And Linear Plasmids are Widespread Among Streptomycetes

Since SCPl behaved in many ways similarly to well-characterised plasmids such as the F-factor of E. coli, it was reasonable to suppose that it would be physically similar as well. It was therefore natural to try to isolate it by CsCl-ethidium bromide density gradient centrifugation. Application of this technique to S. coelicolor indeed yielded CCC DNA, of about 30 kb (Schrempf et al. l975), but this proved to be present even in SCPl-free strains, and was designated SCP2 (Bibb et al. l977). Nevertheless, physical evidence that SCPl existed as DNA came from two kinds of experiments: DNA renaturation using S. coelicolor strains and a strain of another species, S. parvulus, into which SCPl had been transferred by conjugation revealed that the presence of SCPl was associated with a significant amount of DNA (more than 100 kb of unique sequence) (Hopwood et al. l979); and fragments of SCPl were cloned in the early 1980s (Bibb et al. l980; Chater and Bruton l983, l985).

Eventually, the idea that SCPl might be linear instead of circular was given impetus by the ground-breaking discovery of linear plasmids in S. rochei in K. Sakaguchi's laboratory. In the 1970s, the development of restriction enzyme technology, and the resultant recombinant DNA revolution, had been greatly helped by the use of electrophoresis through agarose gels to separate linear DNA fragments. Hayakawa et al. (l979) found that when total DNA preparations from a lankacidin-producing streptomycete (later called S. rochei) were subjected to standard agarose gel electrophoresis, the usual loose band corresponding to sheared chromosomal DNA fragments was accompanied by a sharp band of molecules shown by electron microscopy to be about 15 kb (later recalibrated as 17 kb), and revealed by restriction enzyme analysis to consist of one type of molecule. (Note that the use of a pH-neutral DNA extraction procedure was important for the success of this experiment:

the usual alkaline extraction method for plasmid isolation denatures and precipitates most linear DNAs.) The copy number of this DNA was about 60 per chromosome (Hirochika and Sakaguchi 1982). It was deduced that proteins were bound at or close to the ends, since the plasmid end-fragments were not seen on agarose gels when DNA prepared without the usual help of pronase was digested with restriction enzymes, and subsequent pronase digestion resulted in their full representation. Hirochika et al. (1984) went on to show that only the 5'-ends were linked to a protein. They also sequenced about 800 bp from each end, and found that both ends had the same sequence for 614 bp. The presence of 5'-linked proteins, often at the ends of terminal inverted repeats (TIRs) of various lengths, has turned out to be a general feature of linear replicons of actinomycetes, the terminal proteins playing a key role in the replication of the ends (Bao and Cohen 2001; see Chen 2007, in this volume).

Multicopy linear plasmids of tens of kilobases can be detected in some other streptomycetes by conventional gel electrophoresis (e.g. S. rimosus, Chardon-Loriaux et al. 1986; S. clavuligerus, Keen et al. 1988), but they have not so far been shown rigorously to carry genes for recognisable phenotypic traits, or to interact physically with the host genome. It was not until the advent of various kinds of pulsed field gel electrophoresis (PFGE; Schwartz and Cantor 1984; Carle and Olson 1984) that very large linear DNA molecules were revealed by the work of H. Kinashi. These PFGE techniques all depend on frequent changing of the direction of the current during electrophoresis, which requires linear DNA molecules to realign, a process that takes longer for larger molecules. Shearing of the DNA is minimised by preparing it from mycelium immobilised in an agarose plug. The full preparation in the plugs includes successive treatments with lysozyme, pronase and detergent, before the plugs are embedded in the electrophoresis gel.

The Kinashi laboratory's extraordinary first results were published in two papers (Kinashi and Shimaji 1987; Kinashi et al. 1987). They found one or more linear plasmids ranging from 17 to 590 kb in six out of 12 antibiotic-producing streptomycete (strains) tested (Fig. 3). (Although these have often been called "giant" linear plasmids, it is clear that there is a continuum of sizes of linear plasmids from 17 to hundreds of kb, so we have not retained the term in this chapter.) One of the strains examined was S. coelicolor A3(2), in which it was found that a series of plasmids varying in size from 410 to 590 kb in ca. 30-kb increments was made up of forms of SCP1 (using as probe a cloned fragment of SCP1 carrying resistance to methylenomycin—see below and Fig. 4). No bands were found in a strain genetically defined as lacking SCP1, and strains carrying SCP1, but not SCP2, had only a single SCP1 band of 350 kb. Subsequent work showed that the bands forming the ladder in the A3(2) strain arose from the integration of tandem copies of SCP2-derived sequences into SCP1 (see below).

Further examples of linear plasmids have also been described from other species (Table 1), and they have been found in several other actinomycete

Fig. 3 Separation of linear plasmids from antibiotic-producing Streptomyces strains by orthogonal-field-alteration gel electrophoresis. The figure is from Kinashi and Shimaji (1987). Note that in these early days of PFGE technology, the tracks often showed great distortion, which has been largely overcome by later technical improvements. M, Saccharomyces cerevisiae chromosomes; M2, ÀDNA; M3, ÀDNA digested with HindIII; 1, Streptomyces lasaliensis NRRL3382R; 2, S. violaceoruber JCM4979; 3, S. fradiae C373.1; 4, S. parvulus JCM4068; 5, S. venezuelae JCM4526; 6, S. rochei 7434AN4

genera and other bacteria (see Fetzner et al. 2007, Hertwig 2007, Holsters et al. 2007, all in this volume). Here we briefly expand on a few general aspects of Streptomyces linear plasmids. The first such plasmids to be studied all possessed TIRs hundreds of base pairs in length, leading Hirochika et al. (1984) to propose that the terminal repeats were held together by proteins in a "racket frame-like" structure ("invertrons": Sakaguchi 1990); but some other linear plasmids from streptomycetes have TIRs of only a few tens of base pairs, and in the cases of Rhodococcus opacus plasmids pHG201 and 205 the TIRs are of only 3 bp (Kalkus et al. 1998), so the racket-frame model in its original incarnation does not really apply. However, the ends ("telomeres") of all actinobacterial linear plasmids do show complex secondary structural features, conserved among most of the plasmids, over several tens of bases (see Chen 2007, in this volume). The fact that the plasmid 5'-ends are always linked to a terminal protein leaves open the possibility that the ends may be held together by terminal-protein-mediated interactions. Significantly, Streptomyces chromosomes were also eventually found to be linear, and to have

M12345M 6789 10

Fig. 4 Separation of SCP1 plasmids by PFGE and their Southern blot analysis probed by the methylenomycin resistance gene (mmr). The figure is from Kinashi et al. (1987). In addition to the linear plasmid SCP1, a series of plasmids were detected in S. violaceoruber JCM4979, which was originally Sermonti's A3 (2) strain. The multiple bands are the result of tandemly integrated sequences derived from the circular plasmid SCP2, which is also present in strain A3 (2) (see text for further discussion). M, Saccharomyces cerevisiae chromosomes; 1,6, S. violaceoruber JCM4979, originally Sermonti's S. coelicolor A3(2); 2, 7, S. coelicolor M124; 3, 8, S. coelicolor M130; 4, 9, S. coelicolor M138; 5,10, S. coelicolor M146

Fig. 4 Separation of SCP1 plasmids by PFGE and their Southern blot analysis probed by the methylenomycin resistance gene (mmr). The figure is from Kinashi et al. (1987). In addition to the linear plasmid SCP1, a series of plasmids were detected in S. violaceoruber JCM4979, which was originally Sermonti's A3 (2) strain. The multiple bands are the result of tandemly integrated sequences derived from the circular plasmid SCP2, which is also present in strain A3 (2) (see text for further discussion). M, Saccharomyces cerevisiae chromosomes; 1,6, S. violaceoruber JCM4979, originally Sermonti's S. coelicolor A3(2); 2, 7, S. coelicolor M124; 3, 8, S. coelicolor M130; 4, 9, S. coelicolor M138; 5,10, S. coelicolor M146

similar protein-linked ends (Lin et al. 1993; Chen 1996; Chen et al. 2002). Cytological evidence in support of spatial co-location of the ends of the S. coelicolor chromosome was obtained by Yang and Losick (2001), using fluorescence in situ hybridisation to probes ostensibly specific for each end, but it was subsequently found that the particular strain used in that work (J1508) possessed a duplication compared with the sequenced strain M145 that would have resulted in both probes hybridising to the same end (Weaver et al. 2004; C.W. Chen, personal communication).

At least some of the linear plasmids are evidently transmissible by conjugation (SCP1, Vivian 1971; SLP2, Chen et al. 1993), but the absence of genetic markers on many of them has limited analysis of this aspect. Their copy numbers vary from several tens per chromosome in the case of the small plasmids to just a few-fold for the larger examples; but at least for SCP1, which typically has a copy number of up to seven in S. coelicolor (Yamasaki et al. 2003), the copy number was greatly increased in an SCP1+ derivative of S. parvulus obtained by interspecific conjugation (Hopwood and Wright 1973a; Chater and Bruton 1983).

Table 1 Linear plasmids of streptomycetes

Plasmid (host)

Size (kb)

Terminal inverted repeats

Copy number

Comments

(S. coelicolor)

356

75 kb

c. 7

Encodes methylenomycin production; many interactions with host chromosome

Bentley et al. 2004

SLP2

(S. lividans)

50

44 bp

?

15.7 kb at one end; corresponds Chen et al. to ends of host chromosome 1993

pPZG101 (S. rimosus)

387

95 kb

?

Derived from pPZG102 during strain improvement; many interactions with chromosome including acquisition of oxytetracycline pathway genes

Gravius et al. 1994

pSCLl

(S. clavuligerus)

12

c. 900 bp High

Host produces clavulanic acid

Keen et al. 1988; Wu and Roy 1993

pSLA2-S (S. rochei)

17

614 bp

c. 60

Origin of the "racket-frame" model

Hirochika et al. 1984

pSLA2-M (S. rochei)

100

?

?

Kinashi et al.1994

pSLA2-L (S. rochei)

211

12 bp

?

Mainly occupied by gene sets for production of lankacidin, lankamycin, a type II poly-ketide, carotenoids, gamma-butyrolactone

Mochizuki et al. 2003

pSRM (S. rimosus)

43

?

?

Evidence for pock formation accompanying transfer

Chardon-Loriaux et al. 1986

Unnamed (S. rimosus)

255

?

?

Host produces oxytetracycline

Rathos et al. 1989

pKSL

(S. lasaliensis)

520

?

?

Host produces lasalocid and echinomycin

Kinashi et al. 1987

SAP1

(S. avermitilis)

94

?

?

Discovered during genome sequencing

Ikeda et al. 2003

Unnamed (S. fradiae)

420

?

?

Host produces tylosin

Kinashi and Shimaji 1987

Unnamed (S. parvulus)

520, 560, 580

?

?

Host produces actinomycin D

Kinashi and Shimaji 1987

Table 1 (continued)

Plasmid Size Terminal Copy Comments

(host) (kb) inverted number repeats

Refs.

Unnamed 130 (S. venezuelae)

pSV2

(S. violaceo-) ruber

100 426 bp

Host produces chloramphenicol Kinashi and

Shimaji 1987

Plasmid ends similar to those Spatz et al. of the S. coelicolor chromosome 2002

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