John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK1
Tel: +44 1603 452571. Fax: +44 1603 456844. e-mail: hopwood{at}bbsrc.ac.uk
Keywords: Streptomyces, genetics, antibiotics, chromosome-linear
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Beginnings: the A3(2) strain |
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To get me started, Harold Whitehouse lent me a copy of Waksmans book on the actinomycetes (Waksman, 1950 ), newly purchased from Heffers bookshop, and Lewis Frost handed over half a dozen cultures of Streptomyces that he had obtained from his friend George Floodgate at the then Royal College of Technology in Glasgow (now Strathclyde University). I streaked them out: several grew well, and one produced a striking blue pigment. I could therefore (I thought!) name it Streptomyces coelicolor. As a bonus, pigmentation might in due course make a valuable genetic marker, as it had in Aspergillus nidulans. I chose this culture (strain 204F) and set about isolating auxotrophic mutants in order to look for genetic recombination à la Lederberg & Tatum (1946
). Pretty soon, I could grow pairs of mutants together and select rare prototrophs from the progeny spores. In crosses of two doubly marked strains, non-selected markers from each parent segregated amongst the progeny, indicating a genuine process of gene reassortment rather than simply some kind of heterozygote formation. However, by early 1955 problems had arisen: the cultures started to grow erratically and became friable (Fig. 1
). I diagnosed phage infection, but could not cure it. I therefore wrote to Dagny Erikson, at the University of Aberdeen, to ask for cultures of S. coelicolor. She had published extensively on variation in cultures that she had derived from Staniers agar-liquefying strain (Stanier, 1942
), including some derived by using a micromanipulator to isolate individual spores from the same chain (Erikson, 1948
). Her initial response was disappointing: she had retired to Kincardineshire to raise a family (as the new Mrs A. E. Oxford) and doubted whether any viable cultures remained. However, she would see if Dr D. M. Webley could help. A week later, a further letter arrived: Dr Webley informs me that his culture of Streptomyces coelicolor is not in good condition. I have found an old sterile soil ampoule which was sown with the blue-pigmented, agar-liquefying strain A3(2). I cannot guarantee it, but hope it may still be viable and pure. Indeed it was! I shook some soil particles from the ampoule onto an agar plate and beautiful blue colonies grew out. I made auxotrophic mutants and they were stable, and grew perfectly. They produced prototrophic recombinants (Fig. 2
) when grown in pairs (Hopwood, 1957
). Thus was the A3(2) strain launched on its genetic career. Meanwhile, the true phylogenetic relationships of Streptomyces needed looking into.
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Streptomycetes are bacteria |
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By the early 1950s, few microbiologists regarded the actinomycetes as fungi, but many still thought of them as intermediate between fungi and bacteria. For Streptomyces, there were biochemical pointers to a bacterial affinity the cell-wall composition resembled that of typical Gram-positive bacteria, and they were sensitive to specifically anti-bacterial antibiotics but their cellular architecture was unclear. In Cambridge, through a mutual friend (Martin Canney), I was lucky enough to meet the brilliant electron microscopist Audrey Glauert who, with Ernst Brieger, was studying the fine structure of Mycobacterium strains at the Strangeways Laboratory. This was the start of a long and rewarding collaboration during which we showed, among other things, that S. coelicolor lacks a nuclear membrane (Fig. 3) and so is, by definition, a bacterium (or prokaryote to use a later term). Nevertheless, it was clearly very different morphologically from the simple, rod-shaped bacteria that had hitherto been studied genetically, so the idea that it might reveal novel genetic phenomena was still very much alive.
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Does Streptomyces have a life cycle? |
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Genetic studies in other laboratories |
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Taxonomy of the A3(2) culture |
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Establishing a linkage map |
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My solution to the problem came to me out of the blue one afternoon when I was explaining my project to two German colleagues from St Johns College over tea in the Botany School, one a linguist and the other an economist (thereby doing nothing to dispel the idea prevalent among non-scientists that science advances by the Eureka principle!). This was the four-on-four cross. In it two parents each have two markers, which can be either selected or non-selected. Recombinants are recovered from equal numbers of total progeny on four media, each making one of the four possible selections of one marker from each parent and leaving the second marker of that parent non-selected. Because there are two non-selected markers, four recombinant genotypes can grow on each medium; their frequencies are determined by picking and classifying samples of the colonies. Some of the genotypes grow on more than one of the media, while others are unique to a single medium. Taking them all together, they include one or both members of each of the seven possible complementary pairs of recombinant genotypes, leaving only the parental classes (which are, of course, the same as the vast excess of asexual progeny) unmeasured. In this way relative linkage distances could be measured and used to construct a map.
The first version of the map (Hopwood, 1958 ) had just six loci, in two linkage groups, and this was soon extended to 15 loci, with the (temporary) appearance of a third linkage group represented by a single gene (Hopwood, 1959
). With the discovery of heteroclones colonies deriving from partially diploid plating units, each of which gave rise to a population of haploid recombinants that could be analysed non-selectively (Sermonti et al., 1960
; Hopwood et al., 1963
) the map grew to 39 loci, still in two linkage groups (Hopwood, 1965a
). Soon, the map was firmly enough established for a simpler system of analysis to be employed. Selection was made for recombination between just two known points on the chromosome with other markers unselected. In appropriate crosses, the pattern of marker selection in one linkage group influenced, in very specific ways, the segregation of markers in the other linkage group; the interpretation was a single, circular linkage group that incorporated the two previously separate groups of genes (Hopwood, 1965b
).
The reason it had not been possible earlier to bridge the gaps between the two original linkage groups was that they were separated by long, very sparsely marked segments. These so-called silent regions were devoid even of a general class of temperature-sensitive mutants, as described in a paper (Hopwood, 1966a ) that acknowledged for the first time the input of Helen Ferguson (later Wright, still later Kieser), who has contributed so much to our 35 year collaboration. These regions remained for long a striking feature of the S. coelicolor genome and would be interpreted only some 30 years later (see later section) when Helen was able to map the chromosome physically (Kieser et al., 1992
).
As Frank Stahl (1967 ) had taught us, this map circularity did not necessarily mean that the chromosome itself was a circle: merozygosity, leading to the requirement for even-numbered crossovers to generate recombinants between a complete chromosome from one parent and a fragment from the other (except in the rare case when the fragment included a chromosome end), could have been a sufficient explanation for circularity of the genetic map even if the chromosome were linear (Fig. 6
). For technical reasons, it was not possible to obtain the kind of direct physical evidence that had established chromosome circularity in E. coli (Cairns, 1963
), so I tried at least to distinguish between linearity on the one hand and circularity or circular permutation on the other by analysing several hundred heteroclones selected from the same cross. It turned out that the region of heterozygosity in a particular heteroclone could cover any continuous arc of the linkage map. On the assumption that this region represented an uninterrupted segment of the chromosome, the conclusion was that the chromosome lacked constant ends (Hopwood, 1966b
). It was provisionally assumed to be circular pending direct evidence, but might have been circularly permuted instead. It was a very long time before we knew more than this!
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The three phases of Streptomyces genetics |
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In skimming over developments in Streptomyces genetics since 1965, I have inevitably made subjective choices, not to say parochial ones [there is certainly a bias in favour of S. coelicolor A3(2)], even though I have tried to include in Tables 13 appropriate discoveries from other laboratories. I hope the choice will be viewed indulgently by those whose work I have omitted to mention.
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The plasmid nature of SCP1 was deduced genetically. In spite of some raised eyebrows, because we had not identified the element physically, this did not render the conclusion invalid in view of the original definition of the word plasmid, which did not refer to its physical nature (Lederberg, 1952 ). However, we needed to isolate SCP1, and it long resisted isolation, notwithstanding valiant attempts by Janet Westpheling and others (see Hopwood et al., 1979
). The reason, discovered nearly ten years later by Haruyasu Kinashi (Kinashi et al., 1987
), who exploited the newly invented pulsed-field gel technology, is that SCP1 is both large (350 kb) and linear. With hindsight, we should have taken more seriously the possibility that SCP1 was linear, because linear plasmids small enough to be revealed by conventional technology had been found as early as 1979 (Hayakawa et al., 1979
). This was another momentous development, the full significance of which became apparent only much later when the widespread occurrence in Streptomyces of linear replicons, including the chromosome, was realized.
Alan Vivian (1971 ) had found that SCP1+ strains inhibited SCP1- strains through the agency of a diffusible molecule, but only later was this found to be a small molecule (i.e. not a protein like a bacteriocin) with a wide spectrum of activity (Fig. 10
) and so it could be called an antibiotic (Kirby et al., 1975
), later identified as methylenomycin (Wright & Hopwood, 1976a
). This, the first example of genetic localization of antibiotic-biosynthetic genes, led to much speculation that plasmid determination of antibiotic production might be widespread, if not typical (e.g. Umezawa, 1977
). Ironically, methylenomycin remains, after 20 years, the only unambiguous example of a Streptomyces antibiotic specified by a plasmid. The very next example actinorhodin in S. coelicolor (Wright & Hopwood, 1976b
) implicated chromosomal genes, later shown to be clustered (Rudd & Hopwood, 1979
), and this instead has turned out to be the paradigm.
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Meanwhile, a second plasmid had surfaced in S. coelicolor; this proved to be a conventional, moderate-sized, CCC molecule, amenable to isolation by a fairly standard protocol (Schrempf et al., 1975 ). SCP2 was largely responsible for the fertility in crosses that could not be attributed to SCP1 (Bibb et al., 1977
); a variant form of the plasmid, called SCP2*, was an even better sex factor. The discovery of SCP2* was an essential stepping stone towards introducing plasmid DNA into Streptomyces hosts by transformation, thereby opening the way for gene cloning in these organisms. The transformation procedure devised (Bibb et al., 1978
) depended on the earlier development of protoplast fusion, which gave rise to enormously high frequencies of genetic recombination without the need for a plasmid vector (Hopwood et al., 1977
; Baltz, 1978
). These studies, in turn, could not have been made without the pioneering work of Masanori Okanishi and his colleagues, who painstakingly investigated the factors necessary for good protoplast formation and, even more crucially, their regeneration (Okanishi et al., 1974
).
Plasmid biology was also beginning to develop as the 1970s were coming to an end. The marker used by Bibb et al. (1978 ) to track transformants by SCP2* was pock formation: transformants receiving SCP2* gave rise to small, circular areas of retarded growth in a lawn of the plasmid-free strain. Originally this effect was assumed to be an actual killing of the strain by the mating process, and was likened to lethal zygosis in E. coli (Skurray & Reeves, 1973
). An even earlier notion, when pocks were seen in matings between S. coelicolor carrying SCP1 and S. lividans (Hopwood & Wright, 1973a
), was that inhibition of the S. lividans lawn was due to the diffusible agent that was subsequently identified as methylenomycin. Only later, when plasmid mutants altered in pock formation were isolated (Fig. 11
: Kieser et al., 1982
), was the phenomenon interpreted (we hope correctly, but direct proof is still lacking) as a manifestation of the migration of a conjugative plasmid within the mycelium of the recipient. This process was described as plasmid spreading and became capable of molecular analysis only in the in vitro years.
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As the 1970s came to an end, generalized transduction was added to the other two modes of gene transfer by plasmid-mediated conjugation and by transformation (albeit artificial) when Colin Stuttard described a phage, SV1, that could transduce markers in Streptomyces venezuelae (Stuttard, 1979 ). Unfortunately, SV1 did not work in any other species; and the second example of general transduction, by SF1 in Streptomyces fradiae (Chung, 1982
), was also taxonomically circumscribed. Only very recently has a system of general transduction for S. coelicolor and S. lividans been developed (J. Westpheling, personal communication), thereby promising to fill a significant gap in the genetic tool-kit.
The in vitro years
The major achievements of the in vitro years are summarized in Table 2. The 1980s opened momentously for Streptomyces genetics with the first reports of gene cloning in S. coelicolor and S. lividans (Bibb et al., 1980
; Suarez & Chater, 1980
; Thompson et al., 1980
). Of the genes cloned in these first experiments, several conferring antibiotic resistances soon established themselves as versatile tools in vector development; the tsr gene, for thiostrepton resistance (Thompson et al., 1980
), is perhaps the most famous, having been used as a selective marker on a multitude of Streptomyces cloning vectors (a mixed blessing perhaps, because this antibiotic is not the easiest to come by!).
The developments in Streptomyces in vitro genetics that followed during the 1980s may be divided into two broad groups. One includes the beginnings of an understanding of basic features of the molecular biology and physiology of the organisms, such as gene and operon structure, and the control of transcription, translation and primary metabolism; the other represents the use of gene cloning to understand the molecular genetics of some of the special features of Streptomyces biology, especially antibiotic production and the developmental cycle.
In the first category, developments were coming thick and fast by the mid-1980s. Streptomyces promoter sequences were isolated (Bibb & Cohen, 1982 ) and soon after that, the first Streptomyces gene was sequenced (Thompson & Gray, 1983
). Not surprisingly, this gene sequence revealed an extraordinarily high proportion of codons ending in G or C, and this bias, typical of Streptomyces genes, was exploited in the frame program (Bibb et al., 1984
), which remains an extremely useful tool for finding genes (and frameshift errors!) in DNA sequences. Soon, promoters were sequenced and found to be both heterogeneous and abundant: many genes turned out to have more than one transcription-start site (e.g. Bibb et al., 1985
; Buttner et al., 1987
), a finding that is still not understood in any detail but which may reflect the need for subtly modulated expression at different stages in the life cycle or in different physiological states.
Pioneering studies revealed multiple forms of RNA polymerase holoenzyme in S. coelicolor (Westpheling et al., 1985 ; Buttner et al., 1988
), establishing this as a potential strategy for differential regulation of different subsets of genes in Streptomyces, following the Bacillus subtilis paradigm (Losick & Pero, 1981
). The number of different sigma factors in S. coelicolor has increased steadily over the years, including the remarkable and still only partially understood presence of four sigma factors that strongly resemble the vegetative E. coli and B. subtilis form (Tanaka et al., 1988
); only one of these is essential (Buttner, 1989
). Currently, the number of known or inferred alternative sigma factors in S. coelicolor runs to at least 20 (M. J. Buttner, G. Kelemen & M. Paget, personal communication): even more than in B. subtilis.
Another surprising discovery was the relative abundance of Streptomyces genes that must be translated without a conventional ShineDalgarno sequence, because there is no untranslated leader on the mRNA before the start codon (Janssen et al., 1989 ). This provided one of the rather rare examples of a basic aspect of molecular biology that was brought to prominence in Streptomyces rather than in E. coli.
The first examples of Streptomyces operons to be rigorously defined reflect aspects of primary metabolism, and specify the proteins needed for the utilization of galactose and glycerol (Fornwald et al., 1987 ; Smith & Chater, 1988
). Both are subject to specific induction as well as generalized carbon-catabolite repression by glucose (Mattern et al., 1993
; Hindle & Smith, 1994
). This global regulatory control has itself turned out to differ fundamentally in mechanism from the E. coli paradigm, with an involvement of glucose kinase, but not of cyclic AMP (Hodgson, 1982
; Angell et al., 1994
).
In the second category, the 1980s saw the beginning of our understanding of the molecular genetics of antibiotic production and its manipulation. The first antibiotic-biosynthetic genes were cloned, by a variety of methods (Chater & Bruton, 1983 ; Feitelson & Hopwood, 1983
; Gil & Hopwood, 1983
), and soon genes for a whole pathway, the act genes for actinorhodin biosynthesis, were isolated and expressed in a different Streptomyces host (Malpartida & Hopwood, 1984
). Just as loss of the blue colour of actinorhodin had been instrumental in the isolation and in vivo mapping of antibiotic-pathway genes (Rudd & Hopwood, 1979
), so its reacquisition was crucial to the isolation and in vitro mapping of the genes (Malpartida & Hopwood, 1984
, 1986
). Soon, colour was exploited again, this time to produce a hybrid antibiotic by genetic engineering; when specific segments of the act gene cluster were introduced on a plasmid vector into the producer of the brown antibiotic medermycin, a beautiful purple culture arose and was found to be making mederrhodin, a compound with structural features of both medermycin and actinorhodin (Hopwood et al., 1985a
).
While cloning the act genes demonstrated close linkage of biosynthetic structural genes, the finding that Streptomyces parvulus containing the act cluster produced actinorhodin without killing itself (Malpartida & Hopwood, 1984 ) provided indirect evidence that a resistance gene had also been cloned; the basis of actinorhodin self-resistance, while probably in some way reflecting antibiotic export (Bystrykh et al., 1996
), is still not well understood. In contrast, the methylenomycin gene cluster, on SCP1, was proved to contain resistance as well as biosynthetic and regulatory genes. This followed from the recognition of a specific methylenomycin-resistance gene the very first Streptomyces gene to be cloned in Streptomyces (Bibb et al., 1980
) as part of the DNA that caused methylenomycin production in a non-producing host, and overproduction when a deduced regulatory region was inactivated (Fig. 12
; Chater & Bruton, 1985
).
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Two further techniques were added to the toolbox for Streptomyces genetic manipulation at the end of the 1980s. The first Streptomyces transposon was identified in 1987. This was a Tn3-type element found in S. fradiae when it jumped on to the plasmid prophage of SF1, the transducing phage already referred to (Chung, 1987 ). A second was engineered from a naturally occurring insertion sequence in S. lividans (Solenberg & Burgett, 1989
). The use of transposons in Streptomyces has lagged behind that in many other bacteria. However, the most promising seem now to be those isolated from other bacteria, such as Mycobacterium (Smith & Dyson, 1995
), or even Tn5 (Volff & Altenbuchner, 1997
). The second major advance was the demonstration of conjugal transfer of plasmids from E. coli to Streptomyces (Mazodier et al., 1989
). The trick was to provide E. coli with a bifunctional plasmid containing a copy of oriT, as well as with mobilizing genes on the chromosome or on a second plasmid. This system has become a very useful one for the straightforward transfer of genes into Streptomyces (e.g. Bierman et al., 1992
).
As a manifestation of all the activity in Streptomyces in vitro genetics in the 1980s, as well as the continued use of in vivo techniques, the John Innes Manual was published in 1985 (Hopwood et al., 1985b ). Judging by the number of research papers citing its use over the next decade, it filled a major gap. Now it is badly out of date, but we hope that the second edition, in preparation since 1994, will appear before the next millennium dawns (T. Kieser, M. J. Bibb, M. J. Buttner, K. F. Chater & D. A. Hopwood, unpublished).
The in silico years
The main developments of the in silico years are summarized in Table 3. If we define in silico genetics as the drawing of conclusions about the functions of genes from comparisons between a newly determined DNA sequence and sequences already in the databases, this phase of Streptomyces genetics probably began with the sequencing of the S. coelicolor bldA gene in 1987. Mutations in bldA abolish aerial-mycelium formation (hence the name bald) and also prevent production of the four known S. coelicolor antibiotics under certain nutritional conditions; this phenotype was so pleiotropic that the gene was thought likely to act at a central point in decision-making during the life cycle. Sure enough, the sequence showed that bldA could not encode a protein but rather would produce a typical tRNA with the anti-codon UUA (
TTA in DNA) (Lawlor et al., 1987
). So was born the concept of a developmental switch that is dependent on the absence of the rare TTA codon for leucine from all genes required for vegetative growth, but present in a limited number of genes expressed post-exponentially (Leskiw et al., 1991
). This idea was strikingly illustrated by the finding of a TTA codon in the 5' region of actII-ORF4, the gene for the pathway-specific activator of the actinorhodin cluster: mutation of this codon to TGA restored antibiotic production in a bldA background (Fernández-Moreno et al., 1991
). The genome-sequencing project for S. coelicolor is providing a potentially complete inventory of genes containing TTA codons, and therefore making up a regulon controlled by bldA: the current prediction is about 100 genes (K. F. Chater, personal communication).
A second striking example of the power of in silico comparison came soon after: the sequence of the whiG gene, mutation of which causes the aerial mycelium to continue indeterminate growth and fail to switch to metamorphosis into chains of spores, showed it to encode a sigma factor (Chater et al., 1989 ), thereby establishing RNA polymerase heterogeneity as a strategy in Streptomyces development, recollecting the B. subtilis story (Losick & Pero, 1981
). Again, sequencing and in silico analysis had short-circuited perhaps years of experimental attempts to elucidate the role of a developmental gene. This approach continues to bear fruit (e.g. Chater, 1998
).
In the field of antibiotic biosynthesis, in silico genetics soon began to play an equally decisive role. Sequencing of the DNA regions responsible for the early carbon-chain-building steps towards the aromatic polyketides granaticin and tetracenomycin revealed that the enzymes involved the first bacterial polyketide synthases to be studied by molecular genetics were organized like those of a typical bacterial fatty acid synthase, strikingly confirming the idea that polyketide and fatty acid synthesis are homologous processes. The synthases were clearly so-called type II enzymes, consisting of separate subunits (Sherman et al., 1989 ; Bibb et al., 1989
); this turned out to be the rule for aromatic polyketides (Fig. 13
). In contrast, similar studies on the molecular genetics of erythromycin biosynthesis in Saccharopolyspora erythraea revealed a type I polyketide synthase organization, in which large proteins carry the different enzymic functions as a linear array of domains, an organization hitherto known only in eukaryotes. Even more remarkably, the sequencing revealed multiple sets, or modules, of domains (Fig. 14
) that would together form an assembly line for biosynthesis of the polyketide backbone (Cortes et al., 1990
; Donadio et al., 1991
). I have likened this discovery to that of the deduction of the mode of DNA replication directly from the WatsonCrick structure (Hopwood, 1997
); the assembly line model for complex polyketide biosynthesis had to be experimentally verified, but it arose directly from the determination of the gene sequences. These discoveries about the aromatic and complex polyketides in turn opened the way for the burgeoning field of combinatorial biosynthesis of unnatural natural products by the genetic engineering of polyketide synthases (McDaniel et al., 1993
, 1999
; Khosla & Zawada, 1996
; Hopwood, 1997
).
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Hardly had the Kieser et al. (1992 ) paper been published than things took a dramatic turn. Carton Chen came on sabbatical to the John Innes Centre from the National Yang-Ming University in Taipei in the summer of 1992, with the first evidence for a linear chromosome in S. lividans, and he and Helen Kieser, with constant input from Yi-Shing Lin back in Taipei, clearly established this (Lin et al., 1993
). Pretty soon, linearity was shown also for the S. coelicolor chromosome. How had this been missed? It turned out that a mistake had been made in positioning one of the DraI sites and so a point where apparent DraI and AseI sites coincided had been overlooked (this was the position of the chromosome ends); and long terminal repeats, later estimated to be >60 kb, at either end of the chromosome had led to hybridization of a probe taken from a point near to one end, to both ends, thereby mimicking a physical continuity across the 3 oclock region. Carton Chen has vividly described the whole story, and especially the excitement of the hunt for the chromosome ends (Chen, 1997
). The discovery has helped to change our ideas about bacterial chromosomes. It was also linked to elegant experiments on linear plasmids which proposed a model for Streptomyces chromosome replication in which conventional bidirectional replication takes place from a typical, centrally located oriC (Calcutt & Schmidt, 1992
; Zakrzewska-Czerwinska & Schrempf, 1992
), followed by patching replication primed by proteins attached covalently to the free 5' ends of the chromosome to fill the gap left by removal of the RNA primer for the last Okazaki fragment on each discontinuous strand (Chang & Cohen, 1994
).
The next step towards genomics was the preparation by Matthias Redenbach of a Supercos library of S. coelicolor chromosomal DNA and the selection from it, in a fruitful collaboration involving Kaiserslautern, Hiroshima and Norwich, of a minimal set of some 320 cosmids that form an overlapping library of clones covering the complete chromosome, except for three short gaps (Redenbach et al., 1996 ). More than 170 genes, gene clusters and other genetic elements were located on the set of cosmids, mainly by hybridization to specific cosmids or the overlaps between pairs of adjacent cosmids, to form a detailed genetic and physical map (Fig. 17
). This immediately became a valuable resource to help in gene mapping and gene isolation for the Streptomyces genetics community. Even more importantly, it became the jumping-off point for the genome-sequencing project which was initiated at the Sanger Centre at Hinxton, Cambridge in August 1997 with funding from the UK Biotechnology and Biological Sciences Research Council (BBSRC) (www.sanger.ac.uk/Projects/S_coelicolor). Collaborative funding from the Beowulf Genomics initiative of the Wellcome Trust in October 1998 has ensured that the complete sequence should be available by late 2000.
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Clusters of antibiotic-biosynthetic genes in actinomycetes range in size from about 20 kb or less for a relatively simple aromatic polyketide like actinorhodin to at least 90 or 100 kb for complex polyketides like rifamycin or rapamycin, where encoding the modular polyketide synthases alone requires 5080 kb of DNA (Schwecke et al., 1995 ; August et al., 1998
). Even though a given strain can make several different antibiotics, it is thus likely that genes encoding antibiotic-biosynthetic enzymes, and the associated genes for self-protection and pathway-specific activators, run to a few hundreds, not thousands. However, many other genes are probably involved more indirectly in modulating antibiotic production (Bibb, 1996
).
The number of genes directly involved in determining the life cycle, including those currently recognized by the bld and whi genes, is also not likely to exceed a hundred or two (Chater, 1998 ), even though the range of genes directly involved in the transition to aerial mycelium production has been crucially expanded from the original set of bld loci (Merrick, 1976
) by the isolation of others that seem to act via an extracellular signalling pathway involving a morphogenetic protein, SapB (Willey et al., 1993
). Again, the discovery of two phases of glycogen deposition and hydrolysis, at different stages of the life cycle, and controlled by two sets of paralogous genes (Bruton et al., 1995
), served to identify genes with a presumptive but indirect role in the developmental cycle which would not have been discovered by a simple search for mutants with a clear defect in morphogenesis.
In agreement with these ideas, the CDSs so far annotated in the S. coelicolor genome sequence include a high proportion that would encode putative transcriptional regulators, sensors, transporters and clusters of genes likely to be dedicated to the uptake and utilization of nutrients (J. Parkhill, personal communication; www.sanger.ac.uk/Projects/S_coelicolor). Such genes could help the organism to respond appropriately to a soil environment that is highly variable in terms of physical, chemical, nutritional and biotic (competition) stresses. It will be a major challenge, when in silico genomics of S. coelicolor gives way to global functional analysis, to find out what all these genes are doing, not to mention the ~27% of CDSs with no significant database match. I look forward eagerly to this analysis of the S. coelicolor transcriptome, proteome and metabolome as the next phase of Streptomyces genetics.
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ACKNOWLEDGEMENTS |
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