Sites of action of two ribosomal RNA methylases responsible for resistance to aminoglycosides.

Methylation of either of two residues (G-1405 or A-1408) within bacterial 16 S ribosomal RNA results in high level resistance to specific combinations of aminoglycoside antibiotics. The product of a gene that originated in Micromonospora purpurea (an actinomycete that produces gentamicin) gives resistance to kanamycin plus gentamicin by converting residue G-1405 to 7-methylguanosine. Resistance to kanamycin plus apramycin results from conversion of residue A-1408 to 1-methyladenosine catalysed by the product of a gene from Streptomyces tenjimariensis.

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.

The antibiotic micrococcin acts on protein L11 at the ribosomal GTPase centre.

Micrococcin-resistant mutants of Bacillus megaterium that carry mutations affecting ribosomal protein L11 have been characterised. The mutants fall into two groups. "L11-minus" strains containing an L11 gene with deletions, insertions or nonsense mutations which grow 2.5-fold slower than the wild-type strain, whereas other mutants carrying single-site substitutions within an 11 amino acid residue segment of the N-terminal domain of L11 grow normally. Protein L11 binds to 23 S rRNA within the ribosomal GTPase centre which regulates GTP hydrolysis on ribosomal factors. Micrococcin binding within the rRNA component of this centre was probed on wild-type and mutant ribosomes, in vivo, using dimethyl sulphate where it generated an rRNA footprint indistinguishable from that produced in vitro, even after the cell growth had been arrested by treatment with either kirromycin or fusidic acid. No drug-rRNA binding was detected in vivo for the L11-minus mutants, while reduced binding (approximately 30-fold) was observed for two single-site mutants P23L and P26L. For the latter, the reduced drug affinity alone did not account for the resistance-phenotype because rapid cell growth occurred even at drug concentrations that would saturate the ribosomes. Micrococcin was also bound to complexes containing an rRNA fragment and wild-type or mutant L11, expressed as fusion proteins, and they were probed with proteinases. The drug produced strong protection effects on the wild-type protein and weak effects on the P23L and P26L mutant proteins. We infer that inhibition of cell growth by micrococcin, as for thiostrepton, results from the imposition of a conformational constraint on protein L11 which, in turn, perturbs the function(s) of the ribosomal factor-guanosine nucleotide complexes.

Impact of thioesterase activity on tylosin biosynthesis in Streptomyces fradiae.

BACKGROUND: The polyketide lactone, tylactone, is produced in Streptomyces fradiae by the TylG complex of five multifunctional proteins. As with other type I polyketide synthases, the enzyme catalysing the final elongation step (TylGV) possesses an integral thioesterase domain that is believed to be responsible for chain termination and ring closure to form tylactone, which is then glycosylated to yield tylosin. In common with other macrolide producers, S. fradiae also possesses an additional thioesterase gene (orf5) located within the cluster of antibiotic biosynthetic genes. The function of the Orf5 protein is addressed here. RESULTS: Disruption of orf5 reduced antibiotic accumulation in S. fradiae by at least 85%. Under such circumstances, the strain accumulated desmycosin (demycarosyl-tylosin) due to a downstream polar effect on the expression of orf6, which encodes a mycarose biosynthetic enzyme. High levels of desmycosin production were restored in the disrupted strain by complementation with intact orf5, or with the corresponding thioesterase gene, nbmB, from S. narbonensis, but not with DNA encoding the integral thioesterase domain of TylGV. CONCLUSIONS: Polyketide metabolism in S. fradiae is strongly dependent on the thioesterase activity encoded by orf5 (tylO). It is proposed that the TylG complex might operate with a significant error frequency and be prone to blockage with aberrant polyketides. A putative editing activity associated with TylO might be essential to unblock the polyketide synthase complex and thereby promote antibiotic accumulation.

Multiple regulatory genes in the tylosin biosynthetic cluster of Streptomyces fradiae.

BACKGROUND: The macrolide antibiotic tylosin is composed of a polyketide lactone substituted with three deoxyhexose sugars. In order to produce tylosin efficiently, Streptomyces fradiae presumably requires control mechanisms that balance the yields of the constituent metabolic pathways together with switches that allow for temporal regulation of antibiotic production. In addition to possible metabolic feedback and/or other signalling devices, such control probably involves interplay between specific regulatory proteins. Prior to the present work, however, no candidate regulatory gene(s) had been identified in S. fradiae. RESULTS: DNA sequencing has shown that the tylosin biosynthetic gene cluster, within which four open reading frames utilise the rare TTA codon, contains at least five candidate regulatory genes, one of which (tylP) encodes a gamma-butyrolactone signal receptor for which tylQ is a probable target. Two other genes (tylS and tylT) encode pathway-specific regulatory proteins of the Streptomyces antibiotic regulatory protein (SARP) family and a fifth, tylR, has been shown by mutational analysis to control various aspects of tylosin production. CONCLUSIONS: The tyl genes of S. fradiae include the richest collection of regulators yet encountered in a single antibiotic biosynthetic gene cluster. Control of tylosin biosynthesis is now amenable to detailed study, and manipulation of these various regulatory genes is likely to influence yields in tylosin-production fermentations.

Resistance to macrolides and lincosamides in Streptomyces lividans and to aminoglycosides in Micromonospora purpurea.

Ribosomal (r) resistance to gentamicin in clones containing DNA from the producing organism Micromonospora purpurea is determined by grmA, and not by kgmA as originally reported. The kgmA gene originated in Streptomyces tenebrarius and is identical to kgmB. Both grmA and kgm encode enzymes that methylate single specific sites within 16S rRNA, although the site of action of the grmA product has not yet been determined. In either case, the methylated nucleoside is 7-methyl G. Inducible resistance to lincomycin (Ln) and macrolides in Streptomyces lividans TK21 results from expression of two genes: lrm, encoding an rRNA methyltransferase and mgt, encoding a glycosyl transferase (MGT), that specifically inactivates macrolides. The lrm product monomethylates residue A2058 within 23S rRNA (Escherichia coli numbering scheme) and confers high-level resistance to Ln with much lower levels of resistance to macrolides. Substrates for MGT, which utilises UDP-glucose as cofactor, include macrolides with 12-, 14-, 15- or 16-atom cyclic polyketide lactones (as in methymycin, erythromycin, azithromycin or tylosin, respectively) although spiramycin and carbomycin are not apparently modified. The enzyme is specific for the 2'-OH group of saccharide moieties attached to C5 of the 16-atom lactone ring (corresponding to C5 or C3 in 14- or 12-atom lactones, respectively). The lrm and mgt genes have been cloned and sequenced. The deduced lrm product is a 26-kDa protein, similar to other rRNA methyltransferases, such as the carB, tlrA and ermE products, whereas the mgt product (deduced to be 42 kDa) resembles a glycosyl transferase from barley.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning of tlrD, a fourth resistance gene, from the tylosin producer, Streptomyces fradiae.

In addition to tlrA, tlrB and tlrC, which were previously cloned by others, a fourth antibiotic-resistance gene (tlrD) has been isolated from Streptomyces fradiae, a producer of tylosin (Ty), and cloned in Streptomyces lividans. Like tlrA, tlrD encodes an enzyme that methylates the N6-amino group of the A2058 nucleoside within 23S ribosomal RNA. However, whereas the tlrA protein dimethylates that nucleoside, the tlrD product generates N6-monomethyladenosine. The genes also differ in their mode of expression: tlrA is inducible, whereas tlrD is apparently expressed constitutively, and it has been confirmed that the tlrA-encoded enzyme can add a second methyl group to 23S rRNA that has already been monomethylated by the tlrD-encoded enzyme. Presumably, that is what happens in S. fradiae.

Cloning and characterization of two genes from Streptomyces lividans that confer inducible resistance to lincomycin and macrolide antibiotics.

Inducible resistance to lincomycin and macrolides in Streptomyces lividans TK21 results from expression of two linked genes: lrm, encoding a ribosomal RNA methyltransferase that confers high-level resistance to lincomycin with lower levels of resistance to macrolides, and mgt, encoding a glycosyl transferase that specifically inactivates macrolides using UDP-glucose as cofactor. The lrm and mgt genes have been cloned and sequenced. The deduced lrm product is a 26-kDa protein with much similarity to other ribosomal RNA methyltransferases, such as the carB, tlrA and ermE products, whereas the mgt product (predicted to be 42 kDa) resembles a eukaryotic glycosyl transferase. Macrolides that induce the lrm-mgt gene pair are substrates for inactivation by the mgt product, and the lrm product confers ribosomal resistance to such inducers.

Molecular analysis of tlrD, an MLS resistance determinant from the tylosin producer, Streptomyces fradiae.

The macrolide antibiotic, tylosin (Ty), is produced by Streptomyces fradiae. Two resistance determinants (tlrA, synonym ermSF, and tlrD) conferring resistance to macrolide, lincosamide and streptogramin B type (MLS) antibiotics were previously isolated from this strain, and their products shown to methylate 23S ribosomal RNA (rRNA) at a common site, thereby rendering the ribosomes MLS resistant. However, the TlrA and TlrD proteins differ in their action; the former dimethylates, and the latter monomethylates, the target nucleotide. Here, 2.2 kb of DNA from the tylLM region of the tylosin biosynthetic gene cluster of S. fradiae has been sequenced and shown to encompass tlrD. Comparison of the sequences of tlrA and tlrD (and of their deduced products) with those of related ('erm-type') genes from other actinomycetes suggests that the combined presence of tlrA and tlrD in S. fradiae is not the result of recent gene duplication.

Resistance to pactamycin in clones of Streptomyces lividans containing DNA from pactamycin-producing Streptomyces pactum.

A pactamycin (Pc)-resistance determinant (pct) from Streptomyces pactum has been isolated on a 4.9-kb KpnI fragment. The original construct involving plasmid pIJ702 was highly unstable in Streptomyces lividans, leading to deletion of the pct gene from the vector. Subcloning of pct into an alternative vector (pOJ160) led to the generation of a more stable clone which possessed Pc-resistant ribosomes, and reconstitution analysis established that 16S rRNA was responsible for such resistance. Post-transcriptional modification of rRNA is probably the mechanism of resistance since the cloned DNA fragment did not appear to encode 16S rRNA.

Interplay of novobiocin-resistant and -sensitive DNA gyrase activities in self-protection of the novobiocin producer, Streptomyces sphaeroides.

The novobiocin (Nb)-producing organism, Streptomyces sphaeroides, possesses two gyrB genes: gyrBS and gyrBR (encoding the DNA gyrase B subunit-the normal target for Nb) whose products differ in their response to the drug. Novobiocin-sensitive gyrase is the predominant form of the enzyme in this strain and is produced constitutively but at variable levels, whereas Nb-resistant gyrase appears when growth takes place in the presence of the drug. The promoter isolated from the Nb-resistance determinant responds sharply to changes in DNA topology, being activated when the (negative) superhelical density is reduced and vice versa when the supercoiling of DNA is increased. Thus, resistance to Nb in S. sphaeroides is induced by a reduction in DNA supercoiling due to the action of autogenous drug on the sensitive gyrase.

Characterization and targeted disruption of a glycosyltransferase gene in the tylosin producer, Streptomyces fradiae.

An open reading frame, designated tylN, has been identified by sequence analysis at one end of the tylosin biosynthetic gene cluster of Streptomyces fradiae, alongside a cluster of genes encoding the biosynthesis of dTDP-deoxyallose. This 6-deoxyhexose sugar is converted to mycinose, via bis O-methylation, following attachment to the polyketide lactone during tylosin biosynthesis. The deduced product of tylN is similar to several glycosyltransferases, authentic and putative, and displays a consensus sequence motif that appears to be characteristic of a sub-group of such enzymes. Specific disruption of tylN within the S. fradiae genome resulted in the production of demycinosyl-tylosin, whereas other glycosyltransferase activities involved in tylosin biosynthesis were not affected. Evidently, tylN encodes deoxyallosyl transferase.

Analysis of two capreomycin-resistance determinants from Streptomyces capreolus and characterization of the action of their products.

Two genes encoding capreomycin (Cp)-modifying enzymes have been isolated from the producing organism Streptomyces capreolus. Cp acetyltransferase (CAC), encoded by cac, is active against all four components of the Cp complex, whereas Cp phosphotransferase (CPH), the product of cph, is active against Cp components IA and IIA (and also the related antibiotic, Vm) but not against Cp IB or Cp IIB.

Analysis of four tylosin biosynthetic genes from the tylLM region of the Streptomyces fradiae genome.

The tylLM region of the tylosin biosynthetic gene cluster of Streptomyces fradiae contains four open reading frames (orfs1*-4*). The function of the orf1* product is not known. The product of orf2* (tylM2) is the glycosyltransferase that adds mycaminose to the 5-hydroxyl group of tylactone, the polyketide aglycone of tylosin (Ty). A methyltransferase, responsible for 3-N-methylation during mycaminose production, is encoded by orf3* (tylM1). The product of orf4* (cer) is crotonyl-CoA reductase, which converts acetoacetyl-CoA to butyryl-CoA for use as a 4C extender unit during tylactone production.

Cloning and characterization of gentamicin-resistance genes from Micromonospora purpurea and Micromonospora rosea.

Aminoglycoside-resistance genes (grm) were cloned from a gentamicin producer Micromonospora purpurea and a sisomicin producer Micromonospora rosea. The nucleotide (nt) sequences of both genes were determined and the similarity between them was very high (90.4% identity). In either case, the transcription start point was localised to about 11 nt upstream from the likely translation start codons of grm, which is expressed as a polycistronic transcript. In studies to be reported elsewhere, it has been established that the M. purpurea grm gene encodes a ribosomal RNA methyltransferase. Here, we confirmed that the similarity of the two genes exists not only at the structural but also at the functional level.

Cloning of an aminoglycoside-resistance-encoding gene, kamC, from Saccharopolyspora hirsuta: comparison with kamB from Streptomyces tenebrarius.

An aminoglycoside-resistance-encoding gene (kamC) has been isolated from the sporaricin producer, Saccharopolyspora (Sac.) hirsuta, and expressed both in Streptomyces lividans and Escherichia coli. The pattern of resistance conferred by this gene was identical to that given by another gene (kamB) previously isolated from Streptomyces tenebrarius. In accordance with the known action of the kamB product, the Sac, hirsuta determinant also encodes a methyltransferase that modifies 16S rRNA, thereby rendering ribosomes refractory to certain aminoglycosides. The nucleotide sequences of both genes have been determined and comparison of the deduced amino acid sequences reveals a high degree of similarity.

Ribosomal modification and resistance in antibiotic-producing organisms.

Antibiotic producing organisms defend themselves against their products in a variety of ways including modification of the normal target sites for antibiotic action. In the ribosomal context, some organisms that produce inhibitors of protein synthesis render their own ribosomes refractory to the autogenous drugs via specific methylation of ribosomal RNA. Such hints that antibiotics might normally recognize RNA within the ribosome, complement other data in suggesting that RNA might be intimately associated with--and, in evolutionary terms, might originally have constituted--various functional sites within the ribosome.

On the nature of antibiotic binding sites in ribosomes.

The ribosome is an enzyme and enzymes have active sites. Antibiotics that affect ribosomal function can be considered as enzyme inhibitors (or regulators) and it is therefore pertinent to identify their molecular targets as a means of studying the active sites of the particle. The methods available for doing this are considered and, in general terms, the data are evaluated. The conclusion is reached that there exists virtually no compelling evidence that antibiotics bind primarily to ribosomal proteins. Rather, studies of antibiotic resistance in various systems strongly suggest that ribosomal RNA is the primary target for a number of drugs. Moreover, in at least one case (relating to the antibiotic thiostrepton), such an effect can be demonstrated directly. These conclusions imply a fundamental role for RNA in ribosomal function.

The binding of thiostrepton to 23S ribosomal RNA.

The antibiotic, thiostrepton, binds to 23S ribosomal RNA from E coli with a dissociation constant (KD) of 2.4 x 10(-7) M. The specificity of the interaction was established using 16S rRNA and modified or mutationally-altered 23S rRNA. Thus, no binding was detected with rRNA from the 30S subunit nor with rRNA modified in vitro by the thiostrepton resistance methylase. Mutant 23S rRNA, altered at residue 1067 in each of the 3 possible ways, showed reduced binding affinity for thiostrepton. The KD for the G mutation was 3.5 x 10(-6) M; for the C mutation, 2.4 x 10(-5) M; and for the U mutation, 4.8 x 10(-5) M. This reduction in drug binding is compatible with functional analyses; the C or U mutation results in ribosomal particles which are poorly inhibited by the drug compared with wild-type, whereas the G mutation results in an intermediate response to the drug in protein synthesis. The smallest 23S rRNA fragment used here that was capable of binding thiostrepton, in a nitrocellulose filter binding assay, comprised residues 1052-1112 and the dissociation constant was 3.0 x 10(-7) M, ie virtually indistinguishable from that with intact 23S RNA. However, the drug was incapable of binding to the 5'-moiety of this fragment (ie residues 1052-1084) or to an RNA transcript complementary to 1052-1112.

Structural modifications of anguidin and antitumor activities of its analogues.

Approximately 60 derivatives of anguidin were prepared for evaluation of antitumor activities. Positions 3, 4, 8-10, and 15 were modified, and the resultant derivatives were screened against P-388 leukemia. It was found that introduction of the C3-keto and C3,C8-diketo groups markedly improved the antileukemic activity, whereas epoxidation of the C9-C10 double bond or oxidation of the C15 position diminished its activity. Selected derivatives were further tested in the L1210, B16, Lewis lung, Colon 36, and Colon 38 tumor lines. Among these compounds, 4 beta, 15-diacetoxyscirpene-3,8-dione (54) and 4 beta-(chloroacetoxy)-15-acetoxyscirpene-3,8-dione (55) were found to be most active in various tumors. Inhibitory action of several analogues on protein synthesis was also examined using H-HeLa cells.