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.     

Modulation of 16S rRNA function by ribosomal protein S12

Ribosomal protein S12 is a critical component of the decoding center of the 30S ribosomal subunit and is involved in both tRNA selection and the response to streptomycin. We have investigated the interplay between S12 and some of the surrounding 16S rRNA residues by examining the phenotypes of double-mutant ribosomes in strains of Escherichia coli carrying deletions in all chromosomal rrn operons and expressing total rRNA from a single plasmid-borne rrn operon. We show that the combination of S12 and otherwise benign mutations at positions C1409-G1491 in 16S rRNA severely compromises cell growth while the level and range of aminoglycoside resistances conferred by the G1491U/C substitutions is markedly increased by a mutant S12 protein. The G1491U/C mutations in addition confer resistance to the unrelated antibiotic, capreomycin. S12 also interacts with the 912 region of 16S rRNA. Genetic selection of suppressors of streptomycin dependence caused by mutations at proline 90 in S12 yielded a C912U substitution in 16S rRNA. The C912U mutation on its own confers resistance to streptomycin and restricts miscoding, properties that distinguish it from a majority of the previously described error-promoting ram mutants that also reverse streptomycin dependence.     

Analysis of streptomycin-resistance of Escherichia coli mutants.

We previously reported about Escherichia coli transformation experiments yielding streptomycin-resistant cells carrying a C912 to T transition in a plasmid-born 16S rRNA gene. These experiments were based on results obtained with streptomycin-resistant Euglena chloroplasts bearing an equivalent mutation in the single chloroplast 16S rRNA gene. We extended this study and transformed E. coli with plasmid constructs having a mutated 16S rRNA gene at position 914 (A to C) or a double mutation at positions 912 and 888 (C to T:G to A) or a mutation in the S12 gene (Lys-42 to Thr). We tested the transformed cells before and after a screening procedure in the presence of streptomycin. We find that the plasmid-born mutations protect colonies against a short streptomycin exposure, but ribosomes carrying mutated 16S rRNA do not significantly reduce codon misreading in vitro. However, ribosomes isolated from transformed cells after the screening procedure resist misreading. These ribosomes have acquired a second mutation in the S12 protein as shown in one case by sequencing and by transformation experiments. Furthermore, we show that the A914 to C mutation prevents (strongly reduces) base methylation in the central domain of 16S rRNA.     

Molecular analysis of kanamycin and viomycin resistance in Mycobacterium smegmatis by use of the conjugation system.

We examined the molecular mechanisms of resistance to kanamycin and viomycin in Mycobacterium smegmatis. All of the M. smegmatis strains with high-level kanamycin resistance had a nucleotide substitution from A to G at position 1389 of the 16S rRNA gene (rrs). This position is equivalent to position 1408 of Escherichia coli, and mutation at this position is known to cause aminoglycoside resistance. Mutations from G to A or G to T at position 1473 of the M. smegmatis rrs gene were found in viomycin-resistant mutants which had been designated vicB mutants in our earlier studies. Using the M. smegmatis conjugation system, we confirmed that these mutations indeed contributed to kanamycin and viomycin resistance, and kanamycin susceptibility was dominant over resistance in a heterogenomic strain. Additional experiments showed that three of four Mycobacterium tuberculosis strains with high-level kanamycin resistance had a mutation from A to G at position 1400, which was equivalent to position 1389 of M. smegmatis.     

Loss of a conserved 7-methylguanosine modification in 16S rRNA confers low-level streptomycin resistance in bacteria

Streptomycin has been an important drug for the treatment of tuberculosis since its discovery in 1944. But numerous strains of Mycobacterium tuberculosis, the bacterial pathogen that causes tuberculosis, are now streptomycin resistant. Although such resistance is often mediated by mutations within rrs, a 16S rRNA gene or rpsL, which encodes the ribosomal protein S12, these mutations are found in a limited proportion of clinically isolated streptomycin-resistant M. tuberculosis strains. Here we have succeeded in identifying a mutation that confers low-level streptomycin resistance to bacteria, including M. tuberculosis. We found that mutations within the gene gidB confer low-level streptomycin resistance and are an important cause of resistance found in 33% of resistant M. tuberculosis isolates. We further clarified that the gidB gene encodes a conserved 7-methylguanosine (m(7)G) methyltransferase specific for the 16S rRNA, apparently at position G527 located in the so-called 530 loop. Thus, we have identified gidB as a new streptomycin-resistance locus and uncovered a resistance mechanism that is mediated by loss of a conserved m(7)G modification in 16S rRNA. The clinical significance of M. tuberculosis gidB mutation also is noteworthy, as gidB mutations emerge spontaneously at a high frequency of 10(-6) and, once emerged, result in vigorous emergence of high-level streptomycin-resistant mutants at a frequency more than 2000 times greater than that seen in wild-type strains. Further studies on the precise function of GidB may provide a basis for developing strategies to suppress pathogenic bacteria, including M. tuberculosis.     

Basis for prokaryotic specificity of action of aminoglycoside antibiotics.

The aminoglycosides, a group of structurally related antibiotics, bind to rRNA in the small subunit of the prokaryotic ribosome. Most aminoglycosides are inactive or weakly active against eukaryotic ribosomes. A major difference in the binding site for these antibiotics between prokaryotic and eukaryotic ribosomes is the identity of the nucleotide at position 1408 (Escherichia coli numbering), which is an adenosine in prokaryotic ribosomes and a guanosine in eukaryotic ribosomes. Expression in E.coli of plasmid-encoded 16S rRNA containing an A1408 to G substitution confers resistance to a subclass of the aminoglycoside antibiotics that contain a 6' amino group on ring I. Chemical footprinting experiments indicate that resistance arises from the lower affinity of the drug for the eukaryotic rRNA sequence. The 1408G ribosomes are resistant to the same subclass of aminoglycosides as previously observed both for eukaryotic ribosomes and bacterial ribosomes containing a methylation at the N1 position of A1408. The results indicate that the identity of the nucleotide at position 1408 is a major determinant of specificity of aminoglycoside action, and agree with prior structural studies of aminoglycoside-rRNA complexes.     

Identification of the RsmG methyltransferase target as 16S rRNA nucleotide G527 and characterization of Bacillus subtilis rsmG mutants

The methyltransferase RsmG methylates the N7 position of nucleotide G535 in 16S rRNA of Bacillus subtilis (corresponding to G527 in Escherichia coli). Disruption of rsmG resulted in low-level resistance to streptomycin. A growth competition assay revealed that there are no differences in fitness between the rsmG mutant and parent strains under the various culture conditions examined. B. subtilis rsmG mutants emerged spontaneously at a relatively high frequency, 10(-6). Importantly, in the rsmG mutant background, high-level-streptomycin-resistant rpsL (encoding ribosomal protein S12) mutants emerged at a frequency 200 times greater than that seen for the wild-type strain. This elevated frequency in the emergence of high-level streptomycin resistance was facilitated by a mutation pattern in rpsL more varied than that obtained by selection of the wild-type strain.     

A single base substitution in 16S ribosomal RNA suppresses streptomycin dependence and increases the frequency of translational errors

A C to U substitution at position 1469 of 16S ribosomal RNA (rRNA) from Escherichia coli suppresses streptomycin dependence and causes increased translational error frequencies. Strains containing the rpsL252 or StrM287 streptomycin-dependent alleles are able to grow in the absence of streptomycin when transformed with plasmids containing the U1469 mutation in 16S rRNA. Ribosomes containing wild-type proteins and U1469 mutant 16S rRNA misincorporate leucine in vitro at elevated levels, comparable to that of some typical S4 ram ribosomes. These results provide additional support for the participation of 16S rRNA in maintaining translational accuracy.     

Mutagenesis of 16S rRNA C1409-G1491 base-pair differentiates between 6'OH and 6'NH3+ aminoglycosides

Using a single rRNA allelic Gram-positive model system, we systematically mutagenized 16S rRNA positions 1409 and 1491 to probe the functional relevance of structural interactions between aminoglycoside antibiotics and the A-site rRNA that were suggested by X-ray crystallography. At the structural level, the interaction of the 2-deoxystreptamine aminoglycosides with the rRNA base-pair C1409-G1491 has been suggested to involve the following features: (i) ring I of the disubstituted 2-deoxystreptamines stacks upon G1491 and H-bonds to the Watson-Crick edge of A1408; (ii) ring III of the 4,5-disubstituted aminoglycosides shows hydrogen bonding to G1491. However, we found that mutants with altered 16S rRNA bases 1409 and 1491 discriminated poorly between 4,5-disubstituted and 4,6-disubstituted 2-deoxystreptamines, but differentially affected aminoglycosides with a hydroxyl group versus an ammonium group at position 6' of ring I, e.g. G1491U conferred high-level drug resistance to paromomycin and geneticin, but not to neomycin, tobramycin or gentamicin.     

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.     

Structural insights into the function of aminoglycoside-resistance A1408 16S rRNA methyltransferases from antibiotic-producing and human pathogenic bacteria

X-ray crystal structures were determined of the broad-spectrum aminoglycoside-resistance A1408 16S rRNA methyltransferases KamB and NpmA, from the aminoglycoside-producer Streptoalloteichus tenebrarius and human pathogenic Escherichia coli, respectively. Consistent with their common function, both are Class I methyltransferases with additional highly conserved structural motifs that embellish the core SAM-binding fold. In overall structure, the A1408 rRNA methyltransferase were found to be most similar to a second family of Class I methyltransferases of distinct substrate specificity (m⁷G46 tRNA). Critical residues for A1408 rRNA methyltransferase activity were experimentally defined using protein mutagenesis and bacterial growth assays with kanamycin. Essential residues for SAM coenzyme binding and an extended protein surface that likely interacts with the 30S ribosomal subunit were thus revealed. The structures also suggest potential mechanisms of A1408 target nucleotide selection and positioning. We propose that a dynamic extended loop structure that is positioned adjacent to both the bound SAM and a functionally critical structural motif may mediate concerted conformational changes in rRNA and protein that underpin the specificity of target selection and activation of methyltransferase activity. These new structures provide important new insights that may provide a starting point for strategies to inhibit these emerging causes of pathogenic bacterial resistance to aminoglycosides.     

Mapping of chloroplast mutations conferring resistance to antibiotics in Chlamydomonas: Evidence for a novel site of streptomycin resistance in the small subunit rRNA

Summary A major obstacle to out understanding of the mechanisms governing the inheritance, recombination and segregation of chloroplast genes in Chlamydomonas is that the majority of antibiotic resistance mutations that have been used to gain insights into such mechanisms have not been physically localized on the chloroplast genome. We report here the physical mapping of two chloroplast antibiotic resistance mutations: one conferring cross-resistance to erythromycin and spiramycin in Chlamydomonas moewusii (er-nM1) and the other conferring resistance to streptomycin in the interfertile species C. eugametos (sr-2). The er-nM1 mutation results from a C to G transversion at a well-known site of macrolide resistance within the peptidyl transferase loop region of the large subunit rRNA gene. This locus, designated rib-2 in yeast mitochondrial DNA, corresponds to residue C-2611 in the 23 S rRNA of Escherichia coli. The sr-2 locus maps within the small subunit (SSU) rRNA gene at a site that has not been described previously. The mutation results from an A to C transversion at a position equivalent to residue A-523 in the E. coli 16 S rRNA. Although this region of the E. coli SSU rRNA has no binding affinity for streptomycin, it binds to ribosomal protein S4, a protein that has long been associated with the response of bacterial cells to this antibiotic. We propose that the sr-2 mutation indirectly affects the nearest streptomycin binding site through an altered interaction between a ribosomal protein and the SSU rRNA.     

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.     

Functional dichotomy in the 16S rRNA (m1A1408) methyltransferase family and control of catalytic activity via a novel tryptophan mediated loop reorganization

Methylation of the bacterial small ribosomal subunit (16S) rRNA on the N1 position of A1408 confers exceptionally high-level resistance to a broad spectrum of aminoglycoside antibiotics. Here, we present a detailed structural and functional analysis of the Catenulisporales acidiphilia 16S rRNA (m¹A1408) methyltransferase (‘CacKam’). The apo CacKam structure closely resembles other m¹A1408 methyltransferases within its conserved SAM-binding fold but the region linking core β strands 6 and 7 (the ‘β6/7 linker’) has a unique, extended structure that partially occludes the putative 16S rRNA binding surface, and sequesters the conserved and functionally critical W203 outside of the CacKam active site. Substitution of conserved residues in the SAM binding pocket reveals a functional dichotomy in the 16S rRNA (m¹A1408) methyltransferase family, with two apparently distinct molecular mechanisms coupling cosubstrate/ substrate binding to catalytic activity. Our results additionally suggest that CacKam exploits the W203-mediated remodeling of the β6/7 linker as a novel mechanism to control 30S substrate recognition and enzymatic turnover.     

A mutation in the 530 loop of Escherichia coli 16S ribosomal RNA causes resistance to streptomycin.

Oligonucleotide-directed mutagenesis was used to introduce an A to C transversion at position 523 in the 16S ribosomal RNA gene of Escherichia coli rrnB operon cloned in plasmid pKK3535. E. coli cells transformed with the mutated plasmid were resistant to streptomycin. The mutated ribosomes isolated from these cells were not stimulated by streptomycin to misread the message in a poly(U)-directed assay. They were also restrictive to the stimulation of misreading by other error-promoting related aminoglycoside antibiotics such as neomycin, kanamycin or gentamicin, which do not compete for the streptomycin binding site. The 530 loop where the mutation in the 16S rRNA is located has been mapped at the external surface of the 30S subunit, and is therefore distal from the streptomycin binding site at the subunit interface. Our results support the conclusion that the mutation at position 523 in the 16S rRNA does not interfere with the binding of streptomycin, but prevents the drug from inducing conformational changes in the 530 loop which account for its miscoding effect. Since this effect primarily results from a perturbation of the translational proofreading control, our results also provide evidence that the 530 loop of the 16S rRNA is involved in this accuracy control.     

Structural origins of aminoglycoside specificity for prokaryotic ribosomes

Aminoglycoside antibiotics, including paromomycin, neomycin and gentamicin, target a region of highly conserved nucleotides in the decoding region aminoacyl-tRNA site (A site) of 16 S rRNA on the 30 S subunit. Change of a single nucleotide, A1408 to G, reduces the affinity of many aminoglycosides for the ribosome; G1408 distinguishes between prokaryotic and eukaryotic ribosomes. The structures of a prokaryotic decoding region A-site oligonucleotide free in solution and bound to the aminoglycosides paromomycin and gentamicin C1a were determined previously. Here, the structure of a eukaryotic decoding region A-site oligonucleotide bound to paromomycin has been determined using NMR spectroscopy and compared to the prokaryotic A-site-paromomycin structure. A conformational change in three adenosine residues of an internal loop, critical for high-affinity antibiotic binding, was observed in the prokaryotic RNA-paromomycin complex in comparison to its free form. This conformational change is not observed in the eukaryotic RNA-paromomycin complex, disrupting the binding pocket for ring I of the antibiotic. The lack of the conformational change supports footprinting and titration calorimetry data that demonstrate approximately 25-50-fold weaker binding of paromomycin to the eukaryotic decoding-site oligonucleotide. Neomycin, which is much less active against Escherichia coli ribosomes with an A1408G mutation, binds non-specifically to the oligonucleotide. These results suggest that eukaryotic ribosomal RNA has a shallow binding pocket for aminoglycosides, which accommodates only certain antibiotics.     

Antibiotic Resistance Mutations in the Chloroplast 16s and 23s Rrna Genes of Chlamydomonas Reinhardtii: Correlation of Genetic and Physical Maps of the Chloroplast Genome

Mutants resistant to streptomycin, spectinomycin, neamine/kanamycin and erythromycin define eight genetic loci in a linear linkage group corresponding to about 21 kb of the circular chloroplast genome of Chlamydomonas reinhardtii. With one exception, all of these mutants represent single base-pair changes in conserved regions of the genes encoding the 16S and 23S chloroplast ribosomal RNAs. Streptomycin resistance can result from changes at the bases equivalent to Escherichia coli 13, 523, and 912-915 in the 16S gene, or from mutations in the rps12 gene encoding chloroplast ribosomal protein S12. In the 912-915 region of the 16S gene, three mutations were identified that resulted in different levels of streptomycin resistance in vitro. Although the three regions of the 16S rRNA mutable to streptomycin resistance are widely separated in the primary sequence, studies by other laboratories of RNA secondary structure and protein cross-linking suggest that all three regions are involved in a common ribosomal neighborhood that interacts with ribosomal proteins S4, S5 and S12. Three different changes within a conserved region of the 16S gene, equivalent to E. coli bases 1191-1193, confer varying levels of spectinomycin resistance, while resistance to neamine and kanamycin results from mutations in the 16S gene at bases equivalent to E. coli 1408 and 1409. Five mutations in two genetically distinct erythromycin resistance loci map in the 23S rDNA of C. reinhardtii, at positions equivalent to E. coli 2057-2058 and 2611, corresponding to the rib3 and rib2 loci of yeast mitochondria respectively. Although all five mutants are highly resistant to erythromycin, they differ in levels of cross-resistance to lincomycin and clindamycin. The order and spacing of all these mutations in the physical map are entirely consistent with our genetic map of the same loci and thereby validate the zygote clone method of analysis used to generate this map. These results are discussed in comparison with other published maps of chloroplast genes based on analysis by different methods using many of the same mutants.     

Expression,purification and crystallization of adenosine 1408 aminoglycoside-resistance rRNA methyltransferases for structural studies

High-level resistance to a broad spectrum of aminoglycoside antibiotics can arise through either N7-methyl guanosine 1405 (m⁷G1405) or N1-methyl adenosine 1408 (m¹A1408) modifications at the drug binding site in the bacterial 30S ribosomal subunit decoding center. Two distinct families of 16S ribosomal RNA (rRNA) methyltransferases that incorporate these modifications were first identified in aminoglycoside-producing bacteria but were more recently identified in both human and animal pathogens. These resistance determinants thus pose a new threat to the usefulness of aminoglycosides as antibiotics, demanding urgent characterization of their structures and activities. Here, we describe approaches to cloning, heterologous expression in Escherichia coli, and purification of two A1408 rRNA methyltransferases: KamB from the aminoglycoside-producer Streptoalloteichus tenebrarius and NpmA identified in a clinical isolate of pathogenic E. coli ARS3. Antibiotic minimum inhibitory concentration (MIC) assays and in vitro analysis of KamB and NpmA using circular dichroism (CD) spectroscopy, S-adenosyl-l-methionine (SAM) binding by isothermal titration calorimetry and 30S subunit methylation assays showed both enzymes were soluble, folded and active. Finally, crystals of each enzyme complexed with SAM were obtained, including selenomethionine-derived KamB, that will facilitate high-resolution X-ray crystallographic analyses of these important bacterial antibiotic-resistance determinants.