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Journal of Bacteriology, May 2001, p. 3004-3015, Vol. 183, No. 10
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.10.3004-3015.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Identification and Characterization of a
Developmentally Regulated Protein, EshA, Required for Sporogenic
Hyphal Branches in Streptomyces griseus
Jangyul
Kwak,1,*
Lee Ann
McCue,2
Kristen
Trczianka,1 and
Kathleen E.
Kendrick1,
Department of Microbiology, Ohio State
University, Columbus, Ohio 43210,1 and
Wadsworth Center for Laboratories and Research, New York State
Department of Health, Albany, New York 122012
Received 20 September 2000/Accepted 28 February 2001
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ABSTRACT |
To identify sporulation-specific proteins that might serve as
targets of developmental regulatory factors in
Streptomyces, we examined total proteins of
Streptomyces griseus by two-dimensional gel
electrophoresis. Among five proteins that were present at high levels
during sporulation but absent from vegetative cells, two of the
proteins, P3 and P4, were absent from developmental mutants that
undergo aberrant morphogenesis. The deduced amino acid sequence of the
gene that encodes P3 (EshA) showed extensive similarity to proteins
from mycobacteria and a cyanobacterium, Synechococcus, that
are abundant during nutritional stress but whose functions are unknown.
Uniquely among these proteins, EshA contains a cyclic
nucleotide-binding domain, suggesting that the activity of EshA may be
modulated by a cyclic nucleotide. The eshA gene was
strongly expressed from a single transcription start site only during
sporulation, and accumulation of the eshA transcript depended on a developmental gene, bldA. During submerged
sporulation, a null mutant strain that produced no EshA could not
extend sporogenic hyphae from new branch points but instead accelerated
septation and spore maturation at the preexisting vegetative filaments. These results indicated that EshA is required for the growth of sporogenic hyphae and localization of septation and spore maturation but not for spore viability.
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INTRODUCTION |
Streptomyces is a
gram-positive bacterium that undergoes morphological differentiation.
In a nutritionally favorable condition, Streptomyces spores
germinate to give rise to vegetative hyphae, which are characterized by
filamentous, multigenomic cells called mycelia. Sporulation begins by
the growth of aerial hyphae (or sporogenic hyphae when the sporulation
process is induced in liquid culture; 34), which
subsequently undergo septation to form chains of unicellular spores. We
previously identified two types of sporogenic hyphae during sporulation
of Streptomyces griseus (22): we suggest the
term "distal" sporogenic hyphae for those that grow from the tips
of preexisting vegetative filaments and the term "proximal" sporogenic hyphae for those that grow de novo within the vegetative hyphae at the onset of sporulation. Since both vegetative growth and
spore formation are inherently polar processes similar to those
observed in yeast and filamentous fungi (18), formation of
sporogenic hyphae in Streptomyces is likely to require
proteins that direct the location of sporogenic hyphae, establishment
of polarity, and emergence of the sporogenic hyphae.
To understand the molecular details of Streptomyces
sporulation, researchers have identified developmental genes primarily by complementation of nonsporulating mutants. Most of the
sporulation-specific genes that have been characterized appear to
encode regulatory functions. Some, such as bldA, which
encodes tRNA (38), are required for the production of
aerial hyphae (35, 43) and antibiotics (3, 34,
43). Genes that specifically regulate spore formation include
the whi genes, so named because mutations in these genes
commonly prevent formation of the spore compartments and the
spore-associated pigment that is acquired late in development (11). Included among these proteins are WhiG, a 
subunit of RNA polymerase (12), and WhiH, a putative
transcription regulator of the GntR family (52). A second
sporulation-specific factor is encoded by sigF. WhiG is
required for the intermediate event of sporulation septation, whereas
SigF is required for subsequent maturation of the spores
(49).
Complete understanding of the role of developmentally controlled
regulatory factors requires the characterization of the target genes
they regulate. A few candidates for the targets have recently emerged.
The whiH promoter is recognized by WhiG, a
sporulation-specific factor (52). The whiE
gene cluster encoding grey spore pigment is controlled by several
whi genes in Streptomyces coelicolor (29). Sporulating cultures undergo cycles of synthesis and
degradation of glycogen, and some of the enzymes of glycogen synthesis
are active in aerial hyphae (7). The ftsZ gene,
which is required for septation in S. coelicolor
(41) and is differentially regulated during sporulation in
S. griseus (J. Kwak, unpublished data), would therefore be a
likely target of a factor that regulates development.
Here we describe results from attempts to identify additional
sporulation-specific genes encoding enzymes or structural proteins that
might depend on the Whi proteins or other regulatory factors for their
expression during development. For these studies, we have exploited the
ability of S. griseus to undergo sporulation while submerged
in liquid culture during nutritional downshift (33) or
phosphate starvation (31). Under these conditions sporulation is relatively rapid and synchronous; sporogenic hyphae are
evident at 4 h and continue to elongate for the next 6 h, septation occurs at approximately 10 h, and spores mature during the subsequent 10 to 12 h (34). Our objectives were
to use two-dimensional gel electrophoresis to identify
sporulation-specific proteins, to characterize the genes encoding these
proteins, and to establish the mechanisms of their developmental
regulation. Here we report on one such protein, designated P3, that
contains a cyclic nucleotide-binding domain required for growth of
sporogenic hyphae at an early stage of streptomycete morphogenesis. On
the basis of its role in sporulation, we rename P3 as EshA (extension
of sporogenic hyphae).
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
S. griseus NRRL
B-2682 was used as the wild-type strain. SKK2025, -2026, and -2027 were
three independent isolates containing the eshA null allele.
SKK1015 was used as the class IIIA mutant, SKK1008 was used as the
class IIIB mutant, and SKK1003 was used as the class IIIC mutant, as
described previously (34). Escherichia coli
DH5
was used for routine plasmid construction and preparation. E. coli ET12567 (dam, dcm, and hsd;
39) was used to demethylate plasmid DNA prior to its
methylation in vitro and its introduction into S. griseus
(J. Kwak, unpublished). We used E. coli Top10 (Invitrogen,
Carlsbad, Calif.) to express His-tagged EshA for making antibodies.
pKK842 was constructed by ligating a 2.4-kb SalI fragment of
genomic DNA from S. griseus NRRL B-2682 to pGEM-4Z
(Promega). The EcoRI-HindIII fragment
containing this 2.4-kb SalI fragment was ligated to pIJ2925
(27) to generate pKK847. pKK856 was constructed by
ligating the 2.4-kb fragment from pKK847 as a BglII fragment into the BamHI site of the low-copy vector pXE4
(26). pKK1416 contained eshA on an
approximately 8.0-kb BamHI-HindIII fragment in pXE4. pKK1417 contained the same 8.0-kb fragment in pKK1400 in which
a 1.8-kb Tsr cassette (60) was inserted as a
BamHI fragment into the BglII site of pIJ2920
(27).
To fuse EshA to a hexahistidine tag, we used the vector pTrcHis-A
(Invitrogen). Two oligonucleotides (oligonucleotide 164,
5'-AGTCGGATCCGCTAGCATGACTGTTGACTCGACCTCGGA-3', corresponding
to
nucleotides [nt] 329 to 352 of the 2.4-kb
SalI fragment
and containing
adjacent
BamHI and
NheI sites, and
oligonucleotide 165, 5'-AGAACGTCCAGGGTGACCTCGTC-3',
corresponding to nt 801 to 779) were used to amplify the
N-terminal
coding region of
eshA from pKK842, to introduce
NheI and
BamHI
sites upstream of and in frame
with the
eshA start codon. This
PCR product was ligated to
BamHI-
SacI-digested pUC18 to generate
pKK852. The
1.6-kb
SacI-
HindIII fragment from pKK842,
containing
the C-terminal coding region of
eshA, was ligated
to pTrcHis-A
that had been digested similarly, to yield pKK853. The
0.4-kb
NheI-
SacI fragment from pKK852 was ligated
to similarly digested
pKK853 to form pKK855, which contained the
hexahistidine coding
sequence fused to the N-terminal coding sequence
of
eshA.
To construct the null allele of
eshA, a PCR-amplified
fragment was generated by using an M13 reverse sequencing primer
(5'-TCACACAGGAAACAGCTATGA-3')
as the upstream primer and an
internal primer containing a
BclI
site
(5'-CGGGAGGTGATCACCTGCATCTGCG-3', corresponding to nt 456
to
432 of the 2.4-kb
SalI fragment) as the downstream primer.
The amplification product was digested with
EcoRI and
BclI and
ligated to pKK847 that had been similarly digested.
This ligation
generated pKK2011, which contained a deleted version of
eshA comprising
the first 116 nt and the last 515 nt of the
eshA coding sequence.
This plasmid was digested with
BclI and ligated to a 1.3-kb apramycin
resistance cassette
(obtained from P. Solenberg and R. Baltz in
pCZA263) from pKK974. This
ligation generated pKK2012. The Apr-disrupted
eshA allele
was transferred as a
BglII fragment from pKK2012 to
pKK1400,
which contains the 1.8-kb thiostrepton resistance cassette
(
60) in pIJ2920 (
27). This yielded pKK2014,
which was used
as described below to construct the
eshA null
mutant.
Growth and induction of sporulation.
E. coli
cultures were grown in Luria broth (2) supplemented with
ampicillin (100 µg/ml) or apramycin (100 µg/ml) as needed. Starter
cultures of S. griseus were grown in SpM (31)
for 2 to 5 days to generate spore suspensions that were then used as inoculum for induction of sporulation. Cultures of bald mutants were
treated similarly except that the SpM culture was 36 to 48 h old at the
time of subculture. SpM agar, supplemented as needed with apramycin (20 µg/ml) or thiostrepton (5 µg/ml), was used for maintenance of
streptomycete strains. SpMR (3) was used for protoplast
transformation. Trypticase soy broth (BBL Microbiology Systems), 2XYT
(53), or Luria broth was used for isolation of genomic and plasmid DNA from S. griseus
(24).
Two different methods were used to obtain sporulating submerged
cultures of
S. griseus. To induce sporulation by phosphate
starvation or nutritional downshift, 50 ml of glucose-ammonia
minimal
medium supplemented with 1% casein hydrolysate (U. S.
Biochemicals; salt-free) was inoculated with 0.05 to 0.5 ml of
an SpM
starter culture and was incubated at 30°C on a shaker (250
rpm) until
the absorbance at 500 nm reached 3.0. Then the culture
was harvested by
centrifugation at 22°C, was washed once with
prewarmed medium lacking
casein hydrolysate (nutritional downshift)
and phosphate (phosphate
starvation), and was transferred to 50
ml of identical prewarmed medium
in a 250-ml flask. The time of
transfer was considered 0 h
(equivalent to vegetative growth).
To obtain sporulation as a
consequence of nutrient exhaustion,
0.5 ml of SpM starter culture was
inoculated into 50 ml of SpM,
and incubation at 30°C and 250 rpm was
continued. Typically the
culture ceased exponential growth at an
A500 of 9, and free spores
and spore chains were
first evident 6 h
later.
Protein analysis.
To prepare crude extracts, vegetative or
sporulating cultures were harvested by centrifugation at
14,000 × g for 15 min at 4°C. The cells were washed
once with 1 M KCl and were suspended in 50 mM Tris-HCl, pH 7.5. Cells
were disrupted in a French press (14,000 lb/in2), and the
lysate was centrifuged at 14,000 × g for 5 min. The supernatant was used as the crude extract. Protein concentration was
determined by the ultraviolet absorbance method of Ehresmann et al.
(19). Equal amounts of protein in sodium dodecyl sulfate (SDS) sample buffer were applied, and the gels were run and stained according to standard methods (2, 36).
To examine total proteins by two-dimensional gel electrophoresis
(
48), we extracted proteins by using modifications of
published
methods (
20,
21). Cells harvested from a 50-ml
culture were
suspended in 2 ml of prechilled extraction buffer (50 mM
Tris-HCl,
pH 7.5, 5 mM MgCl
2). After disruption in a French
press (14,000
lb/in
2), the broken cells were centrifuged at
12,000 ×
g for 5 min and
the supernatant was saved.
Deoxyribonuclease I (Sigma; 100 µg/ml)
and ribonuclease A (Sigma; 50 µg/ml) were added to the supernatant,
and the mixture was incubated
at 4°C for 20 min. The digested
extract was combined with an equal
volume of 20% (wt/vol) trichloroacetic
acid in acetone and kept at
20°C for 30 min. The proteins were
collected by centrifugation as
above, were washed twice with cold
acetone, and were dried in vacuo for
5 min. The residue was dissolved
in 1 ml of solubilization buffer
containing 9.8 M urea, 4% Nonidet
P-40 (U. S. Biochemicals), 1%
Ampholine (pH 3.5 to 10; Pharmacia),
1% Pharmalyte (pH 2.5 to 5;
Pharmacia), and 2 mM
dithiothreitol.
The isoelectric focusing gel for the first dimension was prepared
according to the procedure of O'Farrell (
48) with minor
modifications (
20). The gel was prerun in a GT2 tube gel
unit
(Hoefer Scientific Instruments, San Francisco, Calif.) at 400
V
for 30 min and at 800 V for 30 min. Protein (25 µl) was loaded
and
was focused at 800 V for 12 h. The gel either was equilibrated
for
15 min in running buffer (
36) or was frozen at
20°C
and
equilibrated immediately before electrophoresis in the second
dimension. To determine the pI, a tube gel was cut in 5-mm slices,
and
each slice was macerated in 0.5 ml of water. The pH of each
suspension
was then measured with a pH meter. After electrophoresis,
the gel
either was fixed in 50% methanol and 10% acetic acid and
stained with
Coomassie blue R-250 or was immediately prepared
for electrophoretic
transfer (Idea Scientific Co., Minneapolis,
Minn.) onto polyvinylidene
difluoride membrane (Bio-Rad). CAPS-NaOH
(pH 11.0; 10 mM)-10%
methanol was used as a transfer buffer. The
membrane was stained with a
0.1% (wt/vol) solution of Ponceau
S in 1% acetic acid. Protein spots
were excised from multiple
membranes and were sent to the Harvard
Microchemistry Facility
for amino acid analysis and sequence
determination.
Anti-EshA antibodies were made by purification of soluble EshA from the
His tag system (Invitrogen) according to the manufacturer's
instructions. EshA was the only protein evident in the preparation
by
polyacrylamide gel electrophoresis (PAGE) after elution with
500 mM
imidazole in 50 mM Tris-HCl, pH 7.5, and 0.3 M NaCl. A
homogenate of
the pure protein in polyacrylamide was used to immunize
chickens
(Cocalico Biologicals, Reamstown, Pa.). Rabbit anti-chicken
antibodies
conjugated to alkaline phosphatase were from Jackson
Immunoresearch and
were detected by using the chromogenic method
(Boehringer
Mannheim).
To isolate total proteins for immunoblot hybridization, equal biomasses
of cells (0.25 to 1.0 ml of liquid cultures) were
harvested by
centrifugation from different growth stages (during
sporulation by
phosphate starvation, nutritional downshift, and
SpM). The cell pellets
were resuspended in 0.5 ml of 50 mM Tris-HCl
(pH 7.5)-10 mM
MgCl
2, 2 mg of lysozyme was added, and the suspensions
were
incubated for 37°C for 10 min. The extracts were combined
with 2×
SDS sample buffer (
2,
36), and 30 µl of each extract
was
loaded onto the wells for SDS-PAGE. To isolate membrane proteins
from
cytoplasmic and ribosomal fractions, we used a modification
of the
procedure of Mikulik and Janda (
44). One milliliter of
the
starter culture (4-day culture in SpM) was inoculated into
50 ml of
SpM. The culture was harvested at 6 h after the culture
reached an
optical density at 500 mm of 9.0. This time point is
when the culture
enters stationary phase and starts to form sporogenic
hyphae. The cells
were harvested by centrifugation at 14,000 ×
g for 10 min. The cells were washed once and resuspended in 1.5
ml of buffer A
(20 mM Tris-HCl, pH 7.6, 10 mM MgCl
2, 0.5 mM
phenylmethylsulfonyl
fluoride). The cells were disrupted by a French
press at 14,000
lb/in
2. The cell lysate was centrifuged at
14,000 ×
g for 10 min at
4°C in a Beckman JA-20
rotor to remove the cell wall and other
debris. The crude lysate was
centrifuged at 44,000 ×
g for 30
min at 4°C to
remove some larger membrane fragments. The pellet
(P44) was resuspended
in 1.5 ml of buffer A. The supernatant (S44)
was carefully removed and
was centrifuged at 150,000 ×
g for 14
h. After
centrifugation, the supernatant (S150) was removed and
the pellet
(P150) was resuspended in 1.5 ml of buffer B (buffer
A + 1 M
NH
4Cl) at 4°C for 1 h and was layered over 10 ml of
a
50% sucrose cushion (10 mM Tris-HCl, pH 7.4, 500 mM
NH
4Cl, 10
mM MgCl
2, 6 mM 2-mercaptoethanol).
The cushion was centrifuged
at 150,000 ×
g for 10 h at
4°C. Four fractions (upper cushion,
brown layer, lower cushion, and
pellet) were isolated after the
centrifugation. The pellets were
dissolved in 1.5 ml of buffer
B. Thirty microliters of each protein
fraction was loaded onto
a slab gel for SDS-PAGE and Western
hybridization.
DNA manipulation and analysis.
Standard (2, 24,
53) or previously published (35) methods were used
for analysis and manipulation of DNA fragments for colony
hybridization, plasmid DNA minipreparations, and transformations. For
Southern analysis we used the Genius II system after transfer of the
DNA to a positively charged membrane (Boehringer Mannheim). We
generated nested deletions by using exonuclease III digestion (23) of pKK842 to determine the nucleotide sequence of
both strands of the 2.4-kb SalI fragment according to the
dideoxynucleotide method (53). We used Deep Vent
polymerase (New England Biolabs) for PCR amplifications according to
the manufacturer's recommendations.
The single-stranded DNA used in the S1 nuclease protection experiments
was produced by using Deep Vent polymerase with a single
oligonucleotide that was complementary to the coding sequence.
The
downstream primers oligonucleotide 166 (5'-TGGACTGCCGGGGCACTTCCAG-3',
corresponding to nt 380 to
359 of the
SalI fragment) and oligonucleotide
167 (5'-TCCTGCATCTGCGGGGCGGACTT-3', corresponding to nt 444 to
422) were used to produce runoff polymerization. The plasmid pKK842
digested with
EcoRI was used as a template. The reaction
conditions
were similar to those recommended by the manufacturer for
amplification
of double-stranded DNA, except that 2 µl of dimethyl
sulfoxide
and 0.5 µg of linearized template DNA were combined in
reaction
buffer containing 3 mM MgCl
2, and the reaction
proceeded through
60 cycles. The single-stranded fragment was purified
by using
diatomaceous earth resin (
9) after
electrophoresis on a 1%
agarose
gel.
RNA studies.
RNA was purified from vegetative and
sporulating cultures of S. griseus NRRL B-2682, SKK1003,
SKK1008, and SKK1015 by a standard method (24) with some
modifications as follows. Ten microliters of vegetatively growing cells
(A500, 3.0) was harvested by centrifugation at
4°C and then was suspended in 10 ml of modified Kirby mixture (24). The cell suspension was passed through a French
pressure cell (14,000 lb/in2), and the lysate was collected
in 12-ml polypropylene tubes containing 5 ml of Tris-buffered
phenol-chloroform (1:1, pH 8.0). After vigorous vortexing for 1 min,
the mixture was centrifuged at 7,700 × g for 10 min.
The upper phase was reextracted, vortexed, and centrifuged. The aqueous
phase was recovered, and the RNA was precipitated, was digested with
DNase (RNase-free; Boehringer Mannheim) for 2 h at 37°C, and
then was extracted and precipitated as described above and dissolved in water.
To determine the transcription 5' end point, we used the S1 nuclease
protection assay (
24) by combining 50 µg of RNA with
100,000 cpm of end-labeled, single-stranded DNA probe. The sequencing
reactions were prepared with the same primer that had been used
to make
the
probe.
Gene disruption.
pKK2014 was passed through E. coli ET12567 to remove methyl groups from adenine and cytosine
residues and subsequently was methylated with a mixture of commercially
purchased methyltransferases, mAluI and mSssI, and endogenous
methyltransferases from S. griseus NRRL B-2682
(40). After overnight incubation at 30°C, the reaction mixture was extracted once with phenol-chloroform and twice with chloroform and then was precipitated with ethanol. The methylated DNA
was dissolved in water and was introduced into protoplasts of S. griseus NRRL B-2682 suspended in P buffer (24; J. Kwak, unpublished). Apramycin-resistant transformants were selected and
were screened for thiostrepton sensitivity; this phenotype was
indicative of a double-crossover event. Of 150 apramycin-resistant transformants, 3 were sensitive to thiostrepton, indicating that recombination had occurred on both sides of the apramycin cassette. Three such strains, SKK2025, -2026, and -2027, were identified.
Microscopy.
Phase contrast microscopy was performed with a
Zeiss D-7082 microscope equipped with a 35-mm camera. Since
streptomycete filaments grow in all directions, we improved the viewing
quality by applying 1-µl samples to the slides and spreading them as
thinly as possible with coverslips prior to viewing at ×1,000 total
magnification. TMax 400 film was used for the photographs. Either the
prints or the negatives were scanned and were imported as TIFF files into Corel PhotoPaint 8 for cropping and then into CorelDraw 8 for
labeling and assembly of composite photographs. The appearance of each
TIFF file was adjusted with Corel PhotoPaint 8 to resemble the
microscopic view as closely as possible.
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RESULTS |
Identification of sporulation-specific proteins.
To identify
proteins that accumulate to high levels under sporulation conditions
(phosphate starvation and nutritional downshift) but not during
vegetative growth and that may therefore depend on developmental genes,
we compared total proteins of sporulating cells with those of
vegetative cells by two-dimensional PAGE after staining with Coomassie
brilliant blue. By comparing the protein populations from both
sporulation conditions, we sought to exclude proteins that might have
been induced as a result of the phosphate regulatory network. Five
sporulation-specific proteins (named P1, P2, P3, P4, and P5) were
present up to at least 12 h after induction (Fig.
1); after this time, the thick walls made
the maturing spores refractory to breakage at high pressure in a French press (34). The molecular weights and isoelectric points
measured on two-dimensional PAGE are shown in Table
1. These proteins became visible at
different times during the first 12 h of sporulation (Table 1).
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FIG. 1.
Identification of sporulation-specific proteins by
two-dimensional PAGE in the wild-type strain of S. griseus.
Total protein profiles from vegetatively growing cells (A) and from
cells after 12 h of phosphate starvation (B) are shown. Arrowheads
mark five sporulation-specific proteins (P1 through P5). The numbers
indicate molecular mass standards in kDa. The proteins were stained
with Coomassie brilliant blue R-250. The proteins are more horizontally
spread in panel B than in panel A due to longer running during
isoelectric focusing.
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TABLE 1.
Accumulation of developmentally regulated proteins in the
wild-type strain of S. griseus during the first 12 h of
sporulationa
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Since our hypothesis that proteins P1 through P5 were sporulation
specific implied that developmental mutants might lack some
or
all of these proteins, we looked for their production in class
III nonsporulating mutants of
S. griseus (
3).
The class III
mutants, which include
bldA mutants (
34,
35) as well as those
with mutations in other uncharacterized
genes, have complex phenotypes.
The colonies appear bald on an agar
medium that ordinarily supports
luxuriant sporulation. When induced to
sporulate in liquid culture
by phosphate starvation or nutritional
downshift, the class III
mutants prematurely undergo nucleoid
segregation, septation, and
spore maturation throughout the preexisting
vegetative filaments
(
34). Ectopic development of
septation and spore formation in
the mutants is accompanied by
premature fragmentation of the nascent
spore chains. Thick spore walls
form in these mutants by 6 h (class
IIIA and C) to 8 h
(
bldA [class IIIB]) of sporulation, fully 4
to 6 h
earlier than in the wild-type strain (
34). Two-dimensional
gel electrophoresis revealed that extracts prepared from all class
III
mutant strains during the first 6 h of sporulation lacked
P3
(EshA) and P4, whereas P1 and P2 were present in the extracts.
P5 was
produced at high levels by 4 h of phosphate starvation
in the
bldA mutant (Table
2), which
is 4 h earlier than in the
wild-type strain. We chose P3 (EshA)
for further study.
The eshA gene encodes a protein related to
stress-induced proteins of other bacteria but with a likely cyclic
nucleotide-binding domain.
The N-terminal amino acid sequence of
EshA was identified as TVDSTSEARLEVPRQ. From this sequence a
degenerate oligonucleotide (5'-GARGCSMGBCTSGARGTSCC-3',
corresponding to the seventh through thirteenth amino acid of the
sequence) was synthesized and was used to identify the eshA
gene by Southern hybridization. A 2.4-kb SalI fragment of
genomic DNA from wild-type S. griseus hybridized to the
oligonucleotide. Two streptomycete open reading frames (ORFs) were
found in the DNA fragment by the Frame computer program (67). The first ORF encoded a polypeptide of 470 amino
acids with a molecular mass of 52 kDa and an isoelectric point of 5.3. The second one was a partial ORF. The complete ORF (GenBank accession no. L76204) corresponded to the eshA structural gene because (i) the N-terminal sequence of the mature protein was identical to that
deduced from the nucleotide sequence (less the fMet); (ii) the
calculated isoelectric point of the deduced protein (5.3) was close to
that of EshA (5.5) on the basis of its migration in the first dimension
of two-dimensional gel electrophoresis; and (iii) the calculated and
observed molecular masses were identical (52 kDa). The translation
initiation codon of eshA occurred 11 nt downstream of a
strong ribosome-binding site (AGGAG; 32). Two alternative,
secondary structures could be formed from the sequence immediately
downstream of the TAG stop codon: one contained two adjacent, imperfect
inverted repeats (nt 1743 to 1766 and nt 1772 to 1799; 
G = 31
kcal/mol) and the other contained a single, imperfect inverted repeat
(nt 1743 to 1796; G = 35 kcal/mol) resembling a
factor-independent transcription terminator (15).
If EshA is an important protein in streptomycete sporulation, then we
would expect it to be present in diverse species. We
therefore examined
S. coelicolor and its close relative
Streptomyces lividans for the occurrence of
eshA-related DNA
sequences by Southern
hybridization using a 1.5-kb
SphI
fragment that contained the
eshA coding sequence, 31 nt of
upstream DNA, and 93 nt of downstream
DNA. Both species showed
identical patterns: a 4.3-kb
BamHI fragment,
a 7.5-kb
PstI fragment, and a 2.4-kb
SalI fragment
hybridized
at moderate stringency (
75% identity) to the probe
(data not
shown). Additionally, a BLAST search of the partial
genome sequence
of
S. coelicolor
(
www.sanger.ac.uk/Projects/S_coelicolor/blast_server.shtml)
revealed the presence of an ORF that is 76% identical (85% similar)
to the deduced amino acid sequence of
S. griseus eshA (Fig.
2A).
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FIG. 2.
Alignment of the amino acid sequence of S. griseus EshA (Sg EshA) with homologous proteins and the
cNMP-binding domain. (A) Alignment with MMP-1 (SwissProt accession no.
P46841), SrpI (SwissProt accession no. Q55032), and S. coelicolor EshA (Sc EshA;
www.sanger.ac.uk/Projects/S_coelicolor/blast_server.shtml). (B)
Alignment of the cNMP-binding domain. The cNMP-binding domain sequence
given is the most probable amino acid at each position for the
cNMP-binding superfamily described by McCue et al. (42).
The shaded amino acids in rectangles represent amino acid identities,
and bold fonts show similar amino acids (A and G; M, I, L, and V; F, W,
and Y; D and E; N and Q; K, R, and H; S and T) that are present in at
least three of the four proteins. The seven amino acids shown to be
involved in cNMP binding are marked with asterisks.
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|
A BLAST 2.0 search of the nonredundant database at the National Center
for BioTechnology Information (NCBI) (Fig.
2A;
1)
revealed
that the deduced amino acid sequence of the
eshA gene
shared
significant similarity with a mycobacterial protein (MMP-1
from
Mycobacterium leprae [SwissProt accession no.
P46841]
and
a 35-kDa protein from
Mycobacterium avium complex
[SwissProt
accession no.
Q48899]) and with SrpI from the
cyanobacterium
Synechococcus PCC7942 (SwissProt accession
no.
Q55032) (
61).
Although the function of neither
orthologous protein is known,
MMP-1 is immunodominant in infected hosts
(
65) and SrpI is the
deduced product of the third gene of
a three-gene operon that
is strongly expressed during sulfur starvation
(
47). Alignment
of these proteins showed that all three
shared extensive similarity
at the N terminus and within the C-terminal
half (Fig.
2A). The
C terminus of each may contain a 14- to
17-amino-acid transmembrane
anchor (PHDhtm program;
51).
Noteworthy in this alignment was a domain in EshA that was absent from
MMP-1 and SrpI (Fig.
2A). Multiple sequence alignment
(
42)
and domain searches (
4) revealed significant homology
of
this domain with cyclic nucleotide (cNMP)-binding domains from
both
procaryotic and eucaryotic cNMP-binding proteins (Fig.
2B).
This family
of proteins contains those in which the activity is
modulated by a
cNMP, such as
E. coli Crp, cNMP-dependent protein
kinases,
and gated ion channels (
42,
56). These cNMP-binding
proteins have five glycine residues believed to be important for
formation of the cNMP-binding pocket and two conserved residues
that
interact with the cNMP (Fig.
2B;
42). Dot plot analyses
(The University of Wisconsin Genetics Computer Group Program;
16) also showed strong similarity to the cNMP-binding
domains
of the catabolite activator protein from
Haemophilus
influenzae and the cGMP-dependent protein kinase from
Drosophila melanogaster (data not shown). Computer analyses
using PROBE (
46) and Classifier
(
50) also
demonstrated that EshA shared homology with procaryotic
and eucaryotic
proteins containing cNMP-binding
domains.
EshA copurifies with the membrane.
To confirm the expression
of EshA during sporulation, we performed Western hybridization
experiments using a chicken antibody raised against recombinant EshA.
We induced submerged sporulation by using the complex medium SpM
(31), as well as by phosphate starvation and nutritional
downshift. As expected, EshA was detected in cell extracts of all
sporulated cultures (Fig. 3A). EshA was absent in extracts prepared from all class III nonsporulating mutants
that had been sporulated by the same induction methods (data not
shown).
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FIG. 3.
Detection of EshA by immunoblotting. (A) The
accumulation of EshA during phosphate starvation (PS), nutritional
downshift (ND), and SpM culture. The numbers indicate the hours of
culture after shift into the sporulation induction medium. 0 shows the
vegetatively growing cell immediately before the shift. V and S
represent the vegetative cells and sporulating cells in liquid SpM
culture (see Materials and Methods). (B) Detection of EshA using
immunoblotting from crude extract (lane 1), S44 (lane 2), P44 (large
membrane fragments; lane 3), S150 (lane 4), P150 (lane 5), upper
cushion (lane 6), brown layer (lane 7), lower cushion (lane 8), and the
pellet (lane 9; ribosomal fraction; 44).
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|
MMP-1 copurified with membrane fractions (
65), and the
program PHDhtm (
51) showed that EshA, MMP-1 from
mycobacteria,
and SrpI from
Synechococcus contained putative
transmembrane anchor
domains at their C termini. To test whether EshA
is a membrane
protein, we separated the membrane fraction from
cytoplasmic and
ribosomal fractions by using differential
centrifugation and detected
EshA by using Western hybridization. EshA
was much more abundant
in the protein extracts containing membrane
fraction and large
membrane fragments (Fig.
3B, lanes 1, 2, 3, 5, and
8) than in
the cytoplasmic or ribosomal fractions (Fig.
3B, lanes
4, 6, 7,
and
9).
eshA is developmentally regulated and depends on class
III developmental genes.
Two eshA transcripts were
identified by a high-resolution S1 nuclease protection assay using a
single-stranded probe synthesized from oligonucleotide 167 (Fig.
4B). The 5' endpoint of the major transcript mapped 113 nt upstream and the longer one mapped
approximately 300 nt upstream of the translation start codon (Fig. 4A
and B). However, the longer signal was not evident when the probe
synthesized from oligonucleotide 166 was used, suggesting that the
longer signal could be attributed to an artifact. The same site was
identified for the major transcript by using both probes (from
oligonucleotides 166 and 167), and the endpoint was identical for
cultures that were induced to sporulate by either phosphate starvation
or nutritional downshift. The eshA transcript was
undetectable in RNA from vegetative hyphae but was abundant in RNA
extracted at 2 h of sporulation, the earliest time that was
examined. A time course analysis showed that the level of
eshA transcript remained high through 12 h of sporulation (Fig. 4B). The appearance of the eshA transcript
by 2 h of sporulation correlated well with the abundance of the protein at 4 h. PCR amplification and nuclease protection analysis
confirmed that the class III mutants contained the eshA gene
but lacked the transcript (Fig. 4C). There was a putative promoter,
including 10 (TAGTGT) and 35 (TTGGTC) regions
measured from the 5' endpoint of the eshA transcript,
resembling the consensus sequence recognized by
hrdB in Streptomyces (28,
57). The nucleotide sequence in the regions was identical in 16 of 20 positions to the sporulation-specific promoter (Pspo)
of ftsZ in S. griseus (Fig.
5), which is transcribed at high levels
only during sporulation (J. Kwak, unpublished).
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FIG. 4.
Analysis of eshA transcripts by S1 nuclease
protection. A single-stranded 400-nt DNA probe (including 20 nt of
pGEM-4Z polylinker and 328 nt upstream and 52 nt downstream of the
translation start site of the eshA structural gene) was
prepared by using oligonucleotide 166 for a primer extension in panel
A. Oligonucleotide 167, which extended 116 nt downstream of the
initiation codon, was used for the experiments shown in panels B and C. In each case the sequence ladder (A, G, C, T) was extended from the
same primer and used as the size marker. (A) Identification of the 5'
end of the eshA transcript in the wild-type strain of
S. griseus. P, RNA from cells that had been starved for
phosphate for 4 h; N, RNA from cells that had been subjected to
nutritional downshift for 4 h. The nucleotide sequence shown to
the left of the autoradiogram is that of the sense strand. The asterisk
marks the 5' end of the transcript, and the nucleotide numbers refer to
positions in the 2.4-kb SalI fragment (GenBank accession no.
L76204). (B) Time course of eshA transcription during
phosphate starvation. Total RNA was prepared from vegetative cells (V)
and cells that had been starved for phosphate for 2, 4, 6, 8, 10, or
12 h. E. coli tRNA (E) served as the negative control.
(C) Analysis of the eshA transcript in the class III mutants
during phosphate starvation. The sequence ladder and probe were
prepared as described for panel B. Total RNA was prepared from the
wild-type strain of S. griseus and class III developmental
mutants SKK1015 (class IIIA), SKK1008 (bldA; class IIIB),
and SKK1003 (class IIIC) that were growing vegetatively (V) or starved
for 2 or 4 h. The film was deliberately overexposed to demonstrate
the absence of signal in the mutants.
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FIG. 5.
The nucleotide sequence homology of the promoter regions
between eshA and ftsZ. The shaded sequences mark
the similar regions. The bold letters inside the shaded boxes show the
putative 10 and 35 regions. n indicates the spacings between the
putative 10 and 35 regions. +1 indicates the 5' end of transcripts
from both genes detected by high-resolution S1 nuclease mapping.
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|
An eshA mutant is defective in sporulation-specific
growth.
We constructed an eshA null allele by replacing
the coding region from Glu39 to Leu300 with an apramycin resistance
cassette. The disrupted structure of the eshA gene was
confirmed by Southern hybridization analysis; all three isolates
contained the 2.9-kb SalI fragment expected for the
disrupted gene and lacked the 2.4-kb SalI fragment
characteristic of the intact eshA gene (data not shown). As
expected, both fragments were present in a transformant resistant to
both apramycin and thiostrepton. An immunoblot showed that the
wild-type strain but not the null mutants produced EshA within 6 h
after the end of exponential growth in nutrient exhaustion medium (data
not shown).
We were not able to detect any difference in growth and sporulation
between the null mutant and the wild-type strain when
sporulation
occurred on solid media. Like the wild-type strain,
the disrupted
mutant produced streptomycin and grew at a normal
rate (µ = 0.47 ± 0.03 h
1) in the complex medium. On buffered
SpM agar, which permits abundant
sporulation of the wild-type strain,
the mutant sporulated to
the same extent as the wild-type strain: the
colony was as hairy
as the wild-type strain and the mutant produced
abundant spores
when observed with a microscope. Therefore, we turned
to the submerged
sporulation system (nutritional downshift) for more
detailed analysis.
Under this condition, the wild-type strain follows a
reproducible
time course of sporulation (
22,
34). In
liquid culture, the
sporulation yield (sonication resistance units) of
the
eshA mutant
was the same as that of the wild-type strain
(5 × 10
9 to 7 × 10
9 sr/ml;
34). The spores from these mutants did not have any
defects in sonication resistance, lysozyme tolerance, or germination
(
31,
34) when compared to the wild-type strain. The
dramatic
aspect of the mutant phenotype during submerged sporulation
was
the aborted growth of branch sporogenic hyphae (Fig.
6). Phase
contrast microscopy showed that
the defective branch sporogenic
hyphae initiated growth but failed to
extend beyond a length of
about 1 to 2 µm, even after prolonged
incubation. Instead, septation
and spore maturation were accelerated at
the tips of preexisting
vegetative hyphae or de novo synthesized distal
sporogenic hyphae
(Fig.
6). Frequently these stunted branch sporogenic
hyphae became
swollen at their bases, perhaps indicative of isotropic
rather
than polar growth. By 12 h, almost all of the deformed
branch
sporogenic hyphae had detached from the vegetative hyphae (Fig.
6).
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FIG. 6.
Phase-contrast photomicroscopy of the eshA
null mutant (SKK2025) and the wild-type strain of S. griseus
after 6, 8, and 12 h of sporulation induced by nutritional
downshift. Sporogenic hyphae emerging from new branches (Br) and from
preexisting vegetative hyphae (Tip) are marked. SC marks the spore
chains. The bar represents 6.0 µm. No differences were apparent in
vegetative cultures of the mutant and wild-type strains.
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|
The
eshA null mutant formed septate spore chains at the
vegetative hyphal tips at 8 to 10 h. After 12 h of induction
the nascent
spore chains of the mutant were fragmented and released. In
comparison,
the wild-type strain requires 12 h to undergo
septation and an
additional 10 h to form fragmented, mature spore
chains (Fig.
6;
34). The spore chains that formed in the
null mutant were
abnormal, however, because the distal "spore" in
the chain generally
appeared larger when compared to other spores in
the chain (Fig.
6). The septated region was noticeably longer than the
"distal"
sporogenic hyphae of the wild-type
strain.
To demonstrate that the phenotype was caused by the deletion of the
eshA gene, we used two different strategies to complement
the mutant. In the first case, we introduced the wild-type allele
contained in either the 2.4-kb
SalI fragment on pXE4
(pKK856)
or the 8-kb
BamHI-
HindIII fragment
containing
eshA plus approximately
3.5 kb of upstream and
2.5 kb of downstream sequences (pKK1416).
Then we compared sporulation
of these transformants with those
that contained the vector only. In
the second approach, we constructed
the same 8-kb
eshA
fragment in the nonreplicative vector pKK1400
at
BamHI and
EcoRI sites (pKK1417) and generated a single crossover
strain by integrating into the chromosome by homologous recombination
at the
eshA locus. Because the presence of thiostrepton in
the
medium (to select for the maintenance of the plasmid) lengthened
the time required for development of both the wild-type strain
and the
mutant, we excluded thiostrepton from the growth and nutritional
downshift media. At the end of the experiment, the viable count
of
spores (measured as sonication-resistant units per ml;
34)
showed that thiostrepton resistance was maintained in more than
90% of
the spores. This indicated that there was little if any
loss of the
plasmid during the course of the experiment. The null
mutant was
complemented by the
eshA gene in
trans; branch
sporogenic
hyphae formed to the same extent as in the wild-type strain
(data
not shown). Since the complementing fragment lacked a complete
downstream ORF, we conclude that the mutant phenotype was caused
by the
lack of
EshA.
| |
DISCUSSION |
EshA is a stress-response protein.
EshA is an abundant protein
that accumulates during sporulation induced by phosphate starvation and
nutritional downshift in S. griseus. Similar proteins are
present in a cyanobacterium and two species of mycobacteria. Although
the functions of these homologous proteins are not known, they are
produced abundantly when the cells experience nutritional stress: SrpI
when Synechococcus encounters sulfur stress and MMP-1 during
infection by M. leprae, which multiplies in phagosomes of
the infected host (65). Since EshA is also made during
nutritional stress, we have proposed that EshA, SrpI, and MMP-1 define
a new family of bacterial stress-response proteins (61).
MMP-1 is believed to be a membrane protein and is routinely isolated as
multimers in excess of 20 subunits (65; W. J. Britton, personal communication). Our results also showed that EshA
localizes to the cell membrane (Fig. 3B) and possibly exists as large
multimers (J. Kwak, unpublished). A putative transmembrane anchor in
EshA, MMP-1, and SrpI was found by using the program PHDhtm
(51). In light of its abundance and and localization to
the membrane, we speculate that EshA may be part of a structural element. There is no EshA ortholog in E. coli, Bacillus
subtilis, or other procaryotes whose genomes have been completely
sequenced. A gene apparently homologous to EshA was also present in
S. coelicolor and S. lividans, two species that
are not closely related to S. griseus (32).
EshA is a determinant of growth of sporogenic hyphae.
This
study demonstrated that EshA is required for the maintenance of growth
of sporogenic hyphae from de novo branches and for proper formation of
distal sporogenic hyphae, since the absence of EshA resulted in
abortive growth of branch sporogenic hyphae. We do not exclude the
possibility that EshA may function as an inhibitory factor of septum
formation in the preexisting vegetative hyphae until sporogenic hyphae
are fully mature. However, EshA does not seem to be required for
vegetative growth and branching, sporulation septum formation, or spore
maturation. Despite the absence of full-length sporogenic hyphae in the
null mutants, spore chains and mature spores formed from the
preexisting vegetative hyphae. Consistently, the spores from these
mutants did not have any defects in sonication resistance, lysozyme
tolerance, or germination (31, 34) when compared to the
wild-type strain. The premature septation and fragmentation
demonstrated that these spore chains form ectopically by transformation
of the vegetative filaments (Fig. 6 and
7). The ectopic septation and spore
formation from the preexisting vegetative or distal sporogenic mycelia
without mature growth of proximal sporogenic hyphae (34)
are also observed in the class III nonsporulating mutants that do not
accumulate EshA during sporulation. Taking into consideration its
possible existence as a structural component, and the phenotypes
observed in the eshA null mutants and the class III mutants,
it is conceivable that this aspect of the null mutant phenotype could
be caused by a high local concentration of factors required for
septation and spore maturation that might mislocalize to the vegetative hyphae in the absence of sporogenic hyphal growth (Fig. 6 and 7). For
this reason, we named P3 as EshA (extension of sporogenic hyphae). We
speculate that the aberrant phenotype of the mutant observed in
submerged culture also occurs on solid culture. The delicate
morphological changes may not have been detected, since the cells
harvested from solid cultures were more heterogeneous and less
synchronous.
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FIG. 7.
Diagram of alternative ways in which spores could be
made in the null mutant. Initial formation of the sporogenic hyphae is
unaltered (step 1). Subsequently, in the absence of EshA the proximal
sporogenic hyphae form a bulbous structure characteristic of apolar
growth. Simultaneously the apex of the distal sporogenic hypha becomes
deformed (step 2). The spore structures arise either by ectopic
septation and maturation in the preexisting vegetative hyphae (step 3A)
or by proper localization in the distal sporogenic hypha following a
period of new growth (step 3B).
|
|
Our assignment of EshA as a protein required for growth of sporogenic
hyphae in
Streptomyces raises the question of the roles
of
the orthologous proteins in mycobacteria and
Synechococcus.
Limitation of
Synechococcus for sulfur leads to the global
activation
of a number of genes whose products are responsible for the
utilization
of alternative sources of sulfur (
37),
degradation of light
harvesting centers (
17,
55), and
formation of a quiescent
structure that requires extensive remodeling
of the cell membrane
and wall (A. R. Grossman, personal
communication). In view of
the
eshA null phenotype, we
hypothesize that SrpI may be necessary
for the morphological changes
that occur under this condition.
Although mycobacteria are members of
the
Actinomycetales that
grow with rudimentary branching,
whether
M. leprae displays morphological
changes during
infection coincident with synthesis of MMP-1 is
not known (P. J. Brennan and W. J. Britton, personal
communication).
Developmentally regulated expression of EshA.
Because the
eshA transcript was evident only during sporulation,
expression of eshA is regulated at least in part at the
transcriptional level. There was a single transcript 5' endpoint,
regardless of whether sporulation was induced by phosphate starvation
or nutritional downshift. This endpoint could alternatively correspond
to a site at which a longer transcript was processed, but our results
suggest that the 5' endpoint of the eshA transcript marks
the transcription start site since there was a putative promoter
including 10 and 35 regions from the 5' endpoint and there was no
evidence of a transcript 5' end further upstream. Although either
inverted repeat downstream of eshA may act as a
factor-independent transcription terminator, we do not yet know whether
eshA and the downstream ORF are cotranscribed.
The bald colony morphology of the class III mutants suggests that none
of these mutations is allelic to
eshA. Indeed, we know
that
this is true for the class IIIB (
bldA) mutants. Moreover,
the
eshA null mutant produced streptomycin, unlike the class
III
mutants. The absence of the
eshA transcript in the class
III mutants
was a consequence of the lack of gene expression. This
result
shows that the class III mutants are defective in regulatory
factors
that mediate synthesis of EshA and other proteins necessary for
sporulation and antibiotic production. One of these factors is
a tRNA,
the product of
bldA. The requirement of the class III
developmental genes, including
bldA, for transcription of
eshA prompted us to reexamine the morphology of a
bldA mutant of
S. griseus. We confirmed the
previous results (
34) indicating that
these mutants did
not make branch sporogenic hyphae, that the
formation of spore chains
in this strain occurred ectopically,
and that this mutant underwent
premature fragmentation of developing
spore chains. The observation
indicates that the aberrant phenotype
of class III mutants is caused to
a considerable extent by the
absence of
EshA.
We speculate that the known sporulation-specific sigma factors are not
responsible for transcription of
eshA. We can readily
rule
out recognition by WhiG;
whiG mutants are blocked at a later
stage of sporulation because
whiG mutants form lengthy
aerial
hyphae but not spore compartments (
11,
30).
Likewise,
F is not a candidate because it is required
for the relatively
late events of spore maturation (
49).
However, the putative
promoter for
eshA including the
10
and
35 regions is highly
homologous to the consensus sequence
recognized by
hrdB (
28,
57),
which is the essential sigma factor in
Streptomyces (
8). These results suggest that class III developmental
genes,
including
bldA, regulate the transcription of
eshA during sporulation
via a regulatory factor other than a
sigma
factor.
The role of cyclic nucleotides in Streptomyces.
EshA contains a cNMP-binding domain which is absent from the
corresponding proteins from Synechococcus and
Mycobacterium. Such a domain is present in proteins whose
activity is modulated by a cNMP but not in proteins that hydrolyze
cNMPs (56). The eucaryotic cGMP-dependent
phosphodiesterases and cAMP receptor proteins of
Dictyostelium also contain distinctly different cNMP-binding domains. EshA is a member of the cNMP-binding protein superfamily identified using PROBE (46) and Classifier
(50). In E. coli Crp (62) and the
regulatory unit of bovine cAMP-dependent kinase (58), for
which the three-dimensional structure has been solved, seven invariant
amino acid residues are required for interaction with the cNMP: the
stability of an eight-stranded 
-barrel likely depends on the five
Gly residues; the ribose 2'OH of cAMP is hydrogen bonded to Glu72; and
Arg82 interacts with one of the exocyclic oxygens of the phosphate
moiety (42). These seven residues are all conserved in
EshA (42).
Cyclic nucleotides play widespread regulatory roles in eucaryotes,
where they govern processes such as polarized cell growth
in
filamentous fungi (
6) and cation conductance in retinal
photoreceptors (
66). The known role of cyclic nucleotides
in
procaryotes is, up to now, much more limited. The only procaryotic
proteins known to bind cAMP are Crp and DnaA (
25). In
proteobacteria,
which include enteric bacteria and members of
phylogenetically
closely related gram-negative genera, Crp controls a
variety of
regulons, including those involved in catabolism
(
5), virulence
(
14,
64), and development of
competence (
10). With the exception
of Crp, however, many
of the other members of the Crp-like protein
family, such as Fnr and
FixK, have lost one or more of the amino
acids thought to play central
roles in binding to cAMP (
42)
while retaining their
DNA-binding domains. Cyclic AMP has not
been identified as an effector
in the low-GC gram-positive bacteria.
B. subtilis does not
produce cAMP (P. Setlow, personal communication)
and lacks a gene
resembling those encoding either adenylate or
guanylate cyclase. Cyclic
diguanylic acid functions as a reversible
allosteric activator of
cellulose biosynthesis in
Acetobacter xylinum
(
63).
Streptomycetes accumulate cNMPs both intracellularly and
extracellularly. Recent studies have shown that cAMP accumulates
to a
high extracellular level during the second exponential growth
phase of
S. griseus (
45) and
S. coelicolor
(
59). The gene
encoding adenylate cyclase has been
isolated from
S. coelicolor (
13). In addition
to its inability to synthesize cAMP, a
cya null mutant shows
a complex phenotype that includes impaired initiation
of growth during
spore germination and morphogenesis; the latter
defect is brought about
by the inability to overcome the acidic
environment generated by the
extracellular accumulation of organic
acids from glucose at the end of
the first exponential growth
period (
59).
| |
ACKNOWLEDGMENTS |
We thank Keith Chater, Chuck Daniels, and Tina Henkin for
critically commenting on the manuscript. We also thank Tom Thompson for
helpful discussion, R. Baltz and P. Solenberg for providing the
apramycin-resistance cassette, D. MacNeil for providing the E. coli ET12567 strain, Bill Lane of the Harvard Microbiochemistry Lab for amino acid sequence determination, Tim Vojt and Don Ordaz for
photoimaging processing, and Minho Kim for computer drawing.
This work was supported by grant MCB-9724038 from the National Science Foundation.
| |
FOOTNOTES |
*
Corresponding author. Present address: Institute for
Structural Biology and Drug Discovery, 800 E. Leigh St., Virginia
Biotechnology Park, Richmond, VA 23219. Phone: (804) 828-7573. Fax:
(804) 827-3664. E-mail: kwak91{at}hotmail.com.
Deceased.
| |
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Journal of Bacteriology, May 2001, p. 3004-3015, Vol. 183, No. 10
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.10.3004-3015.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
