Structural analysis of kasugamycin inhibition of translation

The prokaryotic ribosome is an important target of antibiotic action. We determined the X-ray structure of the aminoglycoside kasugamycin (Ksg) in complex with the Escherichia coli 70S ribosome at 3.5-A resolution. The structure reveals that the drug binds within the messenger RNA channel of the 30S subunit between the universally conserved G926 and A794 nucleotides in 16S ribosomal RNA, which are sites of Ksg resistance. To our surprise, Ksg resistance mutations do not inhibit binding of the drug to the ribosome. The present structural and biochemical results indicate that inhibition by Ksg and Ksg resistance are closely linked to the structure of the mRNA at the junction of the peptidyl-tRNA and exit-tRNA sites (P and E sites).     

Mutational analysis of the conserved bases C1402 and A1500 in the center of the decoding domain of Escherichia coli 16 S rRNA reveals an important tertiary interaction

Interactions within the decoding center of the 30 S ribosomal subunit have been investigated by constructing all 15 possible mutations at nucleotides C1402 and A1500 in helix 44 of 16 S rRNA. As expected, most of the mutations resulted in highly deleterious phenotypes, consistent with the high degree of conservation of this region and its functional importance. A total of seven mutants were viable under conditions where the mutant ribosomes comprised 100 % of the ribosomal pool. A suppressor mutation specific for the C1402U-A1500G mutant was isolated at position 1520 in helix 45 of 16 S rRNA. In addition, lack of dimethylation of A1518/A1519 caused by mutation of the ksgA methylase enhanced the deleterious effect of many of the 1402/1500 mutations. These data suggest that a higher-order interaction between helices 44 and 45 in 16 S rRNA is important for the proper functioning of the ribosome. This is consistent with the recent high-resolution crystal structures of the 30 S subunit, which show a tertiary interaction between the 1402/1500 region of helix 44 and the dimethyl A stem loop.     

Orientation of the tRNA anticodon in the ribosomal P-site: quantitative footprinting with U33-modified, anticodon stem and loop domains.

Binding of transfer RNA (tRNA) to the ribosome involves crucial tRNA-ribosomal RNA (rRNA) interactions. To better understand these interactions, U33-substituted yeast tRNA(Phe) anticodon stem and loop domains (ASLs) were used as probes of anticodon orientation on the ribosome. Orientation of the anticodon in the ribosomal P-site was assessed with a quantitative chemical footprinting method in which protection constants (Kp) quantify protection afforded to individual 16S rRNA P-site nucleosides by tRNA or synthetic ASLs. Chemical footprints of native yeast tRNA(Phe), ASL-U33, as well as ASLs containing 3-methyluridine, cytidine, or deoxyuridine at position 33 (ASL-m3U33, ASL-C33, and ASL-dU33, respectively) were compared. Yeast tRNAPhe and the ASL-U33 protected individual 16S rRNA P-site nucleosides differentially. Ribosomal binding of yeast tRNA(Phe) enhanced protection of C1400, but the ASL-U33 and U33-substituted ASLs did not. Two residues, G926 and G1338 with KpS approximately 50-60 nM, were afforded significantly greater protection by both yeast tRNA(Phe) and the ASL-U33 than other residues, such as A532, A794, C795, and A1339 (KpS approximately 100-200 nM). In contrast, protections of G926 and G1338 were greatly and differentially reduced in quantitative footprints of U33-substituted ASLs as compared with that of the ASL-U33. ASL-m3U33 and ASL-C33 protected G530, A532, A794, C795, and A1339 as well as the ASL-U33. However, protection of G926 and G1338 (KpS between 70 and 340 nM) was significantly reduced in comparison to that of the ASL-U33 (43 and 61 nM, respectively). Though protections of all P-site nucleosides by ASL-dU33 were reduced as compared to that of the ASL-U33, a proportionally greater reduction of G926 and G1338 protections was observed (KpS = 242 and 347 nM, respectively). Thus, G926 and G1338 are important to efficient P-site binding of tRNA. More importantly, when tRNA is bound in the ribosomal P-site, G926 and G1338 of 16S rRNA and the invariant U33 of tRNA are positioned close to each other.     

Erythromycin resistance mutations in ribosomal proteins L22 and L4 perturb the higher order structure of 23 S ribosomal RNA.

We have used chemical modification to examine the conformation of 23 S rRNA in Escherichia coli ribosomes bearing erythromycin resistance mutations in ribosomal proteins L22 and L4. Changes in reactivity to chemical probes were observed at several nucleotide positions scattered throughout 23 S rRNA. The L4 mutation affects the reactivity of G799 and U1255 in domain II and that of A2572 in domain V. The L22 mutation influences modification in domain II at positions m5U747, G748, and A1268, as well as at A1614 in domain III and G2351 in domain V. The reactivity of A789 is weakly enhanced by both the L22 and L4 mutations. None of these nucleotide positions has previously been associated with macrolide antibiotic resistance. Interestingly, neither of the ribosomal protein mutations produces any detectable effects at or within the vicinity of A2058 in domain V, the site most frequently shown to confer macrolide resistance when altered by methylation or mutation. Thus, while L22 and L4 bind primarily to domain I of 23 S rRNA, erythromycin resistance mutations in these ribosomal proteins perturb the conformation of residues in domains II, III and V and affect the action of antibiotics known to interact with nucleotide residues in the peptidyl transferase center of domain V. These results support the hypothesis that ribosomal proteins interact with rRNA at multiple sites to establish its functionally active three-dimensional structure, and suggest that these antibiotic resistance mutations act by perturbing the conformation of rRNA.     

Kasugamycin methylase modified by ribosomal RNA from group A streptococci]

Kasugamycin sensitivity in Escherichia coli depends on the specific enzyme methylating rRNA. Native group A streptococci (GAS) were found to be sensitive to kasugamycin. After introduction of the erythromycin gene located on the transposon Tn916E into GAS some of the strains obtained kasugamycin resistance together with erythromycin resistance (erm). One of these strains carrying the transposon in its chromosome was tested for methylase activity. It was demonstrated to be deficient in kasugamycin methylase (ksg). The presented data proves the presence of ksg methylase in GAS. Evolutionary relationship between erm and ksg genes is discussed.     

Oxazolidinone resistance mutations in 23S rRNA of Escherichia coli reveal the central region of domain V as the primary site of drug action

Oxazolidinone antibiotics inhibit bacterial protein synthesis by interacting with the large ribosomal subunit. The structure and exact location of the oxazolidinone binding site remain obscure, as does the manner in which these drugs inhibit translation. To investigate the drug-ribosome interaction, we selected Escherichia coli oxazolidinone-resistant mutants, which contained a randomly mutagenized plasmid-borne rRNA operon. The same mutation, G2032 to A, was identified in the 23S rRNA genes of several independent resistant isolates. Engineering of this mutation by site-directed mutagenesis in the wild-type rRNA operon produced an oxazolidinone resistance phenotype, establishing that the G2032A substitution was the determinant of resistance. Engineered U and C substitutions at G2032, as well as a G2447-to-U mutation, also conferred resistance to oxazolidinone. All the characterized resistance mutations were clustered in the vicinity of the central loop of domain V of 23S rRNA, suggesting that this rRNA region plays a major role in the interaction of the drug with the ribosome. Although the central loop of domain V is an essential integral component of the ribosomal peptidyl transferase, oxazolidinones do not inhibit peptide bond formation, and thus these drugs presumably interfere with another activity associated with the peptidyl transferase center.     

Interaction of antibiotics with A- and P-site-specific bases in 16S ribosomal RNA.

We have studied the interactions of the antibiotics apramycin, kasugamycin, myomycin, neamine and pactamycin with 16S rRNA by chemical probing of drug-ribosome complexes. Kasugamycin and pactamycin, which are believed to affect translational initiation, protect bases in common with P-site-bound tRNA. While kasugamycin protects A794 and G926, and causes enhanced reactivity of C795, pactamycin protects G693 and C795. All four of these bases were previously shown to be protected by P-site tRNA or by edeine, another P-site inhibitor. Apramycin and neamine, which both induce miscoding and inhibit translocation, protect A1408, G1419 and G1494, as was also found earlier for neomycin, gentamicin, kanamycin and paromomycin. A1408 and G1494 were previously shown to be protected by A-site tRNA. Surprisingly, myomycin fails to give strong protection of any bases in 16S rRNA, in spite of having an apparently identical target site and mode of action to streptomycin, which protects several bases in the 915 region. Instead, myomycin gives only weak protection of A1408. These results suggest that the binding site(s) of streptomycin and myomycin have yet to be identified.     

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.     

Mutations in rsmG, encoding a 16S rRNA methyltransferase, result in low-level streptomycin resistance and antibiotic overproduction in Streptomyces coelicolor A3(2)

Certain str mutations that confer high- or low-level streptomycin resistance result in the overproduction of antibiotics by Streptomyces spp. The str mutations that confer the high-level resistance occur within rpsL, which encodes the ribosomal protein S12, while those that cause low-level resistance are not as well known. We have used comparative genome sequencing to determine that low-level resistance is caused by mutations of rsmG, which encodes an S-adenosylmethionine (SAM)-dependent 16S rRNA methyltransferase containing a SAM binding motif. Deletion of rsmG from wild-type Streptomyces coelicolor resulted in the acquisition of streptomycin resistance and the overproduction of the antibiotic actinorhodin. Introduction of wild-type rsmG into the deletion mutant completely abrogated the effects of the rsmG deletion, confirming that rsmG mutation underlies the observed phenotype. Consistent with earlier work using a spontaneous rsmG mutant, the strain carrying DeltarsmG exhibited increased SAM synthetase activity, which mediated the overproduction of antibiotic. Moreover, high-performance liquid chromatography analysis showed that the DeltarsmG mutant lacked a 7-methylguanosine modification in the 16S rRNA (possibly at position G518, which corresponds to G527 of Escherichia coli). Like certain rpsL mutants, the DeltarsmG mutant exhibited enhanced protein synthetic activity during the late growth phase. Unlike rpsL mutants, however, the DeltarsmG mutant showed neither greater stability of the 70S ribosomal complex nor increased expression of ribosome recycling factor, suggesting that the mechanism underlying increased protein synthesis differs in the rsmG and the rpsL mutants. Finally, spontaneous rsmG mutations arose at a 1,000-fold-higher frequency than rpsL mutations. These findings provide new insight into the role of rRNA modification in activating secondary metabolism in Streptomyces.     

Isolation of spectinomycin resistance mutations in the 16S rRNA of Salmonella enterica serovar Typhimurium and expression in Escherichia coli and Salmonella

Two single-base mutations in 16S rRNA conferring high-level resistance to spectinomycin were isolated on a plasmid-borne copy of the rrnD operon from Salmonella enterica serovar Typhimurium. Neither of the mutations (C1066U and C1192U) had appreciable effects on cell growth, but each had differential effects on resistance to spectinomycin and fusidic acid. Both mutations also conferred resistance to spectinomycin in Escherichia coli strains containing deletions of all seven chromosomal rrn operons and expressing plasmid-encoded Salmonella rRNA exclusively. In contrast, when expressed in E. coli strains containing intact chromosomal rrn operons, the strains were sensitive to spectinomycin. However, chromosomal mutations arose that allowed expression of the rRNA-dependent spectinomycin resistance phenotype. It is proposed that in heterogeneous rRNA populations, the native E. coli rRNA out-competes the heterologous Salmonella rRNA for binding to ribosomal proteins, translation factors, or ribosome assembly, thus limiting entry of the antibiotic-resistant 30S subunits into the functioning ribosome pool.     

The aminoglycoside resistance methyltransferase Sgm impedes RsmF methylation at an adjacent rRNA nucleotide in the ribosomal A site

Ribosome-targeting antibiotics block protein synthesis by binding at functionally important regions of the bacterial rRNA. Resistance is often conferred by addition of a methyl group at the antibiotic binding site within an rRNA region that is already highly modified with several nucleotide methylations. In bacterial rRNA, each methylation requires its own specific methyltransferase enzyme, and this raises the question as to how an extra methyltransferase conferring antibiotic resistance can be accommodated and how it can gain access to its nucleotide target within a short and functionally crowded stretch of the rRNA sequence. Here, we show that the Sgm methyltransferase confers resistance to 4,6-disubstituted deoxystreptamine aminoglycosides by introducing the 16S rRNA modification m⁷G1405 within the ribosomal A site. This region of Escherichia coli 16S rRNA already contains several methylated nucleotides including m⁴Cm1402 and m⁵C1407. Modification at m⁵C1407 by the methyltransferase RsmF is impeded as Sgm gains access to its adjacent G1405 target on the 30S ribosomal subunit. An Sgm mutant (G135A), which is impaired in S-adenosylmethionine binding and confers lower resistance, is less able to interfere with RsmF methylation on the 30S subunit. The two methylations at 16S rRNA nucleotide m⁴Cm1402 are unaffected by both the wild-type and the mutant versions of Sgm. The data indicate that interplay between resistance methyltransferases and the cell''s own indigenous methyltransferases can play an important role in determining resistance levels.     

Site-directed mutagenesis of Escherichia coli 23 S ribosomal RNA at position 1067 within the GTP hydrolysis centre

Site-directed mutagenesis has been used to change, specifically, residue 1067 within 23 S ribosomal RNA of Escherichia coli. This nucleoside (adenosine in the wild-type sequence) lies within the GTPase centre of the larger ribosomal subunit and is normally the target for the methylase enzyme responsible for resistance to the antibiotic thiostrepton. The performance of the altered ribosomes was not impaired in cell-free protein synthesis nor in GTP hydrolysis assays (although the 3 mutant strains grew somewhat more slowly than wild-type) but their responses to thiostrepton did vary. Thus, ribosomes containing the A to C or A to U substitution at residue 1067 of 23 S rRNA were highly resistant to the drug, whereas the A to G substitution resulted in much lesser impairment of thiostrepton binding and the ribosomes remained substantially sensitive to the antibiotic. These data reinforce the hypothesis that thiostrepton binds to 23 S rRNA at a site that includes residue A1067. They also exclude any possibility that the insensitivity of eukaryotic ribosomes to the drug might be due solely to the substitution of G at the equivalent position within eukaryotic rRNA.     

Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16 S rRNA

Transfer RNA protects a characteristic set of bases in 16 S rRNA from chemical probes when it binds to ribosomes. We used several criteria, based on construction of well-characterized in vitro ribosome-tRNA complexes, to assign these proteins to A or P-site binding. All of these approaches lead to similar conclusions. In the A site, tRNA caused protection of G529, G530, A1492 and A1493 (strongly), and A1408 and G1494 (weakly). In the P site, the protected bases are G693, A794, C795, G926 and G1401 (strong), and A532, G966, G1338 and G1339 (weak). In contrast to what is observed for 23 S rRNA, blocking the release of EF-Tu.GDP from the ribosome by kirromycin has no detectable effect on the protection of bases in 16 S rRNA.     

Induction of ermC requires translation of the leader peptide.

ermC confers resistance to macrolide-lincosamide streptogramin B antibiotics by specifying a ribosomal RNA methylase, which results in decreased ribosomal affinity for these antibiotics. ermC expression is induced by exposure to erythromycin. We have previously proposed a translational regulation model in which erythromycin causes stalling of a ribosome, which is translating a leader peptide. Stalling causes a conformation shift in the ermC mRNA which in turn unmasks the methylase ribosomal binding site. A prediction of this translational attenuation model for ermC induction was tested by replacing the second codon of the putative ermC leader peptide coding region by TAA. As expected, the introduction of this mutation resulted in an uninducible phenotype which was suppressible by two ochre suppressor mutations in Bacillus subtilis. It is concluded that translation through the leader peptide coding region, in frame with the predicted leader peptide, is required for ermC induction.     

Increased kasugamycin sensitivity in Escherichia coli caused by the presence of an inducible erythromycin resistance (erm) gene of Streptococcus pyogenes

Summary An inducible erythromycin resistance gene (erm) of Streptococcus pyogenes was introduced into Escherichia coli by transformation with a plasmid. The recipient E. coli cells were either kasugamycin sensitive (wildtype) or kasugamycin resistant (ksgA). The MIC values of erythromycin increased from 150 g/ml to>3000 g/ml for E. coli. An extract of transformed cells, particularly a high-salt ribosomal wash, contained an enzyme that was able to methylate 23S rRNA from untransformed cells in vitro; however, 23S rRNA from transformed cells was not a substrate for methylation by such an extract. 165 rRNA and 30S ribosomal subunits of either the wild type or a kasugamycin resistant (ksgA) mutant were not methylated in vitro. Transformation of E. coli by the erm-containing plasmid led to a reduction of the MIC values for kasugamycin. This happened in wild-type as well as in ksgA cells. However, in vitro experiments with purified ksgA encoded methylase demonstrated that also in erm transformed E. coli, the ksgA encoded enzyme was active in wild-type, but not in ksgA cells. It was also shown by in vitro experiments that ribosomes from erm ksgA cells have become sensitive to kasugamycin. Our experiments show that in vivo methylation of 23S rRNA, presumably of the adenosine at position 2058, leads to enhanced resistance to erythromycin and to reduced resistance to kasugamycin. This, together with previous data, argues for a close proximity of the two sites on the ribosome that are substrates for adenosine dimenthylation.Abbreviations MLS macrolide, lincosamide, streptogramin B     

Mechanism of Kasugamycin Resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Strains of Escherichia coli are resistant to the antibiotic kasugamycin due to the partial non-methylation of 16S ribosomal RNA. An RNA methylase activity, absent from resistant strains, is shown here to methylate in vitro the 16S RNA of resistant as well as sensitive strains.     

Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes

Binding of tRNAPhe to ribosomes shields a set of highly conserved nucleotides in 16S rRNA from attack by a combination of structure-specific chemical probes. The bases can be classified according to whether or not their protection is strictly poly(U)-dependent (G529, G530, U531, A1408, A1492, and A1493) or poly(U)-independent (A532, G693, A794, C795, G926, 2mG966, G1338, A1339, U1381, C1399, C1400, and G1401). A third class (A790, G791, and A909) is shielded by both tRNA and 50S ribosomal subunits. Similar results are obtained when the protecting ligand is tRNAPhe E. Coli, tRNAPhe yeast, tRNAPhe E. Coli lacking its 3' terminal CA, or the 15 nucleotide anticodon stem-loop fragment of tRNAPhe yeast. Implications for structural correlates of the classic ribosomal A- and P-sites and for the possible involvement of 16S rRNA in translational proofreading are discussed.