SCP1, a 356,023 bp linear plasmid adapted to the ecology and developmental biology of its host, Streptomyces coelicolor A3(2).

The sequencing of the entire genetic complement of Streptomyces coelicolor A3(2) has been completed with the determination of the 365,023 bp sequence of the linear plasmid SCP1. Remarkably, the functional distribution of SCP1 genes somewhat resembles that of the chromosome: predicted gene products/functions include ECF sigma factors, antibiotic biosynthesis, a gamma-butyrolactone signalling system, members of the actinomycete-specific Wbl class of regulatory proteins and 14 secreted proteins. Some of these genes are among the 18 that contain a TTA codon, making them targets for the developmentally important tRNA encoded by the bldA gene. RNA analysis and gene fusions showed that one of the TTA-containing genes is part of a large bldA-dependent operon, the gene products of which include three proteins isolated from the spore surface by detergent washing (SapC, D and E), and several probable metabolic enzymes. SCP1 shows much evidence of recombinational interactions with other replicons and transposable elements during its history. For example, it has two sets of partitioning genes (which may explain why an integrated copy of SCP1 partially suppressed the defective partitioning of a parAB-deleted chromosome during sporulation). SCP1 carries a cluster of probable transfer determinants and genes encoding likely DNA polymerase III subunits, but it lacks an obvious candidate gene for the terminal protein associated with its ends. This may be related to atypical features of its end sequences.

Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2).

Streptomyces coelicolor is a representative of the group of soil-dwelling, filamentous bacteria responsible for producing most natural antibiotics used in human and veterinary medicine. Here we report the 8,667,507 base pair linear chromosome of this organism, containing the largest number of genes so far discovered in a bacterium. The 7,825 predicted genes include more than 20 clusters coding for known or predicted secondary metabolites. The genome contains an unprecedented proportion of regulatory genes, predominantly those likely to be involved in responses to external stimuli and stresses, and many duplicated gene sets that may represent 'tissue-specific' isoforms operating in different phases of colonial development, a unique situation for a bacterium. An ancient synteny was revealed between the central 'core' of the chromosome and the whole chromosome of pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae. The genome sequence will greatly increase our understanding of microbial life in the soil as well as aiding the generation of new drug candidates by genetic engineering.

Beta-ketoacyl acyl carrier protein synthase III (FabH) is essential for fatty acid biosynthesis in Streptomyces coelicolor A3(2).

The Streptomyces coelicolor fab (fatty acid biosynthesis) gene cluster (fabD-fabH-acpP-fabF) is cotranscribed to produce a leaderless mRNA transcript. One of these genes, fabH, encodes a ketoacyl synthase III that is essential to and is proposed to be responsible for initiation of fatty acid biosynthesis in S. coelicolor.

Putative protein serine/threonine kinase genes are located in several positions on the chromosome of Streptomyces coelicolor A3(2).

Seven DNA fragments containing putative protein serine/threonine kinase genes were cloned from Streptomyces coelicolor A3(2) by hybridization with a 165-bp amplified polymerase chain reaction product of pkaB containing kinase subdomains VI, VII and VIII. Among them, the nucleotide sequences of three fragments containing subdomains VI, VII and VIII were identical, while those of one fragment were identical with that of afsK, another protein serine/threonine kinase gene. However, these kinase-like genes with very similar nucleotide sequences were distributed on the chromosome of S. coelicolor A3(2).

A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chromosome.

A Supercos-1 library carrying chromosomal DNA of a plasmid-free derivative of Streptomyces coelicolor A3(2) was organized into an ordered encyclopaedia of overlapping clones by hybridization. The minimum set of overlapping clones representing the entire chromosome (with three short gaps) consists of 319 cosmids. The average insert size is 37.5 kb and the set of clones therefore divides the chromosome into 637 alternating unique and overlapping segments which have an average length of approx. 12.5 kb. More than 170 genes, gene clusters and other genetic markers were mapped to their specific segment by hybridization to the encyclopaedia. Genes could be cloned by direct transformation and complementation of S. coelicolor mutants with cosmids isolated from Escherichia coli, selecting for insertion into the chromosome by homologous recombination. As in other streptomycetes, the ends of the chromosome have long terminal inverted repeats.

Mapping of genes involved in macromolecular synthesis on the chromosome of Streptomyces coelicolor A3(2).

The genes for the beta, beta', and seven sigma factor subunits of RNA polymerase, for elongation factors EF-Tu1 and EF-Tu3, and for six rRNA operons were mapped on the combined genetic and physical map of the Streptomyces coelicolor chromosome. Like the previously mapped tRNA genes, the RNA polymerase and rRNA genes map to scattered positions. The lack of rRNA operons in the immediate vicinity of the origin of replication (oriC) and the absence of tRNA genes in any of the rRNA operons are novel features of the Streptomyces chromosome.

tRNA genes of Streptomyces lividans: new sequences and comparison of structure and organization with those of other bacteria.

Three closely linked Streptomyces lividans tRNA genes encoding two tRNA(Lys)s and a tRNA(Gly) were cloned and sequences. The structure of tRNA(Gly) is unusual for eubacterial tRNAs. Including those in previous reports (R. Sedlmeier and H. Schmieger, Nucleic Acids Res. 18:4027, 1990, and R. Sedlmeier, G. Linti, K. Gregor, and H. Schmieger, Gene 132:125-130, 1993), 18 S. lividans tRNA genes were physically mapped on the chromosome of the closely related strain Streptomyces coelicolor A3(2). The structure and organization of tRNA genes of S. lividans and S. coelicolor are compared with those of Escherichia coli and Bacillus subtilis.

Characterization of two groEL genes in Streptomyces coelicolor A3(2).

Two Streptomyces coelicolor A3(2) groEL-like genes, groEL1 and groEL2, were cloned and characterized. Pulsed-field-gel electrophoresis located these genes, which were not adjacent, in the same segment of the chromosome. Nucleotide sequence analysis revealed that groEL1, but not groEL2, was preceded by a groES-like gene. Northern blots showed that heat shock induced the accumulation of transcripts corresponding to groES (0.7 kb), groES/EL1 (2.3 kb) and groEL2 (2.1 kb). Unique transcription start points and promoters were located for groES/EL1 and groEL2, having -10 and -35 hexamers similar to eubacterial vegetative promoters. Regions located 5' to the groES/EL1 or groEL2 structural genes contain 'GCACTCN9GAGTGC' motifs conserved upstream from the heat-shock genes of other bacteria.

The chromosomal DNA of Streptomyces lividans 66 is linear.

Two copies of a DNA sequence similar or identical to one end of the linear plasmid SLP2 were found on the Streptomyces lividans chromosome. Restriction mapping showed that these sequences represented free ends. Electrophoretic retardation and glass-binding studies indicated that the telomeres carry covalently bound proteins. Moreover, the chromosome migrated as an 8 Mb linear DNA in pulsed-field gel electrophoresis. A similar finding with the chromosomes of six other Streptomyces species suggested that a linear chromosome may be characteristic of the genus. The S. lividans chromosome can be circularized by joining the two ends by artificial targeted recombination or by spontaneous deletions spanning both telomeres. Thus the chromosome appears to be able to exist, in viable bacteria, as a linear or a circular molecule.

The conjugative plasmid SLP2 of Streptomyces lividans is a 50 kb linear molecule.

The SLP2 plasmid had previously been demonstrated genetically to exist in Streptomyces lividans by its ability to promote conjugation and to elicit 'pocks' on recipient (SLP2-) cultures, but it had not been physically detected. Using pulsed-field gel electrophoresis, a 50 kb linear DNA was isolated from SLP2+ but not SLP2- strains of S. lividans, and from Streptomyces coelicolor and Streptomyces parvulus strains to which SLP2 had been transferred by conjugation or transformation. We conclude that this linear DNA is SLP2. The terminal fragments of SLP2 were cloned. The determined sequences revealed a 44bp imperfect terminal inverted repeat. The terminal 12bp sequence of SLP2 was identical to those of two other Streptomyces linear plasmids, pSLA2 and pSCL, and similar to the terminal sequences of another Streptomyces linear plasmid, SCP1. The termini of SLP2 DNA were resistant to digestion by lambda exonuclease and ExoIII. A truncated (probably crippled) copy of Tn4811 is present on the plasmid. While the SLP2 plasmid exists as a free form in the host, a 15.7 kb sequence corresponding to the segment of SLP2 from Tn4811 to the right terminus is also present (at a copy number similar to the free form) elsewhere in the genome of S. lividans. Furthermore, SLP2 is partially homologous to a newly discovered 650 kb linear plasmid in S. parvulus.

A putative two-component regulatory system involved in secondary metabolism in Streptomyces spp.

A DNA fragment stimulating actinorhodin, undecylprodigiosin, and A-factor production in Streptomyces lividans 66 was cloned from Streptomyces coelicolor A3(2). Nucleotide sequencing revealed the presence of an open reading frame of 225 codons, named afsQ1, that showed great similarity in amino acid sequence to the response regulators of typical prokaryotic two-component regulatory systems responsible for adaptive responses. The termination codon, TGA, of afsQ1 overlapped the initiation codon, GTG, of a second open reading frame, afsQ2, of 535 codons. The afsQ2 gene product showed homology with the sensory histidine protein kinases of two-component systems. In agreement with the assumption that the AfsQ1 and AfsQ2 proteins comprise an aspartate-histidine phosphotransfer system, an amino acid replacement from Asp to Glu at residue 52 of AfsQ1, generated by site-directed mutagenesis, resulted in loss of the protein's ability to stimulate antibiotic production in S. lividans. Primer extension experiments indicated that transcription of the afsQ1 and afsQ2 genes initiates at the translational start codon (GTG) of the afsQ1 gene. The afsQ1 and afsQ2 genes were physically mapped at a chromosomal position near the actinorhodin biosynthetic gene cluster (act) by hybridization to Southern blots of restriction fragments separated by pulsed-field gel electrophoresis. Disruption of either afsQ1 or afsQ2 on the S. coelicolor chromosome by use of phage phi C31KC515 led to no detectable change in secondary metabolite formation or morphogenesis. The afsQ1 gene on pIJ922 suppressed the S. coelicolor absA mutation and caused actinorhodin production but did not suppress the absB mutation. Southern blot hybridization showed that sequences homologous to afsQ1 and afsQ2 are present in almost all of the actinomycetes examined.

A combined genetic and physical map of the Streptomyces coelicolor A3(2) chromosome.

The restriction enzymes AseI (ATTAAT), DraI (TTTAAA), and SspI (AATATT) cut the Streptomyces coelicolor A3(2) chromosome into 17, 8, and 25 fragments separable by pulsed-field gel electrophoresis (PFGE). The sums of their lengths indicated that the chromosome consists of about 8 Mb of DNA, some 75% more than that of Escherichia coli K-12. A physical map of the chromosome was constructed for AseI and DraI, using single and double digests, linking clones, cross-hybridization of restriction fragments, and locations of genetically mapped genes, insertion sequences, prophages, and the integrated SCP1 and SLP1 plasmids on the physical map. The physical map was aligned with the previously established genetic map, revealing that the two long opposite quadrants of the genetic map that are almost devoid of markers (the silent regions at 3 o'clock and 9 o'clock) are indeed physically long rather than being hot spots for genetic exchange. They must therefore contain long stretches of DNA different in function from the remainder of the genome. Consistent with this conclusion are the locations of significant deletions in both of the silent regions. Of these, a 40-kb deletion in the 9 o'clock region accompanied or followed integration of the SCP1 linear plasmid to produce the NF fertility state. PFGE analysis of Streptomyces lividans 66, a close relative of S. coelicolor A3(2), was hampered by the previously described susceptibility of its DNA to degradation during electrophoresis. However, ZX7, a mutant derivative of S. lividans lacking the DNA modification responsible for this degradation, yielded good PFGE preparations. Not more than 7 of the 17 S. coelicolor AseI fragments could be shared by the S. lividans strain.

abaA, a new pleiotropic regulatory locus for antibiotic production in Streptomyces coelicolor.

Production of the blue-pigmented antibiotic actinorhodin is greatly enhanced in Streptomyces lividans and Streptomyces coelicolor by transformation with a 2.7-kb DNA fragment from the S. coelicolor chromosome cloned on a multicopy plasmid. Southern analysis, restriction map comparisons, and map locations of the cloned genes revealed that these genes were different from other known S. coelicolor genes concerned with actinorhodin biosynthesis or its pleiotropic regulation. Computer analysis of the DNA sequence showed five putative open reading frames (ORFs), which were named ORFA, ORFB, and ORFC (transcribed in one direction) and ORFD and ORFE (transcribed in the opposite direction). Subcloning experiments revealed that ORFB together with 137 bp downstream of it is responsible for antibiotic overproduction in S. lividans. Insertion of a phi C31 prophage into ORFB by homologous recombination gave rise to a mutant phenotype in which the production of actinorhodin, undecylprodigiosin, and the calcium-dependent antibiotic (but not methylenomycin) was reduced or abolished. The nonproducing mutants were not affected in the timing or vigor or sporulation. A possible involvement of ORFA in antibiotic production in S. coelicolor is not excluded. abaA constitutes a new locus which, like the afs and abs genes previously described, pleiotropically regulates antibiotic production. DNA sequences that hybridize with the cloned DNA are present in several different Streptomyces species.

A mutation of Streptomyces lividans which prevents intraplasmid recombination has no effect on chromosomal recombination.

A mutation (rec-46) of Streptomyces lividans, previously shown to prevent (or greatly diminish) homologous and illegitimate intraplasmid recombination, was shown to have no effect on generalised chromosomal recombination occurring in matings or in protoplast fusions, nor to affect homologous recombination between a recombinant plasmid and the host chromosome. By comparison with Escherichia coli mutants defective in various aspects of recombination, the rec-46 mutation is similar to those in recF, recJ, recO and topA.

Structure and deduced function of the granaticin-producing polyketide synthase gene cluster of Streptomyces violaceoruber Tü22.

A 6.5 kb region of DNA from Streptomyces violaceoruber, which contains polyketide synthase (PKS) genes for production of the benzoisochromane quinone moiety of the antibiotic, granaticin, was cloned and sequenced. Of six open reading frames (ORFs) identified, four (ORFs 1-4) would be transcribed in one direction and two (ORFs 5 and 6) divergently from ORFs 1-4. ORF1 and ORF2, which show evidence for translation coupling, encode (deduced) gene products which strongly resemble each other and the Escherichia coli fatty acid ketoacyl synthase (condensing enzyme), FabB. We conclude that ORF1 (which contains a characteristic cysteine residue) functions as a condensing enzyme, possibly as part of a heterodimeric protein including the product of ORF2. The predicted ORF3 gene product strikingly resembles acyl carrier proteins (ACPs) of fatty acid synthase (FAS), particularly in the region of the active site motif, while the predicted ORF5 and ORF6 gene products resemble known oxidoreductases, suggesting that they function as reductive steps required during assembly of the granaticin carbon skeleton. Comparison of the deduced ORF4 gene product with available protein databases failed to elucidate its potential function. The overall conclusion is that the granaticin-producing PKS would consist of at least six separate enzymes involved in carbon chain assembly, thus resembling a Type II, rather than a Type I, FAS.

Homology between Streptomyces genes coding for synthesis of different polyketides used to clone antibiotic biosynthetic genes.

Many important antibiotics such as tetracyclines, erythromycin, adriamycin, monensin, rifamycin and avermectins are polyketides. In their biosynthesis, multifunctional synthases catalyse iterated condensation of thio-esters derived from acetate, propionate or butyrate to yield aliphatic chains of varying length and carrying different alkyl substituents. Subsequent modifications, including aromatic or macrolide ring closure or specific methylations or glycosylations, generate further chemical diversity. It has been suggested that, if different polyketide synthases had a common evolutionary origin, cloned DNA coding for one synthase might be used as a hybridization probe for the isolation of others. We show here that this is indeed possible. Study of a range of such synthase genes and their products should help to elucidate what determines the choice and order of condensation of different residues in polyketide assembly, and might yield, by in vitro recombination or mutagenesis, synthase genes capable of producing novel antibiotics. Moreover, because genes for entire antibiotic pathways are usually clustered in Streptomyces, cloned polyketide synthase genes are valuable in giving access to groups of linked biosynthetic genes.

Production of new hybrid antibiotics, mederrhodins A and B, by a genetically engineered strain.

Hybrid antibiotics mederrhodins A and B were produced by a recombinant strain consisting of the medermycin-producing Streptomyces sp. strain AM7161 containing part of the gene clusters for actinorhodin biosynthesis of Streptomyces coelicolor A3(2). Mederrhodin A has a hydroxyl group at the C-6 position of the medermycin molecule, and mederrhodin B is dihydromederrhodin A. The antimicrobial activity of mederrhodin A resembled that of medermycin. Mederrhodin B was almost devoid of antimicrobial activity.

Production of 'hybrid' antibiotics by genetic engineering.

The recent development of molecular cloning systems in Streptomyces has made possible the isolation of biosynthetic genes for some of the many antibiotics produced by members of this important genus of bacteria. Such clones can now be used to test the idea that novel antibiotics could arise through the transfer of biosynthetic genes between streptomycetes producing different antibiotics. The likelihood of a 'hybrid' compound being produced must depend on the substrate specificities of the biosynthetic enzymes, about which little is known. In attempts to demonstrate hybrid antibiotic production, we therefore began with strains producing different members of the same chemical class of compounds in order to maximize the chance of success. Here we report the production of novel compounds by gene transfer between strains producing the isochromanequinone antibiotics actinorhodin, granaticin and medermycin. These experiments were made possible by the recent cloning of the whole set of genes for the biosynthetic pathway of actinorhodin from Streptomyces coelicolor A3(2) (ref. 8). We believe that this represents the first report of the production of hybrid antibiotics by genetic engineering.

Cloning of a chloramphenicol acetyltransferase gene of Streptomyces acrimycini and its expression in Streptomyces and Escherichia coli.

A gene (cat) for chloramphenicol (Cm) acetyltransferase (CAT) was cloned from Streptomyces acrimycini into S. lividans 66 on the plasmid vector pIJ61. The cat gene was localized on a 1.7-kb BclI fragment, which probably also carries the cat promoter. This DNA fragment conferred Cm resistance, through CAT activity, on S. lividans, S. coelicolor and S. parvulus, but not on Escherichia coli when inserted in the BamHI site of the tetracycline-resistance(TcR) gene of pBR322. However, when inserted in a particular orientation in this site, spontaneous deletions of 0.7 kb led to CAT activity and Cm resistance. DNA homologous to the 1.7-kb BclI cat fragment was found in most, but not all, of a series of other streptomycetes that have CAT activity. The cat provides a potentially useful screening marker for Streptomyces cloning vectors.