Expression of tylM genes during tylosin production: phantom promoters and enigmatic translational coupling motifs.

In the genome of Streptomyces fradiae, the three tylM genes are codirectional with the upstream gene, tylGV. Although the introduction of transcriptional blocks into the tylM genes revealed that they are normally cotranscribed, expression of tylMI still persisted (albeit at a very low level) when either of the upstream genes, tylMII or tylMIII, was disrupted. Such expression apparently resulted from transcriptional initiation at spurious sites that probably contribute insignificantly, if at all, to promote activity in the wild type. Prior to the onset of tylosin production, tylMIII is transcribed independently of tylGV from an authentic promoter buried within tylGV. This latter observation is interesting given that the TGA stop codon of tylGV overlaps the GTG start codon of tylMIII. Evidently, terminally overlapping genes are not always translationally coupled.

Inactivation of a transcriptional repressor during empirical improvement of the tylosin producer, Streptomyces fradiae.

Remarkably few changes of significance seem to have occurred within the tylosin-biosynthetic gene cluster of Streptomyces fradiae during an extensive portion of the empirical strain improvement programme carried out at Lilly Research Laboratories over many years. None of the promoters for polyketide synthase (PKS) genes or for regulatory elements changed within this part of the lineage, nor were any mutations detected in other tyl promoters, although the full set was probably not analysed. Of five regulatory genes within the tyl cluster, only tylQ was altered, having undergone a single point mutation that inactivated its product (a transcriptional repressor). Also unchanged was a gene with unassigned function. Since point mutations affecting antibiotic-biosynthetic enzymes are unlikely to have played a major role in empirical strain improvement, enhanced tylosin production levels appear to have resulted, in large measure, from uncharacterized mutations occurring outside the tyl cluster.

Influence of ancillary genes, encoding aspects of methionine metabolism, on tylosin biosynthesis in Streptomyces fradiae.

The tylosin-biosynthetic (tyl) gene cluster of Streptomyces fradiae contains ancillary genes that encode functions normally associated with primary metabolism. These can be disrupted without loss of viability, since equivalent genes (presumably used for 'housekeeping' purposes) are also present elsewhere in the genome. The tyl cluster also contains two genes that encode products unlike any proteins in the databases. Two ancillary genes, metF (encoding N5,N10-methylenetetrahydrofolate reductase) and metK, encoding S-adenosylmethionine synthase, flank one of the 'unknown' genes (orf9) in the tyl cluster. In a strain of S. fradiae in which all three of these genes were disrupted, tylosin production was reduced, although this effect was obscured in media supplemented with glycine betaine which can donate methyl groups to the tetrahydrofolate pool. Apparently, one consequence of the recruitment of ancillary genes into the tyl cluster is enhanced capacity for transmethylation during secondary metabolism.

Influence of dimethylsulfoxide on tylosin production in Streptomyces fradiae.

The polyketide aglycone, tylactone (protylonolide), does not normally accumulate during tylosin production in Streptomyces fradiae, suggesting that the capacity of the organism to glycosylate tylactone exceeds the capacity for polyketide synthesis. Consistent with this model, tylosin yields were significantly increased (due to bioconversion of the added material) when exogenous tylactone was added to fermentations. However, tylosin yield improvements were also observed (albeit at lower levels) in solvent controls to which dimethylsulfoxide (DMSO) was added. At least in part, the latter effect resulted from stimulation of polyketide metabolism by DMSO. This was revealed when the solvent was added to fermentations containing the tylA mutant, S. fradiae GS14, which normally accumulates copious quantities of tylactone.

The tylosin-biosynthetic genes of Streptomyces fradiae.

The tylosin-biosynthetic (tyl) gene cluster occupies about 1% of the genome of Streptomycesfradiae and includes at least 43 open reading frames. In addition to structural genes required for tylosin production, the tyl cluster contains three resistance determinants and several regulatory genes. Tylosin production is evidently controlled by pathway-specific and pleiotropic regulators with the likely involvement of y-butyrolactone signalling factors. Accumulation of the polyketide aglycone is controlled by glycosylated macrolides and optimal performance of the complex polyketide synthase enzyme requires the activity of an editing thioesterase.

The mycinose-biosynthetic genes of Streptomyces fradiae, producer of tylosin.

The tylE-J region of the tylosin-biosynthetic gene cluster of Streptomyces fradiae contains six open reading frames. The products of tylJ and tylD are nucleoside diphospho (NDP)-deoxyhexose 3-epimerase and NDP-deoxyhexose 4-ketoreductase, respectively, involved in the synthesis of NDP-6-deoxyallose from NDP-4-keto, 6-deoxyglucose. After incorporation of deoxyallose at C23-OH of the polyketide lactone, tylosin biosynthesis is completed by the products of tylE and tylF, which convert the deoxyallosyl moiety to mycinose via bis-O-methylation at 2-OH and 3-OH, respectively. Hydroxylation of the polyketide lactone at C23 is catalysed by the cytochrome P450 enzyme, TylHl. The product of tylHll is a ferredoxin of unknown specificity that could conceivably act together with TylHl.

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.

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.

Molecular analysis of tlrB, an antibiotic-resistance gene from tylosin-producing Streptomyces fradiae, and discovery of a novel resistance mechanism.

The tlrB gene, which confers inducible resistance to a range of macrolide antibiotics including biosynthetic precursors of tylosin, was isolated and sequenced. In the genome of Streptomyces fradiae, it lies between pbp, which encodes a putative penicillin-binding protein, and tylN, encoding a glycosyltransferase involved in tylosin biosynthesis. The TlrB protein was produced in E. coli as a fusion to MalE. The fusion protein, but not MalE alone, inactivates macrolides in the presence of S-adenosyl-methionine (SAM) but the modified product(s) has not been characterised.

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.

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.

The antibiotic micrococcin is a potent inhibitor of growth and protein synthesis in the malaria parasite.

The antibiotic micrococcin is a potent growth inhibitor of the human malaria parasite Plasmodium falciparum, with a 50% inhibitory concentration of 35 nM. This is comparable to or less than the corresponding levels of commonly used antimalarial drugs. Micrococcin, like thiostrepton, putatively targets protein synthesis in the plastid-like organelle of the parasite.

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.

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.

Structure-activity studies of tylosin-related macrolides.

The effects of tylosin-related macrolide antibiotics were examined in cell-free protein synthesis (using a coupled transcription-translation system derived from Streptomyces lividans) and against whole cells of that organism. Anti-ribosomal potency was determined primarily by the number and nature of the glycosyl substituents, and was not significantly influenced by lactone ring oxidation or sugar methylation. In contrast, uptake of the drugs into S. lividans was influenced, either positively or negatively, by each of these structural parameters. The presence of erm type I or erm type II resistance genes in S. lividans markedly affected the resistance phenotype and studies involving ribosomes from such strains revealed differences in macrolide activity that were not otherwise apparent.

The macrolide-lincosamide-streptogramin B resistance phenotypes characterized by using a specifically deleted, antibiotic-sensitive strain of Streptomyces lividans.

Genes conferring resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics via ribosomal modification are widespread in bacteria, including clinical isolates and MLS-producing actinomycetes. Such erm-type genes encode enzymes that mono- or dimethylate residue A-2058 of 23S rRNA. The different phenotypes resulting from monomethylation (MLS-I phenotype, conferred by erm type I genes) or dimethylation (MLS-II phenotype due to erm type II genes) have been characterized by introducing tlrD or ermE, respectively, into an MLS-sensitive derivative of Streptomyces lividans TK21. This strain (designated OS456) was generated by specific replacement of the endogenous resistance genes lrm and mgt. The MLS-I phenotype is characterized by high-level resistance to lincomycin with only marginal resistance to macrolides such as chalcomycin or tylosin, whereas the MLS-II phenotype involves high-level resistance to all MLS drugs. Mono- and dimethylated ribosomes were introduced into a cell-free protein-synthesizing system prepared from S. lividans and compared with unmodified particles in their response to antibiotics. There was no simple correlation between the relative potencies of MLS drugs at the level of the target site (i.e., the ribosome) and their antibacterial activities expressed as MICs.

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.

Transcriptional attenuation control of the tylosin-resistance gene tlrA in Streptomyces fradiae.

The tylosin producer Streptomyces fradiae contains four known resistance genes, two of which (tlrA and tlrD) encode methyltransferases that act on ribosomal RNA at a common site. Expression of tlrA is regulated via transcriptional attenuation. A short transcript, only 411 nucleotides long, terminates 27 nucleotides into the methylase-coding sequence in the uninduced state. Induction of tlrA is proposed to involve a ribosome-mediated conformational change within the mRNA leader that allows transcription to continue beyond the attenuation site, resulting in a transcript about 1450 nucleotides long. Transplantation of tlrD and/or tlrA into Streptomyces albus revealed that the induction specificity of tlrA depends upon the state of the ribosomes and is significantly altered in strains also expressing tlrD.

Analysis of five tylosin biosynthetic genes from the tyllBA region of the Streptomyces fradiae genome.

The tyllBA region of the tylosin biosynthetic gene cluster of Streptomyces fradiae contains at least five open reading frames (ORFs). ORF1 (tylI) encodes a cytochrome P450 and mutations in this gene affect macrolide ring hydroxylation. The product of ORF2 (tylB) belongs to a widespread family of proteins whose functions are speculative, although tylB mutants are defective in the biosynthesis or addition of mycaminose during tylosin production. ORFs 3 and 4 (tylA1 and tylA2) encode delta TDP-glucose synthase and delta TDP-glucose dehydratase, respectively, enzymes responsible for the first two steps common to the biosynthesis of all three deoxyhexose sugars of tylosin via the common intermediate, delta TDP-4-keto, 6-deoxyglucose. ORF5 encodes a thioesterase similar to one encoded in the erythromycin gene cluster of Saccharopolyspora erythraea.

Expression and analysis of two gyrB genes from the novobiocin producer, Streptomyces sphaeroides.

A novobiocin producer, Streptomyces sphaeroides, contains two genes, designated gyrBS and gyrBR, that encode novobiocin-sensitive and -resistant DNA gyrase B proteins, respectively. The cloning of gyrBR was reported earlier; here, we describe the cloning of gyrBS. Both genes have been sequenced (the deduced products of gyrBS and gyrBR have M(r) values of 87.6K and 86.5K, respectively) and their transcripts have been mapped. Downstream of gyrBS, and co-transcribed with it, is the sole gyrA gene (encoding DNA gyrase A protein). By constructing hybrid gyrB genes, using fragments of gyrBS and gyrBR, a specific portion of the N-terminal domain of the gyrase B protein (corresponding to amino acid residues 134-256 of Escherichia coli gyrase B) has been implicated in the binding of novobiocin.