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Journal of Bacteriology, December 2007, p. 9108-9116, Vol. 189, No. 24
0021-9193/07/$08.00+0     doi:10.1128/JB.00983-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Introduction of the Foreign Transposon Tn4560 in Streptomyces coelicolor Leads to Genetic Instability near the Native Insertion Sequence IS1649{triangledown} ,{dagger}

Elizabeth M. Widenbrant* and Camilla M. Kao

Department of Chemical Engineering, Stanford University, Stanford, California 94305

Received 20 June 2007/ Accepted 9 October 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
We report an altered pattern of genetic instability for Streptomyces coelicolor when the bacterium harbored a foreign transposon, Tn4560. Deletions, amplifications, and circularizations of the linear 8.7-Mb chromosome occurred more frequently at sites adjacent to native insertion elements, notably IS1649. In contrast, deletions, amplifications, and circularizations of a wild-type strain happened at heterogeneous sites within the chromosome. In 50 strains examined, structural changes removed or duplicated hundreds of contiguous S. coelicolor genes, altering up to 33% of the chromosome. S. coelicolor shows a bias toward one type of genetic instability during this particular assault from the environment, the invasion of foreign DNA.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Streptomyces coelicolor belongs to a genus of bacteria that produce many molecules for human and veterinary medicine (2). As of 2002, 8,700 antibiotics originated from actinomycete sources, compared with 2,900 from all other bacteria combined (3). S. coelicolor has served as a model actinomycete for many decades. Its sequenced genome (2) enhances previously available techniques to manipulate S. coelicolor genetically (21).

The linear chromosome of S. coelicolor extends for more than 8 Mb. This size exceeds that of most bacterial chromosomes (6). The two chromosome ends contain the same DNA sequence in an inverted orientation (terminal inverted repeats), to which host proteins bind. The linear structure might confer certain advantages during growth, for example, avoiding a need for decatenation during chromosome replication and recombination (18). Other Streptomyces species have linear chromosomes with similar structures and possess, as well, high rates of instability: chromosomal changes that include large deletions and amplifications occur at rates greater than 10–3 (8, 13, 26). Although genetic instability first was reported for S. coelicolor several decades ago (13), the mechanisms underlying the phenomenon remain poorly understood. The mechanisms likely involve both homologous and nonhomologous recombination, as well as native insertion elements (7, 14, 20, 22, 35). Indeed, about 40% of the putative 109 transposase homologues predicted in the S. coelicolor chromosome lie within 2.3% of the ends, regions that preferentially exhibit genetic instability (7).

Originally, we sought to identify S. coelicolor mutants that produce larger amounts of the blue-pigmented antibiotic actinorhodin. We aimed to identify genes that might shed light on design principles for engineering high-producing strains of Streptomyces bacteria. The use of a transposon for large-scale mutagenesis facilitates the identification of mutations, compared to classical mutagenesis methods, such as UV light and chemical mutagens. In our work, we used a derivative of the 8.6-kb transposon Tn4556 of Streptomyces fradiae, which contains a transposase (~2.7 kb), resolvase (~400 bp), and viomycin resistance gene (~800 bp). Others have used this transposon with Streptomyces and Mycobacterium species (4; K. Fowler, personal communication). A liquid culture screen of over 8,000 transposon insertions yielded 24 high-producing candidates. Subsequently, a chloramphenicol resistance screen, screening for loss of the right chromosome end, and DNA microarray experiments together revealed large deletions and amplifications in the chromosomes of these strains, genetic changes that probably occur in industrial strain improvement programs. Further analysis of these transposon-containing mutants, as well as chloramphenicol-resistant mutants isolated from a wild-type strain, showed that the presence of foreign transposon Tn4560 led to more events of genetic instability near an insertion sequence, IS1649, native to S. coelicolor.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Bacterial strains and media. The S. coelicolor strain M145 (2) was used for all experiments. Strains were grown in 96-well plates in R5 liquid medium (21). M145:Tn4560 strains were cultivated on soy flour mannitol (SFM) medium (21) with 30 µg/ml viomycin. For DNA isolation, strains were grown in 5 ml YEME (yeast extract-malt extract) liquid medium (21) supplemented with 5 mM MgCl2. ET12567/pUZ8002 was used to deliver pKay1 (described below). ET12567/pUZ8002 was grown in LB liquid medium (30) with 50 µg/ml apramycin, 10 µg/ml kanamycin, and 25 µg/ml chloramphenicol. For chloramphenicol screening, M145 spores were plated onto SFM medium and replica plated onto minimal medium (21) with 0.5 mg/liter Casamino Acids with and without 8 µg/ml chloramphenicol. M145:Tn4560 spores were plated onto SFM medium with viomycin and replica plated as the M145 spores were.

M145 spore preparation. M145 was grown on SFM medium, and spores were harvested with glass beads and sterile water (21). To further disrupt spore chains, the spore-water suspension was vortexed for 5 min with glass beads. The remainder of the spore preparation was subjected to a standard protocol (21). The final spore suspension was examined under a microscope to determine if single spores had been obtained.

Chloramphenicol screening. A suspension of M145 spores was plated for single colonies on SFM medium. After 7 days at 30°C, plates were replica plated using sterile velvet onto minimal medium plus Casamino Acids with or without 8 µg/ml chloramphenicol. Replica plates were grown for 5 days at 30°C, and chloramphenicol-sensitive colonies were counted and restreaked onto SFM for spore preparation. These strains were grown in liquid tryptic soy broth to prepare genomic DNA. Since all mutant colonies arising from genomic instability appeared bluer than the M145 wild-type strain, chloramphenicol-resistant bluer colonies also were analyzed.

In a similar experiment to that described above, the M145:Tn4560 mutant library was plated for single colonies on SFM medium and screened for chloramphenicol sensitivity. Chloramphenicol-sensitive colonies were counted, and the frequency of chloramphenicol-sensitive colonies was calculated.

Transposon insertion. A delivery vector, pKay1, was used to introduce Tn4560 into S. coelicolor (a gift from The John Innes Centre). Tn4560 derives from Tn4556, a Tn3-like element from Streptomyces fradiae (4). Previously, pUC1169 was fused with pOJ260 (5), and a 2-bp frameshift was introduced into the pIJ101 rep gene of pUC1169::pOJ260 to generate pKay1 (12). Conjugation between ET12567/pUZ8002/pKay1 and M145 was carried out by mixing ET12567/pUZ8002/pKay1 mycelia with M145 spores previously heat shocked at 50°C. Antibiotic-resistant transformants were selected using SFM at 37°C with 30 µg/ml viomycin and 20 µg/ml nalidixic acid overlaid at 20 h.

Growth in 96-well plates and screening. (i) Initial cultures. Spores were harvested from conjugation plates by scraping a razor blade at a 45° angle to the plate. Spores were suspended in 5 ml water, vortexed 3 min, and spun at 3,000 rpm for 10 min. Supernatants and pellets were stored in 20% glycerol at –80°C. Aliquots of supernatant stock were plated onto SFM plus viomycin (30 µg/ml) plates for single colonies. Single colonies were picked into 96-well seed plates (Axygen 1.1-ml 96-well deep-well plates) containing 170 µl R5 medium and a 3-mm glass bead by using a toothpick. The 96-well plate cultures were grown at 250 rpm, 80% relative humidity, and 30°C for 5 days. Using wide-bore tips, 30-ml aliquots were transferred into 300-µl volumes of R5 containing a 3-mm glass bead in deep-well 96-well production plates (Whatman Uniplate, 2-ml polypropylene round bottom). These production plates were grown at 250 rpm, 80% relative humidity, and 30°C for 5 days. To preserve the seed plates, 135 µl 40% glycerol was added, giving a final concentration of 20% glycerol to seed plates. The plates were frozen on dry ice and stored in a –80°C freezer.

(ii) Actinorhodin measurement. Total actinorhodin production was measured by adding 1,200 µl 1.25 M KOH to each well using an electronic multichannel pipette. The plates were covered with plastic sealing tape and briefly vortexed to ensure even mixing. Plates were spun for 5 min at 3,000 rpm in a benchtop centrifuge (2,000 x g). Supernatant (200 µl) was removed and transferred to flat-bottom 96-well spectrophotometric plates (0.4 ml polystyrene; Nalgene Nunc). A measurement of absorbance at 640 nm was taken in a 96-well plate spectrophotometer, using a well with medium as the blank.

Genomic DNA isolation. A 2-ml culture of YEME supplemented with 5 mM MgCl2 was inoculated with S. coelicolor spores and grown in a test tube at 30°C and 250 rpm for 1 day. A 1-ml sample was harvested by centrifugation for 1 min at 16,000 x g. The pellet was washed with 1 ml 10% sucrose and spun again. The pellet was resuspended by vortexing in 300 µl of lysis buffer (36). An aliquot of 150 µl of lysozyme solution (15 mg lysozyme/ml lysis buffer, made fresh) was added and mixed by inversion. All subsequent mixing of the samples was done by inversion. The sample was incubated 37°C for 45 min, mixing every 10 min. Fifty-five µl of 10% sodium dodecyl sulfate was added, and the sample was mixed. A small scoop (approximately 10 mg) of proteinase K was immediately added and mixed by inversion. The sample was incubated at 50°C for 15 min until a clear, viscous solution was obtained. Subsequently, 85 µl of 5 M NaCl was added and mixed. Then, 500 µl distilled H2O was added and mixed. Buffer-saturated phenol (400 µl) was added and mixed for 5 min, the mixture was centrifuged for 10 min at 13,000 rpm, and the aqueous phase was removed to a new tube. Phenol-chloroform-isoamyl alcohol (400 µl) was added and mixed for 5 min. The mixture was centrifuged as above. This extraction was repeated with phenol-chloroform-isoamyl alcohol until the interface was clear and then repeated once with chloroform only. The aqueous phase was removed, and 100% isopropanol was slowly added to a final volume of 1,500 µl. The sample was mixed slowly until a cloud of DNA appeared. The sample was placed in ice for 30 min and centrifuged for 10 min to pellet the DNA. The supernatant was removed, and the pellet was rinsed twice with 1 ml of 70% ethanol. The pellet was dried in air until it became slightly clear (about 5 min) and then resuspended in 30 µl of Qiagen EB buffer with 50 µg/ml RNase.

Tn4560 location identification (LMPCR). Ligation-mediated PCR (LMPCR) was used to identify TN4560 chromosomal insertion sites, as previously described (25). An existing protocol to identify insertion sites of Tn4560 is described briefly here (4). Oligonucleotides EagI-AD2 (5'-GGCCTCAACTGTCG-3') and UNIV5-AD1 (5'-GACTCGCGAATTCCGACAGTTGA-3') were used to make adapters complementary to EagI sticky ends. Genomic DNA was treated with 5 U of EagI and ligated to the adapters. A 1/20 portion of the ligation volume was used for PCR amplification. PCR conditions were 95°C 10 min for the hot start, 95°C 30 s for the denaturation, 57°C 30 s for the annealing, and 72°C 90 s for the elongation, for 35 cycles. Primers were EagI-AD2 and Tn4560R-2 (5'-CAGAATTCCCCCTTGCCACAGATAACAG-3'). For insertions that failed to amplify, the other side of the transposon was examined as above using, first, primers NEST3 (5'-GTGCTCCAGCCGGGTGTT-3') and EagI-AD2. A 1-µl aliquot of a 1:100 dilution of this product was used in a second PCR as above with NEST6 (5'-AAAAACTCGACCGGCACCCCG-3') and EagI-AD2. The second reaction was conducted to eliminate nonspecific PCR products.

Microarray hybridization. DNA microarrays were fabricated as previously described (19). The microarrays represented 97% of the predicted genes in S. coelicolor. For each microarray experiment, one DNA sample was labeled with Cy3-dCTP and the other with Cy5-dCTP. The samples were mixed and hybridized onto the same microarray. DNA was labeled and hybridized and arrays scanned as previously described (36).

PCR-targeted disruptions in M145. Genes with a transposon insertion were deleted in the M145 strain using a previously described method (16).

PCR amplifications to show genome circularity. PCR primers were designed to detect the circular structures of several mutant chromosomes. These primers were located in the genes immediately adjacent to deletions revealed by microarray results. PCRs were carried out using HotStar Taq. Amplified bands were sequenced and compared to the published S. coelicolor genome sequence.

Generational stability tests. To investigate whether the mutants would change with generations of growth, selected wild-type mutant strains were grown for four generations on solid SFM plates. Genomic DNA was analyzed with DNA microarrays as described above.


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
To identify mechanisms of antibiotic overproduction in S. coelicolor, we constructed a library of mutants using the S. fradiae transposon Tn4560. A screen of 8,000 insertion mutants grown in liquid cultures identified 24 mutants that appeared to produce larger amounts of actinorhodin. It seemed, subsequently, that 12 of these mutants were likely to have large deletions at one end of the chromosome: they grew poorly on a solid medium containing the antibiotic chloramphenicol. Since the right end of the S. coelicolor chromosome encodes two chloramphenicol resistance genes, cmlR1 (SCO7256) and cmlR2 (SCO7662), spontaneous deletions that remove several hundred kilobases of the right chromosome end cause chloramphenicol sensitivity (11).

Genomic microarray analysis of foreign transposon mutants. To study this phenomenon further, we examined the chromosomes of the 24 strains using DNA microarrays. Of the 24 strains, 5 additional strains showed large deletions and/or duplications in the chromosome, bringing the total number of mutants that experienced genetic instability to 17 (71%). Figures 1A and B summarize the deletions and amplifications. The deletions typically occurred, within a strain, at both ends of the chromosome and removed between 112 and 1,339 genes. Only 4 of the 17 strains (CO 16, CO 25, 26, 87/45) experienced a deletion of only one chromosome end (the left), and they all also acquired large amplifications with 630 to 2,204 genes. Five strains (32/62, 80/28, 30.1, 50/17, and 28/11) acquired small amplifications with 5 to 45 genes. For 9 of the 17 mutants examined (66/1, 50/17, 35.2, 17.2, 18.1, CO16, CO25, 26, and 87/45) loss of the left chromosome end occurred near a copy of IS1649 (SCO0091 and SCO0368) (Fig. 1B and C). For three strains (CO25, 26, and CO16), a large duplication terminated near a native insertion element, two with sequences nearly identical to IS1649 (SCO4183 and SCO5641) (Fig. 1B and C). SCO0368, a 1-kb transposase, shares a similar sequence (between 99.7% and 100% identity) with eight genes spread across the S. coelicolor chromosome (Fig. 1D).


Figure 1
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  		FIG. 1. S. coelicolor M145 mutants that contained the foreign transposon Tn4560. A. DNA microarray data of the left and right chromosome ends. In the color scale, green, black, and red represent gene copy numbers less than, equal to, and greater than the wild-type copy numbers, respectively. B. Graphical representation of the microarray data. Blue dashed lines designate IS1649 genes. C. Chromosome locations showing upstream and downstream genes of two IS1649 genes adjacent to deletions or amplifications. D. Locations of all IS1649 genes in the S. coelicolor chromosome. The arrows represent copies of IS1649 in their chromosomal orientation.

 
These results recall associations, reported previously, between insertion elements and genetic instability in Streptomyces bacteria. In S. lividans 66, insertion sequences were found adjacent to a large amplifiable element (10, 34). Spontaneous deletions in a Streptomyces chromosome occurred, most likely, from homologous recombination between multiple integrated copies of a foreign insertion element (32). Introduction of an insertion element, IS6100, into S. lividans amplified chromosomal DNA that included IS6100 (15). Given the proximity of those insertion elements to sites of genetic instability, we next identified the insertion sites of Tn4560 in our mutant strains.

Transposon LMPCR. To determine the proximity of the Tn4560 insertion sites to the deletions and amplifications, we performed LMPCR to identify the chromosomal sequences adjacent to the foreign transposon (25) (see Materials and Methods). Tn4560 had insertion sites throughout the chromosome (Table 1; see also Fig. S1 in the supplemental material). Thus, the chromosomal positions of the transposon did not correlate with sites of genetic instability. This observation contrasts with previous reports of genetic instability occurring at sites of foreign insertion elements (15, 32).


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  		TABLE 1. Mutant strains of S. coelicolor M145 harboring the foreign transposon Tn4560

 
Genomic microarray analysis of M145 mutants. As a control for the data above, we examined 33 chloramphenicol-sensitive and bluer strains derived from the wild-type parent and lacking the foreign transposon. Figure 2A and B summarizes the deletions and amplifications revealed by microarrays. The deletions removed similar numbers of genes as deletions in strains with Tn4560 (between 332 and 1,172 genes) and also occurred simultaneously at both chromosome ends. Only two strains (S3 and S26) experienced a deletion of one chromosome end, and these strains simultaneously acquired an amplification, as in the case of the mutants containing Tn4560. These amplifications duplicated between 87 and 1,186 genes. In strains derived from a wild-type parent, boundaries of deletions and amplifications failed to correlate with sites of native insertion elements, suggesting that the presence of Tn4560 changed the nature of genetic instability in S. coelicolor. This work provides, to our knowledge, the first evidence of the involvement of a foreign transposon and a native insertion element in altering patterns of genetic instability.


Figure 2
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  		FIG. 2. Genomic DNA microarray data of the left and right chromosome ends of S. coelicolor mutants derived from the wild-type strain M145. A. In the color scale, green, black, and red represent gene copy numbers less than, equal to, and greater than the wild-type copy numbers, respectively. B. Graphical representation of the M145 microarray data. C. Plot of microarray data showing gradual and sharp slopes at deletion sites. The log2 scale shows red/green ratios. A log2(red/green) value of 0 indicates gene copy numbers are equal to the wild type.

 
S. fradiae Tn4560 and S. coelicolor IS1649 lack similar DNA coding sequences and inverted repeats, and the S. fradiae genome lacks homologues of IS1649. Moreover, transcript levels of IS1649 in a transposon-bearing strain remain unchanged during growth in liquid cultures (data not shown). These observations suggest no obvious interaction between Tn4560 and native S. coelicolor insertion elements, yet the patterns of genetic instability observed here suggest that the foreign transposase interacts generically with Streptomyces insertion elements or specifically with IS1649. Whether a different transposon foreign to S. coelicolor would alter genetic instability in a similar manner remains unknown.

The microarray data for the two populations of S. coelicolor mutants differed in their variability at chromosomal sites of deletions and amplifications. For all the mutant strains with Tn4560, a sharp transition at deletion boundaries detected by microarrays suggested that a population of cells contained the same deletion (Fig. 3). Similarly, the amplifications showed a sharp transition between genes present in single and multiple copies in the chromosome, suggesting a homogeneous population (Fig. 3). These results suggest a deletion and amplification mechanism which generated a single chromosomal species that a strain stably maintained. Alternatively, genetic instability might have occurred prior to the insertion of Tn4560 into a chromosome, after which selection for Tn4560 (growth on viomycin) selected that single chromosome from a heterogeneous population. In contrast, for mutant strains without a foreign transposon, a gradual slope in microarray data at deletion boundaries suggested a heterogeneous population of cells and a deletion mechanism that generated multiple circular species of the S. coelicolor chromosome (Fig. 2C). For six strains examined further (see below), this heterogeneity, with no change of the deletion boundary, remained after four additional generations of growth, indicating that the population stably maintained heterogeneous chromosomes.


Figure 3
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  		FIG. 3. S. coelicolor mutant CO 25, which contains a foreign transposon. A. Plot of microarray data. The log2 scale shows red/green ratios. A log2(red/green) value of 0 indicates gene copy numbers are equal to the wild type. The data have sharp slopes at sites of the deletions and amplifications. B. Color representation of the microarray data around the duplication junction and IS1649. In the color scale, green, black, and red represent gene copy numbers less than, equal to, and greater than the wild-type copy numbers, respectively.

 
Circular chromosomes in mutants. Streptomyces chromosomes usually become circular when they lose both ends of the chromosome (23, 27). To test whether some chromosomes had circularized and to characterize the newly formed junctions, we attempted to amplify, for several strains derived from wild-type and transposon-bearing parents, the predicted junctions of truncated chromosome ends connected together. PCR amplification using primers flanking the predicted junction (Fig. 4A) demonstrated circularity for several chromosomes. The DNA around these junctions contained few or no rearrangements of the S. coelicolor genome sequence (Fig. 4B). For mutants containing the foreign transposon, the junctions near IS1649 contained little or no overlap (2 bp or less) between DNA from left and right chromosome ends (Fig. 4). For strains derived from a wild-type parent, the junctions contained a slightly greater overlap (4 to 16 bp) between DNA from left and right chromosome ends (Fig. 4). For the transposon mutants, the junctions near IS1649 also (i) occurred at seemingly random distances from the native insertion sequence (i.e., up to two genes away) and (ii) lacked an intact copy of IS1649 (either SCO0368 or a new copy, as determined by PCR, in DNA from the right chromosome end) (data not shown). Thus, replicative transposition of IS1649 to the right chromosome end, followed by intramolecular homologous recombination with SCO0368, fails to explain the junctions observed in the transposon mutants. For mutants derived from a wild-type parent, circular junctions likely formed through nonhomologous recombination rather than homologous recombination, with the exception of, possibly, the junction of strain S22.


Figure 4
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  		FIG. 4. PCR amplification and sequences of newly formed junctions of several deletion mutants with circularized chromosomes. A. Locations of PCR primers (gray arrows). Black bars denote the wild-type chromosomal DNA. Dashed bars denote deletions in the mutant chromosomes. The circle denotes a resulting circularized genome. B. Sequenced junctions of several circularized chromosomes. Gray bars denote DNA from the left chromosomal end. Black bars denote DNA from the right chromosomal end. Diagonal lines denote sequences identical in both chromosome ends. Transposon-bearing mutants with IS1649 adjacent to a deletion junction: 18.1, 17.2, and 35.2. Transposon-bearing mutant without IS1649 adjacent to a deletion junction: 31.1. Mutants derived from the wild-type parent: S22, S30, and MS4.

 
Spontaneous instability frequency. To determine if a foreign transposon changed the rate of genetic instability in S. coelicolor, we measured the frequency of chloramphenicol-sensitive strains generated by a wild-type strain and the original transposon library. Both strains, plated as populations of single colonies on solid medium, yielded spontaneous mutants at similar frequencies: 1.6% and 1.8% for the wild-type strain (n = 1,600) and the transposon library (n = 1,900), respectively. Thus, the foreign transposon appeared to leave unchanged the rate of deletions caused by genetic instability. In contrast, UV mutagenesis is known to increase the frequency of genetic instability 10-fold (13). Technical differences probably caused the slightly higher frequencies of chloramphenicol sensitivity in our strains compared with the value previously reported for S. coelicolor A3(2), 0.5% (13).

Antibiotic production. S. coelicolor mutants with spontaneous deletions often produce higher amounts of the blue-pigmented actinorhodin antibiotic (31). All of the chloramphenicol-sensitive strains derived from a wild-type parent produced more blue pigment on a solid medium (Fig. 5A). The Tn4560 strains that we identified in a screen of 96-well liquid cultures also produced more blue pigment on a solid medium (Fig. 5B and C). The high-throughput assay, which measured actinorhodin yields, likely screened for genetic instability without influencing the pattern of chromosomal changes observed here. To confirm that transposon insertions per se did not increase antibiotic titers, we deleted the corresponding genes in the wild-type strain (see Materials and Methods). The resulting strains produced normal or lesser amounts of blue pigment on a solid medium, indicating that Tn4560 mutations lacked linkage with the bluer phenotype of chloramphenicol-sensitive strains (Fig. 5B and C). Note that genes known to alter yields of actinorhodin have locations in the chromosome distant from the sites of genetic instability observed here (data not shown).


Figure 5
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  		FIG. 5. Antibiotic production of mutants on solid media. (A) Colonies of the wild-type parent (M145) and the library of transposon mutants (M145:Tn4560). Circles show mutants that produced more antibiotic. (B and C) Growth of wild-type (I), transposon-bearing mutants (II), and the corresponding strains with targeted deletions of the single genes disrupted by Tn4560 (III) for transposon-bearing mutant 66/1 and M145{Delta}0103 (B) and transposon-bearing mutant 26 and M145{Delta}5065 (C). The transposon-bearing strains produce more antibiotic, while the strains with targeted single-gene deletions do not. The fronts and backs of the plates are shown.

 
Stable propagation of chromosome alterations in strain lineages. Spontaneous deletion mutants of S. coelicolor have been shown to have chromosomes with decreased stability (1, 11, 13). Figure 6 illustrates the retention of altered chromosomes by wild-type-derived strains that experienced genetic instability. To investigate whether a heterogeneous mutant population would become homogeneous with additional generations of growth, 11 of the 33 bluer and/or chloramphenicol-sensitive mutants spontaneously derived from the wild-type parent were grown for several generations (generation 2 [G2] to G4) on solid plates with chromosome monitoring on DNA microarrays. Eleven mutants derived from M145 (G1) included two chloramphenicol-resistant mutants with a deletion of the left chromosome end (Fig. 6, left side), six chloramphenicol-sensitive mutants with "gradual" deletions of both chromosome ends (Fig. 6, middle), and three chloramphenicol-sensitive mutants with "sharp" deletions of both chromosome ends (Fig. 6, right side). The figure labels these strains as "populations."


Figure 6
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  		FIG. 6. Generational stability of selected M145 mutants, monitored on DNA microarrays. Green dashed boxes show where a mutant lost or gained an amplification; orange dashed boxes indicate a change in the deletion. See text for further details.

 
Two strains with "gradual" deletions of both chromosome ends (i.e., mixed populations) were streaked for single colonies and analyzed with microarrays for several generations. The figure labels the resulting single chromosome strains with "sharp" deletion ends as "individuals" (Fig. 6, middle; G2).

Both chloramphenicol-resistant mutants with a deletion of the left chromosome end retained the same deletion over time (Fig. 6, left side). In contrast, two of the six chloramphenicol-sensitive mutants with "gradual" deletions changed with time. In particular, mutant S2 lost its amplification, and mutant S7 acquired "sharp" deletions of both chromosome ends. For only one strain, S7, heterogeneity within a population's chromosomes became homogeneous in subsequent generations, suggesting a predominance of a single mutant during growth (Fig. 6). Thus, mutant strains derived from a wild-type parent appear to stably propagate a population of heterogeneous chromosomes. Of the three mutants with "sharp" deletions of both chromosome ends (Fig. 6, right side), two changed with time: S20 acquired a "gradual" deletion of both chromosome ends, and B6 gained an amplification. All other mutants' deletion boundaries remained in their original shape ("sharp" or "gradual") with no change in deletion boundary location.

In general, chloramphenicol-sensitive mutants maintained amplifications less stably than deletions, perhaps due to a loss of amplified segments by "looping out" of tandem DNA (29). Chloramphenicol-resistant mutants with deletions of the left chromosome end and right end amplifications, which likely have linear chromosomes (37), had more stable amplifications than mutants with circular chromosomes formed from truncated ends. Overall, the composition of deletions and amplifications possessed by most genetic instability mutants seemed unchanged with time. In particular, strain populations with multiple species of circular chromosomes persisted through several generations of growth without changing.

We observed an altered pattern of genetic instability when S. coelicolor harbored a foreign transposon in its genome. Perhaps the foreign transposon interacted with a native insertion element or mimicked a stress condition in S. coelicolor. In other bacteria, stress conditions activate native insertion elements. In Escherichia coli K-12, transposition of insertion elements increases with UV irradiation (9), heat shock (28), protein overexpression (17), and nutrient limitations (33). Indeed, the potential for genetic instability caused by insertion elements motivated the construction of a smaller genome of E. coli K-12 that lacked these insertion sequences and displayed stable growth properties (24). A Streptomyces sp. strain that lacked insertion sequences might serve more reliably in experimental studies and as a production strain for pharmaceutical molecules. Note that mycobacteria have multiple homologues of IS1649, and the use of Tn4560 as a mutagen (4) might cause chromosomal changes similar to those found in this study.


   ACKNOWLEDGMENTS
 
We thank Kay Fowler for providing pKay1 and Tobias Kieser for assistance in generating a transposon mutant library. We thank David Hopwood for helpful comments on the manuscript. Also, we thank the John Innes Centre for their generous support and, in particular, Mark Buttner, Mervyn Bibb, David Hopwood, Tobias Kieser, and Maureen Bibb for their scientific guidance. We also thank Carton Chen for his scientific guidance.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Chemical Engineering, Stanford University, Stanford, CA 94305. Phone: (650) 723-4906. Fax: (650) 723-9780. E-mail: ewidenbrant{at}gmail.com Back

{triangledown} Published ahead of print on 19 October 2007. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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Journal of Bacteriology, December 2007, p. 9108-9116, Vol. 189, No. 24
0021-9193/07/$08.00+0     doi:10.1128/JB.00983-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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