From the National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan
Received for publication, August 21, 2002, and in revised form, December 16, 2002
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ABSTRACT |
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The 52-kDa protein, EshA, whose expression is
controlled developmentally, is produced during the late growth phase of
Streptomyces spp. We found that disruption of the
eshA gene, which encodes the EshA protein, abolishes the
aerial mycelium formation and streptomycin production in
Streptomyces griseus when grown on an agar plate. The
eshA disruptant KO-390 demonstrated a reduced amount of
expression of the transcriptional activator strR, thus accounting for the failure to produce streptomycin. KO-390 was found to
accumulate deoxynucleoside triphosphates at high levels, including
dGTP, at late growth phase. The accumulation of dGTP was a cause for
the impaired ability of KO-390 to produce aerial mycelium, because the
ability to form aerial mycelium was completely repaired by addition of
decoyinine, an inhibitor of GMP synthetase. The accumulation of
dNTP in KO-390 coincided with a reduced rate of DNA synthesis. The
developmental time frame of these phenomena in KO-390 matched a burst
of EshA expression in the wild-type strain. In contrast to
S. griseus, the eshA disruption did
not affect the ability for Streptomyces coelicolor to form
aerial mycelium and did not result in the aberrant accumulation of dNTP accompanied by arrest of DNA synthesis, implying qualitative
differences in addition to quantitative differences between the two
EshA proteins. We propose that the S. griseus
EshA protein somehow positively affects (or regulates) the replication
of DNA in wild-type cells at late growth phase but leads to aberrant
phenotypes in mutant cells due to the disturbed DNA replication. The
EshA protein was found to exist as a multimer (~20-mers) creating a
cubic-like structure with a diameter of 27 nm and located predominantly
in cytoplasm.
The Gram-positive, filamentous, soil bacterial
streptomycetes are a unique group of prokaryotic organisms
that display complex morphological differentiation and the ability to
produce a wide variety of secondary metabolites (called physiological
differentiation), including antibiotics and other useful medicinal
compounds (1-3). In some bacteria, especially the genus
Streptomyces, morphological and physiological
differentiation often commence at the same time in response to
environmental signals such as nutrient limitation. Only a limited
number of Streptomyces species, for example
Streptomyces griseus, can produce spores when cultured in
liquid medium (4, 5). Although a molecular based mechanism that
connects antibiotic production with morphological differentiation has
not yet been fully elucidated, it is apparent that the stringent
response plays an essential role in detecting and responding to adverse
environmental conditions, eventually leading to the activation (or
suppression) of various biological functions. The stringent response, a
general and ubiquitous response to nutritional environmental stress in prokaryotic microorganisms, is mediated by a unique nucleotide, guanosine 5'-diphosphate 3'-diphosphate (ppGpp) (reviewed by Cashel et al. (6)). By analyzing mutants with an impaired ability to elicit the stringent response, Ochi (7-10) has proposed that ppGpp
plays a role in triggering the onset of antibiotic production in
Bacillus subtilis and Streptomyces spp., whereas
morphological differentiation is triggered by detecting a reduced level
of GTP.
Several genera of bacteria produce spores when they encounter adverse
environmental conditions such as nutrient deficiency (reviewed by
Freese and Heinze (11)). A distinguishing characteristic of
Streptomyces spp. is the ability to form an aerial mycelium from a substrate mycelium when cultured on solid media, eventually leading to the formation of spores by creating synchronous and regularly spaced septations in the aerial mycelium (for reviews see
Refs. 1, 2, and 12). Insight into the mechanisms by which GTP functions
came from studying the development of bacteria that provided several
clues for understanding the onset mechanism for bacterial
differentiation on a molecular level (13, 14). We (9, 10, 15, 16) and
Freese and co-workers (11, 17-19) have demonstrated that a decrease in
the GTP pool size correlates with the initiation of morphological
differentiation in B. subtilis, Streptomyces
spp., and Saccharomyces cerevisiae. Thus these organisms can
be induced to differentiate in nutritionally rich media, in which cells
normally do not sporulate, when the GTP pool size is reduced
artificially. Although the role of GTP pool size variations in
Streptomyces is unclear, it seems logical that a GTP-binding protein(s) could be involved in detecting a decreased GTP pool level as
a signal for differentiation (13).
Recently, Kendrick and co-workers (20) and our laboratory (21)
independently found a novel 52-kDa protein, which has been demonstrated
to be required for initiating several developmental processes in
Streptomyces. Specifically, in the course of studying ribosomes isolated from Streptomyces coelicolor
A3(2) and S. griseus, we found a protein that is
produced during the late growth phase (21). The disruption of the gene
(eshA), which codes for this 52-kDa protein, was shown to
abolish the antibiotic production in S. coelicolor A3(2). At
the same time, Kwak et al. (20) reported that EshA is
required for the extension of sporogenic hyphal branches (thus the
origin of the gene designation, eshA) in S. griseus. Although EshA may be a good tool for uncovering and
analyzing still unknown biological events that take place in stationary phase cells, the function of the EshA remains obscure. In this paper,
we report the results from genetic and biochemical analysis of the EshA
protein working with S. griseus, because this organism produces 10-fold more EshA protein than S. coelicolor A3(2).
Eventually, we found that the EshA exists as a large multimer with a
characteristic shape. This is also discussed in terms of protein chemistry.
Bacterial Strains and Culture Conditions--
S.
griseus IFO13189, a prototrophic streptomycin-producing wild-type
strain, was used as the parental strain. Escherichia coli
DH5 Plasmid Construction--
General DNA manipulations such as
plasmid isolation and transformation in E. coli were
employed as described by Sambrook et al. (24).
Characteristics and construction of plasmids used in this study are
summarized in Table I. Plasmid pV52SG,
which contains a 3.5-kb S. griseus eshA region, was
constructed as follows. A 3.2-kb MluI fragment and a 1.3-kb
NcoI fragment, which contains the upstream and the
downstream region of eshA gene respectively, were excised
from pSGRp52 which carries a 8.0-kb fragment containing the
eshA gene on pBluescript SK(+) (21). Then the remaining 3.5-kb BamHI fragment was ligated to pV1, resulting in
pV52SG.
In order to construct a vector to prepare a probe for S1 mapping, a
304-bp fragment containing the 5' part of the S. griseus strR gene was amplified by PCR with genomic DNA isolated from strain 13189 as the template using the primers strR-FS1
(5'-TCGGCAATCAAACTGCGGTTTATTTTG-3') and strR-RS1
(5'-CTCGGGGCTGTTCCCTGAAATATGC-3'). The fragment generated was
cloned into the plasmid pGEM-T, resulting in pGEMstrRSG. A 351-bp
fragment containing the 5' part of the S. griseus hrdB gene
was amplified by PCR with genomic DNA isolated from strain 13189 as the
template using the primers SghrdB-F02
(5'-AAGGATCCGCGCCGCGCGAGCACTGAC-3'; the underline indicates
the cleavage site for BamHI) and SghrdBR1 (5'-GGAACGATGGAAACGGCTTC-3'). The fragment generated was cloned into
the plasmid pGEM-T, resulting in pGEMhrdBSG.
Gene Disruption of eshA--
The plasmid pSGRp52 was digested at
the unique BstPI site (456th base position) within the
eshA-coding region. The ends of the
BstPI-digested fragment were flush-ended with the Klenow
fragment of DNA polymerase I and then ligated resulting in the plasmid pSGR Measurement of the Intracellular Nucleotide Pools--
For plate
culture assays, spores (~2 × 107) were spread on
TSB agar medium covered with a cellophane sheet. Methods for extraction of intracellular nucleotides from mycelia, which had been grown on a
cellophane sheet or in liquid medium, were described previously (26,
27). Nucleotide pool sizes were analyzed using a high performance
liquid chromatography system (Hitachi D-7000
HPLC1 series with L-7100 pump
and L-7400 UV detector) with an ion-exchange column (Partisil-10 SAX,
4.6 mm × 25 cm; GL Science). Elution was performed at a flow rate
of 1 ml/min by a gradient made up of either a low ionic strength buffer
(7 mM KH2PO4, adjusted to pH 4.0 by
H3PO4) or a high ionic strength buffer (0.5 M KH2PO4 + 0.5 M
Na2SO4, adjusted to pH 5.4 by KOH). UV
detection was done at 260 nm. Gradient conditions used in this study to
separate NTP and dNTP were as follows. The percentage of high ionic
strength buffer was increased during the initial 5 min from 0 to 15%,
increasing again for the next 60 min from 15 to 60%, and again for 3 min from 60 to 100%, remaining at 100% for 20 min and then reduced from 100 to 0% in 3 min, then remaining at 0% for 10 min.
Intracellular concentrations of nucleotides are expressed as pmol per
mg wet cell weight (in agar plate culture) or pmol per
AM500 (in liquid culture).
Assay for Streptomycin--
For streptomycin production, strains
were inoculated on TSB agar medium. After incubation for the indicated
time, agar pieces (diameter 8 mm) were cut from agar plates and were
used to assay for streptomycin as described previously (10). B. subtilis ATCC6633 was used as a test organism.
RNA Isolation and S1 Nuclease Protection Assay--
Methods for
RNA preparation and S1 mapping were carried out as described by Kieser
et al. (28). Mycelia grown on the surface of agar medium
covered with cellophane were harvested and immediately dispersed in 5 ml of modified Kirby mix solution and mixed thoroughly by vigorous
vortexing with glass beads. Total RNA (100 µg, as estimated
spectrophotometrically) was used in S1 nuclease protection assay. The
hybridization probes were prepared by PCR using linearized plasmid DNA
as a template and 32P-labeled primer, resulting in an
unique 5'-end-labeled antisense single strand DNA fragment. For
constructing the 329 nucleotides strR probe,
SphI-digested pGEMstrRSG (as a template) and the primer 5'-CTCGGGGCTGTTCCCTGAAATATGC-3' (nucleotides +85 to +61 from the transcriptional start point (29)), which was 32P-labeled at
the 5' end, were used. For constructing the 349 nucleotides hrdB probe, BamHI-digested pGEMhrdBSG (as a
template) and the primer 5'-GGAACGATGGAAACGGCTTC-3' (nucleotides +110
to +91 from the transcriptional start point (30)), which was
32P-labeled at the 5' end, were used.
Expression and Purification of the EshA Protein--
Plasmid
pAH0 used for the overexpression of the EshA protein in E. coli was constructed as follows. An insert sequence encoding the
entire eshA gene was produced by PCR using the S. griseus 13189 genomic DNA as the template and the synthetic
oligonucleotides (5'-AGAGCCTGCCATATGACTGTTGAC-3') and
(5'-CCGGATCCCTACCTGGGCCAGTT-3') as primers. These primers
contain sequences designed to create an NdeI site
immediately prior to the initiation codon in the EshA open reading
frame and a BamHI site just after the termination codon as
designated by underlines. The 1.8-kb PCR product was digested with
NdeI and BamHI and then inserted into the
expression vector pPROEX-1 previously digested with NdeI and
BamHI. The recombinant EshA protein that possesses an
N-terminal His tag sequence was purified on nickel-nitrilotriacetic
acid matrices (Qiagen). The EshA protein was further purified using an
8% SDS-PAGE and extracted with phosphate-buffered saline containing
0.1% SDS. Purified EshA protein (0.2 mg) was used to raise antibodies
in rabbit.
Antiserum and Western Blotting--
Polyclonal antiserum against
the EshA protein from S. griseus was prepared in rabbit
using the recombinant protein prepared as described above. Crude
extracts for Western blotting were prepared as follows. Cells collected
from the cellophane sheet were suspended in buffer A (containing 20 mM Tris-HCl (pH 8.0), 10 mM
Mg(CH3COO)2, 20 mM
NH4Cl, 5 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, and 3 µg/ml of
antipain, chymostatin, and leupeptin) and then disrupted by sonication.
After centrifugation at 15,000 × g for 20 min at
4 °C, the resultant supernatant was used as a crude extract. For
Western blotting, the crude extracts were subjected to SDS-PAGE
(containing 8-10% acrylamide). Western blotting was performed as
described previously (13).
Determination of DNA Synthesis--
Strains were grown in 10 ml
of TSB medium in a 100-ml flask. When A500
reached 0.5, 1.5, 2.5, or 5.0, [2-14C]thymidine
(PerkinElmer Life Sciences) was added to a final concentration of 20 µM (0.025 µCi/ml), and incubation was continued with
shaking. Samples (0.5 ml) were withdrawn at the indicated time, and DNA synthesis was determined by measuring the incorporation of
[2-14C]thymidine into the acid-precipitable portion as
described previously (26).
Determination of Total DNA--
Strains were grown in TSB medium
for the indicated time, and an aliquot of cultured broth (0.5 to 4 ml)
was acidified with HClO4 to give a final concentration of
0.25 M. After standing in an ice bath for 30 min, samples
were centrifuged, and the resulting precipitant (containing total DNA)
was hydrolyzed two times by heating at 70 °C for 15 min in 0.5 M HClO4. The amount of total DNA was determined
according to the method described by Burton (31) using diphenylamine reaction.
Isolation and Purification of Intact EshA--
The S. griseus wild-type strain 13189 was grown in GYM medium
supplemented with 15% (w/v) sucrose for 30-40 h (late growth phase),
and then cells were collected by filtration with filter paper
(ADVANTEC, number 2). The resultant cells were suspended in buffer A
(see above) without 2-mercaptoethanol, but containing lysozyme (2 mg/ml), and was incubated at 30 °C for 20 min, followed by a gentle
sonication for 40 s. After cell debris was removed by
centrifugation (8,000 × g for 10 min), EshA was
precipitated by ultracentrifugation at 100,000 × g for
2.5 h. The precipitant was then suspended in buffer A and
subjected to density gradient centrifugation using 10-30% linear
sucrose gradient in buffer A. Finally, EshA protein was isolated by
ion-exchange chromatography using a Mono-Q HR 5/5 column (0.5 × 5 cm, Amersham Biosciences).
Gel Filtration with EshA--
Gel filtration was carried out
using the AKTA chromatography system equipped with a Superose 6 HR
10/30 column (1.0 × 30 cm, Amersham Biosciences). A molecular
weight curve was obtained using the elution profiles of thyroglobulin,
ferritin, catalase, aldolase (Gel Filtration HMW calibration Kit,
Amersham Biosciences), and bovine serum (Wako) as standard proteins.
Electron Microscopy--
A drop of native EshA sample was placed
on a carbon film and stained with uranyl acetate. Electron microscopy
was performed with TEM (2010, JEOL) at an acceleration voltage of 80 kV
and a magnification of 50,000.
Immunoelectron Microscopy--
Cells, which were grown on GYM
agar, were fixed with 0.2% glutaraldehyde and 4% paraformaldehyde,
dehydrated, and embedded in LR White resin (Nisshin EM) at 60 °C for
20 min. The resin was cooled and cut into thin sections using an
ultramicrotome. Immunolabeling was performed using anti-EshA antiserum
as the first antibody and anti-rabbit IgG conjugated to colloidal gold (diameter 10 nm) using the method described by Roth (32). Labeled cells were stained with 2% uranyl acetate and visualized by electron microscopy.
Disruption of eshA Gene--
Because Kwak et al. (20)
reported previously that EshA is required for the extension of
sporogenic hyphae during submerged spore formation in liquid culture,
we assumed that disruption of the eshA gene may cause an
impairment of aerial mycelium formation. Thus, we attempted to clarify
the function of EshA in S. griseus by disrupting the
eshA gene. Gene disruption by frameshift mutation was
carried out using the wild-type strain 13189 as briefly illustrated in
Fig. 1A. The resultant
eshA disruptants grew normally, suggesting that EshA is not
essential for viability. Disruption of the eshA gene was
confirmed by sequence analysis and also by the lack of EshA protein
production as determined by Western blotting (Fig. 1B).
Furthermore, we detected the reappearance of EshA protein upon
transforming disrupted cells with the plasmid pV52SG, which contains
the wild-type eshA gene (Fig. 1B). Strain KO-390
was used for further study as a representative eshA
disruptant.
First, the ability of KO-390 to form aerial mycelium was studied.
Wild-type strain 13189 produced abundant aerial mycelium 2 days after
incubation on TSB agar medium. In contrast, disruptant KO-390 almost
completely lacked the ability to form aerial mycelium (Fig.
1C). The defects in aerial mycelium formation was, however, completely repaired (or even enhanced compared with the wild-type strain) when plasmid pV52SG was introduced into KO-390 (data not shown). KO-390 cells also failed to form aerial mycelium when cells
were grown on sporulation agar medium; however, cells could produce
aerial mycelium as abundantly as the parental strain when grown on GYM
or SPY agar medium (data not shown). Thus, the ability of KO-390 to
form aerial mycelium is conditional based on culture conditions.
Previous studies (10) have demonstrated that a reduction of
intracellular GTP pool can induce aerial mycelium and spore formation
in S. griseus. To study whether a forced reduction of the
intracellular GTP pool size could lead to restoration of aerial mycelium formation, decoyinine (a GMP synthetase inhibitor) was used.
Notably, the ability for KO-390 to form aerial mycelium was completely
restored when 0.2 mM decoyinine was added to the medium
(Fig. 1C). Decoyinine was effective only when it was added at the early (until 16 h) growth phase. The supplementation of guanosine or deoxyguanosine together with decoyinine (each 0.2 mM) abrogated the ability of decoyinine to restore aerial
mycelium formation (data not shown).
Second, the ability to produce antibiotic was studied (Fig.
1D). The wild-type strain produced streptomycin after a
36-48-h incubation on TSB agar medium, but the eshA
disruptant KO-390 was unable to produce the antibiotic. This indicates
that the EshA protein somehow plays a role in initiating the secondary metabolism in addition to morphological differentiation. The deficiency in producing streptomycin by the eshA disruptant was
confirmed in liquid culture using TSB, SPY, and synthetic medium (data
not shown). Interestingly, introduction of the plasmid pV52SG into KO-390 restored or even enhanced streptomycin productivity compared with the wild-type strain (Fig. 1D). Addition of decoyinine
did not restore streptomycin production. The impaired ability to
produce streptomycin may be the result from a failure of inducing the strR gene, which is a transcriptional activator of genes for
streptomycin biosynthesis (33, 34). Therefore, we compared the
strR transcription level between the wild-type and
eshA disruptant strains. Transcription of hrdB
encoding one of the major The eshA Disruptant Accumulates dNTP--
Morphological
differentiation of B. subtilis and Streptomyces
spp. is known to result from a decrease in intracellular GTP levels
(18, 19, 26). Therefore, we speculated that the impaired ability of
KO-390 to form aerial mycelium might result from an insufficient
reduction of GTP (or dGTP) level. To examine this possibility, we
monitored the levels of intracellular GTP and dGTP, together with the
level of EshA protein, during growth on TSB agar medium. Expression of
EshA (in wild-type cells) began at mid-growth phase (24 h) and then
decreased at the end of growth (74 h) (Fig.
3). The GTP levels in the wild-type
strain and the mutant strain both decreased sharply during the first
30 h of incubation, where the mutant strain displayed the GTP
levels only slightly higher than did the wild-type strain. In contrast,
it was striking that the eshA disruptant KO-390 accumulated
deoxynucleoside triphosphate (dNTP) within the cell. An HPLC profile of
extracted nucleotides from wild-type and mutant cells grown to late
growth phase on TSB agar was shown in Fig.
4 as a representative example. It is
evident from Fig. 4 that dGTP, dCTP, and dTTP all increased significantly in the mutant cells. The amount of dATP was not determined because ATP and dATP have the same retention time. The
changes in the level of dNTP were monitored by cultivating the
wild-type and mutant cells on TSB agar, and the results for dGTP are
shown in Fig. 3 together with the results for GTP. It is apparent that
the increase of dGTP (and also dCTP and dTTP (not shown)) typical for
mutant KO-390 is temporary and that this accumulation occurs at
the mid-growth phase (30 h). Thus, we concluded that the cell's
excessive accumulation of dGTP can be a cause at least in part (if not
entirely) for the impaired ability of the eshA disruptant to
undergo morphogenesis.
To confirm the above conclusion, we investigated the effect of
decoyinine and guanosine on the levels of intracellular GTP and dGTP.
For this purpose, we used liquid culture instead of agar plate culture,
because nucleotide levels can be studied in more detail in liquid
culture. Like the agar plate cultures, wild-type cells grown in TSB
liquid medium displayed a more accentuated decrease of GTP compared
with the eshA disruptant KO-390 at the middle to late growth
phase (Fig. 5). However, when KO-390
cells were treated with 0.5 mM decoyinine at the middle
growth phase, there was a significant reduction of GTP and dGTP,
accounting for the observed restoration of morphogenesis in this mutant
(see Fig. 1C). The addition of guanosine together with
decoyinine resulted in an increase of GTP to a level higher than
non-treated cells, although dGTP level did not increase (Fig. 5).
Addition of 0.2 mM deoxyguanosine, however, was accompanied
by an elevated amount of dGTP as determined by HPLC analysis (data not
shown). Thus, these results are all in agreement with the observed
abolishment of decoyinine-induced aerial mycelium formation by addition
of either guanosine or deoxyguanosine.
Disruption of eshA Causes Arrest of DNA Synthesis--
dNTP serve
as a substrate in DNA synthesis. Therefore, the elevated levels of dNTP
in the eshA disruptant imply a disturbance of DNA synthesis
at the late growth phase. To assess this possibility, we first
investigated the effect of novobiocin, a known inhibitor for DNA
synthesis, on intracellular dNTP level. When the wild-type strains
grown in TSB liquid medium were treated with a low concentration (20 µg/ml) of novobiocin, dNTP levels were found to increase about 5-fold
1 h after the addition. Growth was inhibited only by 20% (data
not shown).
The results of using novobiocin suggest that DNA synthesis in the
eshA disruptant is somehow arrested to some extent,
eventually leading to elevation of dNTP levels. This was confirmed by
measuring directly the rate of DNA synthesis using
[2-14C]thymidine. Wild-type (13189) and eshA
disruptant (KO-390) strains were grown in TSB liquid medium (Fig.
6A). Both strains grew at the
same growth rate until they reached stationary growth phase. Like agar
plate cultures (see Fig. 3), the level of intracellular dGTP (and also
other dNTP (not shown)) increased sharply at the late (20-24 h) growth
phase in KO-390 cells (Fig. 6B). Wild-type cells show an
abrupt increase in the amount of EshA protein at the time point b
(which corresponds to 20 h in cultivation time) (Fig.
6C). Therefore, we monitored the rate of DNA synthesis at various growth phases using [2-14C]thymidine. Strikingly,
13189 and KO-390 displayed a similar rate of DNA synthesis during the
early growth phase (time point a), but later KO-390 exhibited a
significantly lower rate compared with 13189 (Fig. 6D). The
reduction of the rate of DNA synthesis in KO-390 was most pronounced at
the end of the growth phase (time point d). Similar results were
obtained when a 5-fold higher concentration of
[2-14C]thymidine (100 µM) was used to avoid
the effect which pre-existing endogenous dTTP may have had (data not
shown). Thus, failure to produce EshA protein in disruptant cells
resulted in a reduced DNA synthesis during the middle to late growth
phase, accompanied by an increase in dNTP levels. This conclusion was
supported by the fact that the disruptant cells had a reduced content
of DNA (13.4 versus 15.1 µg/AM500) at late
growth phase (time point d), as determined by the method of Burton (31)
(Table II).
S. coelicolor eshA Disruptant Does Not Accumulate dNTP--
Our
next interest was to know if EshA protein of S. coelicolor
can replace in function the S. griseus EshA protein, because these two proteins share an amino acid sequence homology as high as
76% (21).
First, we found that, unlike S. griseus, the S. coelicolor eshA disruptant KO-350 (21) did not accumulate dNTP
throughout whole growth cycle. Moreover, there was no substantial
difference in the rate of DNA synthesis even at late growth phase when
compared between the wild-type strain 1147 and the eshA
disruptant KO-350 (data not shown). Thus, arrest of DNA synthesis by
disrupting the eshA gene is a property characteristic for
S. griseus.
Second, in order to see the effect of high level expression of the
S. coelicolor flavor of EshA in an S. griseus
eshA mutant, we cloned the S. coelicolor eshA gene into
low copy number and high copy number plasmids, and we transformed the
S. griseus eshA disruptant KO-390 with the resulting
plasmids (pV52SC and pNS52SC; see Table I). However, there was no
expression of EshA at all throughout the whole growth cycle as
determined by Western analysis, possibly due to inactivity of the
S. coelicolor eshA promoter in S. griseus cells.
Isolation and Characterization of the EshA Protein--
We next
attempted to isolate the EshA protein in its native form.
S. griseus cells (50 g wet wt) grown to the late
growth phase (30-40 h) were harvested, disrupted by sonication, and
centrifuged to remove cell debris. The supernatant was subjected to
ultracentrifugation. The precipitant (containing EshA) was applied to
sucrose gradient centrifugation (Fig.
7A), in which the EshA was
fractionated with ribosomal 50 S particles as determined by Western
blotting using EshA antibody (Fig. 7B). Finally, the EshA
fraction was subjected to ion-exchange chromatography to separate EshA
from the 50 S particles (Fig. 7C), eventually resulting in
28 mg of purified EshA protein. The purity of EshA protein was
confirmed by SDS-PAGE in which EshA was shown to be 52 kDa in size
(Fig. 7D) and by native gel electrophoresis in which EshA
behaved as a macromolecule as well as ribosomal 30 S, 50 S, or 70 S
particles (data not shown). The size of the EshA multimer was estimated
to be 1100-1200 kDa as determined on the basis of results from gel
filtration. This result indicates that the EshA multimer consists of
about 20 EshA molecules.
We performed electron microscopy on the EshA multimers. Surprisingly,
the EshA multimer was found to be a cubic-like structure with a
diameter of 27 nm (Fig. 8). The shape of
each particle is uniform as seen from the photograph. The purified
particles were found not to be associated with nucleic acid, as
determined by staining with ethidium bromide. The characteristic shape
was no longer detected when the EshA sample had been subjected to pH
5.0 or pH 10 before electron microscopy, indicating that this EshA
multimer is fragile under physical stress.
Localization of the EshA Protein--
Finally, we studied the
localization of EshA proteins using immunoelectron microscopy. The
results clearly indicated that the majority (if not all) of EshA
protein is localized in the cytoplasm but not in the membrane (Fig.
9). To confirm this conclusion, we
prepared protoplasts, burst the cells, and then subjected the sample to
ultracentrifugation at 30,000 × g for 30 min. Majority (80%) of EshA was found in supernatant (cytoplasmic fraction), whereas
only 20% was found in precipitate (membrane fraction), as determined
by Western blotting (data not shown). Thus, we concluded that the EshA
protein exists as a cytoplasmic protein at least in the experimental
conditions used here.
One of the most intriguing challenges in biology is to elucidate
mechanisms by which cells detect and respond to extracellular nutritional conditions. Among prokaryotes, B. subtilis and
Streptomyces spp. offer a feasible system for studying such
mechanisms by displaying a wide range of adaptations to nutrient
limitations, including production and secretion of antibiotics and
enzymes and formation of aerial mycelium (in Streptomyces
spp.) and endospores (in B. subtilis) as an extreme response
to nutrient limitation (35). In this regard, nutritional status and
sporulation have been successfully linked in B. subtilis
(14), especially CodY has been shown to be able to detect and respond
to nutrient limitations. Recent works by Inaoka and Ochi (36) support
its proposal. In this paper we described a novel function for the
S. griseus EshA protein in DNA synthesis. Apparently, EshA
is required to sustain the rate of DNA synthesis during the late growth
phase, even though the lack of EshA does not affect the growth rate
(i.e. increase in biomass) (Fig. 6A). Also, it is
possible that the inability to form aerial mycelium (and thus
sporulation) in the eshA disruptant mutant is a result of an
unusual elevation of intracellular dGTP due to reduced DNA synthesis.
Moreover, when the disruptant mutant was treated with decoyinine, a
decrease of GTP sufficient to fully induce aerial mycelium formation
was demonstrated (Figs. 1C and 5). Therefore, we conclude
that disruption of the eshA gene indirectly affects
morphogenesis of S. griseus. Rather, the EshA protein may
function to regulate directly DNA synthesis. It is worth stressing that
the aberrant phenotypes mentioned above for S. griseus are not the case for S. coelicolor; namely,
the eshA disruption did not affect the ability for S. coelicolor to form aerial mycelium as examined using a wide
variety of media (Ref. 21 and this study) and did not result in the
aberrant accumulation of dNTP. Although we could not demonstrate the
effect of expression of the S. coelicolor EshA protein
within the S. griseus eshA disruptant cells, the
differences in the behavior of the two hosts implicate qualitative
differences in addition to quantitative (10-fold) differences between
the two EshA proteins. Further work is required to elucidate this point.
Although we have demonstrated that disruption of eshA
abolishes the aerial mycelium formation (in S. griseus) and
antibiotic production (in S. griseus and S. coelicolor) (Ref. 21 and this study), it should be noted that
these aberrant phenotypes are conditional. Apparently, this led to a
discrepancy between our results and previous results reported by Kwak
et al. (20), who stated that eshA disruptant from
S. griseus is able to produce aerial mycelium and
streptomycin normally. It should be pointed out that the expression of
eshA commenced at late growth phase in both S. griseus and S. coelicolor as determined by Western blotting (21; and this study), indicating that EshA is a
developmentally regulated protein. These results are in agreement with
the previous works reported by Kwak et al. (20). Also
important is the fact that propagation of wild-type eshA
with low copy number plasmids caused overproduction of antibiotics in
both S. griseus (Fig. 1D) and S. coelicolor (21). Therefore, eshA may offer a feasible target for strain improvement, because genes homologous to
eshA appear to be widely distributed among streptomycetes
(21).
Most interesting is the fact that EshA exists as a multimer in its
native state (Fig. 7, A and B), possibly forming
an icosahedron as indicated from the results of electron microscopy
(Fig. 8), which is an architecture known for coat proteins of certain
viruses. Molecular self-assembly is the spontaneous association of
molecules into stable, structurally well defined aggregate joined by
noncovalent bonds. Understanding self-assembly and the associated
noncovalent interactions that connect complementary interacting
molecules into aggregates is a central concern in structural
biochemistry (37). A recent breakthrough has been reported describing
the supramolecular fabrication of large complexes from small molecules by self-assembly, which is called "supramolecular chemistry" (38, 39). As discussed in detail previously by Kwak et al. (20), the EshA protein shows extensive similarity to proteins from
mycobacteria and cyanobacterium, namely MMPI in Mycobacterium
leprae and SrpI in Synechococcus sp. (40-42). However,
there is no EshA homologue in E. coli, B. subtilis, or other prokaryotes whose genomes have been completely
sequenced. EshA proteins in S. griseus and S. coelicolor (sharing 76% amino acid identity to one another) are characterized by the presence of a cyclic nucleotide-binding domain, which is absent from the corresponding proteins from mycobacteria and
cyanobacterium. Therefore, EshA (termed P3 in the former article) can
be classified as a member of the cyclic nucleotide-binding protein
superfamily (20). The activity of this family of proteins is modulated
by a cyclic nucleotide (43, 44). In prokaryotes, only Crp and DnaA are
known to bind cAMP (45-47). The dnaA gene in E. coli has been shown to be an essential element in the initiation of DNA replication at the chromosomal origin, oriC (48). The DnaA protein with a size of 52.5 kDa interacts with cAMP, and this
interaction stimulates the DnaA protein to bind to the chromosomal replication origin (49). It has also been shown that the DnaA protein
possesses a high affinity for ATP, and ATP activates DnaA protein for
the initiation of DNA replication (50). cAMP may also play a role in
the reactivation of the DnaA protein by ensuring a rapid release of ADP
after each ATP hydrolysis event (45). Although it is unknown whether
the EshA protein exerts its influence via the nucleotide-binding
domain, recent studies (51, 52) with Streptomyces spp. have
demonstrated the existence of cAMP at a high extracellular level during
the late growth phase. It is intriguing to investigate whether the
nucleotide-binding domain (in the aid of a cyclic nucleotide(s)) plays
a role in forming the characteristic multimer as shown in Fig. 8.
Previous studies (20, 42) from several laboratories have emphasized
that EshA (and also the MMPI protein of M. leprae) may be a
membrane protein. This conclusion is based on the fact that (i) EshA
and MMPI were copurified with membrane fractions as determined by
ultracentrifugation of crude extract, and (ii) EshA, MMPI, and SrpI all
contain putative 14-17-amino acid transmembrane anchor domain at their
C termini. However, results presented here indicate that EshA in
S. griseus exists predominantly in cytoplasm as
demonstrated by both immunoelectron microscopy (Fig. 9) and ultracentrifugation (see "Results"). This apparent discrepancy might be a result of EshA having two different forms, i.e.
monomers and multimers. Therefore, localization of EshA within the cell may be directed according to these different conformational forms.
EshA is produced abundantly and accumulates during sporulation
induced by phosphate starvation and nutritional downshift in S. griseus (20). Our results demonstrate that EshA in S. griseus is produced even in normal culture conditions when
cells reach the middle to late growth phase (Fig. 3). Likewise, the
SrpI protein is also induced under sulfur deprivation (53) and MMPI
during infection by M. leprae (42). According to these
facts, Triccas et al. (53) and Kwak et al. (20)
proposed that EshA, SrpI, and MMPI may define a new family of bacterial
stress-response proteins. Our present observations may be helpful to
provide clues for understanding the mechanism of stress response in
relation to protein chemistry and regulation of DNA synthesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was used routinely for plasmid construction. E. coli GM2163 (New England Biolabs), a non-methylating strain, was used to
prepare DNA for transformation into S. griseus. Strains of S. griseus were grown at 30 °C using GYM medium (22) or
TSB medium. TSB medium contained (per liter): 10.3% (w/v) sucrose; 3%
(w/v) tryptic soy broth (Difco); 5 mg of
CuSO4·5H2O; 7.5 mg of
FeSO4·7H2O; 3.6 mg of
MnSO4·5H2O; 15 mg of
CaCl2·2H2O; 9 mg of
ZnSO4·7H2O; and 5 mM
MgCl2. The regeneration medium, SpRR (23), was used for
transformation of S. griseus. SPY medium, synthetic medium,
and sporulation medium were described previously (10).
Bacterial strains and plasmids used in this study
p52, which contains a frameshift mutation in the eshA
gene. This frameshift mutation makes cells to produce a protein
containing about one-third (152 amino acid residues) of the native EshA
protein (470 amino acids). A 1.4-kb EcoRI fragment from
pKK1000 containing the apramycin acetyltransferase gene
(aac(3)IV) was introduced into the plasmid pSGR
p52 to
generate pSGR
p52Ap. pSGR
p52Ap was passed through E. coli GM2163 (New England Biolabs) to remove methyl groups and
subsequently was methylated with methyltransferases, mAluI
and mHapII. The methylated DNA was introduced into S. griseus 13189 protoplasts. Transformants that contain pSGR
p52Ap
in the chromosome as a result of single crossover were selected with apramycin (50 µg/ml). The apramycin-resistant strains were cultured at 30 °C for 72 h in TSB liquid medium without apramycin to
allow double crossover. The cells recovered were then spread on GYM agar without apramycin to screen for apramycin-sensitive clones, which
represent eshA disruptant. The frameshift mutation in the eshA gene was confirmed by DNA sequencing.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (61K):
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Fig. 1.
Gene disruption of eshA and
its consequence. A, schematic of the strategy used for
the disruption of eshA. Plasmid pSGR p52Ap contains the
eshA gene with a frameshift mutation at the unique
BstPI site and the apramycin resistance gene (aac
(3)IV) in the pBluescript SK(+) vector. The designation
B represents BamHI. B, Western blot
analysis of the EshA protein. Crude extracts were prepared from cells
grown in SPY medium for 24 h. C, effect of
eshA disruption on aerial mycelium formation. Spores were
spread on TSB agar medium or TSB agar medium containing 0.2 mM decoyinine. The plates were incubated at 30 °C for 3 days. White color represents the development of aerial
mycelium. D, effect of eshA disruption on
streptomycin production. Agar pieces (diameter 8 mm) cut from each of
the TSB agar plates after incubation for the indicated time were put on
the assay plate, followed by 12 h of incubation.
factors (30) was monitored as a positive
internal control. S1 nuclease protection assay revealed that
strR mRNA was transcribed growth
phase-dependently in both strains, but the level of
strR mRNA was 2-fold less in KO-390 than the wild-type
strain (Fig. 2), accounting for the
impaired ability to produce streptomycin in the eshA
disruptant. There was no substantial difference in the level of
hrdB mRNA between the wild-type and KO-390 strains (data
not shown). The reduced expression of strR in the mutant is
possibly due to the less accentuated accumulation of ppGpp at late
growth phase (0.3-0.5 pmol/mg wet weight for the wild-type strain and
0.2-0.3 pmol/mg wet weight for KO-390) as determined using HPLC.
View larger version (33K):
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Fig. 2.
S1 nuclease protection assay for the
strR transcript. RNA was isolated from wild-type
(WT) and mutant cells grown for the indicated time on TSB
agar medium. 32P-Labeled, MspI-digested pBR322
size marker is shown at right. nt,
nucleotide.
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Fig. 3.
Changes in the intracellular concentrations
of GTP and dGTP and levels of the EshA protein during growth on agar
medium. Strains were grown on TSB agar medium covered with a
cellophane sheet. Cells were harvested at the indicated time to
determine biomass, nucleotide concentrations, and the amount of EshA
protein produced. Nucleotides were separated by HPLC, whereas EshA
protein was determined by Western blot analysis (see "Experimental
Procedures"). , wild-type strain 13189;
, eshA
disruptant KO-390.
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Fig. 4.
Elution profile from HPLC of nucleotides
extracted from cells. The strains were grown on TSB agar covered
with a cellophane sheet for 28 h. The samples injected were
equivalent to 3 mg wet weight of cells. Elution from HPLC was done as
described under "Experimental Procedures." The numbered
arrows indicate the retention times for: 1, dGDP;
2, GDP; 3, dTTP; 4, UTP; 5,
dCTP; 6, CTP; 7, ATP and dATP; 8,
dGTP; 9, GTP.
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Fig. 5.
Changes in the concentration of intracellular
GTP and dGTP during growth in liquid medium. Wild-type strain
13189 (closed circles) and eshA disruptant KO-390
(open circles) were grown in TSB liquid medium. Decoyinine
(0.5 mM; squares) and decoyinine plus guanosine
(each 0.5 mM; triangles) were added 19 h
after inoculation as indicated by the arrows. Samples were
taken at the indicated times to determine culture density
(A500) and GTP and dGTP concentrations.
View larger version (25K):
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Fig. 6.
Growth and DNA synthesis of the wild-type
(13189) and eshA disruptant strain (KO-390).
Closed circles and open circles represent the
wild-type strain and KO-390, respectively. A, growth of
wild-type strain and KO-390 in TSB medium at 30 °C. The designation
a-d indicate the specified time points in growth.
B, changes in intracellular dGTP level during growth.
C, Western blot analysis of EshA protein. Cells grown to the
time points of a-d were harvested, and crude extracts (each
5 µg of total protein) were subjected to analysis. D,
incorporation of [2-14C]thymidine into DNA during growth.
[2-14C]Thymidine was added into the culture at the time
points of a d (see "Experimental Procedures"). The
incorporation rate of [2-14C]thymidine is expressed as a
specific activity (cpm × 102/AM500).
Changes in DNA contents during growth of S. griseus
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Fig. 7.
Purification of EshA by density gradient
centrifugation and ion-exchange chromatography. A,
ultracentrifuge precipitant containing EshA was fractionated on a
10-30% sucrose gradient. B, fractions containing the EshA
protein were detected by Western blot analysis using the EshA antibody.
C, the ion-exchange chromatography was performed using a
Mono-Q HR 5/5 column. D, purity of EshA was determined by
SDS-PAGE. Each lane contained the following: lane 1, low
range SDS-PAGE standard (Bio-Rad); lane 2, crude extract;
lane 3, fraction 12 after the sucrose gradient; lane
4, EshA protein purified by ion-exchange chromatography.
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Fig. 8.
Transmission electron microscopy
visualization of EshA multimers. EshA protein was stained with
uranyl acetate and observed by transmission electron microscopy.
B represents a simple expansion of a particle shown in
A.
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Fig. 9.
Localization of EshA proteins determined by
immunostained electron micrograph of vegetative cells. EshA
protein localized in a vegetative cell section was immunolabeled using
anti-EshA antibodies and anti-rabbit IgG conjugated to 10 nm
gold.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to T. Yoshinaga, A. Lezhava, A. Hesketh, and L. Skhirtladze for preliminary work done in several of the experiments.
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FOOTNOTES |
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* This work was supported in part by a grant from the Organized Research Combination System of the Science and Technology Agency of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Translational Research Department, Mitsubishi
Kagaku Institute of Life Sciences, Yokohama Research Center, 1000, Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 227-8502, Japan.
§ Present address: Dept. of Biological Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuda-Cho, Midori-ku, Yokohama 226-8501, Japan.
¶ Present address: Dept. of Microbiology, School of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8501, Japan.
To whom correspondence should be addressed: National Food
Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
Tel.: 81-29-838-8125; Fax: 81-29-838-7996; E-mail:
kochi@affrc.go.jp.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M208564200
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ABBREVIATIONS |
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The abbreviation used is: HPLC, high performance liquid chromatography.
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REFERENCES |
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1. | Chater, K. F. (1984) in Microbial Development (Losick, R. , and Shapiro, L., eds) , pp. 89-116, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
2. | Hopwood, D. A. (1988) Proc. R. Soc. Lond. Ser. B. Biol. Sci. 235, 121-138[Medline] [Order article via Infotrieve] |
3. | Chater, K. F. (1993) Annu. Rev. Microbiol. 47, 685-713[CrossRef][Medline] [Order article via Infotrieve] |
4. | Ensign, J. C. (1978) Annu. Rev. Microbiol. 32, 185-219[Medline] [Order article via Infotrieve] |
5. | Kendrick, K. E., and Ensign, J. C. (1983) J. Bacteriol. 155, 357-366[Medline] [Order article via Infotrieve] |
6. | Cashel, M., Gentry, D. R., Hernandez, V. J., and Vinella, D. (1996) in Escherichia coli and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C. , Curtiss, R., III , Ingraham, J. L. , Lin, E. C. C. , Low, K. B. , Magasanik, B. , Reznikoff, W. S. , Riley, M. , Schaechter, M. , and Umbarger, E., eds), 2nd Ed. , pp. 1458-1496, American Society for Microbiology, Washington, D. C. |
7. | Ochi, K., and Ohsawa, S. (1984) J. Gen. Microbiol. 130, 2473-2482[Medline] [Order article via Infotrieve] |
8. | Ochi, K. (1986) J. Gen. Microbiol. 132, 299-305[Medline] [Order article via Infotrieve] |
9. | Ochi, K. (1987) J. Gen. Microbiol. 133, 2787-2795 |
10. | Ochi, K. (1987) J. Bacteriol. 169, 3608-3616[Medline] [Order article via Infotrieve] |
11. | Freese, E., and Heinze, J. (1984) in The Bacterial Spores (Hurst, A. , Gould, G. W. , and Dring, J., eds), Vol. 2 , pp. 101-172, Academic Press, London |
12. | Chater, K. F. (1989) in Regulation of Procaryotic Development (Smith, R. I. , Slepecky, A. , and Setlow, P., eds) , pp. 227-299, American Society for Microbiology, Washington, D. C. |
13. | Okamoto, S., and Ochi, K. (1998) Mol. Microbiol. 30, 107-119[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Ratnayake-Lecamwasam, M.,
Serror, P.,
Wong, K. W.,
and Sonenshein, A. L.
(2001)
Genes Dev.
15,
1093-1103 |
15. | Ochi, K., Inatsu, Y., Okamoto, S., Penyige, A., Kudo, T., and Hotta, K. (1994) Actinomycetologica 8, 79-84 |
16. | Ochi, K., Zhang, D., Kawamoto, S., and Hesketh, A. (1997) Mol. Gen. Genet. 256, 488-498[CrossRef][Medline] [Order article via Infotrieve] |
17. | Freese, E., Heinze, J. E., and Galliers, E. M. (1979) J. Gen. Microbiol. 115, 193-205[Medline] [Order article via Infotrieve] |
18. | Lopez, J. M., Dromerick, A., and Freese, E. (1981) J. Bacteriol. 146, 605-613[Medline] [Order article via Infotrieve] |
19. |
Ochi, K.,
Kandala, J. C.,
and Freese, E.
(1981)
J. Biol. Chem.
256,
6866-6875 |
20. |
Kwak, J.,
McCue, L. A.,
Trczianka, K.,
and Kendrick, K. E.
(2001)
J. Bacteriol.
183,
3004-3015 |
21. |
Kawamoto, S.,
Watanabe, M.,
Saito, N.,
Hesketh, A.,
Vachalova, K.,
Matsubara, K.,
and Ochi, K.
(2001)
J. Bacteriol.
183,
6009-6016 |
22. | Kawamoto, S., Watanabe, H., Hesketh, A., Ensign, J. C., and Ochi, K. (1997) Microbiology 143, 1077-1086[Abstract] |
23. | Kawamoto, S., and Ensign, J. C. (1995) Actinomycetologica 9, 124-135 |
24. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
25. | Kawamoto, S., Zhang, D., and Ochi, K. (1997) Mol. Gen. Genet. 255, 549-560[CrossRef][Medline] [Order article via Infotrieve] |
26. | Ochi, K. (1986) J. Gen. Microbiol. 132, 2621-2631[Medline] [Order article via Infotrieve] |
27. | Ochi, K. (1988) in International Symposium on the Biology of Actinomycetes, Tokyo, May 22-26, 1988 (Okami, Y. , Beppu, T. , and Ogawa, H., eds) , pp. 330-337, Japan Scientific Societies Press, Tokyo, Japan |
28. | Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F., and Hopwood, D. A. (2000) Practical Streptomyces Genetics , The John Innes Institute, Norwich, UK |
29. | Vujaklija, D., Ueda, K., Hong, S. K., Beppu, T., and Horinouchi, S. (1991) Mol. Gen. Genet. 229, 119-128[Medline] [Order article via Infotrieve] |
30. | Shinkawa, H., Hatada, Y., Okada, M., Kinashi, H., and Nimi, O. (1995) J. Biochem. (Tokyo) 118, 494-499[Abstract] |
31. | Burton, K. (1956) Anal. Biochem. 62, 315-323 |
32. | Roth, J. (1982) in Techniques in Immunocytochemistry (Bullovk, G. R. , and Petrusz, P., eds), Vol. 1 , pp. 107-133, Academic Press, New York |
33. | Distler, J., Mansouri, K., Mayer, G., Stockmann, M., and Piepersberg, W. (1992) Gene (Amst.) 115, 105-111[Medline] [Order article via Infotrieve] |
34. | Retzlaff, L., and Distler, J. (1995) Mol. Microbiol. 18, 151-162[Medline] [Order article via Infotrieve] |
35. |
Dworkin, J.,
and Losick, R.
(2001)
Genes Dev.
15,
1051-1054 |
36. |
Inaoka, T.,
and Ochi, K.
(2002)
J. Bacteriol.
184,
3923-3930 |
37. | Whitesides, G. M., Mathias, J. P., and Seto, C. T. (1991) Science 254, 1312-1319[Medline] [Order article via Infotrieve] |
38. | Lehn, J. M. (1993) Science 260, 1762-1763[Medline] [Order article via Infotrieve] |
39. | Ebina, S., Matsubara, K., Nagayama, K., Yamaki, M., and Gotoh, T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7367-7371[Abstract] |
40. | Nicholson, M. L., and Laudenbach, D. E. (1995) J. Bacteriol. 177, 2143-2150[Abstract] |
41. | Nicholson, M. L., Gaasenbeek, M., and Laudenbach, D. E. (1995) Mol. Gen. Genet. 247, 623-632[Medline] [Order article via Infotrieve] |
42. | Winter, N., Triccas, J. A., Rivoire, B., Pessolani, M. C., Eiglmeier, K., Lim, E. M., Hunter, S. W., Brennan, P. J., and Britton, W. J. (1995) Mol. Microbiol. 16, 865-876[Medline] [Order article via Infotrieve] |
43. |
Shabb, J. B.,
and Corbin, J. D.
(1992)
J. Biol. Chem.
267,
5723-5726 |
44. |
McCue, L. A.,
McDonough, K. A.,
and Lawrence, C. E.
(2000)
Genome Res.
10,
204-219 |
45. | Hughes, P., Landoulsi, A., and Kohiyama, M. (1988) Cell 55, 343-350[Medline] [Order article via Infotrieve] |
46. | Botsford, J. L., and Harman, J. G. (1992) Microbiol. Rev. 56, 100-122[Abstract] |
47. | Chandler, M. S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1626-1630[Abstract] |
48. | von Meyenburg, K., and Hansen, F. G. (1987) in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (Neidhardt, F. C. , Ingraham, J. L. , Low, K. B. , Magasanik, B. , Schaechter, M. , and Umbergar, E., eds) , pp. 1555-1577, American Society for Microbiology, Washington, D. C. |
49. | Fuller, R. S., Funnell, B. E., and Kornberg, A. (1984) Cell 38, 889-900[Medline] [Order article via Infotrieve] |
50. | Sekimizu, K., Bramhill, D., and Kornberg, A. (1987) Cell 50, 259-265[Medline] [Order article via Infotrieve] |
51. | Susstrunk, U., Pidoux, J., Taubert, S., Ullmann, A., and Thompson, C. J. (1998) Mol. Microbiol. 30, 33-46[CrossRef][Medline] [Order article via Infotrieve] |
52. | Kang, D. K., Li, X. M., Ochi, K., and Horinouchi, S. (1999) Microbiology 145, 1161-1172[Abstract] |
53. |
Triccas, J. A.,
Winter, N.,
Roche, P. W.,
Gilpin, A.,
Kendrick, K. E.,
and Britton, W. J.
(1998)
Infect. Immun.
66,
2684-2690 |