Actinomycetologica (2008) 22:20–29 Copyright Ó 2008 The Society for Actinomycetes Japan VOL. 22, NO. 1 Award Lecture Antibiotic production, linear and linear in Streptomyces

Haruyasu Kinashi Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima 739-8530, Japan (Received May 12, 2008 / Accepted May 12, 2008 / Published Jun. 25, 2008)

INTRODUCTION pSLA2 from Streptomyces rochei 7434AN4, which produces two structurally unrelated polyketide antibiotics, Streptomyces species are saprophytic soil that lankacidin and lankamycin (Fig. 1). They succeeded in have been found to carry an 8–9 Mb linear . isolating pSLA2 using a neutral DNA extraction method, Preceding the finding of linear chromosomes from Strep- the usual alkaline methods for circular plasmids being tomyces, linear plasmids with extensively similar structural unable to recover linear DNA. Physical analysis revealed features were isolated. Streptomyces linear chromosomes that pSLA2 is 17 kb in size and is structurally similar to and plasmids have terminal inverted repeats (TIRs) at both adenovirus and bacteriophage 29 DNA; TIRs are present ends and the 50 ends are blocked by a terminal protein. at both ends and a terminal protein is bound to the 50 Streptomyces linear plasmids are proving to be widely ends (Hirochika & Sakaguchi, 1982; Hirochika et al., distributed and involved in antibiotic production, degrada- 1985). However, it was later found that pSLA2 was not tion of aromatic compounds, phytopathogenicity and other involved in antibiotic production. functions. It appears that linear plasmids may have con- tributed to horizontal transfer of secondary metabolism in Detection of giant linear plasmids by pulsed-field gel microorganisms. electrophoresis Streptomyces linear chromosomes display dynamic re- In 1981 I started to study the function of pSLA2 arrangements, which frequently result in the formation of in antibiotic production in Dr. Sakaguchi’s laboratory, circular chromosomes by fusion of both deletion ends. In Mitsubishi-Kasei Institute of Sciences, where pSLA2 addition, a single crossover of the linear plasmid SCP1 and had been isolated and analyzed. We constructed many the linear chromosome of Streptomyces coelicolor A3(2) mutants by various mutagenic treatments of the parent generated two chimeric linear chromosomes. These rear- strain 7434AN4, and analyzed the production of lankacidin rangements have provided important hints on the evolution and lankamycin and the presence/absence of pSLA2 using of bacterial chromosomes. Since I have been involved conventional agarose gel electrophoresis. However, we in many of these processes, I would like to review here could not find any correlation between them. the story focusing on our own results on the occasion of In 1984 I had a chance to study abroad in Prof. receiving the SAJ Award. Hutchinson’s laboratory, Wisconsin University, where I investigated biosynthesis of the polyether antibiotic Plasmids in antibiotic production lasalocid in Streptomyces lasaliensis NRRL3382R. This It has long been suggested that plasmids are involved in project was related to the theme of my doctoral work, in antibiotic production in Streptomyces species, based on the which I determined the chemical structure of the polyether genetic instability of antibiotic-producing ability. In partic- antibiotic salinomycin by X-ray crystallography (Kinashi ular, extensive genetic studies of Prof. Hopwood, John et al., 1973) and studied its mass spectrometry and Innes Centre, confirmed that the plasmid SCP1 carried the structure-activity relationship under the supervision of biosynthetic for methylenomycin (mmy)inStrepto- Prof. Otake in Tokyo University. Although we constructed myces coelicolor A3(2) (Kirby et al., 1975; Kirby & many lasalocid-nonproducing mutants, attempted cosyn- Hopwood, 1977). Furthermore, the mmy cluster was thesis experiments with many combinations all failed to cloned by mutational cloning and analyzed in detail (Chater give positive lasalocid production (Kinashi et al., 1988). & Bruton, 1985), even though SCP1 itself was still resistant This result suggested that all of the mutants might have the to isolation and its physical identity had not been clarified. same genetic defect in antibiotic production, for example, This situation changed in 1987 when the application of the loss of a plasmid carrying the biosynthetic gene cluster pulsed-field gel electrophoresis (PFGE) to Streptomyces for lasalocid. However, we could not detect such a plasmid DNA revealed that SCP1 was a giant linear plasmid of with conventional agarose gel electrophoresis. about 350 kb (Kinashi et al., 1987). After I returned to Japan, Dr. Sakai, who was using one Molecular studies of Streptomyces linear plasmids began of the first OFAGE (orthogonal-field-alternation gel elec- in 1979 when Hayakawa et al. (1979) isolated a linear trophoresis) machines in Japan for yeast molecular biology,

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Fig. 1. Secondary metabolites produced by S. rochei 7434AN4 and its mutant. suggested that I should use PFGE. I applied this method a sheared purine-purine (typically, guanine-adenine) pair. to S. lasaliensis NRRL3382R and detected a giant linear Eventually, the complete nucleotide sequence (356,023 bp) plasmid pKSL of about 520 kb (Kinashi & Shimaji, 1987). of SCP1 was determined (Bentley et al., 2004), which In due course, I collected several antibiotic-producing confirmed the size of the TIRs (75,122 bp) and located strains whose antibiotic production was suggested to be the mmy gene cluster, a replication origin, two partition plasmid-determined, and detected many giant linear plas- genes (parA and parB) and others. However, a conserved mids (Kinashi & Shimaji, 1987; Kinashi et al., 1994; terminal protein gene (tpg) (Bao & Cohen, 2001; Yang et Kinashi, 1994; Kinashi et al., 1995) including SCP1 from al., 2002) could not be identified from the sequence data, S. coelicolor A3(2) (Kinashi et al., 1987). In this con- because of the unique sequence of SCP1. Later, nection, I later talked with Prof. Schrempf, Osnabruck Huang et al. (2007) isolated the terminal protein as a University, who said ‘‘You were lucky to use S. coelicolor complex with SCP1; finally identifying the unique tpc A3(2) strains, because we used Streptomyces lividans and (terminal protein of SCP1, orf127) and tac (terminal could not detect SCP1 due to DNA degradation in this associated protein of SCP1, orf125) genes of SCP1. species.’’ This DNA degradation problem in S. lividans was solved by changing the buffer for electrophoresis (Zhou Secondary metabolism-related genes on pSLA2-L et al., 1988) and was finally found to be caused by site- As mentioned above, the linear plasmid pSLA2 proved specific phosphorothioation of DNA (Wang et al., 2007). not to be involved in antibiotic production. The application of PFGE to S. rochei 7434AN4 revealed two linear Physical characterization of SCP1 plasmids, pSLA2-L (210 kb) and pSLA2-M (110 kb) in Among the many linear plasmids detected in antibiotic- addition to pSLA2-S (=pSLA2, 17 kb) (Kinashi et al., producing Streptomyces strains, we at first chose SCP1 1994). We analyzed plasmid profiles of all mutants derived for detailed analysis, because it had been studied the from strain 7434AN4 and found a complete correlation most extensively. Treatment of SCP1 with exonuclease III between the presence of the largest plasmid pSLA2-L and caused DNA digestion, while  exonuclease treatment did the production of both lankacidin and lankamycin. South- not, suggesting that the 50 ends are blocked by a terminal ern hybridization using two polyketide biosynthetic probes, protein (Kinashi & Shimaji-Murayama, 1991). Restriction eryAI for erythromycin and actI for actinorhodin, revealed mapping with EcoRV (Kinashi & Shimaji-Murayama, two homologous regions to each probe (Kinashi et al., 1991) and then EcoRI (Redenbach et al., 1998) revealed 1998), the eryAI–related sequences being identified as that SCP1 contained TIRs of about 80 kb. At the inside end lankamycin biosynthetic genes (Suwa et al., 2000). How- of TIR-R, an insertion sequence (IS466) was identified, ever, the location of the lankacidin cluster was not which suggested its involvement in the formation of the identified. TIRs (Kinashi et al., 1991). Thus, we started to sequence pSLA2-L, beginning from a Cloning and sequencing of the terminal fragments library of strain 51252 that carried only pSLA2-L. revealed that the telomere sequence of SCP1 could not Nucleotide sequencing was done using five ordered form a Y-shaped foldback structure (Kinashi et al., 1991), a and four plasmids containing a left or right feature later shown to be conserved in most Streptomyces terminal fragment. This revealed the sizes of pSLA2-L linear replicons (Huang et al., 1998) (Fig. 2). However, (210,614 bp) and the TIRs (1,992 bp), the GC content many hairpin loop structures could still be made. It is (72.8%), and 143 open reading frames (orfs) (Mochizuki et noteworthy that all of the loops consist of four nucleotides al., 2003). pSLA2-L has a conserved telomere sequence, rather than the usual three nucleotides, and are stabilized by which can make a typical Y-shaped foldback structure

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Fig. 2. Y-shaped foldback structures and hairpin loops that can be formed at the 30 ends of Streptomyces linear chromosomes and plasmids. Purine-purine sheared pairings are indicated by dots.

1 3 4 lkc cluster 18 Left end 40 kb lkcO lkcG lkcF lkcE lkcC lkcA

80 24 lkm cluster 53 120 srrW 62 srrX srrE 70 160 roc cluster srrZ srrCsrrY srrB srrA 110 200 srrD 104 crt cluster 116 srrF

Right end 143

Fig. 3. Gene organization of the linear plasmid pSLA2-L. Biosynthetic gene clusters for lankacidin (lkc), lankamycin (lkm), a cryptic type-II polyketide (roc), and carotenoid (crt) are shown. An afsA homolog (srrX), six tetR family repressor genes (srrA, srrB, srrC, srrD, srrE, and srrF), and three SARP family activator genes (srrW, srrY, and srrZ) are also indicated.

(Hiratsu et al., 2000) (Fig. 2). Homology searching of the SARP (Streptomyces antibiotic regulatory protein) genes 143 Orfs along with gene disruption experiments delimited (srrW, srrY, and srrZ). Thus, two thirds of pSLA2-L DNA the ranges of the biosynthetic gene clusters for lankacidin is occupied by secondary metabolism-related genes. (lkc, orf4-orf18), lankamycin (lkm, orf24-orf53), a cryptic Gene disruption of the regulatory genes indicated that type-II polyketide (roc, orf62-orf70) and carotenoid (crt, srrX has a positive effect on the production of both orf104-orf110) (Fig. 3). In addition, many regulatory genes lankacidin and lankamycin and a negative effect on spore for secondary metabolism were found, for example, a - formation (Arakawa et al., 2007). This effect is different butyrolactone (GB) biosynthetic gene (srrX, Streptomyces from that of afsA in Streptomyces griseus, which shows rochei regulatory gene X, an afsA homologue), six tetR positive effects on both streptomycin production and spore family repressor genes (srrA, srrB, srrC, srrD, srrE, and formation (Horinouchi & Beppu, 2007). The receptor gene srrF) including the GB receptor gene (srrA), and three srrA has counteracting functions against both effects of

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produced lankacidin at a similar level to the parent strain, suggesting that an iterative function of the LkcF protein is unlikely. These results are consistent with our hypothesis that LkcC is used four times and LkcA, LkcF and LkcG are used modularly to accomplish eight condensation reactions leading to the lankacidin skeleton.

Streptomyces chromosomes are linear Several years after the isolation of SCP1 and other linear plasmids, Prof. Chen, Yang-Ming University, revealed the physical organization of Streptomyces chromosomes. During the analysis of a Tn4811 in Fig. 4. A possible regulatory cascade for secondary metabolism S. lividans, they found two copies of Tn4811, one near the in S. rochei 7434AN4, deduced by hitherto obtained results. right end of the 50-kb linear plasmid SLP2. Another copy Pathways drawn by broken lines are speculative and have not been was also located near the end of a linear DNA. The 16-kb analyzed in detail. LC, lankacidin; LM, lankamycin. restriction maps from Tn4811 to the end were identical in both copies (Chen et al., 1993). Eventually, the linear DNA carrying the second copy was identified as a chromosome, srrX. On the other hand, srrB has a negative effect only on which revealed that the S. lividans chromosome is linear antibiotic production, while srrC has a positive effect on (Lin et al., 1993). They were lucky because the terminal spore formation without affecting antibiotic production. structures of SLP2 and the S. lividans chromosome are Transcriptional experiments including RT-PCR, S1 nucle- identical, which made them notice a chromosomal end. In ase mapping, gel retardation and foot printing have contrast we had been unlucky this time, since the terminal confirmed the main signaling pathway from srrX via srrA structures of SCP1 and the S. coelicolor A3(2) chromo- to srrY (Yamamoto et al., 2008) (Fig. 4). Expression of the some are totally different due to the unique telomere SARP gene srrY may lead to lankamycin production via sequence of SCP1. another SARP gene srrZ and to lankacidin production At about that time, I became involved in the directly or via an unidentified gene (unpublished results). project of S. coelicolor A3(2). Dr. Redenbach, However, the regulatory pathway from srrX to spore Kaiserslautern University, stayed in our laboratory from formation has not been clarified. Studies to reveal the 1994 to 1995 as a foreign researcher supported by the Japan whole regulatory cascade in S. rochei 7434AN4 are now in Society for the Promotion of Science (JSPS) and almost progress, the results of which will be described in future. completed the ordering of cosmids covering the chromo- Lankacidin C (2a) is a 17-membered macrocyclic some of the M145 (plasmid-free) derivative of S. coelicolor antibiotic different from the usual even-membered macro- A3(2). The ordered cosmid map combined with the genetic lides (Fig. 1). This unique structure and the gene organ- and restriction map constructed by Dr. Kieser, John Innes ization of the lankacidin synthase (lkc) cluster raised two Centre, by extensive hybridization experiments was pub- interesting questions. (i) How is the carbon-carbon linkage lished in 1996 (Redenbach et al., 1996). This work was formed between C2 and C18, which generates the lankacidin of great use to the first genome sequencing project in skeleton. (ii) How can the five ketosyntase domains in the Streptomyces, which was completed in 2002 (Bentley cluster accomplish eight condensation reactions necessary et al., 2002). The M145 chromosome is 8,667,507 bp long, for lankacidin synthesis. Biosynthetic studies led by Dr. contains 21,653-bp TIRs, and encodes 7,825 Orfs. Its Arakawa answered both questions. Regarding the first telomere sequence can form a typical Y-shaped foldback question, we found that the amine oxidase (LkcE, Orf14) structure as shown in Fig. 2. converts the acyclic amine (C18-N) intermediate LC-KA05 (3) to an unisolated imine (C18=N), which receives a Rearrangements of Streptomyces linear chromosomes nucleophilic attack by an enolic carbon (C2) to form the C2- It is well known that Streptomyces chromosomes are C18 linkage in lankacidinol A (2b) (Arakawa et al., 2005). unstable and often undergo rearrangements including large In regards to the second question, we proposed a hypothesis deletions and amplifications (Volff & Altenbuchner, 1998). of modular-iterative mixed polyketide biosynthesis for However, molecular understanding of these events was lankacidin (Arakawa et al., 2005). This hypothesis was difficult, because everyone was prepossessed with an idea supported by the following two pieces of experimental that Streptomyces chromosomes are circular. The finding evidence (Tatsuno et al., 2007). Heterologous expression of of the linear chromosome structure immediately opened the lankacidin cluster (lkcA-lkcO)inS. lividans resulted in a new insight into genetic instability in Streptomyces: lankacidinol A production, indicating that the gene cluster many phenomena could now be explained by deletion is sufficient for the synthesis of the lankacidin skeleton. of chromosomal ends and subsequent rearrangements In addition, a fusion of two PKS genes, lkcF and lkcG, (Leblond & Decaris, 1994).

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Fig. 5. Dynamic chromosomal rearrangements occurring concomitantly with terminal deletions in S. griseus. Linear chromosomes are drawn in racket frame structure (Sakaguchi, 1990). The terminal protein bound to the 50 ends is shown by a solid circle.

Before the finding of the linear chromosome of S. coleli- deletion ends (Kameoka et al., 1999; Inoue et al., 2003). color A3(2), I had moved to Hiroshima University in 1990, Thus, it was proved that chromosomal circularization where Prof. Nimi had been studying A-factor-independent occurred by nonhomologous recombination of deletion streptomycin production in S. griseus strain 2247. Con- ends, and Streptomyces chromosomes could be replicat- sequently, we started to analyze the chromosome structure ed and maintained in both linear and circular forms of this strain and revealed that it also carried a 7.8-Mb (Fig. 5). Chromosomal circularization was also indicated linear chromosome (Lezhava et al., 1995). It is noteworthy in S. lividans (Lin et al., 1993; Redenbach et al., 1993) that, as in the case of SCP1, the telomere sequence of strain and S. ambofaciens (Leblond et al., 1996) by detection of 2247 does not form a tertiary foldback structure (Goshi a fused macrorestriction fragment. In addition, Qin and et al., 2002) (Fig. 2). However, in this case all of the loops Cohen (2002) reported circularization of artificial pSLA2 near the end were composed of three nucleotides and plasmids in S. lividans by nonhomologous recombination contained a sheared purine-purine pair. of deletion ends. Therefore, circularization may be a To analyze chromosomal rearrangements in S. griseus, common strategy of Streptomyces linear replicons, when we isolated many mutants from strain 2247 and selected both of the two have been deleted. mutants with different macrorestriction patterns. All of the Analysis of the deletion in mutant MM9 at first mutants except one (mutant 402-2) were found to suffer suggested that only the right end was deleted. However, chromosomal deletion at both or either end(s). In mutant precise analysis revealed that the right arm was replaced by 402-2, we found a large , which is the left arm, and generated unusually long TIRs (Uchida et the first example of such rearrangements in Streptomyces al., 2003) (Fig. 5). At the inside ends of the newly formed species (unpublished results). It is noteworthy that the long TIRs, lipoprotein-like genes were identified, which chromosomal inversion in mutant 402-2 has not taken place indicated that homologous recombination between two over evolutionary time, but occurred during mutation lipoprotein-like genes caused arm replacement and recov- processes of strain 2247 and its mutant in our laboratory. ered a telomere on the right end. Thus, arm replacement The deletion sizes (30–550 kb) were different from mutant may be a strategy of Streptomyces to recover a telomere, to mutant and also at the left and right ends, indicating no when one of the two TIRs is completely deleted. Prior to hot spot for deletion. our work, Fischer et al. (1998) reported chromosomal arm Chromosomal circularization was suggested for four replacement in S. ambofaciens. mutants, 404-23, N2, No. 9 and No. 83, by generation of a In mutants 301-22-L and M, we detected arm replace- fused fragment that hybridized to both the left and right ment at the same lipoprotein-like genes. However, in these deletion end probes (Lezhava et al., 1997). The fused mutants the newly formed long TIRs again suffered fragments were cloned and sequenced, which revealed terminal deletion, and were fused at different points in microhomology or no homology between the left and right the TIRs, resulting in the formation of a circular chromo-

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Fig. 6. Generation of two chimeric chromosomes in S. coelicolor 2106 by single crossover of SCP1 and the wild-type linear chromosome. some with an unusually large palindrome (Uchida et al., When both arms are deleted, the telomeres cannot 2004) (Fig. 5). This unique structure was named ‘closed be recovered and therefore the chromosome should be racket frame’ from its shape. The fact that these 301-22 circularized by nonhomologous recombination to survive. mutants grow normally indicates that large palindromic Long TIRs formed by chromosomal arm replacement, also sequences are replicated without difficulty in S. griseus. suffer telomere deletions. If a second recombination occurs This is very surprising since one would not expect bacteria at the deletion ends in the long TIRs, a circular chromo- to be able to replicate such large palindromes. Together some with a large palindrome is generated. with the finding of symmetric linear plasmids with a large palindrome in Streptomyces (Kinashi et al., 1994; Qin Two chimeric linear chromosomes in S. coelicolor 2106, & Cohen, 2000), it was suggested that the replication and the implications for genome evolution machinery of this genus is somewhat different from that of A large linear DNA of about 1850 kb was found in other bacteria. S. coelicolor 2106, a cysD donor, which transfers a cysD phenotype in the mating with an SCP1 strain (Hopwood & Survival strategy of the S. griseus linear chromosome Wright, 1976). Consequently, we considered that this linear and function of TIRs DNA was a presumed plasmid, SCP10-cysD. Macrorestric- Dynamic rearrangements of the S. griseus linear chro- tion analysis and partial sequencing of this linear DNA and mosome are summarized in Fig. 5, which shows a the 7.2-Mb linear chromosome in strain 2106 revealed that remarkable fluidity of Streptomyces chromosomes and both DNA elements were formed by single crossover of the suggests the following hypothesis on chromosomal rear- wild type chromosome and SCP1, with deletion of 15-bp rangements and the function of TIRs (Kinashi, 2007). chromosomal DNA and 55-bp SCP1 DNA (Yamasaki & When one of the two telomeres is deleted inside the Kinashi, 2004) (Fig. 6). Since the telomere sequences of TIR region, recombinational DNA repair may function SCP1 and the S. coelicolor chromosome are totally differ- between the intact and truncated TIR sequences, and ent, these two linear DNAs do not have TIRs. Since we regenerate an intact telomere. However, we usually can- failed to cure the 1850-kb DNA by various mutagenic not detect this event, because an identical TIR structure treatments, it is indispensable for survival. Thus, both the is reproduced. Thus, TIRs guarantee homologous se- 7.2-Mb chromosome and the 1850-kb linear DNA are quences for recombination, and this may be the reason chimeric chromosomes and were named chromosomes I why most of the Streptomyces linear replicons have TIRs at and II, respectively. both ends. Streptomyces linear chromosomes contain two replica- When the deletion in one telomere extends beyond the tion mechanisms; bidirectional replication from a centrally TIR region, an intact TIR structure cannot be recovered in located replication origin (Musialowski et al., 1994) and this way. However, if nonallelic but similar sequences are terminal replication primed by a terminal protein (Qin & present on the left and right arms, recombination between Cohen, 1998). The replication origins of Streptomyces them causes chromosomal arm replacement and reproduces chromosomes are quite similar to those of typical bac- a telomere. In this case, some chromosomal sequences are terial circular chromosomes (Zakrzewska-Czerwinska & lost and longer TIRs are formed. This may be the reason Schrempf, 1992; Calcutt & Schmidt, 1992), while the why the sizes of TIRs vary greatly in Streptomyces strains, telomere sequences of most such chromosomes can make a and homologous genes or insertion elements are frequently secondary foldback structure like those of adenovirus and found at the inside ends of TIRs. parvovirus (Huang et al., 1998; Qin & Cohen, 1998). Thus,

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their ends (Pandza et al., 1998). The linear plasmid SLP2 was shown to be a composite of a chromosome end and a linear plasmid (Huang et al., 2003). Thus, end exchanges between linear replicons with different telomere sequences produce chimeric replicons without TIRs. The frequent occurrence of end exchange in Streptomyces gives addi- tional hints on the recovery mechanism of telomeres and horizontal transfer of secondary metabolism. Without TIRs at both ends, how can Streptomyces linear replicons recover an intact telomere after terminal deletion? Qin and Cohen (2002) demonstrated that when a linear plasmid, which contained a pSLA2 telomere at one end and a damaged chromosomal telomere at the other, was introduced into S. lividans, the damaged telomere was repaired by intermolecular recombination between the plasmid and the chromosome. Thus, if identical telomere sequences are present on two separate linear replicons in the same , they may compensate each other by Fig. 7. Generation of a Streptomyces linear chromosome by intermolecular recombinational DNA repair. recombination of a bacterial-type and a Microarray analysis showed that the chromosome struc- linear plasmid or phage with TIRs and terminal proteins. tures of S. coelicolor A3(2) and S. lividans are similar in the 5-Mb core region, but they differ in their left and right terminal regions (R. Kirby, personal communication). This it was proposed that Streptomyces linear chromosomes fact may also reflect frequent end exchange of Streptomy- might have been generated by recombination of a bacterial- ces linear chromosomes. Since essential genes are not type circular chromosome and a linear plasmid or phage, coded in the terminal regions, they are exchangeable except which contained TIRs at both ends and a terminal protein for the extreme ends that are necessary for terminal bound to the 50 ends (Volff & Altenbuchner, 2000; Chen replication. Even the ends are exchangeable, as long as et al., 2002) (Fig. 7). the genes for terminal replication are linked to the ends. In addition, two chimeric chromosomes in S. coelicolor Thus, linear plasmids such as SCP1 and pSLA2-L could 2106 suggested another evolutionary possibility: duplica- transfer an antibiotic-producing ability initially by conjugal tion and multiplication of a linear chromosome might have transfer into other cells to be maintained extrachromoso- occurred by single crossover of two linear chromosomes mally, and subsequently either by end exchange with the or of a linear chromosome and a linear plasmid (Fig. 6). chromosome or, perhaps less frequently, by integrating into Multiple linear chromosomes can now accommodate a the chromosome as, for example, in the case of NF strains large amount of genetic information that facilitates evolu- of S. coelicolor in which SCP1 has inserted into a more tion. In contrast, the survival of multiple versions of or less central location in the chromosome (Hanafusa & a circular chromosome in a common cytoplasm through Kinashi, 1992; Yamasaki et al., 2001). cycles of growth would normally be prevented by incom- pSLA2-L is a promising subject to study the mechanism patibility. of horizontal transfer of secondary metabolism-related genes, because it is occupied by many such genes. End exchange of linear replicons and horizontal Interestingly, we could not detect any transposition-related transfer genes or sequences surrounding the biosynthetic clusters Most of the linear chromosomes and plasmids isolated on pSLA2-L, except for several truncated genes. This is from Streptomyces thus far have TIRs. The sizes of the true for the methylenomycin biosynthetic cluster on SCP1 TIRs of linear replicons are quite different, even going up (Bentley et al., 2004). Based on these results, I hypothesize to about 6500 kb in the end-to-end fused chromosome that in most cases horizontal transfer has not occurred by a in S. ambofaciens mutant NSA65 (Wenner et al., 2003). direct transposition of the cluster but has involved multiple However, linear replicons without TIRs have also been recombinational events. Several cycles of single crossover isolated: for example, plasmid pHG201 in Rhodococcus at both sides of the cluster generate a gene organization that opacus has only 3-bp homology at both ends (Kalkus et al., gives a probably misleading impression of having been 1993). The two chimeric chromosomes in S. coelicolor generated by a direct transposition of the cluster. Truncated 2106 contain an SCP1 telomere and a chromosomal genes frequently found around the cluster may be traces of telomere at each end and therefore do not have TIRs. nonhomologous recombination. Recombination between plasmid pPZG101 and the chro- Since the finding of SCP1 in S. coelicolor A3(2), studies mosome of Streptomyces rimosus led to an exchange of on linear plasmids have not progressed rapidly. However,

26 ACTINOMYCETOLOGICA VOL. 22, NO. 1 although the biosynthetic gene clusters for antibiotic actinomycete Streptomyces coelicolor A3(2). Nature 417, 141– production are frequently chromosomally located, recent 147. studies have revealed that linear plasmids, in addition to Calcutt, M. & Schmidt, F. J. (1992). Conserved gene arrange- SCP1 and pSLA2-L, carry the genes for; oxytetracycline ment in the origin region of the Streptomyces lividans in S. rimosus (Pandza et al., 1998), lasalocid and echino- chromosome. J. Bacteriol. 174, 3220–3226. Chater, K. F. & Bruton, C. J. (1985). Resistance, regulatory and mycin in S. lasaliensis (Kinashi et al., 1988; Watanabe production genes for the antibiotic methylenomycin are et al., 2006; Migita et al., 2007), neocarzinostatin in clustered. EMBO J. 4, 1893–1897. S. carzinostaticus, leinamycin in S. globispora and C-1027 Chater, K. F. & Kinashi, H. (2007). Streptomyces linear in S. atroolivaceus (B. Shen, personal communication), and plasmids: their discovery, functions, interactions with other chlorothricin in S. antibioticus (Jia et al., 2006). Linear replicons, and evolutionary significance. In Microbiology plasmids are also involved in phytopathogenicity (Goethals Monographs, Vol 7, Microbial Linear Plasmids, pp. 1–31. et al., 2001) and degradation of compounds with low Edited by F. Meinhardt & R. Klassen. Berlin/Heidelberg, degradability such as polychlorinated biphenyls (Warren et Germany: Springer. al., 2004). Thus, linear plasmids are widely distributed in Chen, C. W., Yu, T.-W., Lin, Y.-S., Kieser, H. M. & Hopwood, microorganisms and may be contributing to horizontal D. A. (1993). The conjugative plasmid SLP2 of Streptomyces transfer of secondary metabolism, directly and indirectly. lividans is a 50 kb linear molecule. Mol. Microbiol. 7, 925–932. Chen, C. W., Huang, C. H., Lee, H. H., Tsai, H. H. & Kirby, R. Reflecting these situations, Microbiology Monographs, (2002). Once the circle has been broken: dynamics and Vol. 7, Microbial Linear Plasmids, was recently published, evolution of Streptomyces chromosomes. Trends Genet. 18, in which Prof. Chater, John Innes Centre, and I wrote the 522–529. first chapter on Streptomyces linear plasmids (Chater & Fischer, G., Wenner, T., Decaris, B. & Leblond, P. (1998). Kinashi, 2007). Chromosomal arm replacement generates a high level of intraspecific polymorphism in the terminal inverted repeats of ACKNOWLEDGEMENTS the linear chromosomal DNA of Streptomyces ambofaciens. Proc. Natl. Acad. Sci. USA 95, 14296–14301. I would like to thank Profs. N. Otake, K. Sakaguchi, Goethals, K., Vereecke, D., Jaziri, M., Montagu, M. V. & C. R. Hutchinson, O. Nimi for their guidance and en- Holsters, M. (2001). Leafy gall formation by Rhodococcus couragement, Prof. D. A. Hopwood for S. coelicolor A3(2) fascians. Annu. Rev. Phytopathol. 39, 27–52. Goshi, K., Uchida, T., Lezhava, A., Yamasaki, M., Hiratsu, K., strains and suggestions, Prof. K. F. Chater for collaboration Shinkawa, H. & Kinashi, H. (2002). Cloning and analysis of and reviewing this manuscript, and Prof. T. Beppu for his the telomere and terminal inverted repeat of the linear support and encouragement. I also would like to thank chromosome of Streptomyces griseus. J. Bacteriol. 184, many collaborators and students who contributed to the 3411–3415. studies described above, especially, Drs. and Profs. A. Hanafusa, T. & Kinashi, H. (1992). The structure of an Sakai, M. Shimaji-Murayama, T. Hanafusa, M. Redenbach, integrated copy of the giant linear plasmid SCP1 in the S. Horinouchi, A. Lezhava, H. Sugino, H. Shinkawa, M. chromosome of Streptomyces coelicolor 2612. Mol. Gen. Yamasaki, T. Uchida, K. Hiratsu, S. Mochizuki, S. Genet. 231, 363–368. Tatsuno, Y. He, S. Yamamoto and K. Arakawa. Hayakawa, T., Tanaka, T., Sakaguchi, K., Otake, N. & Yonehara, H. (1979). 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