New Sporulation Loci in Streptomyces
coelicolor A3(2)
N. Jamie Ryding, Maureen J. Bibb, Virginie Molle, Kim C.
Findlay, Keith F. Chater and Mark J. Buttner
J. Bacteriol. 1999, 181(17):5419.
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JOURNAL OF BACTERIOLOGY, Sept. 1999, p. 5419–5425
0021-9193/99/$04.00⫹0
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Vol. 181, No. 17
New Sporulation Loci in Streptomyces coelicolor A3(2)
N. JAMIE RYDING,† MAUREEN J. BIBB, VIRGINIE MOLLE, KIM C. FINDLAY, KEITH F. CHATER,
AND MARK J. BUTTNER*
John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom
Received 1 April 1999/Accepted 24 June 1999
transcripts (22, 31). Mutation in the remaining locus, whiD,
causes the formation of spores of highly irregular size and
shape which are defective in wall thickening and lyse extensively (6, 25, 28). Two other loci, whiC and whiF, are no longer
included: the only known whiC mutant has been lost, and
whiF99 was shown to be an unusual allele of whiG (5, 31). The
eight known whi loci have all been cloned and sequenced (1,
11–13, 27, 31, 32).
Chater (6) suggested that the genetic map of whi loci might
not be saturated on the grounds that several loci are poorly
represented in the original collection of whi mutants analyzed;
examples are whiD (one allele), whiB (two alleles), whiE (two
alleles) and whiJ (two alleles). The subsequent discovery by
reverse genetics of the involvement of the sigF locus in sporulation supported this viewpoint (21, 22, 29). As a consequence,
we have attempted to identify novel whi loci through the isolation of new whi mutants and their complementation, taking
advantage of previously cloned whi genes to exclude known
loci from the screen. We report the identification, phenotypic
characterization, mapping, and cloning of five new whi loci.
In the filamentous, gram-positive bacterium Streptomyces
coelicolor, dispersal is achieved through a simple differentiation process that results in the release of exospores (7). During
this process, multigenomic aerial hyphae divide into unigenomic prespore compartments by synchronized multiple septation at regular intervals along their length. These cylindrical
prespore compartments subsequently mature to give rise to
chains of 50 to 100 ovoid, thick-walled spores. During this
maturation phase, colonies develop a grey color, due to the
synthesis of a polyketide spore pigment.
This pigmentation has been exploited to isolate developmental mutants; all of the original sporulation-deficient mutants were identified by virtue of their inability to synthesize
wild-type levels of the spore pigment, resulting in colonies that
remained white, even on prolonged incubation (18). Fifty of
these white (whi) mutants were analyzed genetically, and current information suggests that they represent eight separate
loci, whiA, whiB, whiD, whiE, whiG, whiH, whiI, and whiJ (6, 9,
31). Of these, whiE is a complex locus which encodes the
enzymes that synthesize the spore pigment itself, and whiE
mutants do not appear to be morphologically defective (6, 12,
25). whiA, whiB, and whiG mutants are completely blocked in
sporulation septation and are white in appearance. whiH and
whiI mutants produce some sporulation septa; while whiI mutants are completely white, whiH mutants are pale grey and
make low but detectable levels of the whiE transcripts that
specify the spore pigment (6, 7, 9, 22, 25, 31, 32). whiJ mutants
produce low numbers of apparently normal spore chains, are
pale grey, and again make low but detectable levels of the whiE
MATERIALS AND METHODS
Bacterial strains, plasmids, growth conditions, protoplast transformation,
and chemical mutagenesis. S. coelicolor A3(2) strains used were the wild-type
strain 1147 (prototrophic, SCP1⫹ SCP2⫹ Pgl⫹ [19]), M145 (prototrophic, SCP1⫺
SCP2⫺ Pgl⫹ [19]), J1501 (hisA1 uraA1 strA1 SCP1⫺ SCP2⫺ Pgl⫺ [10]), 1258
(proA1 hisC9 argA1 cysD18 uraA1 strA1 SCP1NF [SCP2 status uncertain] Pgl⫹
[19]), and J243 (uraA1 strA1 whiD16 SCP1⫹ [SCP2 status uncertain] Pgl⫹ [8]). S.
coelicolor strains were cultured on minimal medium MM (19) containing 0.5%
(wt/vol) mannitol as the carbon source or on MS (mannitol plus soya flour) agar
(17). Protoplasts were made and transformed as described by Hopwood et al.
(19). S. coelicolor M145 spores were mutagenized with nitrosoguanidine (NTG)
as described previously (14, 18). Plasmids used were pIJ698 (23) and pSET152
(4).
Construction of a genomic library and complementation of mutants. Total
chromosomal DNA from wild-type S. coelicolor was partially digested with
Sau3AI and size fractionated on a sucrose gradient, and fragments in the size
range 15 to 22 kb were treated with calf intestinal alkaline phosphatase and
ligated with the self-transmissible, single-copy SCP2*-derived plasmid, pIJ698,
* Corresponding author. Mailing address: John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom. Phone (44) 1603 452571.
Fax: (44) 1603 456844. E-mail [email protected].
† Present address: Department of Microbiology, Michigan State
University, East Lansing, MI 48824-1101.
5419
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Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the
grey polyketide spore pigment, and such white (whi) mutants had been used to define eight sporulation loci,
whiA, whiB, whiD, whiE, whiG, whiH, whiI, and whiJ (K. F. Chater, J. Gen. Microbiol. 72:9–28, 1972; N. J.
Ryding, Ph.D. thesis, University of East Anglia, 1995). In an attempt to identify new whi loci, we mutagenized
S. coelicolor M145 spores with nitrosoguanidine and identified 770 mutants with colonies ranging from white
to medium grey. After excluding unstable strains, we examined the isolates by phase-contrast microscopy and
chose 115 whi mutants with clear morphological phenotypes for further study. To exclude mutants representing
cloned whi genes, self-transmissible SCP2*-derived plasmids carrying whiA, whiB, whiG, whiH, or whiJ (but not
whiD, whiE, or whiI) were introduced into each mutant by conjugation, and strains in which the wild-type
phenotype was restored either partially or completely by any of these plasmids were excluded from further
analysis. In an attempt to complement some of the remaining 31 whi mutants, an SCP2* library of wild-type
S. coelicolor chromosomal DNA was introduced into 19 of the mutants by conjugation. Clones restoring the
wild-type phenotype to 12 of the 19 strains were isolated and found to represent five distinct loci, designated
whiK, whiL, whiM, whiN, and whiO. Each of the five loci was located on the ordered cosmid library: whiL, whiM,
whiN, and whiO occupied positions distinct from previously cloned whi genes; whiK was located on the same
cosmid overlap as whiD, but the two loci were shown by complementation to be distinct. The phenotypes
resulting from mutations at each of these new loci are described.
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RYDING ET AL.
RESULTS
Isolation of new whi mutants. S. coelicolor M145 spores were
mutagenized with NTG and plated directly on MM at a density
of approximately 200 colonies per plate; 1,500 mutant colonies
with aerial surface color varying from white to medium grey
were picked from a total population of approximately 30,000.
From the 1,500 patches, 770 strains were streaked for single
colonies, and those that showed unstable phenotypes (approximately one-third of the total) were discarded. A large majority
of the unstable mutants exhibited a hypervariable phenotype
similar to that described for strains of Streptomyces ambofaciens that have undergone large chromosomal rearrangements
(24, 34). Coverslip impressions of the remaining strains were
examined, and 431 isolates with stable phenotypes distinguishable from that of the wild type were identified. These strains
could be divided into three groups: 71 that failed to produce
abundant aerial hyphae, 144 that produced abundant aerial
hyphae but showed clear defects in sporulation; and 216 mutants that sporulated abundantly but produced either spores
with aberrant size or shape or spores of normal appearance but
with reduced pigmentation. Primary interest was in the second
group (144 strains).
Complementation with known whi genes. In an attempt to
exclude mutants that represented whi genes that had already
been cloned, self-transmissible SCP2*-derived plasmids carrying whiA, whiB, whiG, whiH, or whiJ (but not whiD, whiE, or
whiI) were introduced into 115 of the 144 whi mutants by
mating. The morphological defects shown by the mutants
made it unlikely that they were caused by mutations in the
whiE spore pigment biosynthesis cluster, and self-transmissible
clones were not available for whiD or whiI. Complementation was judged initially by looking for the restoration of grey
pigmentation to the aerial surface of exconjugants and subsequently by inspection of coverslip impressions in a phase-contrast microscope. Of the 115 strains, 6 were fully complemented by whiA, 15 were fully complemented by whiB, 19 were
fully complemented by whiG, 16 were fully complemented by
whiH, and 3 were fully complemented by whiJ. A striking observation was that in two of the strains complemented by whiB,
five of the strains complemented by whiG, and seven of the
strains complemented by whiH, one or more of the other whi
genes also increased the level of grey pigmentation. In some
cases, there was also some degree of morphological correction
as well. In a further 25 strains, no fully complementing whi
gene was identified, but one or more of the five whi genes
increased the level of grey pigmentation.
All strains in which the wild-type phenotype was restored,
either partially or completely, by whiA, whiB, whiG, whiH, or
whiJ were excluded from further analysis. This left 31 whi
mutants.
Complementation and phenotypes of novel whi mutants.
Attempts were made to complement 19 of the remaining 31
whi mutants. An SCP2* library of chromosomal DNA isolated
from wild-type S. coelicolor was constructed in the auxotrophic
strain J1501. Four thousand clones from this library were arrayed on master plates and mated with each mutant in turn by
replica plating, and transconjugants were isolated by a second
round of replica plating to a selective medium as described in
Materials and Methods. Potentially complementing clones
were identified initially by looking for grey patches and subsequently by inspection of coverslip impressions in a phase-contrast microscope. In each case, we confirmed plasmid linkage
of the phenotype by reintroduction of the cloned DNA into the
corresponding whi mutant(s), either by protoplast transformation using the primary SCP2* clone itself or by mating from E.
coli, having first subcloned the insert from SCP2* into the
intergeneric, conjugative vector pSET152, which integrates site
specifically into the S. coelicolor chromosome at the phage
␾C31 attB site (4). In all, complementing clones were found for
12 of the 19 strains, and the mutations were allocated to five
loci, according to their complementation groups. A summary
of information on these 12 strains is given in Table 1.
whiK. One clone, pIJ6700, complemented three of the new
whi mutants, R273, R318, and R655. Microscopic examination
showed that the plasmid restored wild-type levels of sporula-
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cut with BglII. The ligation mix was introduced into the histidine and uracil
auxotroph S. coelicolor J1501 by protoplast transformation, and 4,000 of the
resulting transformants were arrayed on master plates (MM containing histidine,
uracil, and 50 ␮g of hygromycin per ml), with 100 patches per plate.
For preparation of an inoculum for mating, developmental mutants were
grown on cellophane discs on MM agar, scraped into 3 ml of 10.3% (wt/vol)
sucrose, and homogenized in a manual glass homogenizer. This suspension was
diluted with 10.3% (wt/vol) sucrose to 9 ml of final volume, and 200 ␮l was
spread and dried on each mating plate (MM containing histidine and uracil).
Genomic library master plates were replicated onto the mating plates, and after
incubation to allow mating and growth, exconjugants of the developmental mutant carrying the plasmid were selected by replica plating to MM lacking auxotrophic supplements but containing 50 ␮g of hygromycin per ml.
Complementation of mutants with known whi genes. Self-transmissible
SCP2*-derived plasmids carrying whiA (pIJ6204 [31]), whiB (pIJ2157 [13]), whiG
(pIJ597 [31]), whiH (pIJ6201 [32]), or whiJ (pIJ6205 [31]) were transferred by
conjugation from S. coelicolor J1501 into new whi mutants by replica plating and
subsequent counterselection of the auxotrophic donor strain as described for the
genomic library.
Conjugation from Escherichia coli into Streptomyces developmental mutants.
Because many whi mutants do not sporulate, the method of Flett et al. (16) was
adapted to promote conjugal transfer from E. coli into Streptomyces by using
mycelial fragments, rather than spores, as an inoculum; high frequencies of
exconjugants were obtained without difficulty. pSET152 and its derivatives were
introduced by transformation or electroporation into the dam dcm hsdS E. coli
strain ET12567 containing the RK2 derivative pUZ8002 (35). pUZ8002 supplies
transfer functions to oriT-carrying plasmids, such as pSET152, but is not efficiently transferred itself because of a mutation in its own oriT. E. coli containing
pSET152 or its derivatives was grown in L broth to an A600 of 0.4, washed twice
with an equal volume of fresh medium, and resuspended in 1/10 the volume of
L broth. Four-day-old lawns of the whi mutants, grown on MS agar, were
harvested by pipetting 3 to 4 ml of 20% (vol/vol) glycerol onto the surface and
gently dislodging the aerial mycelium with a sterile loop. The resulting suspension of mycelial fragments was vortexed for 1 min, and 0.5 ml was mixed with 0.5
ml of washed E. coli cells. After harvesting by centrifugation, the pellet, containing Streptomyces mycelial fragments and E. coli cells, was resuspended in the
residual medium and plated on MS agar containing 10 mM MgCl2. Following
incubation for 16 to 20 h at 30°C, each plate was overlaid with 1 ml of water
containing 0.5 mg of nalidixic acid (to kill E. coli) and 1 mg of apramycin (to
select Streptomyces exconjugants).
Physical mapping of cloned DNA. Inserts from complementing clones were gel
isolated, 32P radiolabelled by random priming, and used to probe Qiabrane
membranes on which the entire minimal, ordered cosmid library of Redenbach
et al. (30) had been arrayed.
Genetic mapping of whiL mutants. A suspension of mycelial fragments was
prepared from each of the mutants R214, R349, and R491 in the same way as the
inoculum for the library matings. Each suspension was mixed with spores of S.
coelicolor 1258 and plated on appropriately supplemented MM. After incubation
to allow mating and growth, serial dilutions of harvested mycelium and spores
were plated on MM containing uracil, proline, arginine, cystine, and 10 ␮g of
streptomycin per ml, selecting for His⫹ streptomycin-resistant recombinants.
One hundred recombinants from each cross were arrayed on master plates of the
same medium, incubated for several days, and then replicated to MM lacking one
of the auxotrophic supplements (uracil, proline, arginine, or cystine). Recombinants were scored for the whiL phenotype by phase-contrast microscopy and for
growth on each medium.
Scanning electron microscopy. For scanning electron microscopy, colonies
were mounted on the surface of an aluminum stub with O.C.T. compound (BDH
Laboratory Supplies, Poole, United Kingdom), plunged into liquid nitrogen slush
at approximately ⫺210°C to cryopreserve the material, and transferred to the
cryostage of a CT1500HF cryotransfer system (Oxford Instruments, Oxford,
United Kingdom) attached to a Philips XL30 FEG scanning electron microscope
(Philips Electron Optics, FEI UK Ltd., Cambridge, United Kingdom). Surface
frost was sublimated at ⫺95°C for 3 min before sputter coating the sample with
platinum for 2 min at 10 mA, at below ⫺110°C. Finally, the sample was moved
onto the cryostage in the main chamber of the microscope, held at approximately
⫺140°C, and viewed at 1.2 to 5.0kV. Photographs were taken with Ilford FP4 120
roll film in a Linhof camera.
J. BACTERIOL.
NEW SPORULATION LOCI IN S. COELICOLOR
VOL. 181, 1999
5421
TABLE 1. Characteristics of the novel S. coelicolor whi mutants
Cosmid
Allele
Aerial surface
color
whiK
D63
whiL
4A7
whiM
I51
R273
R318
R655
R139
R214
R349
R491
R514
R432
White
White
White
Pale grey
Very pale grey
Medium grey
Pale grey
Pale grey
White
whiN
E68
R112
White
whiO
6G4
R650
R589
Medium grey
Medium grey
Phenotype
Long tight coils but with septation
Long tight coils but with septation
Undifferentiated aerial hyphae with very rare chains of curved spores
Long curved and straight spores
Long curved and straight spores
Short coils and long and short fragments
Long curved and straight spores
Long curved and straight spores
Undifferentiated hyphae with occasional spore chains of normal appearance; aerial hyphae
lyse rapidly
Undifferentiated hyphae with rare spore chains sometimes showing irregular septum
placement
Slightly oligosporogenous; spores longer than those of the wild type
Undifferentiated hyphae with frequent spore chains of normal appearance
tion to all three strains. Hybridization, using the insert from
pIJ6700 as a probe, localized the complementing DNA to the
overlap between cosmids 6G4 and D63, in the 7 o’clock region
of the chromosome (Fig. 1). whiD also maps to this overlap
(27, 30), but further analysis showed that whiD did not complement R273, R318, or R655 and that whiK did not complement the whiD strain J243 (26). Accordingly, the new locus was
designated whiK.
The phenotypes of R273 and R318 were indistinguishable.
Both mutants produced white colonies with long, tightly coiled
aerial hyphae with frequent septation (Fig. 2B). In contrast,
the third whiK mutant, R655, showed a more severe phenotype; although very occasional short chains of spores were
detected, R655 produced, almost entirely, straight, undifferentiated aerial hyphae (Fig. 2C).
FIG. 1. Locations of developmental genes on the combined physical and
genetic map of S. coelicolor. Positions of the five new whi loci are shown on the
outside of the circle, and positions of previously identified developmental genes
are shown on the inside (adapted from reference 30). Sizes of the AseI fragments
are in kilobases. }, oriC; F, telomeres.
whiL. Three clones, pIJ6701, pIJ6702, and pIJ6703, complemented five of the new whi mutants, R139, R214, R349, R491,
and R514. Southern hybridization showed that these three
clones overlapped and that they mapped to the unique region
of cosmid 4A7, in the 10 o’clock region of the chromosome
(Fig. 1). This locus was designated whiL.
There was significant phenotypic variation among the whiL
mutants. R139, R491, and R514 had pale grey colonies that
produced long, curved and straight spores (Fig. 2D). R214 was
morphologically indistinguishable from these three strains but
produced less spore pigment. The most striking phenotype
among the whiL mutants was that of R349. R349 produced
medium grey colonies with tightly coiled aerial hyphae that had
septa at long intervals, giving rise to corkscrew-like fragments
of irregular length, often several times longer than a wild-type
prespore compartment (Fig. 2E). Although the cloned DNA
restored the wild-type phenotype to four of the whiL strains, it
did not completely complement R349; introduction of the
cloned DNA restored wild-type levels of colony pigmentation
and regular septation, but the spores produced were still noticeably longer than those of the wild type (Fig. 2F). One
possible explanation for this observation is that R349 carries a
second mutation that contributes to its morphological phenotype, in addition to the mutation in whiL.
The variation in the phenotypes of the strains apparently
complemented by whiL, and the fact that whiL clones did not
restore a full wild-type phenotype to R349 raised the possibility
that not all of these strains carried allelic mutations and that
some of the effects observed might represent suppression. In
an attempt to address this question, we determined approximate genetic map locations for the whi mutations carried by
R214, R349, and R491, three strains with distinguishable phenotypes, to see if the genetic map locations of these mutations
were compatible with the physical map location of the complementing DNA. In all three cases, the mutation mapped
between proA and hisC but closer to proA (e.g., Fig. 3). proA
has been mapped physically to the unique region of cosmid
C123 (30). Cosmid 4A7, which carries whiL, lies between proA
and hisC, 10 cosmids distant from proA and 18 cosmids distant
from hisC. The approximate genetic map locations of the mutations in R214, R349, and R491 are therefore consistent with
the physical map location of the DNA that complements these
strains.
whiM. One clone, pIJ6704, complemented R432, and hybridization showed that the cloned DNA mapped to the unique
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Locus
5422
RYDING ET AL.
J. BACTERIOL.
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FIG. 2. Scanning electron micrographs of the aerial hyphae of the morphologically wild-type strain M145 (A), the whiK mutant R273 (B), the whiK mutant R655
(C), the whiL mutant R491 (D), the whiL mutant R349 (E), the whiL mutant R349 carrying pIJ6710 (a derivative of the vector pSET152 carrying the whole whiL insert
derived from the original SCP2* clone pIJ6702 [3]) (F), the whiM mutant R432 (G), the whiN mutant R112 (H), and the whiO mutant R589 (I). All strains were grown
on MM containing 0.5% (wt/vol) mannitol. Scale bars are shown in each panel.
VOL. 181, 1999
NEW SPORULATION LOCI IN S. COELICOLOR
5423
region of cosmid I51, in the 12 o’clock region of the chromosome (Fig. 1). This locus was designated whiM.
R432 colonies were white and produced undifferentiated
aerial hyphae with occasional spore chains of normal appearance (Fig. 2G), a phenotype similar to that of whiJ mutants.
However, unlike those of whiJ mutants, the aerial hyphae of
R432 lysed very rapidly (data not shown), and presumably as a
consequence, the strain showed very poor viability on prolonged incubation on plates in comparison to the other whi
mutants examined.
whiN. Two clones, pIJ6705 and pIJ6706, complemented
R112 and R650. Both clones restored wild-type levels of sporulation. Southern hybridization showed that these clones overlapped and that they mapped to the unique region of cosmid
E68, in the 9 o’clock region of the chromosome (Fig. 1). This
locus was designated whiN.
The morphological phenotypes of the two whiN mutants
were different. On MM, colonies of R650 were pale to medium
grey and produced frequent spores that were longer than those
of the wild type. In contrast, colonies of R112 were white and
produced long, straight, undifferentiated hyphae, although occasional spore chains, sometimes showing highly irregular septum placement, were observed (Fig. 2H). The R112 mutation
also showed clear signs of pleiotropic effects; in addition to the
defects in sporulation, on some media, such as MM, R112
produced significantly less aerial mycelium than the parental
strain M145. In this respect, R112 does not fit the classical
definition of a whi mutant, which should be solely defective in
sporulation (6). All aspects of the pleiotropic phenotype of
R112 were fully complemented by both clones.
whiO. One clone, pIJ6707, complemented R589. Southern
hybridization showed that the cloned DNA mapped to the
unique region of cosmid 6G4 in the 7 o’clock region of the
chromosome (Fig. 1). whiD and whiK map very close by, in the
overlap between cosmids 6G4 and D63, but further Southern
analysis confirmed that the insert from pIJ6707 did not overlap
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FIG. 3. Genetic mapping of the whiL mutant R214. R214 (inner circle) was
crossed with strain 1258 (outer circle), and hisC⫹ and strA1 (indicated by solid
triangles) were selected. Numbers in the diagram are the frequencies of alleles
among the recombinant progeny. The segregation of the whi mutation with
respect to proA1 is tabulated below. Of the three whiL mutants analyzed, R214
showed the largest number of Pro⫹ grey (multiple-crossover class) recombinants.
The crosses with R349 and R491 gave 3 and 0 such recombinants, respectively.
5424
RYDING ET AL.
either of these genes. Therefore, this locus was designated
whiO.
R589 colonies were medium grey in color and had an oligosporogenous phenotype; by scanning electron microscopy,
some undifferentiated aerial hyphae, but with fairly frequent
spore chains of normal appearance, were observed (Fig. 2I).
DISCUSSION
alternative strategy to complement the remaining strains would
be to derive an approximate genetic map position for each
mutation and then to use the minimal, ordered cosmid library
of Redenbach et al. (30) to “walk” across the corresponding
interval of the combined physical and genetic map. Although
these cosmids cannot replicate autonomously in S. coelicolor,
selection for kanamycin resistance after protoplast transformation results in the recovery of isolates in which the cosmid has
integrated into the chromosome via insert-directed homologous recombination. The cosmids can therefore be used to
clone genes by complementation (30), an approach that has
been used to isolate whiD (27, 28), whiI (1), and bldC (20).
The new screen showed a wide variation in the number of
mutations isolated at each whi locus (whiA, 6; whiB, 15; whiG,
19; whiH, 16; whiJ, 3; whiK, 3; whiL, 5; whiM, 1; whiN, 2; whiO,
1), as was found in the previous screen (6). Part of this disparity
can be explained by the fact that some of the mutants (particularly whiA, whiB, and whiG) yield colonies that are snowy
white and have a raised aerial mycelium, but others (especially
some of those representing the new loci described here) produce some spore pigment and so were less likely to be picked
out from a field of colonies.
Of the 115 mutants that were checked for complementation
by clones of whiA, whiB, whiG, whiH, and whiJ carried on
self-transmissible plasmids, 59 showed full complementation
by one of the clones, 25 showed partial complementation by
one or more of the clones, and 31 strains were unaffected,
although many of the strains that were fully complemented by
one of the existing clones were also partially complemented by
another. The 25 mutants that were only partially complemented by one of the five clones could, in principle, represent
partially dominant alleles that do not allow full restoration of
the wild-type phenotype, but it seems much more likely that
they contain mutations in genes other than whiA, whiB, whiG,
whiH, and whiJ which are partially suppressed by the introduction of the cloned gene on the SCP2* low-copy-number plasmid. In cases where more than one of these five genes were
able partially to restore the wild-type phenotype, there can be
no doubt that suppression is involved. Suppression effects have
not previously been reported in developmental work in Streptomyces, although the ability of an additional copy of whiG
partially to suppress the spore pigment defect of whiH mutants
without affecting their morphological phenotype has recently
been noted (15). Suppression effects caused by additional copies of genes have been both informative and problematic in the
analysis of the regulation of antibiotic biosynthesis in Streptomyces (2).
In the first genetic screen for whi loci, most of the mutants
assigned to a given locus were similar in phenotype (6), as
might be expected if most of the alleles were null or close to
null. The one notable exception was a mild allele of whiG that
caused the formation of long spores, rather than the more
characteristic block in sporulation septum formation, and indeed this mutant was originally designated whiF because of this
wide phenotypic difference (5, 6, 31). In contrast, in the work
described here, there was wide phenotypic variation within
each of the three loci represented by multiple alleles, whiK,
whiL, and whiN. This again raised the possibility that some of
these effects might represent suppression rather than true
complementation (although the three clones complementing
whiL mutants overlapped, as did the two clones complementing whiN mutants). However, in genetic mapping experiments
using three whiL strains each having a distinct phenotype
(R214, R349, and R491), the genetic map locations of the
three mutations were consistent with the physical map location
of the complementing DNA.
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The poor representation of several loci among the original
collection of 50 whi mutants analyzed suggested that the genetic map of whi loci was not saturated (6, 31), a conclusion
borne out by the results presented here. The primary criterion
for the selection of sporulation mutants in the new screen
remained the loss of production of the grey spore pigment,
because, in contrast to the endospores of Bacillus and Clostridium, the exospores of S. coelicolor are not particularly resistant
to high temperatures or chemical attack, and screens for sensitivity to these kinds of stresses therefore could not be used.
However, we recognized that some developmental mutants
could have severe morphological defects but be only partially
blocked in production of spore pigment (as is true for whiH
and whiJ mutants), and so during the selection process we
attempted to include all mutants in which spore pigmentation
was reduced to a noticeable extent. Two important variables
between the first and second screens whose influence cannot
readily be assessed are that (i) in the first screen (18) a rich
medium was used, whereas in this screen MM was used
throughout for the assessment of phenotype; and (ii) some of
the mutations in the earlier screen were induced by UV light
(18), whereas all the mutations in this screen were induced by
NTG. Here, genetic mapping was not relied on heavily as a
means of determining the positions of mutations, because the
library-based complementation required less time and effort
and ultimately led to the physical mapping of the new loci. Like
the old whi loci, the new loci are located in the 6-Mb central
region of the linear S. coelicolor chromosome where virtually
all genes identified by classical genetics have mapped (Fig. 1),
in contrast to the two ⬃1-Mb regions at the chromosome ends
that are almost devoid of classical markers (30).
From the 770 mutants that were streaked for single colonies,
431 were stable and could be distinguished from the wild type
by spore pigmentation, and the vast majority of these also
showed morphological defects. As in the first whi mutant analysis (6), emphasis was placed on mutants showing clear morphological defects that could easily be distinguished from the
wild type by phase-contrast microscopy, thereby making the
scoring of complementation relatively straightforward. However, this left as subjects for further study a large number of
more subtle mutants that showed variation in spore size or
shape. This group contained strains that showed phenotypes
similar to those of sigF (29) and whiD (6) and could be used to
expand the number of genes known to be involved in the later
stages of spore development. Both whiL (Fig. 2D and E) and
whiN (Fig. 2H) mutants showed unusual intervals between
sporulation septa. The control of septal placement in bacteria
is mostly studied in rod-shaped unicellular bacteria, but further
studies of these Streptomyces mutants may provide distinctive
and novel insights into this process.
Of the 19 whi strains that received the library, 12 were
complemented. The 12 mutants were grouped by complementation into five loci, named whiK to whiO. The failure to complement the other seven strains, and the small numbers of
complementing clones isolated for each of the new loci (whiK,
one; whiL, three; whiM, one; whiN, two; and whiO, one) indicated that the library was not fully representative. An attractive
J. BACTERIOL.
NEW SPORULATION LOCI IN S. COELICOLOR
VOL. 181, 1999
The approach taken here has enabled us simultaneously to
identify, map, and clone five new whi loci and should facilitate
further rapid progress. Our prospects in these endeavors will
undoubtedly be enhanced by the current S. coelicolor genome
sequencing project (33).
ACKNOWLEDGMENTS
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Downloaded from http://jb.asm.org/ on April 27, 2014 by PENN STATE UNIV
We thank Gabriella Kelemen, David Hopwood, and Mervyn Bibb
for helpful comments on the manuscript, Sue Bunnewell for handprinting the scanning electron micrographs, and Tobias Kieser for help
in preparing Fig. 1.
This work was funded by BBSRC grants CAD 04380 (to K.F.C.) and
83/P07658 (to M. J. Buttner), by a Lister Institute research fellowship
(to M. J. Buttner), by a John Innes Foundation studentship (to V.M.),
and by a grant-in-aid to the John Innes Centre from the BBSRC.
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