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Chapter 1 General introduction to actinomycete biology Chapter 1 General introduction to actinomycete biology CONTENTS Taxonomy of Streptomyces ………………………………………………………………………………2 The genera of actinomycetes ……………………………………………………………………2 The genus Streptomyces………………………………………………………………………….2 Ecology of Streptomyces ………………………………………………………………………………….5 Streptomycetes as pathogens ……………………………………………………………………………6 Some physiological features of primary metabolism in streptomycetes ……………………………...7 Carbon sources …………………………………………………………………………………..7 Nitrogen sources …………………………………………………………………………………8 Amino acid catabolism ………………………………………………………………………….9 Biosynthesis ………………………………………………………………………………………9 Some physiological novelties ………………………………………………………………...…10 Antibiotic production by Streptomyces …………………………………………………………….…..10 Streptomycetes as antibiotic producers ……………………………………………………….10 Antibiosis in soil ………………………………………………………………………………...10 Physiology and regulation of antibiotic production …………………………………………..16 Developmental biology of Streptomyces ………………………………………………………………..17 The Streptomyces chromosome and its genetic elements ……………………………………………...18 DNA base composition ………………………………………………………………………….18 The chromosome ………………………………………………………………………………..18 Plasmids …………………………………………………………………………………………20 Transposable elements …………………………………………………………………………21 Phages ……………………………………………………………………………………………21 Restriction and modification of Streptomyces DNA ………………………………………….22 Genetic studies with streptomycetes and their near relatives ……………………………………….22 Actinomycetes used for genetical studies …………………………………………………….22 Genetics and strain improvement for antibiotic and enzyme production ………………….23 Safety guidelines for recombinant DNA experiments with Streptomyces …………………33 This chapter contains an introduction to actinomycete biology as a background to the molecular biology and genetics. Books dealing in more detail with some of these areas include two edited collections of chapters (Goodfellow et al., 1984, 1988). The proceedings of the triennial International 1

Chapter 1 General introduction to actinomycete biology Symposia on the Biology of Actinomycetes are also rich sources of information. The last four volumes are: Szabo et al. (1986); Okami et al. (1988); Ensign et al. (1992); and Debabov et al. (1995). There is also Volume 4 of Bergey's Manual of Systematic Bacteriology (Williams et al., 1989) devoted to the actinomycetes. Taxonomy of Streptomyces The genera of actinomycetes The Gram-positive bacteria include two major branches: the low G+C organisms, containing genera such as Bacillus, Clostridium, Staphylococcus and Streptococcus; and the high G+C organisms referred to as the actinomycetes. Many of the latter develop a mycelial habit - originally regarded as the hallmark of the actinomycetes - at least at some stage in their life cycle, but others do not. A whole array of taxonomic tools has been used to define genera and suprageneric groups of actinomycetes (Goodfellow, 1989; Embley and Stackebrandt, 1994), but partial sequence analysis of 16S ribosomal RNA is the most significant. An abridged tree based on this analysis is shown in Fig. 1.1 to give a flavour of the diversity of the actinomycete families and the place of the genus Streptomyces in the group. The genus Streptomyces The genus is defined by both chemotaxonomic and phenotypic ("phenetic") characters. The major emphasis is now on 16S rRNA homologies, in addition to cell wall analysis and fatty acid and lipid patterns (Williams et al, 1989; Wellington et al., 1992). One of the quickest methods for preliminary identification to genus level was the presence of the LL isomer of diaminopimelic acid (LL-DAP) as the diamino acid in the peptidoglycan. This feature, when combined with the characteristic substrate and aerial mycelium, was diagnostic for Streptomyces. However, it turned out that some strains, previously classified as Kitasatosporia, may contain major amounts of DL-DAP in the vegetative mycelium only (Wellington et al., 1992); species from this former genus were found by Wellington et al. (1992) to lie within the 16S rRNA clade for Streptomyces, but this was recently contradicted when more strains were studied (Zhang et al., 1997). The genus Streptomyces has been subjected to numerous systematic studies during the past 30 years but it is still difficult to identify unknown isolates. Many type species have been described, but there has been much over-speciation resulting from antibiotic patents and the consequent need to assign a name to the producing organism. Since the International Streptomyces Project in 1964, an attempt was made to produce valid species descriptions with at least a minimal number of standard phenotypic criteria. However, the criteria turned out to be too minimal and the proliferation of species continued, without any real attempt to compare species thoroughly with each other. The first study to do this relied on numerical phenetic techniques to define clusters of strains or species based on comparison of many phenotypic traits (Williams et al., 1983). This established 23 major clusters and some 20 minor groups; the clusters were equated with species, except for the largest, cluster 1, which was split into three species (Williams et al., 1989). Because the Williams et al. (1983) study included only one representative for each species, very many species were reduced to synonyms. However their specific epithets are still in 2

Chapte er 1 General in ntroduction to actinomycete e biology Fig. l. sequen outgro to fam familie .l. Abridged nces. The tree oup. There are milies. About h es. Kindly pro phylogenetic e was construc e now over 100 half of the ge ovided by M. G tree of acti cted by using t 0 validly desc enera omitted Goodfellow. inomycetes b the neighbour cribed actinom from this ab based on alm r-joining meth mycete genera ridged tree be most full 16S hod with Baci , but not all ha elong to one ribosomal R llus subtilis as ave been assig of the recogn RNA s the gned nised 3

Chap pter 1 Genera al introduction to actinomyce ete biology Ssm Fig. sim arith We Stre . 1.2. Dendro mple matching hmetic averag llington. (Not eptomyces by ogram obtaine g coefficient ges (UPGMA) te that species Williams et a ed using phen (Ssm) and cl ). Data were d s of Chainia, K al., 1989 and W notypic charac lustering by derived from W Kitasatoa and Witt and Stack cters for selec the unweight Williams et al d Streptovertic kebrandt, 1990 cted major cl ted pair grou l. (1983). Kind cillium, reduce 0, are included lusters derived up method ba dly provided b ed to synonym d.) d using ased on by E. M. my with 4

Chapter 1 General introduction to actinomycete biology constant use; for example S. griseus is synonymous with S. anulatus, S. lividans with S. violaceoruber, and S. hygroscopicus with S. violaceoniger. The major species groups (Williams et al, 1983), are given in Fig. 1.2, with some minor groups of interest also included. The names are those of the oldest extant type species within each cluster. A second numerical phenetic analysis was made by Kampfer et al. (1991); it differed from the earlier study in including many more species, and more than one strain of each species when available. Many of the clusters defined by Williams et al. (1983) were recognised; a striking example is the albidoflavus/anulatus/griseus/halstedii group appearing as cluster 1 in both studies, in which 28 of the S. griseus strains were grouped. Despite the problems associated with phenotypic characterisation, most of the strains sharing the same specific epithet grouped together, indicating previously reliable identification, but there were some notable exceptions; for example S. hygroscopicus strains were recovered in cluster 1 subcluster 6, 8, 9, 10, 13, 24, 25, 35, 53, 54, 55, 56, 57 and 85. This may indicate either problems in identification of this group or considerable phenotypic variation. Several studies have attempted to use sequence data from variable regions of 16S rRNA to establish taxonomic structure within the genus, but the variation was regarded as too limited to help resolve problems of species differentiation (Witt and Stackebrandt, 1990; Stackebrandt et al., 1991, 1992). For example, species with a phenotype characteristic of the streptoverticillia grouped as a clade, but were not distinct from other species in the genus, in contrast to the results of the phenotypic analysis (clusters 55, 56, 58 and 59). The close phenotypic relationship between S. lavendulae and the streptoverticillia species (Fig. 1.2) was also confirmed by 16S rRNA sequence comparisons (Witt and Stackebrandt, 1990). The type species of the genus, S. albus, retained a distinct position in the phylogenetic trees and had unique sequences in the variable a and P regions of the gene (Stackebrandt et al., 1991). Total DNA homology studies (Labeda, 1992) have indicated genetic heterogeneity within some of the large phenotypic species groups defined by numerical taxonomy. The S. cyaneus cluster 18 was studied in detail; strains showed DNA relatedness of 20-85% with the majority of values around 50% (Labeda and Lyons, 1991). Selected species within the cluster were reduced to synonymy with others if the comparisons gave homology values >70%. Ecology of Streptomyces Streptomycetes are ubiquitous in nature. Their ability to colonise the soil is greatly facilitated by growth as a vegetative hyphal mass which can differentiate into spores that assist in spread and persistence. The spores are a semi-dormant stage in the life cycle that can survive in soil for long periods (Mayfield et al., 1972; Ensign, 1978): viable Streptomyces cultures were recovered from 70 year old soil samples (Morita, 1985). The spores impart resistance to low nutrient and water availability, whereas the mycelial stage is sensitive to drought (Karagouni et al., 1993). The relatively high numbers of streptomycetes in soil exist largely as inactive spores for most of the time. When laboratory-grown spores were added to nonsterile soil, they exhibited very low germination efficiencies, probably because of competition with indigenous microorganisms, but pre-germinated spores grew for a short time and then re-sporulated (Lloyd, 1969). Germination can be partially density-dependent, but the interaction did not cross species boundaries (Triger et al., 1991), suggesting special signalling factors between spores of the same strain, causing inhibition of germination above a certain concentration. The advantage would be 5

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