding capacity of the 2.4-Mdal pE194 plasmid. One of these proteins (29,000 daltons) is inducible by erythromycin in the wild type pE194 but is synthesized constitutively in a mutant derivative which also expresses antibiotic resistance constitutively. Several other proteins are detected only in copy control mutants. pUB110, a kanamycin resistance plasmid, expresses three major proteins which comprise 50% of the coding capacity of this 3.0-Mdal plasmid. Two additional minor proteins are occasionally observed. pC194 (2.0 Mdal), which confers chloramphenicol resistance, expresses two polypeptides comprising about 25% of its coding capacity. One of these polypeptides (22,000 daltons) is inducible by chloramphenicol. pBD9, an in vitro composite of pUB110 and pE194, probably expresses all of the major parental plasmid proteins with the exception of one from pUB110 and one from pE194.     

Antimicrobial susceptibilities of Propionibacterium acnes isolated from patients with acne vulgaris

Antibiotic susceptibilities of Propionibacterium acnes in Japan were determined. Erythromycin‐resistance was found in 10.4% (5/48) of the strains, and four of these were cross‐resistance to clindamycin. Although the erythromycin ribosome methylase gene erm(X) was looked for, no strain carrying erm(X) was found. Sequencing analysis revealed that all of the erythromycin‐resistant strains had a mutation in the peptidyl transferase region of the 23S rRNA gene: G2057A, A2058G, or A2059G. Consequently, our results show that P. acnes resistance to macrolides is caused by a mutation in the 23S rRNA gene, and has been increasing in Japan.     

Molecular characterization of erm(B)- and mef(E)-mediated erythromycin-resistant Streptococcus pneumoniae in China and complete DNA sequence of Tn2010

Aims: To characterize the erm(B)‐ and mef(E)‐mediated erythromycin‐resistant Streptococcus pneumoniae clinical isolates obtained from ten hospitals located different cities in China. Methods and Results: Totally 83 S. pneumoniae were collected, and eighteen representative strains of 66 strains that exhibited erythromycin resistance were used for further characterization by antibiograms, serotyping, PFGE, MLST, DNA sequencing of the macrolide‐resistance elements and mapping of the elements on the chromosome. Twelve isolates showed a high‐level resistance to erythromycin, and six other isolates showed a low‐level resistance to erythromycin. Thirteen isolates harboured a Tn2010 transposon (26·4 kbp) encoding the erm(B), tet(M) and mef(E) genes and were classified into three types by Tn2010 structures. The remaining five isolates harboured a Tn6002 transposon (20·9 kbp) encoding the erm(B) and tet(M) genes and were classified into three types by Tn6002 locations on the chromosome. Three of the Tn6002 elements were located within the Tn5252‐like element, implying that these composed a large mobile element. The MLST analyses showed that several clones had been disseminated and that the CC271 strains carrying the Tn2010 element expressing the high‐level resistance to erythromycin were predominant in China. Four new MLST strains, which were designated as ST3262, ST3263, ST3397 and ST3398 were also identified. Conclusions: The erythromycin resistance determinant of S. pneumoniae that had been isolated in China was located in Tn2010 or the Tn6002 element and several clones had been disseminated, and the CC271 strains carrying the Tn2010 element expressing the high‐level resistance to erythromycin were predominant in China. Significance and Impact of the Study: This is the first molecular analysis of erythromycin‐resistant Streptococcus pneumoniae clinical isolates in China, and the first report of the complete nucleotide sequence of Tn2010 (26 390 bp).     

Functional interdependence among ribosomal elements as revealed by genetic analysis

Summary Escherichia coli strains with preexisting ribosomal mutations were used in order to isolate further ribosomal mutations. The ribosomal mutations used were resistance to erythromycin, spectinomycin, streptomycin or kasugamycin. These mutations cause alteration of specific ribosomal elements, L4, S5, S12 proteins and 16S rRNA respectively. Mutations have been introduced into strains carrying one, two or three of these mutations. Strains with all possible combinations of these four mutations were constructed. The phenotypes of all isolated mutants were tested, and frequently the strains lost one or more of their pre-existing resistances.Thus, functional interactions were revealed among proteins, as well as RNA and proteins within the 30 S ribosomal subunit and as well as between the 30 S and the 50 S ribosomal subunits.     

Action of synergimycins and macrolides on in vivo and in vitro protein synthesis in archaebacteria

Summary Single synergimycins (virginiamycin M or VM, type A component, and virginiamycin S or VS, type B component) inhibit reversibly growth and protein synthesis in Bacillus subtilis; a mixture of VM and VS produces viability loss and irreversible halting of peptide bond formation. In vitro, VM produces a five- to tenfold increase of the affinity of Escherichia coli ribosomes for VS, and erythromycin, which competes with VS for binding to eubacterial 50S subunits, is ineffective in the presence of VM. In the present work, the action of synergimycins and macrolides has been explored in vivo and in vitro on methanogenic and sulphurdependent archaebacteria. Multiplication of Methanococcus vannielii was synergistically inhibited by VM plus VS (for technical reasons, the action of synergimycins on growth and viability of most archaebacteria was unverifiable). When assayed on cell-free systems for protein synthesis from methanogens, both macrolides and single synergimycins were found ineffective. However, a mixture of VM and VS strongly inhibited poly(U)-directed polyphenylalanine synthesis. Binding of erythromycin to archaebacterial ribosomes and subunits was 10% (Mc. vannielii) or less than the control value (E. coli), and was not competed for by tylosin. The association constant of VS-50S complex formation, although low in the case of Mc. vannielii (as compared to enterobacteria), underwent a 100-fold increase in the presence of VM and was unaffected by macrolides. These data further stress the difference of the organization for protein synthesis of eubacteria and archaebacteria.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Improvement of Bacillus thuringiensis δ-endotoxins Synthesis Yields Through Acquisition of Erythromycin Resistance

The acquisition of the erythromycin resistance by Bacillus thuringiensis kurstaki improved yields of δ-endotoxins in sporulating cells ranging from 134 to 215%. Resistance to erythromycin decreased the final spore count by at least 50%. Consequently, erythromycin resistance is an efficient tool for the improvement of bioinsecticides yields with a high ratio of δ-endotoxins to spores. Revisions requested 31 October 2005; Revisions received 28 November 2005     

Erythromycin resistant mutants of Bacillus subtilis

Summary Erythromycin resistant (ery ^r) mutants were isolated from Bacillus subtilis ATCC 6633. The composition of ribosomal proteins were analyzed for thirteen such ery ^r-mutants with chromatography on a carboxymethyl cellulose (CMC) column. The 50s subunit from all of the ery ^r-mutants was found to contain the altered 50d protein. The ribosomes prepared from the ery ^r-mutants did not show in vitro alteration of the ability to combine with erythromycin.     

酸枣果提取物在体内外对抗生素的增效作用及机制研究

作者研究团队先前从酸枣果的氯仿提取物中精制得到其低极性范围的活性组合物Fr.2a,发现Fr.2a与多种抗生素联用显示出广泛的协同抗菌作用。该研究在Fr.2a的基础上利用硅胶柱层析对酸枣果氯仿提取物中其他极性范围内的活性成分进行了分离纯化,得到精制物Fr.B,并对精制物Fr.B进行GC MS、核磁共振氢谱、红外光谱分析,以确定Fr.B的组成成分;通过抗菌谱分析和细胞通透性分析,以明确Fr.B的抗菌增效谱和抗菌增效机制;采用熔和法将精制物Fr.B制备成软膏,通过小鼠伤口感染模型评价该软膏对抗生素的增效效果。结果表明：(1)由酸枣果氯仿提取物进一步精制得到的Fr.B组分,主要包含反油酸、油酸、顺 10 十六碳烯醇、棕榈酸等脂肪酸类化合物。(2)Fr.B分别与庆大霉素、妥布霉素、氨苄青霉素、氯霉素、红霉素、夫西地酸、制霉菌素、酮康唑和两性霉素B等多种抗生素联用时显示出广泛的协同抗菌作用。(3)Fr.B可破坏细胞膜和细胞壁的完整性而增强细菌细胞的通透性。(4)在体内和体外Fr.B均能显著增强红霉素对耐甲氧西林金黄色葡萄球菌(MRSA)的杀菌作用,从而提高红霉素对MRSA菌株引起的伤口感染的治疗效果。研究表明,本研究所得到的Fr.B具有广谱的抗菌增效活性,能够增强红霉素对伤口耐药菌感染的治疗效果。该研究结果为克服微生物对抗生素的耐药性提供了新的思路和解决方案。