Biosci. Biotechnol. Biochem., 67 (5), 1101–1108, 2003
Chromosomal Circularization in Streptomyces griseus by Nonhomologous
Recombination of Deletion Ends
Shoichi INOUE, Koji HIGASHIYAMA, Tetsuya UCHIDA, Keiichiro HIRATSU,
and Haruyasu KINASHI†
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter,
Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan
Received December 24, 2002; Accepted January 29, 2003
Streptomyces linear chromosomes frequently cause
deletions at both ends spontaneously or by various
mutagenic treatments, and concomitantly display dynamic structural changes such as circularization and
arm replacement. We have cloned and sequenced the
fusion junctions of circularized chromosomes in two
deletion mutants of Streptomyces griseus. No homology
and a 1-bp overlap were found between the deletion
ends of the mutant chromosomes. Taking this together
with previous results, we concluded that chromosomal
circularization in Streptomyces occurs by nonhomologous recombination between deletion ends.
Key words:
Streptomyces; chromosomal circularization; chromosomal deletion; genome
‰uidity; nonhomologous recombination
lividans 1,13) and Streptomyces ambofaciens.4) It was
ˆnally conˆrmed in Streptomyces griseus by cloning
and sequencing of the fusion junctions of the circularized chromosomes.14) No homology and a 6-bp
microhomology were detected between the right and
left deletion ends of the mutant chromosomes. This
result suggested that chromosomal circularization
occurred by nonhomologous recombination between
both deletion ends. Qin and Cohen15) reported that
nonhomologous recombination also generated circular plasmids from telomere-damaged linear plasmids
in S. lividans. To reveal if nonhomologous recombination is general in circularization of Streptomyces
linear replicons, we analyzed two additional circularized chromosomes of S. griseus mutants.
Materials and Methods
Filamentous soil bacteria, Streptomyces, are unusual in carrying an 8–9 Mb linear chromosome:1–7)
the genome project for Streptomyces coelicolor
A3(2), a model strain for Streptomyces genetics, has
been recently completed.8) Streptomyces species are
also known to frequently have linear plasmids, which
range from 12 to 1700 kb in size.9) These Streptomyces linear replicons have principally the same
structural features; terminal inverted repeats (TIRs)
are present at both ends and the 5? ends are linked
covalently to a terminal protein. Streptomyces linear
chromosomes easily undergo terminal deletions
spontaneously or by various mutagenic treatments.10,11) The sizes of deletions sometimes reach up
to 2 Mb.12) The deleted chromosomes subsequently
show several types of rearrangements; circularization, arm replacement, and ampliˆcation. The last
phenomenon may be an intermediate state to reach a
ˆnal stable state.
Chromosomal circularization in Streptomyces was
ˆrst indicated by detection of a macrorestriction
fusion fragment in deletion mutants of Streptomyces
Bacterial strains, cosmids, plasmids, and media. S.
griseus strain 2247, used for mutation, was described
previously.2) The cosmid library was previously
constructed for strain 22472) and ordered at both
chromosomal end regions.16) E. coli XL1-Blue and
pUC19 were used for cloning and sequencing of
DNA fragments. Glucose-meat extract-peptone
(GMP) medium contains 1.0z glucose, 0.4z
peptone, 0.2z meat extract, 0.2z yeast extract,
0.5z NaCl, and 0.025z MgSO4 7H2 O (pH 7.0). MB
plates contain 1.0z mannitol, 0.1z yeast extract,
0.2z Polypepton (Wako Chemicals, Osaka), 0.1z
meat extract, and 1.5z agar (pH 7.0).
Mutation of S. griseus 2247. A spore suspension of
strain 2247 in distilled water was UV-irradiated at a
distance of 60 cm under a germicidal lamp (15W) for
0.5–5 min with constant agitation. Portions were
taken out every 30 sec, kept in the dark for 2 hr to
prevent photoreactivation, and spread and grown on
MB plates at 289C for several days. The spore sus-
To whom correspondence should be addressed. Fax: ＋81-824-24-7869; E-mail: kinashi＠hiroshima-u.ac.jp
Abbreviation: TIR, terminal inverted repeat
†
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S. INOUE et al.
pension, which was irradiated for 2.5–3.5 min, gave a
survival of 0.1–1z and used for isolation of deletion
mutants.
DNA isolation and pulsed-ˆeld gel electrophoresis.
S. griseus strains were reciprocally cultured at 289C
in a 500-ml Sakaguchi ‰ask containing 100 ml of
GMP medium and total DNAs were isolated as
described by Suwa et al.17) Gel samples for PFGE
were prepared by the mycelium method as described
previously,2,18) digested in gels, and separated by contour-clamped homogeneous electric ˆelds (CHEF).19)
CHEF was done in 0.5×TBE buŠer using 1.0z
agarose at 159
C. E. coli strains were cultured at 379
C
in LB medium, and cosmid and plasmid DNAs were
extracted as described by Sambrook et al.20)
Southern hybridization. DNA fragments were
separated by CHEF or conventional agarose gel
electrophoresis and transferred to nylon membrane
ˆlters by the capillary method. Hybridization was
done using the DIG system (Roche Diagnostics
GmbH, Mannheim, Germany) overnight at 709
C in
standard buŠer according to the supplier's protocol.
After hybridization, washing was done twice for
5 min each in 2×wash solution at room temperature,
and then twice for 15 min each in 0.1×wash solution
C.
at 709
DNA sequencing. Nucleotide sequencing was done
by the dideoxy termination method using a dye terminator cycle sequencing kit (Amersham Pharmacia
Biotech, Uppsala, Sweden) and the ABI-373S
sequencing system (PE Biosystems, Foster City, CA).
Genetyx-Mac 10.2 (Software Development, Tokyo)
and FramePlot 2.3.221) were used for analysis of
sequence data.
Results
Chromosomal deletion in mutants, No. 9 and No.
83
Surviving colonies of S. griseus after UV irradiation were picked up randomly. Their total DNAs
were digested with Bam HI, separated by conventional agarose gel electrophoresis, and analyzed by
Southern hybridization using pSGE1 as a probe,
which contains the 2.7-kb end Sal I fragment of the
2247 chromosome.22) Among 60 colonies tested, two
colonies, No. 9 and No. 83, showed no hybridizing
signals, which indicated that both chromosomal ends
were deleted in these mutants. Colonies of mutant
No. 9 have a bald appearance on solid MB medium,
while mutant No. 83 sporulates as the parent strain
2247 does. Both mutants grow normally in solid and
liquid cultures.
To measure the sizes of deletions, the ordered terminal cosmids of the 2247 chromosome16) were hybri-
Fig. 1. Southern Hybridization Analysis of the Deletion Sizes in
Mutants No. 9 and No. 83.
The Bam HI digest of total DNAs from strain 2247 and
mutants No. 9 and No. 83 were hybridized with the deletion endpoint cosmids; 6E12 (A), F1B6 (B), 6C8 (C), and F2D2 (D).
For the left deletion end of mutant No. 9, the SacI digest is also
shown to reveal a fusion fragment. Fusion fragments are indicated by an arrow. Ba, Bam HI; SI, SacI.
dized to the Bam HI digest of total DNAs of the two
mutants. As shown in Figs. 1A and 1B, mutant No. 9
showed fewer signals compared with strain 2247,
when probed by the right end cosmid 6E12 and cosmid F1B6, which is more than 500 kb from the left
end. These results showed that the right and left deletion endpoints in mutant No. 9 are located on the
cosmids 6E12 and F1B6, and their deletion sizes are
30 and 550 kb, respectively (Fig. 2). By the same
analysis shown in Figs. 1C and 1D, the right and left
deletion endpoints in mutant No. 83 were on cosmids
6C8 and F2D2, and their deletion sizes were 130 and
170 kb, respectively (Fig. 2).
Chromosomal circularization in mutants, No. 9
and No. 83
To see widely the structural changes that occurred
at the chromosomal ends of the two deletion
mutants, Southern hybridization analysis was done
using macrorestriction fragments of strains, 2247,
No. 9, and No. 83 (Fig. 3). When the right end cosmid 6E12 was used as a probe, the SpeI digest of
strain 2247 showed the 280-kb right end fragment as
well as the 520-kb left end fragment, due to the
presence of TIRs at both ends, while the SpeI digest
of mutant No. 9 gave a new 450-kb fragment. When
the left deletion end cosmid F1B6 was used a probe,
strain 2247 gave a 350-kb fragment, while mutant
No. 9 gave the same 450-kb fragment, as detected by
cosmid 6E12. These results indicated that in mutant
No. 9, the right and left deletion ends of the chromosome were combined to generate the 450-kb fusion
Spe I fragment; namely, the linear chromosome was
circularized.
Chromosomal Circularization in Streptomyces griseus
1103
Fig. 2.
Restriction and Cosmid Maps at Both Chromosomal Ends of S. griseus 2247 and the Deleted Regions in Mutants No. 9 and No. 83.
The deleted regions in mutants No. 9 and No. 83 as well as the previously analyzed mutants 404–23 and N2 are indicated by dashed
lines. All of the four mutants were found to contain a circular chromosome. Af, Af l II; As, Ase I; Sp, Spe I; Ss, Ssp I; L, linear; C, circular.
Fig. 3.
Southern Hybridization Analysis of Chromosomal Circularization in Mutants No. 9 and No. 83.
Gel samples of strain 2247 and mutants No. 9 and No. 83 were digested with Spe I or Ase I, separated by CHEF electrophoresis, and
hybridized with the deletion endpoint cosmids; 6E12 (A), F1B6 (B), 6C8 (C), and F2D2 (D). CHEF was done at 150 V with 24-s pulses
for 30–35 h.
Similarly, cosmids 6C8 and F2D2, which contain
the right and left deletion endpoints of the No. 83
chromosome, hybridized to Ase I fragment E
(550 kb) and Ase I fragment F1 (480 kb) of the 2247
chromosome,2) respectively, and hybridized to the
same 800-kb fusion fragment of the No. 83 chromosome (Figs. 3C and 3D). Therefore, circularization
occurred in this mutant, too.
Restriction and hybridization analysis of fusion
junctions
To analyze the chromosomal circularization more
precisely, we tried to clone the fusion junctions from
the two mutants and corresponding right and left
deletion endpoints from strain 2247. In the case of
mutant No. 9, an 8.1-kb fusion fragment was clearly
seen in its Bam HI digest, when probed by the right
end cosmid 6E12 (Fig. 1A). When probed by the left
deletion end cosmid F1B6, the 8.1-kb fusion frag-
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S. INOUE et al.
Fig. 4.
Location of the Fusion Junction in Mutant No. 9 by Restriction and Hybridization Analysis.
(A) The Bam HI digest of the 2247 and No. 9 DNAs was probed by plasmid p2F9, which showed the right and left deletion end fragments at 13.6 and 1.0 kb, respectively, and the fusion fragment itself at 8.1 kb. (B) The fusion junction in mutant No. 9 was located on a
0.5-kb Af l II-Bam HI fragment. The deleted and fused regions are indicated by dashed and shaded lines, respectively. Af, Af l II; Ba,
Bam HI; Ec, Eco RI; SI, SacI.
ment was not detected (Fig. 1B), due to their short
homology. Therefore, the No. 9 DNA was digested
with another enzyme, SacI, and probed by F1B6,
which revealed an fusion fragment at 3.8 kb
(Fig. 1B). The two fusion fragments were cloned and
plasmid p2F9 was obtained from the former. When
the Bam HI digest of the 2247 DNA was probed by
p2F9, two positive signals were detected at 13.6 and
1.0 kb (Fig. 4A), which correspond to the right and
left endpoints, respectively. These fragments were
also subcloned from cosmids, 6E12 and F1B6, and
put through restriction and hybridization analysis.
Comparison of the three restriction maps showed
that the fusion junction is on a shaded 0.5-kb Af l IIBam HI fragment (Fig. 4B).
In the case of mutant No. 83, the fusion fragment
was not easily identiˆed among many hybridizing signals in Figs. 1C and 1D. However, precise restriction
and hybridization analysis ˆnally located the fusion
junction on a 3.6-kb Bam HI fragment of the No. 83
DNA, the right deletion endpoint on a 2.7-kb Bam HI
fragment of cosmid 6C8, and the left deletion
endpoint on a 3.3-kb Bam HI fragment of cosmid
F2D2. These fragments were cloned from the No. 83
DNA or subcloned from cosmids 6C8 and F2D2,
respectively. As shown in Figs. 5A and 5B, the right
(Probe A, 2.7 kb) and left (Probe B, 3.3 kb) deletion
end fragments hybridized to the same 3.6-kb fusion
fragment. Restriction maps were constructed for
these three clones and compared, which located the
fusion junction on a central 0.8-kb ApaI-PvuI fragment (Fig. 5C).
Nucleotide sequences of fusion junctions in
mutants, No. 9 and No. 83
The nucleotide sequences around the fusion junctions in mutants No. 9 and No. 83 and those around
their corresponding regions in strain 2247 were determined. By sequence comparison, the fusion junction
in mutant No. 9 was located at nt 291–292 in Fig. 6.
No homology was found between the right and left
deletion end sequences, and no ampliˆcation was
detected around the junction. No initiation and stop
codons were found in the determined sequences, but
two ORFs were identiˆed based on the unique codon
usage of the high GC Streptomyces DNAs.23) Homology search for databases found that the ORF at the
right deletion end codes a hypothetical protein and
the ORF at the left deletion end may code glycosyl
hydrolase (pfam02837). The two ORFs, which have
opposite directions, were completely destroyed by
deletion and circularization in mutant No. 9.
The nucleotide sequence around the fusion junction in mutant No. 83 is compared with those around
the right and left deletion endpoints (Fig. 7). In this
case, a G residue is present at both the right and left
deletion ends, which form a fusion junction at nt
222. No ampliˆcation was found near the fusion
junction. Two ORFs were identiˆed at the right and
left deletion ends, both of which are coded on the
complementary strand. Homology search revealed
that the ORF at the right deletion end codes a
hypothetical protein, and the ORF at the left deletion
end code phosphate acyltransferase (smart00563).
Both ORFs were completely destroyed by deletion
Chromosomal Circularization in Streptomyces griseus
1105
Fig. 5.
Location of the Fusion Junction in Mutant No. 83 by Restriction and Hybridization Analysis.
(A, B) The Bam HI digest of the 2247 DNA showed the same junction fragment (3.6 kb), when probed by the right (Probe A, 2.7 kb)
and left (Probe B, 3.3 kb) deletion end probes. (C) The fusion junction in mutant No. 83 was located on a 0.8-kb ApaI-PvuI fragment.
All sites are shown for ApaI (Ap), Bam HI (Ba), Bgl II (Bg), Pvu I (Pv), and Sal I (Sa). For SacII (SII) and SmaI (Sm), only the sites
‰anking the determined sequences in Fig. 7 are shown.
Fig. 6. Nucleotide Sequences of the Fusion Junction (J) in Mutant No. 9 and Corresponding Right (R) and Left (L) Deletion Ends in Strain
2247.
The fusion junction is located at nt 291–292. Putative two ORFs, which have opposite directions, are shown in the one-letter code.
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S. INOUE et al.
Fig. 7. Nucleotide Sequences of the Fusion Junction (J) in Mutant No. 83 and Corresponding Right (R) and Left (L) Deletion Ends in
Strain 2247.
The fusion junction is composed of a G residue at nt 222. Putative two ORFs, both of which are coded on the complementary strand,
are shown by the one-letter code.
and circularization, and a possible fused ORF in
mutant No. 83 has a frame shift and therefore cannot
encode a functional protein.
Discussion
In this study, we have elaborated chromosomal
circularization in two S. griseus deletion mutants,
No. 9 and No. 83, by restriction and hybridization
analysis and ˆnally by nucleotide sequencing. No
homology was detected between the right and left
deletion endpoints in mutant No. 9 and only a 1-bp
overlap was found in mutant No. 83. In addition, no
ampliˆcation was detected around the fusion
junctions in both mutants. We previously reported
chromosomal circularization in S. griseus mutants,
404–23 and N2.14) In these cases, no homology and a
6-bp microhomology were found between the right
and left deletion ends. In any of the four cases, no
hotspot was detected as a deletion endpoint. From
these results, we concluded that chromosomal circularization occurs by nonhomologous recombination and the sequences at both deletion ends play no
signiˆcant roles in circularization. It should be noted
that all of the four circularized mutants grow normally in spite of their chromosomal arm deletions,
up to 550 kb in mutant No. 9.
Chromosomal circularization was also indicated in
S. lividans 1,13) and S. ambofaciens 4) by detection of a
fused macrorestriction fragment. In addition, Qin
and Cohen15) reported the circularization of artiˆcial
pSLA2 plasmids in S. lividans by nonhomologous
recombination of deletion ends. Therefore, circularization may be a common strategy of Streptomyces
linear replicons, when both of the two telomeres were
deleted.
In addition to circularization, Streptomyces chromosomes display several structural changes to survive from the loss of the extreme ends that may be
essential for terminal replication.24,25) Fischer et al.26)
reported that homologous recombination of two
sigma factor-like ORFs caused chromosomal arm
replacement in S. ambofaciens, which generated
unusually long TIRs in the mutants. We also found
that in S. griseus mutant MM9, homologous recombination between two similar lipoprotein-like ORFs
caused chromosomal arm replacement and generated
450-kb TIRs in place of the original 24-kb TIRs.27) If
recombinational DNA repair occurs between the
intact and deleted TIR regions on the same chromosome, intact TIRs could be reproduced. We proposed
a hypothesis that this type of repair may occur frequently in Streptomyces and TIRs may guarantee
homologous sequences for recombination at both
chromosomal ends.27) Only when recombination occurred outside the TIRs can we recognize it as chromosomal arm replacement. Furthermore, recombination between the ends of a linear chromosome and
a linear plasmid was observed, which caused chromosomal end exchange in Streptomyces rimosus 28)
and repair of a damaged telomere in S. lividans.15)
Qin and Cohen29) reported another strategy of
Chromosomal Circularization in Streptomyces griseus
Streptomyces, in which duplication of the right half
of pSLA2 generated symmetric linear plasmids, when
the left telomere was damaged. Nucleotide sequencing of the centers of symmetry revealed that small
palindromic sequences were originally present in
pSLA2. We previously isolated a similar symmetric
plasmid pSLA2-L1 from S. rochei.30) All of these
rearrangements may be strategies of Streptomyces to
recover an extreme end when one of the two telomeres was deleted.
As described above, Streptomyces linear chromosomes and plasmids display dynamic structural
changes to survive from lethal deletions; circularization, arm replacement, repair of intact TIRs, and
formation of symmetric linear plasmids. The
mechanisms underlined in these rearrangements are
diŠerent; nonhomologous recombination for circularization, homologous recombination for arm
replacement and repair of TIRs, and misreplication
at a small palindrome sequence for formation of a
symmetric linear plasmid.15) These dynamic structural changes of Streptomyces linear replicons are quite
interesting in respect of the evolution of genome
structures. Most of these deletion mutants grow normally. Therefore, terminal regions carry dispensable
genes and are deletable except for the extreme ends,
and could accept various foreign DNAs. This
genome ‰uidity of Streptomyces may give it an
extraordinary ability to produce many secondary
metabolites.
5)
6)
7)
8)
Acknowledgment
We thank A. Hara for her technical assistance.
This work was supported by Grant-in-Aid for
Scientiˆc Research on Priority Areas (C) ``Genome
Biology'' from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan.
9)
10)
11)
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