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Journal of Bacteriology, March 2000, p. 1286-1295, Vol. 182, No. 5
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
WhiD and WhiB, Homologous Proteins Required for
Different Stages of Sporulation in Streptomyces
coelicolor A3(2)
Virginie
Molle,1,*
Wendy J.
Palframan,1
Kim C.
Findlay,2 and
Mark J.
Buttner1
Departments of Molecular
Microbiology1 and Cell
Biology,2 John Innes Centre, Colney, Norwich
NR4 7UH, United Kingdom
Received 4 October 1999/Accepted 5 December 1999
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ABSTRACT |
The whiD locus, which is required for the
differentiation of Streptomyces coelicolor aerial hyphae
into mature spore chains, was localized by map-based cloning to the
overlap between cosmids 6G4 and D63 of the minimal ordered library of
Redenbach et al. (M. Redenbach et al., Mol. Microbiol. 21:77-96,
1996). Subcloning and sequencing showed that whiD encodes a
homologue of WhiB, a protein required for the initiation of sporulation
septation in S. coelicolor. WhiD and WhiB belong to a
growing family of small (76- to 112-residue) proteins of unknown
biochemical function in which four cysteines are absolutely conserved;
all known members of this family are found in the actinomycetes. A
constructed whiD null mutant showed reduced levels of
sporulation, and those spores that did form were heat sensitive, lysed
extensively, and were highly irregular in size, arising at least in
part from irregularity in septum placement. The whiD null
mutant showed extreme variation in spore cell wall deposition; most
spores had uniformly thin (20- to 30-nm) walls, but spore chains were
frequently observed in which there was irregular but very pronounced
(up to 170 nm) cell wall thickening at the junctions between spores.
whiD null mutant spores were frequently partitioned into
irregular smaller units through the deposition of additional septa,
which were often laid down in several different planes, very close to
the spore poles. These "minicompartments" appeared to be devoid of
chromosomal DNA. Two whiD promoters, whiDp1 and
whiDp2, were identified, and their activities were analyzed
during development of wild-type S. coelicolor on solid
medium. Both promoters were developmentally regulated;
whiDp1 and whiDp2 transcripts were detected
transiently, approximately at the time when sporulation septa were
observed in the aerial hyphae.
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INTRODUCTION |
Streptomycetes are gram-positive
soil bacteria with a mycelial growth habit (7). Germination
of spores gives rise to a vegetative mycelium consisting of a branching
network of multigenomic hyphae. In order to disperse themselves,
streptomycetes develop specialized aerial hyphae that grow out of the
aqueous environment of the vegetative mycelium into the air, giving the
colonies a characteristic fuzzy appearance (23). These
aerial hyphae differentiate into chains of exospores, a process that
begins with the synchronous deposition of 50 or more sporulation septa
at ~1 µm intervals at the tips of the hyphae (7). The
formation of sporulation septa creates, for the first time in the
developing colony, unigenomic cells, referred to as prespores. These
cylindrical prespore compartments subsequently mature to form chains of
thick-walled, ovoid spores, during which time the colonies develop a
characteristic color, gray in the case of the model species
Streptomyces coelicolor A3(2), due to the synthesis of a
polyketide spore pigment (11).
Hopwood et al. (19) identified sporulation-deficient mutants
of S. coelicolor by virtue of their inability to synthesize the gray spore pigment, thereby remaining white, even on prolonged incubation on plates. Fifty of these white (whi) mutants
have been mapped genetically, and current information suggests that they represent eight separate loci: whiA, whiB,
whiD, whiE, whiG, whiH,
whiI, and whiJ (6, 8, 38). Mutations
in six of these loci, referred to as the early whi genes
(whiA, whiB, whiG, whiH, whiI, and whiJ), essentially abolish the
formation of sporulation septa (1, 6, 8, 9, 12, 13, 38, 39).
whiE specifies the spore pigment itself, and whiE
mutants do not appear to be morphologically defective (6, 11, 26,
32). Although other loci required for sporulation have
subsequently been described in S. coelicolor (31, 35,
40), some of which affect the late stages of sporulation, of the
originally described whi strains only the whiD
mutant formed sporulation septa but failed to go on to produce mature
spores. The whiD mutant formed spores at wild-type abundance
but these were unpigmented, were thin-walled, and showed frequent lysis
(6, 32). Here we describe the map-based cloning of
whiD, show that WhiD is a member of a growing family of
actinomycete proteins of unknown biochemical function that includes
WhiB (in which four cysteine residues are absolutely conserved),
describe the phenotype of a constructed whiD null mutant,
and show that whiD is developmentally regulated at the level
of transcription.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, growth conditions, and protoplast
transformation.
S. coelicolor strains used are summarized in
Table 1 and were cultured on R5
(20), MM (20) containing 0.5% (wt/vol) mannitol as the carbon source, or MS agar (mannitol plus soya flour)
(18), supplemented with uracil and histidine where
necessary. For transformation of S. coelicolor, strains were
cultured in YEME liquid medium (20) and protoplasts were
prepared and transformed as described previously (20). To
bypass the methyl-group specific restriction system of S. coelicolor during protoplast transformation, unmethylated plasmid
and cosmid DNA was isolated from the dam dcm hsdS E. coli strain ET12567 (30). To stimulate integration by homologous recombination when using cosmid or plasmid replicons that do not replicate in Streptomyces, double-stranded DNA was alkaline
denatured before protoplast transformation (33). The vectors
used were pDH5 (17), pKC1132 (4), and pSET152
(4).
Conjugation from E. coli into
Streptomyces.
Because whiD mutant spores were
found to be temperature sensitive (see Results), the method of Flett et
al. (14) was adapted to avoid heat shocking; high
frequencies of exconjugants were obtained without difficulty. pSET152,
pKC1132, or their derivatives were introduced by transformation into
E. coli ET12567 containing the RK2 derivative pUZ8002
(34). pUZ8002 supplies transfer functions to
oriT-carrying plasmids, such as pSET152 and pKC1132, but is not efficiently transferred itself because of a mutation in its own
oriT. E. coli containing pSET152, pKC1132, or their
derivatives was grown in L broth to A600 of 0.4 to 0.6, washed twice with an equal volume of fresh medium, and
resuspended in 1/10th the volume of L broth. S. coelicolor
J243 was grown on one plate of MS agar and spores were harvested after
5 days and resuspended in 2 ml of 20% (vol/vol) glycerol. Then, 0.5 ml
of fresh spores and 0.5 ml of fresh E. coli suspension were
mixed and spread on one plate of MS agar containing 10 mM
MgCl2. After incubation for 16 to 20 h at 30°C,
exconjugants were selected by overlaying the plate 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).
Construction of a whiD null mutant.
A 1.3-kb
BamHI-SphI fragment carrying whiD was
cloned into pUC19 digested with BamHI and SphI,
and a whiD null mutant allele was created by blunt-end
cloning a 1.8-kb hyg cassette (46) into the
StyI site internal to whiD (see Fig. 3A and 4).
Additional flanking sequences were added to this plasmid by inserting a
2-kb SphI-HindIII fragment upstream of
whiD and a 3-kb BamHI-KpnI fragment downstream to create pIJ6628. This reconstructed a contiguous 6.3-kb
segment of the chromosome but carrying the 1.8-kb hyg
insertion in whiD. The entire 8.1-kb insert was removed from
pIJ6628 as a PvuII fragment and cloned into the
EcoRV site of pKC1132. Finally, a 1.3-kb BglII
fragment carrying the counterselectable glkA gene was
ligated into the unique BglII site in the vector to create pIJ6629.
pIJ6629 was introduced into
S. coelicolor J1915
(
glkA119) by mating from
E. coli, and
exconjugants in which the plasmid had
presumptively integrated at the
whiD locus by single-crossover
homologous recombination were
selected with apramycin. After one
round of nonselective growth,
putative
whiD::
hyg null mutants
in
which the delivery plasmid had been lost were selected on MM
containing
100 mM 2-deoxyglucose and 50 µg of hygromycin per
ml.
RNA isolation and S1 nuclease protection analysis.
The
developmental time course of RNA samples described by Kelemen et al.
(25) was used to analyze expression of whiD. The whiD promoter region was mapped with a 0.6-kb probe
generated by PCR from pIJ6626, using a 5'-end-labeled oligonucleotide
primer internal to whiD (5'-CCAGGAGCTGCCAGTCCCAC-3')
and the universal sequencing primer. Oligonucleotide primers were
labelled and S1 nuclease protection assays were set up as described
earlier (25).
Microscopy.
For light microscopy, coverslips were placed
gently on the surface of S. coelicolor colonies grown on
solid medium for 4 days and then lifted and analyzed by phase-contrast
light microscopy with oil immersion (Carl Zeiss; 100/2.0 objective).
For transmission electron microscopy, colonies were fixed in 2%
(vol/vol) glutaraldehyde in 0.05 M sodium cacodylate (
16),
stained with osmic acid, and embedded in LR White resin according
to
the manufacturer's instructions (The London Resin Co.). Sections
were
cut on an Ultracut Microtome (Reichart) and examined in a
JEOL 1200 EX
electron
microscope.
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 cryo-transfer system
(Oxford
Instruments, Oxford, United Kingdom), attached to a Phillips
XL30 FEG
scanning electron microscope (Phillips 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 colder than
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 3 kV. Photographs
were taken using Ilford FP4
120 roll film in a Linhof
camera.
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RESULTS |
Localization of the whiD gene on the physical map of
the S. coelicolor chromosome.
Redenbach et al.
(36) constructed a minimal, ordered cosmid library covering
the S. coelicolor chromosome by using the E. coli
vector Supercos-1. 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 (36), and we used this approach to
isolate whiD.
Chater (
6) mapped
whiD genetically to a position
between
strA and
uvsB, in the "7-o'clock"
region of the chromosome, and
while
strA has been mapped
physically (to the overlap between
cosmids D31 and D74
[
36]),
uvsB has not. However,
hisD, the
next locus counterclockwise of
uvsB,
has also been mapped physically
(to the overlap between cosmids 6F2 and
7E4 [
36]). Therefore,
we reasoned that
whiD
must reside on one or two of the twenty
overlapping cosmids covering
the
strA-hisD interval (Fig.
1).
Each of these cosmids was passaged
through the methylation-deficient
E. coli strain ET12567 and
introduced individually into the
whiD strain J243 by
protoplast transformation. Transformants were patched
onto MS agar, and
potentially complementing cosmids were identified
initially by
restoration of spore pigment production and confirmed
by phase-contrast
examination of spore morphology. Only the overlapping
cosmids 6G4 and
D63 complemented the
whiD phenotype of J243; a
small
percentage of transformants carrying D63 remained white,
presumably due
to cosmid instability or gene conversion.
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FIG. 1.
A simplified version of the combined physical and
genetic map of the S. coelicolor chromosome in the
7-o'clock region, showing the locations of developmental genes and the
21 ordered cosmids that span the strA to hisD
interval (adapted from references 36 and
40). The location of whiD is shown on the
overlap between cosmids 6G4 and D63, as determined by complementation.
The locations of other cloned developmental genes on the cosmid contig
are shown on the inside of the circle. The cosmids and their overlaps
are arbitrarily shown to be of equal length (the average insert size of
the cosmids is 37.5 kb and of the overlaps 12.5 kb
[36]). The sizes of the AseI fragments are
given in kilobases (27). Symbols:  , oriC;  ,
T3 end;  , T7 end.
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Construction of a whiD-whiD+ congenic
pair.
Because the original whiD mutant, C16, has been
lost, and J243 arose from a cross used in the original genetic mapping
of whiD, there existed no whiD-whiD+
congenic pair to allow valid phenotypic comparison. To construct such a
pair, we took advantage of the slight instability of J243 carrying
cosmid 6G4 or D63 in the absence of selection. Such strains carry
tandem duplications of approximately 40 kb of DNA, and we found that
they gave rise to kanamycin-sensitive (Kans) colonies at
readily detectable frequencies, presumptively due to the excision of
the cosmid by homologous recombination. A gray 6G4 transformant of J243
was cultured nonselectively through two rounds of growth and
sporulation, plated for single colonies on MS agar, and replica plated
to identify Kans derivatives. Both gray and white
Kans derivatives were identified, presumably reflecting the
nature of the remaining whiD allele; gray colonies retained
the wild-type allele originally present on the cosmid, while white
colonies retained the mutant allele from J243. The structure of the
whiD region of the chromosome of a representative gray
colony was confirmed by Southern blot analysis, and this strain was
designated J1942. It subsequently turned out that, although the insert
in cosmid 6G4 is approximately 41 kb in size, whiD lies only
1 kb from the end of the insert (see below), making it relatively
unlikely that homologous recombination would occur at a significant
frequency in the short interval. It therefore seems likely that gene
conversion, in which the wild-type allele acted as a template for
repair synthesis of the whiD16 allele, might have occurred
in the generation of J1942 and the other gray Kans
derivatives of J243/6G4.
whiD spores are heat sensitive.
Our initial
attempts to introduce plasmids into J243 from E. coli by
mating yielded extremely low frequencies of exconjugants. Preliminary
control experiments designed to determine the reason for these low
frequencies suggested that whiD spores might be less able to
survive the 10-min 50°C heat shock used to induce germination in our
standard mating protocol (14). To investigate this
possibility, spores of J243 and the congenic
whiD+ strain J1942 were incubated in 2xYT or
20% (vol/vol) glycerol at 50°C for various times, and the spore
survival was assessed by plating serial dilutions on MS agar containing
uracil. Whereas spore viability in J1942 (whiD+)
was virtually unaffected by a 30-min incubation at 50°C, J243 (whiD) spore viability dropped by a factor of
108 in the same period (Fig.
2A). This loss of viability was not simply an osmotic effect, since spores of J243 (whiD) showed
no loss of viability when incubated in 2xYT or 20% (vol/vol) glycerol at 25°C (data not shown).
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FIG. 2.
Heat inactivation curves for spores of J243
(whiD) and J1942 (whiD+) (A) and
spores of J1979 (sigF) and J1508
(sigF+) (B). Spores were incubated in 20%
(vol/vol) glycerol at 50°C.
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One possible explanation for the heat sensitivity of
whiD
spores is that they fail to develop the thick spore wall characteristic
of the wild type (
32). To investigate this possibility, we
examined
the heat sensitivity of another strain that has thin-walled
spores.
sigF encodes a spore-specific RNA polymerase sigma
factor,
F, that is required for spore maturation and,
like
whiD mutants,
sigF mutants are defective in
spore wall thickening (
25,
35,
44). However, when spores of
the
sigF null mutant J1979 were
subjected to the same heat
treatment as those of
whiD, we found
that they were as heat
resistant as those of the congenic
sigF+ parent
strain, J1508 (Fig.
2B).
Subcloning of whiD from cosmid 6G4.
Cosmid 6G4 was
digested with BamHI, generating seven fragments ranging in
size from 2 to 16 kb. Each fragment was subcloned into the E. coli vector pDH5, introduced into J243, and thiostrepton-resistant transformants were selected in which the plasmid had presumptively integrated into the chromosome via insert-directed homologous recombination. Only the largest BamHI fragment from cosmid
6G4, fragment I, complemented J243. Further analysis showed that
fragment I comprised the entire Supercos-1 vector with 1.8 kb of
Streptomyces DNA from the "T3 end" of the 6G4 insert and
7 kb of DNA from the "T7 end." Self-ligation of fragment I (in the
absence of pDH5) gave rise to pIJ5900, which also complemented J243.
Because the T3 end and not the T7 end of cosmid 6G4 overlaps D63
(36), the other cosmid that complemented the whiD
mutant, we reasoned that the 1.8-kb fragment of DNA must contain
whiD, and this was confirmed by deleting the 7-kb fragment
from the T7 end of pIJ5900 to create pIJ5908, which also complemented J243.
Because the
whiD mutant formed spore chains, albeit of a
somewhat irregular nature, it was not trivial to judge complementation
of J243 in the phase-contrast microscope. The discovery that
whiD spores were heat sensitive gave us a useful additional
phenotype
by which to assess complementation of the mutant. Exploiting
this
discovery, we went back to J243 carrying cosmid D63 or cosmid
6G4
and through all the successive rounds of subcloning of
whiD to assess complementation by the heat sensitivity of the spores.
In
every case, the fragments that complemented the morphological
defects
of J243 also restored wild-type levels of heat resistance
to the
spores. Apart from its utility in scoring complementation,
these
results provided strong evidence that both phenotypes arise
from the
same
mutation.
whiD is a member of a family of genes that includes
whiB.
The nucleotide sequence of the 1.8-kb fragment
containing whiD was determined, and protein-coding sequences
were predicted with the aid of the FRAME program (3). One
incomplete (orf1) and two complete potential protein coding
sequences (orf2 and orf3) were identified (Fig.
3A). orf2 and orf3
were each subcloned into the vector pSET152, which integrates site
specifically into the S. coelicolor chromosome at the phage
C31 attB site (4), to create pIJ6627 and
pIJ6602, respectively (Fig. 3A), and these two plasmids were introduced
into J243 by mating from E. coli. orf3 had no effect on the
whiD phenotype of J243, whereas orf2 fully
complemented both the morphological defects and the spore temperature
sensitivity of the strain. As a consequence, orf2 was
designated whiD. To confirm that a significant base change was indeed present in whiD in J243, the entire 1.8-kb region
was amplified from J243 by PCR and sequenced; a single nucleotide difference from the wild type was identified. whiD16 has a
CG to TA transition (at position 700 in database submission AJ010601), giving rise to an alanine to threonine substitution at position 21 in
the primary amino acid sequence of WhiD (Fig. 3B). In addition, the
whiD allele of J1942, the morphologically wild-type strain derived from J243/6G4 by homogenotisation, was amplified by PCR and
shown by sequencing to have lost the whiD16 mutation.
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FIG. 3.
(A) Genetic organization of the 1.8-kb segment of DNA
containing whiD. The positions of the three protein-coding
regions are indicated by arrows, and restriction sites referred to in
the text are marked. The extent of the subclones used in
complementation tests is shown below. (B) Alignment of the predicted
amino acid sequence of WhiD with related proteins. The four absolutely
conserved cysteines are marked by asterisks, and the highly conserved
C-terminal motif G(V/I)WGGLSE is underlined. The alanine-to-threonine
substitution arising from the whiD16 mutation is indicated.
The proteins and their corresponding gene accession numbers are as
follows: sco_whid, S. coelicolor WhiD (AJ010601); mle_whib,
M. leprae WhiB (U00015); mtu_whib1, M. tuberculosis WhiB1 (Z95120); mtu_whib2, M. tuberculosis
WhiB2 (AL021840); mtu_whib3, M. tuberculosis WhiB3 (Z77165);
mtu_whib4, M. tuberculosis WhiB4 (AL022121); rop_whib,
Rhodococcus opacus WhiB (AF030176); sau_whib,
Streptomyces aureofaciens WhiB (L22864); sco_whib, S. coelicolor WhiB (X62287); sgr_whib, Streptoverticillium
griseocarneum WhiB (X68708); tm4_gp49, mycobacterial phage TM4
GP49 (AF068845).
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Global similarity searches of the NCBI databases showed that the
incomplete open reading frame,
orf1, encodes the C-terminal
118 residues of a member of the LysR family of transcriptional
regulators, with highest similarity to GltC, involved in the regulation
of glutamate biosynthesis in
Bacillus subtilis. The
predicted
product of
orf3 is 203 amino acids long and is a
typical member
of the two-component response regulator family of
proteins, showing,
for example, 32% identity with DegU, which is
involved in the
control of competence and degradative enzyme
biosynthesis in
B. subtilis (
10).
WhiD is 112 amino acids long and is a member of a family of proteins
that includes WhiB, required for sporulation septum formation
in
S. coelicolor and
Streptomyces aureofaciens
(
12,
28,
29),
six proteins revealed by genome sequencing in
the related actinomycete
pathogen
Mycobacterium
tuberculosis, proteins from two other actinomycetes,
Rhodococcus opacus (
41) and
Streptoverticillium griseocarneum (
42), and one
protein encoded by the mycobacterial phage TM4
(
15). Several
genes encoding WhiD-related proteins are also
present in the unfinished
genome sequence of
Mycobacterium leprae.
An alignment of
some of these proteins, shown in Fig.
3B, reveals
several striking
features. First, all of the proteins are small,
varying from 76 to 112 residues in length. Second, four cysteine
residues are completely
conserved in all the members of the family
and, third, the motif
G(V/I)WGGLSE is highly conserved close to
the C
terminus.
Construction and phenotypic characterization of a whiD
null mutant, J2152.
A whiD null mutant allele was
constructed in vitro by inserting a hygromycin resistance gene
(hyg) at a unique StyI restriction site internal
to whiD (Fig. 3A and 4B). This mutant allele was used to
replace the wild-type allele in J1915, a plasmid-free, glkA
derivative of the wild-type strain, using the method of Buttner et al.
(5). This method makes use of the counterselectable glucose
kinase gene (glkA) which allows a positive selection to be
made for gene replacement, provided that the mutations are constructed
in a strain carrying a deletion of glkA. The genomic structures of five independently isolated whiD mutants were
confirmed by Southern blot analysis (e.g., Fig.
4C), and one was designated J2152.
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FIG. 4.
Construction of a whiD null mutant. (A and B)
Restriction maps of the wild-type and
whiD::hyg null mutant alleles,
respectively; the black arrow represents the whiD gene. (C)
Southern blot analysis of chromosomal DNA from J1915
(whiD+; lanes 1 and 3) and J2152
(whiD::hyg; lanes 2 and 4) digested
with BamHI and HindIII (lanes 1 and 2) or
BamHI and SphI (lanes 3 and 4). The size markers
(lane 5) are the 1-kb ladder (Bethesda Research Laboratories). The
probe used was the 1.3-kb BamHI-SphI
whiD fragment (A).
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On plates, colonies of J2152
(
whiD::
hyg) produced wild-type levels
of aerial mycelium but remained completely white, even
on prolonged
incubation. In the scanning and transmission electron
microscopes, it
was immediately apparent that the phenotype of
J2152
(
whiD::
hyg) was more severe than that
of J243 (
whiD16),
implying that
whiD16 is not a
null allele (consistent with the
relatively conservative substitution
of threonine for alanine
in WhiD16). However, it should be noted that
the
glkA119 allele,
present in the genetic background of
J2152 (
whiD::
hyg) but absent
from that
of J243 (
whiD16), causes ectopic sporulation in the
substrate hyphae (the Esp phenotype) on certain media (
24).
Spore chains in Esp mutants are morphologically normal, and the
available evidence suggests that the
glkA119 deletion
simply
relieves suppression of sporulation in substrate hyphae
(
24).
However, the formal possibility that the
glkA119 genetic background
could potentiate the
whiD null mutant phenotype has not been
excluded.
Although J243 (
whiD16) forms defective spores, the level of
sporulation in this strain is the same as in its congenic
whiD+ parent, J1942. In contrast, based on
visual inspection in the
scanning electron microscope, the level of
sporulation in J2152
(
whiD::
hyg) was
approximately only 25% of that found in its congenic
whiD+ parent, J1915. In addition, there was
considerable irregularity
in spore size (Fig.
5), arising at least in part from
irregularity
in sporulation septum placement (Fig.
6B) but perhaps also from
spore swelling
and lysis (Fig.
6C). Strikingly, we frequently
observed additional,
aberrant sporulation septa within spores,
deposited close to the poles
and often laid down in several planes
(Fig.
6A). These additional septa
further divided the spores into
irregular "minicompartments"
apparently devoid of chromosomal
DNA (Fig.
6A). In young spore chains
these minicompartments were
usually box-like in appearance (Fig.
6A),
but in "mature" spore
chains they rounded and lysed to form ghost
minicompartments analogous
to the larger ghost spores seen in the same
chains (Fig.
6C).
Another striking feature was the extreme variation in
cell wall
deposition between and within spores. In most mature J2152
spores
the walls were uniformly thin (20 to 30 nm, compared to 60 to
80 nm in J1915; Fig.
6D), but spore chains were frequently observed
in
which there was pronounced but irregular cell wall thickening
at the
junctions between spores (Fig.
6B and C). In certain cases
these
deposits were 170-nm thick (Fig.
6B). After 5 days of growth,
the vast
majority of spores that had formed were lysed and often
swollen (Fig.
6C). Thermosensitivity tests were carried out on
J2152 spores and
yielded results very similar to those obtained
with spores from the
whiD point mutant J243 (data not shown).
J2152 was fully
complemented for all aspects of its phenotype
by pIJ6627, the pSET152
derivative carrying
whiD alone.
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FIG. 5.
Scanning electron micrograph showing the variability in
spore size and spore lysis associated with the constructed
whiD null mutant J2152. Colonies were grown on MS agar.
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|
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|
FIG. 6.
Transmission electron micrographs showing young (A),
intermediate (B), and mature (C) spore chains of the constructed
whiD null mutant J2152 and a mature spore chain of the
congenic whiD+ strain J1915 (D). Colonies were
grown on MS agar.
|
|
Transcription of whiD is developmentally
regulated.
High resolution S1 nuclease mapping of the
whiD promoter region was performed by using a PCR-generated
probe and RNA isolated from wild-type S. coelicolor grown
for 72 h on solid medium. Two promoters were identified,
initiating transcription 62 to 63 bp (whiDp1) and 84 to 85 bp (whiDp2) upstream of the whiD ATG start codon
(Fig. 7A and C).
View larger version (56K):
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|
FIG. 7.
Transcriptional analysis of whiD. (A)
High-resolution S1 nuclease mapping of the 5' ends of the
whiDp1 and whiDp2 transcripts. The lane labeled
"S1" represents the DNA fragments protected by RNA initiating from
whiDp1 and whiDp2. The most likely transcription
start points are indicated by the asterisks. Lanes labeled G, C, A, and
T represent a dideoxy sequencing ladder generated by using the same
oligonucleotide that was used to generate the S1 mapping probe. The RNA
used was from the 72-h time point from panel B. (B) S1 nuclease
protection analysis of transcription from whiDp1,
whiDp2, and hrdBp during development. RNA was
isolated from wild-type S. coelicolor grown on cellophane
discs on MM containing mannitol as carbon source. The time points (in
hours) at which mycelium was harvested for RNA isolation, and the
presence of vegetative mycelium, aerial mycelium, and spores as judged
by microscopic examination, are shown. The hrdB panel from
this figure was published previously (25, 26, 39) and is
shown here for comparison with the whiD data. (C) Nucleotide
sequence of the whiD promoter region indicating the
whiDp1 and whiDp2 transcription start points, the
putative ribosome binding site (RBS), and the start of the
whiD coding sequence.
|
|
The pattern of transcription from
whiDp1 and
whiDp2 during development of wild-type
S. coelicolor on solid medium was monitored
by S1 nuclease
protection. Following previous work (
25,
26,
39), we used a
time course of RNA samples that had already been
used to assess the
developmental pattern of transcription of a
number of sporulation
genes, including the early sporulation gene
whiH
(
39), the early sporulation-specific sigma factor gene
whiG (
25), the late sporulation-specific sigma
factor gene
sigF (
25), and
whiE
(
26), the locus specifying the polyketide spore
pigment. As
a positive internal control for these experiments,
transcription of
hrdB, encoding the principal (essential) sigma
factor of
S. coelicolor, was also analyzed. The data for
hrdB have been published previously (
25,
26,
39)
and are reproduced
here for comparison. No attempt was made to
fractionate the harvested
cell material used for RNA preparation; thus,
for example, the
late samples contained vegetative and aerial mycelium
as well
as spores (
25,
26).
The
whiDp1 and
whiDp2 promoters were found to be
developmentally regulated: both transcripts first appeared at 72 h, the time
at which sporulation was first detected in the culture, and
were
present at similar levels 24 h later (Fig.
7B). The
whiD transcripts
were not seen during vegetative growth or
during aerial mycelium
formation and were almost undetectable at
120 h in mature colonies.
At the low level of resolution of these
experiments, the developmental
profiles of the two
whiD
transcripts were very similar to those
of the
sigF
(
25) and
whiE transcripts (
26), but
the
whiD transcripts
clearly appeared later than the
whiH transcript (
39).
whiD
transcription
was undetectable in RNA isolated from submerged culture,
conditions
that do not support sporulation of
S. coelicolor
(data not
shown).
| |
DISCUSSION |
Taking advantage of the minimal, ordered cosmid library of
Redenbach et al. (36), we have used a map-based cloning
strategy to isolate the whiD gene. This strategy
(36) relies on the fact that, although these cosmids cannot
replicate autonomously in S. coelicolor, they integrate
reasonably efficiently into the chromosome via insert-directed
homologous recombination. As an alternative to the construction of
shotgun libraries, this is an attractive approach for isolating any
S. coelicolor locus for which an approximate genetic map
position has been determined, and it has also been used to clone two
other developmental genes, whiI (2) and
bldC (G. H. Kelemen and A. C. Hunt, personal communication).
Sequencing of whiD showed that it encodes a homologue of
WhiB, another protein required for sporulation in S. coelicolor and also in S. aureofaciens. S. coelicolor
whiB mutants produce abnormally long, tightly coiled aerial hyphae
and are completely unable to form sporulation septa (6, 12,
13). Genome sequencing and directed approaches have identified a
growing number of whiD-related genes in the actinomycete
genera Streptomyces, Mycobacterium, Streptoverticillium, and Rhodococcus (Fig. 3B
[43]). This includes, in addition to whiB
and whiD, at least four further genes in S. coelicolor itself (43). To date, no members of the
WhiB-WhiD family of proteins have been identified outside the
actinomycetes. It is interesting to note that, although it does not
sporulate, Mycobacterium tuberculosis has likely orthologues
of both WhiD and WhiB. This conclusion is based on the level of amino
acid sequence similarity between the S. coelicolor and
M. tuberculosis proteins and the conservation of gene
organization around the corresponding genes. The M. tuberculosis WhiD orthologue (mtu_whib3, Fig. 3B) shows 60% amino
acid sequence identity to WhiD, and the M. tuberculosis WhiB
orthologue (mtu_whib2, Fig. 3B) shows 71% amino acid sequence identity
to WhiB. As in S. coelicolor, genes encoding an
extracytoplasmic function (ECF) sigma factor and
inosine-5'-monophosphate dehydrogenase are found upstream of the
M. tuberculosis whiD orthologue and groELS is
found downstream, although other genes intervene in one species or the
other such that the absolute gene organization is not identical.
Similarly, the two genes upstream of whiB in S. coelicolor, encoding proteins of unknown function, are also present upstream of the M. tuberculosis whiB orthologue.
Apart from whiB and whiD in S. coelicolor, there is only one other report of the disruption of a
whiD-related gene in any organism; inactivation of
whiB3, the whiD orthologue of Mycobacterium
smegmatis (64% identity), did not affect growth or the dormancy
response (21).
The biochemical function of the WhiB-WhiD family of proteins remains
unknown, although it has been suggested that they may function as
transcription factors (12, 43). The most striking aspect of
their primary amino acid sequence is the perfect conservation of four
cysteines. This conservation suggests that these residues may act as
ligands for a metal cofactor such as zinc, copper or an iron-sulfur
cluster or, alternatively, that they may be involved in intramolecular
disulfide bond formation. Some of these speculative possibilities would
be consistent with redox regulation of the activity of WhiD and other
members of the family.
The constructed whiD null mutant, J2152, had a more severe
phenotype than J243, the strain carrying the whiD16 point
mutation that originally defined this locus. In contrast to the
situation in J243, the level of sporulation in J2152 was considerably
lower than in the congenic whiD+ parent,
suggesting that whiD not only influences prespore maturation but also affects the initiation of sporulation septation. Consistent with this, there was considerable variation in spore size in the whiD null mutant (Fig. 5). This variation arose in part from
the swelling that seemed to accompany the lysis of "mature" spores (Fig. 6C), but it also clearly reflected irregularity in septum placement (Fig. 6B). In addition to variation in spore size, one of the
most striking aspects of the whiD null mutant phenotype was
that spore-sized compartments were frequently partitioned into
irregular, smaller units through the deposition of additional septa,
often laid down in several different planes. The additional septa that
defined these minicompartments were usually found very close to the
poles of the spores and the minicompartments appeared to be devoid of
chromosomal DNA (Fig. 6A). These observations are somewhat reminiscent
of minicell formation in unicellular bacteria. In E. coli
(1) and B. subtilis (37) a system for preventing division at old cell poles was discovered through the characterization of min mutants. These mutants often divide
correctly at midcell to produce equal-sized daughter cells, but with
approximately equal frequency they divide close to an existing cell
pole to produce a small, usually anucleoidal minicell (45).
| |
ACKNOWLEDGMENTS |
We thank Maureen Bibb, Mervyn Bibb, Keith Chater, and David
Hopwood for their helpful comments on the manuscript, Gabriella Kelemen
for the gift of the developmental time course of RNA samples, Sue
Bunnewell for hand-printing the transmission electron micrographs, Tobias Kieser for help in preparing Fig. 1, and Julian Parkhill for
helpful discussion.
This work was supported by a John Innes Foundation studentship (to
V.M.), by a BBSRC studentship (to W.J.P.), by a Lister Institute
Research Fellowship (to M.J.B.), and by grants-in-aid to the John Innes
Centre from the BBSRC and the John Innes Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Microbiology, John Innes Centre, Colney, Norwich NR4 7UH,
United Kingdom. Phone: (44) 1603-452571. Fax: (44) 1603-456844. E-mail: virginie.molle{at}bbsrc.ac.uk.
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Abstract
Introduction
