Microbiology (1999), 145, 2209–2220
Printed in Great Britain
Streptomyces genomes : circular genetic maps
from the linear chromosomes
Shih-Jie Wang,† Hua-Mei Chang, Yi-Shing Lin,‡ Chih-Hung Huang
and Carton W. Chen
Author for correspondence : Carton W. Chen. Tel : j886 2 2826 7040. Fax : j886 2 2826 4930.
e-mail : cwchen!ym.edu.tw
Institute of Genetics,
National Yang-Ming
University, Shih-Pai,
Taipei 112, Taiwan
Streptomyces chromosomes are linear DNA molecules and yet their genetic
maps based on linkage analysis are circular. The only other known examples of
this phenomenon are in the bacteriophages T2 and T4, the linear genomic
sequences of which are circularly permuted and terminally redundant, and in
which replication intermediates include long concatemers. These structural
and functional features are not found in Streptomyces. Instead, the circularity
of Streptomyces genetic maps appears to be caused by a completely different
mechanism postulated by Stahl & Steinberg (1964, Genetics 50, 531–538) – a
strong bias toward even numbers of crossovers during recombination creates
misleading genetic linkages between markers on the opposite arms of the
chromosome. This was demonstrated by physical inspection of the telomeres
in recombinant chromosomes after interspecies conjugation promoted by a
linear or circular plasmid. The preference for even numbers of crossovers is
probably demanded by the merozygosity of the recombining chromosomes,
and by the association between the telomeres mediated by interactions of
covalently bound terminal proteins.
Keywords : Streptomyces, conjugation, recombination, genetic map, plasmid
INTRODUCTION
An emerging enigma from the discovery of the linear
chromosomes of the filamentous bacteria Streptomyces
(Lin et al., 1993) is the circularity of their genetic maps
(reviewed in Hopwood, 1967).
The circularity of the genetic maps was first established
in Streptomyces coelicolor A3(2) through extensive
conjugation analysis (Hopwood, 1965, 1966). Subsequently, it was extended to other species (e.g. Friend &
Hopwood) and to protoplast fusion analysis. All the
linkage analyses in different Streptomyces species have
been consistent with the circularity of genetic maps,
without exceptions.
In his papers, Hopwood made these discerning remarks :
‘ … while the linkage map of S. coelicolor is circular, it
appears at present to be impossible to decide by this kind
of formal analysis whether the chromosome is circular
or linear ’ (Hopwood, 1965), and ‘ … whether, like that
.................................................................................................................................................
† Present address : Department of Family Medicine, Taipei Veterans
General Hospital, Shih-Pai, Taipei, Taiwan.
‡ Present address : Food Industrial Research Institute, Hsin-Chu, Taiwan.
Abbreviation : TP, terminal protein.
of E. coli, the genome of S. coelicolor can actually be a
closed loop, remains to be determined ’ (Hopwood,
1966). These statements were based on the careful
conclusion that there was a lack of constant genome
ends in S. coelicolor, which did not rule out the
possibility that the S. coelicolor chromosome is a linear
DNA molecule with circularly permuted sequences and
variable ends, as in the closely related T2 and T4 phages
(Streisinger et al., 1964).
The T2\T4 model has been the only other known case
where a linear replicon exhibits a circular genetic map.
The sequences of these linear genomes are circularly
permuted and contain terminal direct repeats generated
by formation of linear concatemers during replication
followed by ‘ headful ’ packaging of more than one
genome equivalent of DNA. The circular permutation
of the T2 and T4 DNA sequences means that the linkage
of any pair of proximal markers on the circular genetic
map is interrupted only in rare molecules, where they
occupy opposite ends of the DNA. This ‘ lack of constant
genomic ends ’ (Hopwood, 1966) leads to a circular
genetic map.
The T2\T4 prototype is, however, not applicable to the
Streptomyces chromosomes, which, although also lin-
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Table 1. Streptomyces strains used in this study
Strain
Genotype
Source/reference
S. coelicolor
M130
M145
M146
1190
hisA1 uraA1 strA1 SCP1− SCP2−
SCP1− SCP2−
hisA1 uraA1 strA1 SCP1+ SCP2−
hisA1 uraA1 strA1 SCP1− SCP2+
Hopwood et al. (1985)
Hopwood et al. (1985)
Hopwood et al. (1985)
Hopwood et al. (1985)
S. lividans
ZX7
ZX7(SLP2)
pro-2 str-6 rec-46 ∆dndA SLP2− SLP3−
ZX7 containing SLP2 plasmid
Zhou et al. (1988)
Chen et al. (1993)
ear, have very different basic structures. The chromosomal DNA of Streptomyces (mostly about 8 Mb)
contains proteins covalently bound at the 5h ends (Lin et
al., 1993). The overall sequences are not circularly
permuted as in T2\T4 (Leblond et al., 1993 ; Lin et al.,
1993). The chromosomal ends are fixed and contain
long (24–500 kb) inverted repeats (Leblond et al, 1993 ;
Lezhava et al., 1995 ; Lin et al., 1993 ; Pandza et al., 1997 ;
Redenbach et al., 1993).
A completely different model was proposed more than
30 years ago by Stahl & Steinberg (1964), and was also
taken into account by Hopwood (1965, 1966) and Stahl
(1967) in linkage analyses of S. coelicolor. In a hypothetical scenario, the linear replicons recombine with
a bias toward even numbers of crossovers, such that
markers at opposite ends of each mating molecule tend
to finish in the same recombinant molecule (instead of
segregating independently to different ones). The result
of this would be that the terminal markers exhibit
genetic linkage, which would then close the genetic map
into a circle.
There have been no hints whether this scenario may
apply to Streptomyces (Hopwood, 1965, 1966 ; Stahl,
1967). Little is known about the biochemical mechanisms of conjugal transfers and recombination of
these linear chromosomes. Conjugation in Streptomyces
occurs in surface culture when two mating strains are
mixed and grown together. Conjugation is mediated by
naturally occurring linear or circular plasmids, both of
which are abundant in Streptomyces. Some naturally
integrated plasmids also appear to be conjugative, such
as the integrated plasmid SLP1 in S. coelicolor (Bibb et
al., 1981), which on transfer to Streptomyces lividans
can be circularized or integrated into the chromosome
via a site-specific recombination system.
Conjugative plasmids are transferred from the donor
mycelium to recipient mycelia (intermycelial transfer ;
Kieser et al., 1982), and presumably spread along the
recipient mycelia. The transfer of chromosomes during
Streptomyces conjugation cannot be studied biochemically, and can be detected only by the appearance
of recombinants. The transfer has been assumed (without proof) to be essentially the same as that of the
conjugative plasmid – from donor to recipient, although
there are circumstances in which back transfers of the
chromosome from the recipient to the donor are also
indicated (Hopwood, 1984).
Recombination has been postulated to occur mostly in
the merozygote (partial diploid) state during conjugation in most bacteria studied, including Streptomyces
(Hopwood, 1967), but probably not in protoplast fusion
in which complete genomes are expected to be involved
in recombination (Hopwood & Wright, 1978). Studies
of recombination during protoplast fusion in Streptomyces have also been consistent with circular genetic
maps.
In this study, taking advantage of the possibility of
distinguishing between the termini of the chromosomes
of S. coelicolor and S. lividans by hybridization (Huang
et al., 1998), we examined the terminal sequences of the
recombinant chromosomes resulting from interspecies
mating. From the results, we inferred the numbers of
crossovers that had taken place between the parental
chromosomes. An odd number of crossovers would
have given a recombinant chromosome with one
telomere from each parental chromosome, whereas an
even number of crossovers would have led to a recombinant chromosome with both telomeres from the same
parent. Here we present the physical evidence that
supports the Stahl & Steinberg (1964) model, i.e. the
circular genetic maps of the Streptomyces are generated
by a strong bias for even numbers of crossovers.
METHODS
Bacterial strains and plasmids. S. coelicolor and S. lividans
strains used are listed in Table 1 with their respective genetic
markers and plasmid status. Streptomyces cultures were
maintained on YEME agar (Hopwood et al., 1985) at 30 mC.
Conjugation analysis. Conjugation between S. coelicolor and
S. lividans was performed according to Hopwood et al. (1985).
About 10(–10) spores of each parental strain were mixed and
plated on ISP medium 2 [ATCC culture medium 196 containing (g l−") : yeast extract, 4 ; malt extract, 10 ; dextrose, 4 ;
agar, 20 ; pH 7n3]. Spores were collected after incubation at
30 mC for about 7 d, and recombinant cultures were isolated
on minimal medium containing the appropriate supplements.
Each parental culture alone was plated in the same fashion to
determine the background frequency of spontaneous mutation
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Circular genetic maps from linear Streptomyces DNA
giving rise to the selected recombinant phenotype. Agarase
activities were assessed by direct visualization of the degradation of agar surrounding the colonies.
Physical and chemical analyses of Streptomyces chromosomes. For BamHI restriction analysis, chromosomal DNA
was isolated, digested and probed by Southern hybridization
according to Hopwood et al. (1985). The hybridization probes
were the terminal 1n4 kb BamHI fragment of the S. coelicolor
chromosome (Huang et al., 1998) and the terminal 0n65 kb
BamHI fragment of the S. lividans chromosome. For AseI
restriction analysis, preparation, PFGE and Southern hybridization of the genomic DNA were as described previously
(Lin et al., 1993).
RESULTS
Chromosomes of the strains used in heteroclone
analysis are linear
One piece of conclusive evidence for connecting the
(three) linkage groups in S. coelicolor into a circle was
from the analysis of ‘ heteroclones ’ (Hopwood, 1966)
found among recombinant colonies when a pair of very
close markers was selected. The heteroclones are unstable merodiploids that, on subsequent subculturing,
segregate into parental and various recombinant genotypes that are convenient subjects for conventional
linkage analysis. The results of the analysis distinctly
connected the previously separate linkage groups, including the two at the opposite arms of the S. coelicolor
A3(2) chromosome, into a circle (Hopwood, 1966).
Because Streptomyces cultures occasionally undergo
spontaneous deletions, leading to circularization of the
chromosomes (Lin et al., 1993), there exists a possibility
that the mutant strains used in the heteroclone experiments had circular chromosomes, and that these
accounted for the final circular linkage map. To examine
this possibility, the chromosomal DNA of two parental
strains studied by Hopwood (1966), 773 and 928 (D. A.
Hopwood, personal communication), was isolated and
subjected to restriction and PFGE analysis. The AseI
restriction patterns (not shown) of these strains were
identical to that of S. coelicolor M145 except for a
fragment that contained the integrated SCP1 (Kieser et
al., 1992). The terminal sequences of these two chromosomes were shown to be intact by hybridization analysis
using the telomere DNA of S. coelicolor chromosome as
the probe (not shown). These results indicated that the
773 and 928 chromosomes were linear, and thus
circularity of the parental chromosomes in the heteroclone experiment is not a possible explanation for the
‘ lack of constant genomic ends ’ (Hopwood, 1966).
The basic strategy of crossover analysis
To test the Stahl & Steinberg (1964) model for the
generation of circular genetic maps of Streptomyces, one
would need to differentiate between odd and even
numbers of crossovers that have occurred between the
linear chromosomes. A simple strategy is to isolate
recombinant chromosomes and examine their ends. The
presence of both ends from the same parent would
indicate an even number of crossovers, whereas the
presence of ends from different parents (mixed ends)
would indicate an odd number of crossovers.
There are two practical prerequisites for the application
of this strategy. Firstly, in order to reach a definite
conclusion, ideally one must examine the very ends of
chromosomes, not merely certain genetic markers
located near the termini. Secondly, it is necessary to have
a tool to inspect and distinguish the parental chromosomal ends experimentally. For recombination between
two homologous chromosomes, such as in intraspecies
conjugation of Streptomyces, it is generally impossible
to distinguish the ends of the two mating chromosomes,
which are usually identical.
We have recently cloned terminal DNA from the
chromosomes of several species of Streptomyces (Huang
et al., 1998). These terminal sequences, being highly
conserved for only the first 166–168 bp, could be
distinguished by restriction and hybridization analysis.
This makes it plausible after interspecies conjugation to
examine directly the inheritance of the different parental
telomeres in the recombinants.
For interspecies conjugation analysis we chose two
model species, S. lividans 66 and S. coelicolor A3(2), of
which many strains with defined genetic markers and
plasmid status (circular or linear plasmids) are available.
The other important consideration was the relative
global arrangements of essential and housekeeping genes
in the mating pairs. Gross deviations in the gene orders
would demand complex or even illegitimate recombination schemes to achieve viable recombinant chromosomes with the selected phenotypes. The general orders
of the genes that have been characterized in S. coelicolor
and S. lividans are in relatively good correspondence
(Hopwood et al., 1983, 1985 ; Leblond et al., 1993). The
overall similarity in chromosomal sequences in the two
species was further demonstrated by the similarity in
their macro-restriction maps, in which most of the
restriction sites (AseI and DraI) not only are similarly
located (Fig. 3c), but also contain homologous neighbouring sequences as revealed by cross-hybridization
(Leblond et al., 1993).
Wild-type S. coelicolor A3(2) contains two free plasmids
– the linear 350 kb SCP1 plasmid (Kinashi & ShimajiMurayama, 1991) and the circular 31 kb SCP2 plasmid
(Bibb & Hopwood, 1981) – and an integrated SLP1
plasmid, which is occasionally excised and becomes a
circular form on transfer to other species (Bibb et al.,
1981). Wild-type S. lividans 1326 contains a free 50 kb
linear SLP2 plasmid (Chen et al., 1993 ; Hopwood et al.,
1983). The right 15 kb of this plasmid is homologous to
the first 15 kb at both ends of the S. lividans chromosome
(Chen et al., 1993). Another conjugative plasmid, SLP3,
whose presence in S. lividans has only been determined
genetically (Hopwood et al., 1983), was absent from all
the strains used in this study.
Hybridization probes for the respective telomeres were
the 1n4 kb BamH1 terminal fragment of the S. coelicolor
chromosome and the 0n65 kb BamH1 terminal fragment
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of the S. lividans chromosome (Huang et al., 1998). Each
probe hybridized to its own chromosomal DNA, but not
to the other under our conditions (Figs 1 and 2). The S.
lividans probe also hybridized to the right end of SLP2
plasmid.
(a)
In this study, one of the mating pairs (‘ donor ’) contained
a free conjugative plasmid, while the other (‘ recipient ’)
did not possess any free plasmid. Thus, ‘ donors ’ and
‘ recipients ’ are defined with respect to the free conjugative plasmids, for which the direction of transfer is
unambiguous. Transfer of the chromosome is assumed
to proceed along the same lines as transfer of the
plasmid, but may not do so (see below).
The integrated plasmids contribute little, if at all, to
interspecies recombination
No recombination was detectable in conjugation between SLP2− SLP3− strains of S. lividans (Hopwood et
al., 1983). On the other hand, in matings between the
SCP1− SCP2− strains of S. coelicolor the chromosomes
recombine at a low but real frequency of about 10−),
presumably mediated by the integrated SLP1 plasmid
present in all S. coelicolor A3(2) strains (Bibb &
Hopwood, 1981). We set out to determine the extent to
which integrated SLP1 (and any other plasmid) might
contribute to chromosome recombination in our interspecies situation. Two strains devoid of free plasmids, S.
lividans ZX7 (pro-2 str-6 rec-46 ∆dndA SLP2− SLP3−)
and S. coelicolor M145 (SCP1− SCP2− ; Hopwood et al.,
1985) were crossed. The ∆dndA mutation in ZX7
removed a DNA modification system that causes DNA
degradation during PFGE (Zhou et al., 1988), and the
rec-46 mutation decreased plasmid recombination (Tsai
& Chen, 1987), but not chromosome recombination
during conjugation (Kieser et al., 1989). M145 is a
prototroph cured of two intrinsic plasmids, SCP1 and
SCP2, but still possessing the integrated plasmid SLP1
(Bibb et al., 1981).
Proline prototrophic (Pro+) and streptomycin-resistant
(Strr) cultures were isolated at a frequency of about
8n0i10−) by screening the spores recovered after mixed
growth of the two parents (Hopwood et al., 1985). This
was not significantly different from the spontaneous
occurrence of the same phenotype in M145 alone
(4n3i10−( ; presumably through a mutation to streptomycin resistance) or in ZX7 alone (5n4i10−) ; presumably through a reversion to proline prototrophy).
Therefore, the contribution of SLP1 to chromosomal
recombination in our interspecies mating system was
negligible and could be safely ignored in the following
analyses.
Chromosomes recombine with a bias toward even
numbers of crossovers in linear-plasmid-mediated
conjugation
We next analysed conjugation mediated by the linear
plasmid SCP1. From matings between S. coelicolor
M146 (hisA1 uraA1 strA1 SCP1+ SCP2− ; Hopwood et
(b)
.................................................................................................................................................
Fig. 1. Inheritance of telomeres during interspecies conjugation
mediated by linear plasmids. His+ Pro+ recombinants were
isolated following conjugation between S. coelicolor M146
(SCP1) and S. lividans ZX7 (a), and Pro+ Strr recombinants
following conjugation between ZX7(SLP2) and S. coelicolor
M145 (b). Chromosomal DNA from these recombinant cultures
was digested with BamHI and hybridized with the 1n4 kb BamHI
terminal DNA of S. lividans chromosome and the 0n65 kb BamHI
terminal DNA of S. coelicolor chromosome (shown in enlarged
views). The parental chromosomes are represented by the
horizontal lines showing respective genetic markers and TPs
(filled circles). The solid triangles depict the selected alleles and
the open triangles, the non-selected markers found in all or
most of the recombinants. ‘ dag+ ’ represents the agarase gene
present in S. coelicolor, but not in S. lividans (dag0). The two
minimum crossovers necessary for the observed recombination
are indicated by the trace line between the two chromosomes.
In this orientation, the terminal AseI J fragment of S. coelicolor
and the terminal H1 fragment of S. lividans are to the left.
al., 1985), and plasmidless S. lividans ZX7, His+ Pro+
recombinants were isolated at a frequency (1n5 i 10−')
comparable to SCP1-mediated intraspecies recombination in S. lividans (3n5–6n8i10−' ; Hopwood et al.,
1983) or S. coelicolor (about 10−' ; Bibb & Hopwood,
1981). In contrast, spontaneous mutations giving rise
to the selected His+ Pro+ phenotype appeared at
much lower frequencies in M146 (5n3i10−)) or ZX7
(9n2i10−)). Analysis of the chromosomal DNA from 10
recombinants by hybridization revealed that all had
inherited both telomeres from M146 (Fig. 1a). No
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Circular genetic maps from linear Streptomyces DNA
(a)
(b)
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Fig. 2. Inheritance of telomeres during interspecies conjugation mediated by circular plasmids. (a) His+ Pro+ recombinants
were isolated following conjugation between S. coelicolor 1190(SCP2) and ZX7, and their chromosomal DNA was
analysed as described in Fig. 1. Two recombinants (5 and 9) harbouring mixed telomeres are shaded in black. The least
additional crossover necessary for the appearance of the mixed ends in these two recombinants is indicated by the
dashed line. (b) Chromosomal DNA from three reisolates each of recombinants 5 (strains 5–1 to 5–3) and 9 (stains 9–1 to
9–3) was purified and subjected to the same restriction and hybridization analysis.
recombinant chromosomes with mixed ends were
observed. This result indicated that an even number of
crossovers had occurred in these recombinants. Since
only one crossover was necessary to generate His+ Pro+
recombinants, an additional crossover must have occurred between the hisA1 marker and the left telomere.
Whilst the extra crossover might have occurred between
pro-2 and the right telomere, giving rise to recombinant
chromosomes with both ends from ZX7, these alternative recombinant products were not found.
Further inspection of the non-selected phenotypes
among His+ Pro+ recombinants revealed that all of them
(15\15) were uracil-auxotrophic and agarase-positive,
both of which were present only in S. coelicolor M146
(S. lividans does not possess the agarase gene).
A similar result was obtained using another linear
plasmid, SLP2. Conjugation between S. lividans
ZX7(SLP2) and S. coelicolor M145 yielded Pro+ Strr
recombinants at a frequency of 2n7i10−&, comparable
to SLP2-mediated intraspecies recombination in S.
lividans (1n7–3n2i10−% ; Hopwood et al., 1983) or S.
coelicolor (5i10−& ; Bibb & Hopwood, 1981), and much
higher than the frequencies of 1n0i10−( and 4n3i10−(,
respectively, for spontaneous appearance of Pro+ Strr in
ZX7(SLP2) and M145. DNA was isolated from 11 of
these recombinants and hybridized with the terminal
probes (Fig. 1b). No hybridization was observed with
the terminal probe of S. coelicolor, whereas the terminal
probe of S. lividans hybridized to all recombinant
chromosomes. In 10 out of the 11 samples, it hybridized
to presumably both the terminal DNA of the recombi-
nant chromosomes and the right end of SLP2. In the
remaining sample (no. 4), there was a weaker
hybridization to a 10 kb BamHI fragment. This indicated that the chromosomal termini in this strain had
undergone deletions. The chromosomal deletions might
have caused the loss of SLP2 (Denapaite & Cullum,
1998 ; Lin, 1998 ; Wu, 1994).
In another cross between S. lividans ZX7(SLP2) and
plasmidless S. coelicolor M130 (hisA1 uraA1 strA1
SCP1− SCP2−), Pro+ Ura+ recombinants were isolated at
a frequency of 1n0i10−' against background mutation
frequencies of about 10−) in the parents. Chromosomal
DNA from 10 recombinants analysed contained only the
telomeres of the donor S. lividans (data not shown).
Thus, the observed non-reciprocity in recombination
did not appear to be limited to a particular selection
scheme.
Of the unselected markers in these two crosses
[ZX7(SLP2)iM145 and ZX7(SLP2)iM130], most
(9\12) were of the donor type, and the remainder (3\12)
were of a mixed type (agarase-positive and His−).
Chromosomes recombine with a bias toward even
numbers of crossovers in circular-plasmid-mediated
conjugation
The preference for even numbers of crossovers was also
observed in conjugation promoted by a circular plasmid.
In crosses between S. coelicolor 1190 (hisA1 uraA1 strA1
SCP1− SCP2+) and plasmidless S. lividans ZX7, His+
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S.-J. W A N G a n d O T H E R S
(a)
(c)
(d )
(b)
(e)
(f)
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Fig. 3. Restriction patterns of recombinant chromosomes. The genomic DNA from selected recombinants from the
M146(SCP1)iZX7 cross (a) and the 1190(SCP2)iZX7 cross (b) was digested by AseI and separated by PFGE. The
designations of the AseI fragments of the parental chromosomes are indicated on both sides. The two recombinants (5
and 9) with mixed ends are designated by the shaded numbers. In (a), S, S1 and S2 indicate the intact SCP1 (350 kb) and
its two larger (180 and 140 kb) AseI fragments, respectively (Kinashi & Shimaji-Murayama, 1991). Of these, S2 appeared
to co-migrate with the M fragment of the S. coelicolor chromosome (Kieser et al., 1992), resulting in elevated fluorescent
intensities. The smallest (18 kb) AseI fragment of SCP1 might also co-migrate with the O fragment of the chromosome. In
(b), the presence of mixed ends in recombinants 5 and 9 was demonstrated by the presence of the fragment
corresponding to the J fragment of S. coelicolor, which hybridized to the S. coelicolor end probe, and the largest
fragment corresponding to the A fragment of S. lividans, which hybridized to the S. lividans end probe (hybridization
data not shown). The larger fragments in the same cultures were under-represented in these experiments, probably due
to breakdown and/or entrapment. Panels (c)–(f) depict the putative recombination events leading to the His+ Pro+
recombinants in the M146iZX7 cross (c), the His+ Pro+ recombinants with same telomeres (d) and with mixed ends (e) in
the 1190iZX7 cross, and the Ura+ Pro+ recombinants in the same cross (f). The chromosomes of S. coelicolor (SC) and S.
lividans (SL) are aligned with respect to their corresponding AseI fragments and genetic markers (h, hisA1 ; p, pro-2 ; d,
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Circular genetic maps from linear Streptomyces DNA
Pro+ recombinants were isolated at a frequency of
1n1i10−' against spontaneous mutation frequencies of
3–9i10−) in the parent strains. SCP2-mediated intraspecies recombination occurred at about the same
frequencies (10−') in S. coelicolor (Bibb & Hopwood,
1981). Chromosomal DNA from 12 recombinants was
isolated and examined : 10 of them contained only the
telomeres of ZX7, while the other two (nos 5 and 9)
contained one telomere from each parent (Fig. 2a). The
two recombinant cultures with mixed ends grew normally, and the observed mixed telomeres persisted on
extended subculturing and in each reisolated culture
(Fig. 2b). This indicated that (i) the mixed ends observed
were on the same chromosome and did not reflect a
mixed population of two chromosomes with the same
telomeres from different parents, and (ii) the recombinant chromosomes with the mixed telomeres did not
suffer from an obvious growth disadvantage.
All the non-selected genetic markers in these recombinants were those of the recipient ZX7 (Ura+ and
agarase-negative). While a single crossover (between
hisA1 and pro-2) was sufficient to give rise to the selected
His+ Pro+ phenotype, for the 10 recombinants with both
recipient telomeres an additional crossover must have
occurred between pro-2 and dag. For the two recombinants with mixed ends, at least one (or a larger odd
number of) crossover(s) must have occurred either to the
left of hisA1 or to the right of uraA1. The AseI restriction
analysis (see later) indicated the former event.
In the repeat of the same cross, Pro+ Ura+ recombinants
were isolated at a frequency of 2n1i10−'. Eighteen
recombinants were picked and their chromosomes
examined. In all of them, only the termini of the S.
lividans chromosome were present (data not shown). All
the non-selected genetic markers were those of the
recipient ZX7 (His+ and agarase-negative). No recombinants with mixed ends were found.
From these results we conclude that (i) there was a
strong bias for even numbers of crossovers between the
recombining chromosomes during conjugation, and (ii)
there was a surprising correlation between the topology
of the conjugative plasmids and the parental telomeres
inherited by the recombinants – the telomeres and
genetic markers from the plasmid donor were predominantly inherited in linear-plasmid-driven conjugation ; whereas the telomeres and genetic markers from
the recipients were preferentially maintained in circularplasmid-driven transfer.
PFGE analysis of the recombinant chromosomes
The recombinant chromosomes of two crosses were
examined by AseI digestion and PFGE (Fig. 3). The
restriction patterns of these chromosomal DNA
molecules confirmed the foregoing conclusions regarding the inheritance patterns of the recombination
chromosomes. The origins of many fragments in the
recombinants could be readily assigned to one or the
other parent. No obviously new fragments resulting
from recombination were present, indicating that relatively few crossovers had taken place.
Most of the AseI fragments in the four His+ Pro+
recombinants in the M146(SCP1)iZX7 cross appeared
to originate from the donor M146 (Fig. 3a). While
several fragments from the two parents could not be
readily distinguished, all those that could corresponded
to the fragments of S. coelicolor, such as fragments C, D,
E, I, J, L and K (Kieser et al., 1992). In contrast,
fragments B, C, E , E and G of S. lividans (Leblond et
" # Tracing the organization of the
al., 1993) were absent.
recombinant chromosomes from the arrangement of
genetic markers and restriction fragments revealed that
achieving this organization required a minimum of two
crossovers – one between hisA1 and pro-2. and the
other close to the left side of his-2 (Fig. 3c). Although the
two events presumably occurred on fragment C (and
probably I) of S. coelicolor, the sizes of these two
fragments appeared not to have changed, probably
because the corresponding restriction sites in the S.
lividans chromosome were similarly located. Overall,
the prevalence of the donor sequences in these recombinants was apparent.
In the 1190(SCP2)iZX7 cross, both the His+ Pro+ and
Ura+ Pro+ recombinants were analysed (Fig. 3b, d, e, f).
The three His+ Pro+ recombinant chromosomes (nos 3,
4, 7) with both recipient ends were almost in perfect
contrast to their counterparts in the M146(SCP1)iZX7
cross (panels a, c) : the S. coelicolor fragments D, E, I, J
and L were absent and the S. lividans fragments C, E ,
"
E , E , F, H , and H were present (Fig. 3b). This pattern
#
$
"
#
suggested that, in addition to the crossover between
hisA1 and pro-2 (on fragment C of S. coelicolor and
fragment B of S. lividans), another one had occurred
between pro-2 and dag (Fig. 3d). This placed the dag!
and uraA1+ markers and the S. lividans telomeres on the
recombinant chromosomes. The terminal probe of S.
lividans hybridized to the terminal H fragment of S.
" (presumably
lividans and one of the largest fragments
agarase ; u, uraA1). The homology between the AseI linking clones of the two chromosomes (Leblond, 1993) is indicated
by the connecting dashed lines. Those AseI fragments whose origins could be putatively assigned size-wise without
ambiguity are boxed – filled boxes for presence and open boxes for absence in the recombinant chromosomes. In (c), the
presence of the C fragment of S. coelicolor was clear in recombinant 5, but not in the other three. In (e), the presence of
the intact A fragment of S. lividans could not be established due to lack of sufficient resolution (b), but the presence of
its end was identified by hybridization (not shown) and was so indicated. The genetic markers selected in the crosses are
indicated by the filled triangles, and the unselected markers predominant in the recombinants are indicated by the open
triangles. Based on the restriction fragments and markers identified, the structures of the recombinant chromosomes are
traced along the parental chromosomes with the putative crossovers between the parental chromosomes indicated by
the vertical lines.
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S.-J. W A N G a n d O T H E R S
the A fragment of S. lividans ; data not shown),
supporting the deduction and the results of the BamHI
analysis (Fig. 2). The presence of the H fragment of S.
coelicolor and the absence of the G fragment of S.
lividans (both of which contained the uraA1 locus)
suggested the possible occurrence of an additional
double crossover in this region (Fig. 3d).
In the two His+ Pro+ recombinant chromosomes with
mixed ends (nos 5, 9), both parents appeared to
contribute multiple fragments, such as fragments H, I, J
and L from S. coelicolor, and fragments C and F from S.
lividans (Fig. 3b, e). Most significant was the presence of
the J fragment at the left end of the S. coelicolor
chromosome. This fragment hybridized to the terminal
probe of S. coelicolor. The presence of the S. lividans
end on the A fragment (right end of chromosome) was
also demonstrated by hybridization to the S. lividans
terminal probe (not shown). The H fragment con"
taining the left end of the S. lividans chromosome
was
absent, based on the lack of hybridization of the S.
lividans terminal probe to the DNA corresponding to
the co-migrating H and H fragments (not shown) and
"
#intensity of this population
the diminished fluorescent
(Fig. 3b). These results, consistent with the mixed ends
observed among the BamHI fragments (Fig. 1), indicated
one (or an odd number of) additional crossover(s) to the
left of the hisA1 marker in these two recombinants (Fig.
2a). Like the recombinant chromosomes with the same
ends, these recombinant chromosomes appeared to
contain the H fragment of S. coelicolor but not the G
fragment of S. lividans, suggesting a possible double
crossover in this region.
The Ura+ Pro+ recombinants of the 1190(SCP2)iZX7
cross exhibited a very similar pattern to those of the
His+ Pro+ recombinants. Compared to the His+ Pro+
recombinants in the same cross, the most notable
difference was the preservation of the G fragment of S.
lividans and absence of the H fragment of S. coelicolor.
The reason for the difference is not clear. It may
presumably reflect the difference in selection schemes.
The terminal probe of S. lividans hybridized to fragments A and H , whereas no hybridization was observed
" probe of S. coelicolor (not shown).
with the terminal
This is consistent with the previous conclusion based on
BamHI analysis (Fig. 2a).
DISCUSSION
This study provides the first experimental demonstration of the hypothetical model of Stahl & Steinberg
(1964) for generating circular genetic maps operationally
from linear replicons by a constraint in the recombination process. The biased production of recombinant
chromosomes with even-numbered crossovers predicted
in this model was demonstrated in interspecies conjugation of Streptomyces mediated by either a linear or
a circular plasmid.
The even-numbered crossovers, however, produced only
one of the two types of products expected from
reciprocal recombination, and the type of recombination
products recovered appeared to depend on the topology
of the conjugative plasmids. In circular-plasmidmediated conjugation (with only one plasmid tested),
most of the recombinant chromosomes possessed the
recipient-type termini. The reciprocal products, i.e.
recombinant chromosomes with the donor-type ends,
were not recovered. The preponderance of unselected
markers as well as the AseI restriction fragments of the
recipient type in these recombinants suggested that the
recipient chromosomes had picked up only a relatively
small internal segment(s) of the donor chromosome that
was presumably mobilized from the donor cell. This
merozygous state of recombination resembles that in
Escherichia coli conjugation (also mediated by circular
plasmids), in which the donor chromosome rarely enters
the recipient cell in its entirety. Bibb & Hopwood (1981)
did not observe this strongly preferred inheritance of
recipient markers in intraspecies mating between SCP2+
and SCP2− S. coelicolor. It has been proposed that the
inheritance of donor chromosomal markers depends on
recombination in the immediate recipient ‘ cells ’ as well
as subsequent recombination in adjacent cells on further
(intramycelial) transfer of the recombinant chromosomes (Lydiate et al., 1985). The partial donor chromosome in the recipient is not or is rarely transferred
intramycelially unless incorporated into an intact
chromosome through homologous recombination.
Thus, the shortage of donor markers in the recombinant
chromosomes in the interspecies SCP2+iSCP2− crosses
may simply reflect the lower efficiency of recombination
between the chromosomal sequences of S. coelicolor
and S. lividans due to lower sequence homology.
In contrast, in the linear-plasmid-mediated conjugation
of Streptomyces (with two plasmids tested), double
crossovers gave rise to recombinant chromosomes consisting mainly of donor chromosome plus an internal
stretch(es) of the recipient chromosome. Again, the
reciprocal products, i.e. recombinant chromosomes with
both ends of the recipient type, were not found. This
predominant presence of donor markers in the recombinants has also been observed in the classical
SCP1+iSCP1− crosses in S. coelicolor by Hopwood et
al. (1973). Although the SCP2 status in this earlier study
was not yet established, the absence of the tendency
for progeny to inherit donor markers in SCP1−
SCP2+iSCP1− SCP2− crosses of S. coelicolor (Hopwood, 1984) indicated that the linear plasmid SCP1
was involved in the skewed inheritance pattern. These
authors proposed that some high-frequency donors (like
Hfr strains of E. coli) present in the SCP1+ culture
contributed markers preferentially to the progeny. An
alternative conjecture is presented below.
The selectivity in recombinant types recovered was
probably not due to inhibition exerted by one particular
parental species on the other during conjugation.
Although SCP1 encodes genes for biosynthesis of the
antibiotic methylenomycin A (Wright & Hopwood,
1976), which may inhibit SCP1− strains, there appears to
be no or little effect of the methylenomycin A on the
patterns of chromosome recombination during con-
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Circular genetic maps from linear Streptomyces DNA
Recombination in merozygotes
(a)
(b)
z+
z+
a–
a+
a+
Recombination between intact chromosomes
(c)
a+
(d )
a–
z–
z+
z+
z–
a+
a–
.................................................................................................................................................................................................................................................................................................................
Fig. 4. Models for obligatory double crossovers during recombination of Streptomyces chromosomes. (a) Recombination
between an intact linear chromosome (left) and a partial chromosome with a telomere (right). Both odd numbers
(shown) and even numbers of crossover are legitimate, generating recombinant chromosomes with mixed ends or the
same ends, respectively. (b) Recombination between an intact linear chromosome (left) and an internal chromosomal
sequence (right). Even-numbered crossovers are necessary to give rise to a complete recombinant chromosome. (c)
Recombination between two linear chromosomes with free ends. Both odd and even numbers of crossovers are allowed,
giving rise to mixed ends or the same ends, respectively. (d) Recombination between two intact linear chromosomes with
strongly interacting telomeres. Even numbers of crossovers are necessary for separation of the recombinant
chromosomes. Two pairs of terminally located alleles, a+/a− and z+/z−, are indicated to illustrate linkage analysis. The TPs
are represented by the filled circles.
jugation or protoplast fusion (Hopwood, 1984 ; Kirby &
Hopwood, 1977). Furthermore, the other linear plasmid,
SLP2, which does not encode any detectable antibiotic
activities, displays the same skewed inheritance. Thus,
the plasmid status (donor\recipient), rather than the
species, appeared to determine the inheritance of nonselected sequences (including the telomeres) on the
recombinant chromosomes selected.
While the contrasting effect exerted by plasmid topology
on chromosome inheritance is intriguing, the very
limited number of plasmids tested (one circular and two
linear) may not justify formulating a general rule.
Further studies employing more plasmids of both
categories are necessary. Nevertheless, whether the
hypothesis is confirmed or not does not affect the
support our results lend to the scenario postulated by
Stahl & Steinberg (1964) – a circular genetic map
generated by preferred even numbers of crossovers
between linear genomes.
If the preference for an even number of crossovers is
real, what is the biochemical basis for this constraint ?
An even number of crossovers is necessary for linear
chromosomes if recombination occurs in merozygotes
consisting of an intact chromosome and an internal
chromosomal sequence (Fig. 4b). An odd number of
crossovers would result in two partial chromosomes
each lacking a telomere. This scenario probably
represents the recombination events in circular-plasmidmediated conjugation of Streptomyces. The partial
exogenote is likely mobilized from the donor by the
circular plasmid from an internal origin on the chromosome. Occasionally, the transfer may reach one end of
the donor chromosome, which would make odd-numbered crossovers possible (Fig. 4a), resulting in a
recombinant chromosome with mixed ends.
The same merozygote situation may also apply to
chromosome recombination in linear-plasmid-mediated
conjugation, but the roles of the mating partners would
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S.-J. W A N G a n d O T H E R S
have to be reversed to be consistent with the absence of
recombinant chromosomes with both recipient ends :
the intact chromosome would have to be that of the
donor, and the fragment would be of the recipient. On
the other hand, if we assume that this recombination
step takes place in the recipient (or plasmid), this would
imply that the chromosomes are transferred in total (or
nearly so) from the plasmid donor, and this was
demonstrated in the restriction analysis (Fig. 3). The
complete transfer of donor chromosomes in linearplasmid-mediated conjugation agrees with the model
proposed by Chen (1966), in which the chromosome
transfer mediated by linear plasmids starts from the
ends. The principle underlying the unilateral disappearance of the recipient chromosomal sequences can
only be speculated on at present, but it suggests the
devastation of the recipient chromosomes by a selfish
and invasive action of the donor chromosomes. The
alternative scenario, in which part or most of the
recipient chromosome is transferred into the donor cell
(in the opposite direction from plasmid transfer), would
also be a remarkable and uncommon phenomenon.
Although protoplast fusion was not used directly to
address the topology of the genetic maps of Streptomyces, a constraint for even numbers of crossovers has
always been imposed successfully in the formal analysis
of genetic linkage (e.g. Hopwood & Wright, 1978). The
rationale is based on the assumption that intact circular
chromosomes are involved in recombination, and an
odd number of crossovers would result in dimeric
chromosomes. Interestingly, the circular genetic maps
based on this imposed condition have been essentially
consistent for S. coelicolor and other species. Now that
the assumption of circular chromosomes is known to
have been incorrect, a different reason is needed for the
demand for even-numbered crossovers. Whether the
mating linear chromosomes are intact or not in protoplast fusion, there is no a priori topological constraint
that demands even numbers of crossovers (Fig. 4c).
Odd-numbered crossovers give rise to a recombinant
chromosome with mixed ends, whereas even-numbered
crossovers give the same ends. An even number of
crossovers would be necessary if the ends of the
chromosomes were not free (Fig. 4d). This is true even if
the chromosomes are fragmented as long as complete
genomes are to be restored. A likely constraint is a
strong interaction between the telomeres, mediated by
the covalently bound terminal proteins (TPs), as has
been observed in adenoviruses (Robinson et al., 1973)
and φ29 (Ortin et al., 1971), the other linear replicons
with TPs. Circular configurations of these linear viral
DNA molecules were observed when they were released
from the virion particles without proteolytic or detergent treatment.
During electrophoresis, the TP-capped DNA of these
linear replicons appears to form a proteinase- and
detergent-sensitive complex that cannot enter the gel.
The linear chromosomes and plasmids of Streptomyces
also exhibit the same behaviour, suggesting strong
interactions between the TPs (Lin et al., 1993), which
may be implicated in important biological functions,
such as replication, conjugal transfer and structural
stability (Chen, 1996).
The strength of the constraint imposed by terminal
interactions on chromosome recombination should depend on the strength of the terminal interactions. The
existence of rare recombinants with mixed ends
indicates that the terminal interactions are not irreversible during recombination and exchange of telomeres is still possible.
The scheme portrayed in Fig. 4(c) implies that both
products of reciprocal recombination, i.e. recombinant
chromosomes with both ends from either parent, may be
recovered from protoplast fusion. This hypothesis
remains to be tested experimentally. Since plasmids are
not necessary for recombination in protoplast fusion, it
would be interesting to investigate their effect on the
outcome of the recombination, if any.
An alternative to the imposed even-numbered crossovers
scenario is that there is no constraint on the numbers of
crossovers allowed, but that the products of evennumbered crossovers are selectively recovered. This
would imply that the products of odd-numbered crossovers, i.e. chromosomes with mixed ends, are biologically disadvantaged. The rarity of recombinant
chromosomes with mixed ends suggests that the selection against them would be severe in this hypothesis.
Thus the stable maintenance of the S. coelicolor\S.
lividans mixed ends in the recombinant cultures (nos 5
and 9) and their normal growth characteristics do not
support this view.
A third theoretical possibility exists that is independent
of the number of crossovers : it is possible that identical
telomeres are preserved on the recombinant chromosomes through conversion of one terminal sequence
into the other. Again, the stable maintenance of mixed
ends in the recombinant chromosomes on prolonged
subculturing without signs of conversion reduces the
likelihood of conversions. Moreover, if conversion is
involved, it must exert a specific directionality in a very
selective way, i.e. donor to recipient conversion of
telomere sequences in circular-plasmid-mediated conjugation, and conversion in the opposite direction
in linear-plasmid-mediated conjugation. The range
spanned by this homogenotization would also have to
be relatively long – at least 1n4 kb (to reach the restriction sites used in the analysis). Qin & Cohen (1998) in
their study of the replication of the linear plasmid
pSLA2 of Streptomyces rochei failed to observe any
homogenotization between the two termini.
Of the three possible hypotheses – (i) even-numbered
crossovers constrained by the demand for merozygosity
and by terminal interactions, (ii) selective recovery of
even-numbered crossovers and (iii) homogenotization –
the first, with its in vitro evidence and interesting
biological implications, is currently the working model
of our choice.
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Circular genetic maps from linear Streptomyces DNA
While our systematic analysis was performed using only
one free conjugative plasmid in each cross, the classical
conjugation studies of Streptomyces often involve more
than one free plasmid (circular and linear) and sometimes in both parental strains. The directionality of
transfer (of the plasmids and of the chromosomes)
would be more difficult to predict or establish, and the
molecular genetic analysis would be more complex.
However, there are no obvious reasons why the constraints for even-numbered crossovers would be absent
in multi-plasmid schemes.
Considering the broader scope, the validation of the
Stahl & Steinberg (1964) hypothesis indicates that the
danger of equating the topology of genetic maps to that
of genomes is not limited to the special case of T2\T4
phages. The well-founded prudence displayed by
Hopwood (1965, 1966) more than three decades ago
should still be observed vigorously in the physical and
sequence analyses of microbial genomes. For the latter,
the popular shotgun sequencing approach in assembling
whole genome sequences, if applied to T2\T4 genomes,
would certainly give a circular physical map. Therefore,
not only is the topology of circular genetic maps not a
reliable guideline, the topology derived from assembled
sequence contigs is also not free from similarly hidden
pitfalls and traps.
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ACKNOWLEDGEMENTS
We thank Nora S. Chen and Caroline Hu for technical
assistance, Helen Kieser, Tobias Kieser and David A.
Hopwood for the Streptomyces strains, and D. A. Hopwood
and Franklin W. Stahl for reading the manuscript and making
suggestions for improvement. Examination of the cultures
used in heteroclone analysis was suggested by D. A.
Hopwood. This work is supported by a research grant from
the National Science Council (NSC87-2316-B010-M44) and
from the National Health Research Institute (DOH87-HR713). C. W. C. was a recipient of a research award from the
Medical Research and Advancement Foundation in memory
of Chi-Shuen Tsou.
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.................................................................................................................................................
Received 23 March 1999 ; revised 1 June 1999 ; accepted 2 June 1999.
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