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J. Biol. Chem., Vol. 277, Issue 30, 26753-26760, July 26, 2002
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From the Departments of
Received for publication, November 26, 2001, and in revised form, April 8, 2002
Two genes, fruA and csgA,
encoding a putative transcription factor and C-factor, respectively,
are essential for fruiting body formation of Myxococcus
xanthus. To investigate the role of fruA and
csgA genes in developmental gene expression, developing cells as well as vegetative cells of M. xanthus wild-type,
fruA::Tc, and csgA731 strains were
pulse-labeled with [35S]methionine, and the whole cell
proteins were analyzed using two-dimensional immobilized pH
gradient/SDS-PAGE. Differences in protein synthesis patterns among more
than 700 protein spots were detected during development of the three
strains. Fourteen proteins showing distinctly different expression
patterns in mutant cells were analyzed in more detail. Five of the 14 proteins were identified as elongation factor Tu (EF-Tu), Dru,
DofA, FruA, and protein S by immunoblot analysis and mass spectroscopy.
A gene encoding DofA was cloned and sequenced. Although both
fruA and csgA genes regulate early development
of M. xanthus, they were found to differently regulate
expression of several developmental genes. The production of six
proteins, including DofA and protein S, was dependent on
fruA, whereas the production of two proteins was dependent
on csgA, and one protein was dependent on both
fruA and csgA. To explain the present findings,
a new model was presented in which different levels of FruA
phosphorylation may distinctively regulate the expression of two groups
of developmental genes.
Myxobacteria, Gram-negative soil bacteria, provide an
excellent model system for studying multicellular morphogenesis and cell differentiation since nutrient starvation on a solid surface induces their multicellular development. One example is the formation of fruiting bodies in Myxococcus xanthus (1, 2). M. xanthus cells grow vegetatively in nutrient medium with a doubling
time of 4 h. Upon nutrient starvation on a solid surface, they
begin to gather in an aggregation center by gliding. Within 4-12 h
post-starvation, they form mounds that eventually convert into fruiting
bodies. Within the fruiting bodies, the motile, rod-shaped vegetative cells differentiate into non-motile, spherical myxospores. Rippling, a
synchronous cell movement resembling traveling waves, occurs during the
early phase of aggregation (3).
Analysis of programmed gene expression and protein synthesis during
development is important to understand the M. xanthus developmental process. Using one-dimensional polyacrylamide gel electrophoresis, Inouye et al. (4) showed that at least 15 proteins were specifically synthesized during the development of
M. xanthus, suggesting that development is governed by a
highly ordered program. They identified several major developmental
proteins including proteins S and U, which were subsequently
extensively characterized (5, 6). Kroos et al. (7) generated
many M. xanthus strains containing random insertions of
Tn5 lac, a Tn5-derived transposon
carrying a promoterless lacZ gene. A set of developmental
gene fusions to Tn5 lac has been identified and used as developmental markers to analyze the developmental process. Each marker Tn5 lac insertion strain initiates
The exchange of five extracellular signals, A-, B-, C-, D-, and
E-signal, is required to execute normal development of M. xanthus (8-11). Mutations defective in C-signaling resulted in a
developmental arrest at 6 h post-starvation and diminished
expression of Tn5 lac fusions that are normally expressed
after 6 h (12). C-signal mutants are unable to aggregate and are
defective in sporulation (3). All C-signal mutations were mapped to the csgA gene encoding a homologue of short chain alcohol
dehydrogenase (13). A 25-kDa protein encoded by the entire
csgA gene was produced at a basal level during vegetative
growth and induced during development, whereas a 17-kDa protein was
produced after 6 h post-starvation (14, 15). The 17-kDa
csgA protein is a cell surface associated-protein called
C-factor and carries an activity that extracellularly complements the
csgA mutations during development (16-18).
The fruA gene encoding a putative transcription factor
essential for the development of M. xanthus has been
identified (19). The amino acid sequence of FruA protein contains a
DNA-binding motif and shares similarity with response regulators of
two-component His-Asp phosphorelay signal transduction systems (19,
20). FruA protein possesses an aspartic acid residue that can be
potentially phosphorylated. Analysis through the introduction of
mutations into the aspartic acid residue suggested the possibility of
C-signal-dependent phosphorylation of FruA (20). The
expression of the fruA gene initiates at 6 h
post-starvation and increases to reach a maximal level after 12 h
(19). The fruA mutant exhibited a phenotype similar to that
of the csgA mutant (19, 21). A model in which C-signal
transduction requires FruA function was proposed (20, 21). However,
little is known about the C-signaling system and the FruA signal
transduction cascade.
To investigate the effects of csgA and fruA
mutations on developmental gene expression in M. xanthus,
developing cells as well as vegetative cells were pulse-labeled with
[35S]methionine, and whole cell proteins were analyzed
using two-dimensional immobilized pH gradient/sodium dodecyl
sulfate-polyacrylamide gel electrophoresis
(GE),1 which has been used to
analyze heat-shock-induced proteins in M. xanthus (22). At
least 14 proteins exhibited apparently different expression patterns
among the wild-type, fruA::Tc, and
csgA731 strains during development. Five of the 14 proteins
were identified by immunoblot analysis and mass spectroscopy. We
succeeded in cloning a novel gene, dofA, whose expression
was dependent on FruA function. These proteins are useful as
developmental markers for the analysis of M. xanthus
fruiting body formation.
Bacterial Strains and Media--
M. xanthus strains
DZF1 sglA1 (4), MO1 sglA1
fruA::Tc
For the cloning of M. xanthus DNA, Escherichia
coli DH5 Protein Labeling of Vegetative and Developing Cells with
[35S]Methionine--
To label cellular proteins during
vegetative growth, M. xanthus DZF1 cells were grown in CTT
medium at 30 °C and harvested in the exponential phase of growth.
The cells were resuspended in A1 medium containing 67 µM
[35S]methionine (0.33 mCi/mmol) and further grown at
30 °C for 30 h.
To induce development, concentrated M. xanthus DZF1, MO1
fruA::Tc, and DK731 csgA731 cells were spotted on
TM agar plates as described previously (4). After 1, 4, 8, 12, 18, and
24 h, cells were labeled for 2 h by adding 0.5 mCi of
[35S]methionine under the TM agar.
Two-dimensional GE of Cell Proteins--
Two-dimensional
GE was performed as described by Otani et al. (22) with
slight modifications. Briefly, the labeled cells were disrupted by
sonication in L buffer (7 M urea, 2 M thiourea, 50 mM Determination of Amino Acid Sequences of Tryptic Peptides Derived
from Protein Spots--
Cell proteins were separated by
two-dimensional GE and detected by staining of the gel with Coomassie
brilliant blue R-250 (CBB). Protein spots of interest were excised and
digested in situ with trypsin (26). The tryptic peptides
generated were purified on reversed-phase resin (POROS R2; Applied
Biosystems) and administered into a collision-induced
dissociation tandem mass spectrometer (MS/MS) using a quadrupole
time-of-flight mass spectrometer (Micromass) equipped with nanospray
tip. Amino acid sequences of the peptides were determined by manual
de novo interpretation of their y-type production series
(27).
Cloning of the dofA Gene--
Recombinant DNA techniques were
performed as described previously (24, 25). To clone a gene encoding
the spot 11 protein, four degenerate PCR primers were designed from the
amino acid sequences of two peptides determined by MS/MS sequencing
(see Table I). Using the primer set of
5'-GT(G/C)GAGGG(G/C)AACGAC(A/C)T(G/C)(A/C)AGTACAA-3' and
5'-CTTCTC(G/C)AC(G/C)A(G/T)GTT(G/C)A(G/T)(G/C)CC-3', a 69-bp PCR
product was amplified from the chromosomal DNA of M. xanthus DZF1. Since the amino acid sequence deduced from the PCR product was
consistent with those of two peptides from the spot 11 protein, the PCR
product was labeled with [ Comparison of Protein Synthesis Patterns in Vegetative and
Developing M. xanthus DZF1 Cells--
To compare the patterns of
protein synthesis during vegetative growth and development, M. xanthus DZF1 cells were labeled with [35S]methionine
during vegetative growth and during development (after 12 h
post-starvation). Whole cell proteins were separated by
two-dimensional GE followed by fluorography (Fig.
1, A and B).
Protein spots were profiled and quantified using Melanie II 2-D PAGE
software. Both of the two-dimensional GE images contained 700-1200
distinct protein spots depending on the gel exposure time to x-ray film
and the amount of proteins applied to the gels. The patterns of protein synthesis differed greatly between vegetative and developing cells. The
synthetic rates of more than 150 proteins in developing cells (after
12 h post-starvation) of M. xanthus DZF1 were
down-regulated as compared with those in vegetative cells, whereas
those of more than 100 proteins were up-regulated. The down-regulated
or up-regulated protein spots are described in the supplemental
materials.
Comparison of Patterns of Protein Synthesis among M. xanthus DZF1,
MO1 fruA::Tc, and DK731 csgA731 Strains during
Development--
In the present study, M. xanthus
development was induced by spotting vegetative cells on TM agar to
maximize [35S]methionine incorporation. Under these
conditions, M. xanthus DZF1 cells formed fruiting bodies in
a process similar to that on CF agar, generally used for development
(Fig. 2A). M. xanthus fruA::Tc cells arrested development
after formation of abnormal mounds, whereas M. xanthus
csgA731 cells arrested development at the stage of early
aggregation (Fig. 2A). The DZF1 cells formed rigid
aggregates at 18 h, whereas the fruA and
csgA mutants did not.
To analyze the effects of the fruA and csgA
mutations on the expression of other developmental genes, M. xanthus DZF1, MO1 fruA::Tc, and DK731
csgA731 cells were pulse-labeled with
[35S]methionine at 1, 4, 8, 12, 18, and 24 h
post-starvation. The whole cell proteins were analyzed by
two-dimensional GE followed by fluorography. Fig. 1, B-D,
compares the two-dimensional GE images of three strains at 12 h
post-starvation. At this stage of development, fruA and
csgA mutants produced fewer numbers of proteins than the
wild-type strain, whereas the protein synthesis patterns in
fruA and csgA mutants were similar. This result
may be due to the fact that developmental arrest in fruA and
csgA mutants occurs at a similar period during development.
At least 50 proteins were produced only in the wild-type strain in
comparison with the fruA and csgA mutants after
12 h (see supplemental materials), suggesting that expression of
these proteins is dependent on fruA and csgA
genes. Detailed comparisons of Fig. 1, C and D,
as well as the gel patterns of the other developmental stages in
fruA and csgA mutants (Fig. 2), however,
indicated that the syntheses of several proteins were differently
regulated during development in fruA and csgA
mutants. We have selected 14 of these proteins, which are indicated by
the numbers 1-14 in Fig. 1, for further analysis.
Identification of FruA, Elongation Factor Tu (EF-Tu), Dru, DofA,
and Protein S--
Five of the 14 selected proteins were
identified by Western blot analysis or mass spectroscopy. The spot 12 protein was identified as FruA protein since anti-FruA antibody reacted
with the spot 12 protein by Western blot analysis (data not shown).
Production of the spot 12 protein was not detected in the
fruA mutant, whereas FruA production was observed in the
csgA mutant (Fig. 1, C and D).
Among the 14 protein spots detected by [35S]methionine
labeling (Fig. 1B), four spots were visible on the
CBB-stained two-dimensional gel of M. xanthus DZF1 proteins
at 12 h post-starvation, whereas the remaining 10 spots (including
FruA) were undetectable by CBB staining (data not shown). Amino acid
sequences of the tryptic peptides from the four spots were determined
by nanospray tandem mass spectrum analysis after in-gel digestion with
trypsin. Fig. 3 shows a typical mass
spectrum for a tryptic peptide derived from the spot
13 protein. The amino acid sequence and relative molecular mass
of the peptide were determined to be AA(L/I)G(L/I)ENNT(L/I)SSVK and 1727.50, respectively (Table I).
Amino acid sequences and molecular masses of three additional
tryptic peptides from the spot 13 protein were also determined
(Table I). Computer analysis of the observed sequences and masses
revealed that the tryptic peptides corresponded to those of protein S,
indicating that spot 13 is protein S. The molecular mass and pI of
protein S are estimated to be 19 kDa and 4.4, respectively,
which are in good agreement with the location of spot 13 on the
two-dimensional gel (Fig. 1).
Amino acid sequences and masses of four tryptic peptides from the spot
3 protein corresponded to those for putative EF-Tu of M. xanthus (Table I), indicating that spot 3 is EF-Tu. Spot 3 was one
of the largest spots in the vegetative cells (Fig. 1A). Amino acid sequences and molecular masses of three tryptic peptides from the spot 10 protein corresponded to the product of a hypothetical gene tentatively designated dru (downstream of
rpsU) located in the rpsU operon of
M. xanthus (28). The rpsU operon consists of, in
order, rpsU, dru, dnaG,
rpoD, and ogt genes. The dru gene encodes a conserved protein of unknown function. It is of interest that
genes encoding homologues of M. xanthus Dru are also located downstream of rpsU in many bacterial genomes.
Since expression of the spot 11 protein was dependent on
fruA, tryptic peptides from the spot 11 protein were also
analyzed by mass spectroscopy, although spot 11 was rather faint on the CBB-stained two-dimensional gel. The amino acid sequences of two peptides were determined (Table I). Degenerate primers corresponding to
the two peptides were constructed and used for PCR amplification with
M. xanthus chromosomal DNA as a template. A PCR product of 69 bp was produced and used to clone an M. xanthus gene
encoding the spot 11 protein. The cloned DNA contained a gene
tentatively designated dofA (dependent
on fruA). The dofA gene encodes a
protein of 148 amino acids with a calculated molecular mass of 16.6 kDa (Fig. 4). There is no known protein
showing significant similarity to DofA in the protein databases. The
observed amino acid sequences of the two tryptic peptides correspond to
those of the dofA product (Fig. 4,
underline).
Effects of fruA and csgA Mutations on the Expression of Various
Developmental Genes of M. xanthus--
In Fig. 2, changes in the
synthesis of the spot 1-11 proteins during the development of M. xanthus wild-type, fruA::Tc, and csgA731 strains are indicated as three specific
windows a, b, and c (as originally
delineated in Fig. 1) of every gel. The synthetic rates of 14 proteins
including the spot 12, 13, and 14 proteins were estimated as changes in
the percent spot volume of each protein by Melanie II 2-D PAGE software
and summarized as 14 graphs illustrated in Fig. 2.
Synthesis of EF-Tu (spot 3) and Dru (spot 10) was active during
vegetative growth of M. xanthus wild type (percentage
of volume: EF-Tu, 3.9% and Dru, 0.6%) (Fig. 1A), whereas
it decreased to a very low level shortly after the onset of development
(Fig. 2, B and D, graphs 3 and
10). RelA-dependent accumulation of ppGpp (and
pppGpp) at the early stage of M. xanthus development has been reported (29-32). In Escherichia coli, the synthesis
of stable RNA and protein synthesis machinery including EF-Tu and RpsU
is strongly inhibited by (p)ppGpp, which is known as the stringent response (33). Therefore, it is most likely that the synthesis of EF-Tu
and Dru (the product of the second gene of the rpsU operon) is also regulated by the stringent response in M. xanthus.
In the wild-type strain and the csgA mutant, EF-Tu and Dru
synthesis recovered at a later stage of development, whereas minimal
recovery was observed in the fruA mutant.
Under the present conditions, synthesis of the 12 remaining proteins
was not detected during vegetative growth, suggesting that these
proteins are the products of developmental genes. The synthetic rate of
the spot 14 protein was very high shortly after the onset of
development in the wild-type, fruA::Tc, and
csgA731 strains, whereas its synthesis could not be detected
during vegetative growth, indicating that the spot 14 protein is the
product of an early developmental gene (Fig. 2, graph 14).
In the wild-type M. xanthus strain, the synthetic rate of
the spot 14 protein decreased gradually and reached a minimal level
after 18 h, whereas the decrease in its synthetic rate was slow
in the fruA and csgA mutants. Changes in
the synthetic rate of the spot 6 protein during development were
similar to those of the spot 14 protein except that its synthetic rate
in the fruA mutant decreased at a rate similar to the wild type but different from that observed for the csgA mutant
(Fig. 2C, graph 6).
In the wild-type M. xanthus strain, synthesis of three
developmental gene products, including the spot 5 and 7 proteins and FruA, started at 1-4 h post-starvation (Fig. 2C,
graphs 5, 7, and 12). During
development, the synthetic rate of the spot 7 protein increased to
reach a peak at 12 h and then decreased. Synthetic rates of the
spot 5 protein and FruA increased to reach a maximal level at 12 h, and their high synthetic rates were nearly maintained until 24 h. FruA synthesis observed in this work is consistent with the previous
results of fruA transcript and product accumulation (19).
The effects of the fruA and csgA mutations on the
expression of these proteins were different from each other. In the
fruA mutant, FruA production was not observed, as expected. The fruA mutation did not affect spot 5 protein
synthesis, whereas it severely suppressed spot 7 protein synthesis. In
contrast, the csgA mutation severely suppressed spot 5 protein synthesis, whereas the effects of the csgA mutation
on spot 7 protein synthesis were not so marked. In the csgA
mutant, the increase in the synthetic rate of FruA was slower than that
observed in the wild type, but FruA expression was similar to that of
the wild type at 18 h.
In the wild-type M. xanthus strain, synthesis of the spot 1, 2, and 4 proteins, DofA, and protein S started at 4-8 h
post-starvation (Fig. 2, B-D, graphs 1,
2, 4, 11, and 13). The
effects of the fruA and csgA mutations on the
expression of such developmental proteins also differed among them.
Synthesis of the spot 1 and 2 proteins, DofA, and protein S was
dependent on FruA function, whereas residual synthesis of these
proteins was observed in the csgA mutant. In contrast,
synthesis of the spot 4 protein was dependent on CsgA function, whereas
even higher synthesis of spot 4 was observed in the fruA
mutant. The delayed synthesis of FruA in the csgA mutant
(Fig. 2B, graph 12) may result in the delayed synthesis of the spot 1 protein, DofA, and protein S in the
csgA mutant (Fig. 2, B and D,
graphs 1, 11, and 13).
In wild-type M. xanthus strain, synthesis of the spot 8 and
9 proteins started at 18 h post-starvation (Fig. 2C,
graphs 8 and 9). Synthesis of the spot 8 protein
was dependent on both FruA and CsgA functions, whereas that of the spot
9 protein was not dependent. The fruA mutation even enhanced
the synthesis of the spot 9 protein.
The defects in fruiting body formation in M. xanthus
fruA::Tc and csgA731 strains have been
reported to be complemented by the introduction of wild-type
fruA and csgA alleles into the M. xanthus chromosomal Mx8 attB site and csgA
locus, respectively (19, 34). The complemented strain, MO2
sglA1 fruA::Tc In the present work, the patterns of protein synthesis during
vegetative growth and development of M. xanthus have been
analyzed by two-dimensional GE. Comparison of the two-dimensional GE
image of vegetative cells with that of developing cells after 12 h
post-starvation revealed that synthesis of more than 250 protein spots
was up- or down-regulated. The effects of the fruA and
csgA mutations on gene expression during development were
also analyzed by two-dimensional GE, and several proteins were found to
be differently regulated during development in the fruA and
csgA mutants.
The effects of A- to E-signals on the expression of various Tn5
lac fusions have been extensively studied during the development of M. xanthus (11). When In the present work, we found that the synthesis of the spot 4, 5, and
8 proteins was dependent on csgA, similar to the
C-signal-dependent Tn5 lac fusions described
above. The production of six proteins including the spot 1, 2, 7, and 8 proteins, DofA, and protein S was demonstrated to be dependent on FruA
function. Production of protein S and expression of Tn5 lac
The finding that the expression of several genes including
tps and dofA are dependent on FruA function but
independent of CsgA function cannot be explained by the previous
proposal, in which the activation of FruA by phosphorylation requires
the association of C-signal located on the cell surface with a
membrane-bound receptor-type histidine kinase of neighboring cells
(20). This model suggests that the expression of all
fruA-dependent genes would be also dependent on
C-signaling. Postulation of the existence of two distinct levels of
FruA phosphorylation, low level and high level phosphorylation, may
explain this apparent discrepancy (Fig.
5). For the expression of
fruA-dependent/csgA-independent genes, weakly phosphorylated FruA protein is sufficient. Low level FruA
phosphorylation may be accomplished by a putative histidine protein
kinase (HPK) X in the absence of C-signal. For the expression of many
fruA-dependent/csgA-dependent
genes, highly phosphorylated FruA protein is required. High level FruA
phosphorylation may be accomplished by HPK X in the presence of
C-signal. Alternatively, C-signal may inhibit dephosphorylation of
phosphorylated FruA protein. It is known that the expression of
ompF and ompC, encoding outer membrane porins, is
differently regulated by the phosphorylation level of OmpR response
regulator via the EnvZ-OmpR phosphorelay in the E. coli
osmoregulation system (38).
Role of fruA and csgA Genes in Gene
Expression during Development of Myxococcus xanthus
ANALYSIS BY TWO-DIMENSIONAL GEL ELECTROPHORESIS*,
§,
, and Biology and
¶ Chemistry, Tokyo Metropolitan University, Minamiohsawa,
Hachioji, Tokyo 192-0397, Japan and the § Department of
Biochemistry, Robert Wood Johnson Medical School,
Piscataway, New Jersey 08854
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase expression at a specific time during development.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5, MO2 sglA1
fruA::Tc 5 Mx8
attB::pMFA05 (fruA+) (19),
and DK731 csgA731 (9) were used throughout these studies.
For the growth of M. xanthus, CTT and A1 media were
prepared as described previously (23). For the development of M. xanthus, TM agar (10 mM Tris-HCl (pH 7.6), 8 mM MgSO4, and 1.5% agar) was used.
supE44 
lacU169 (ø80
lacZM15) hsdR17 recA1
endA1 gyrA96 thi-1 relA1 (24) was used as a recipient strain for transformation. E. coli cells were grown in Luria-Bertani medium (25) supplemented with 100 µg/ml ampicillin when necessary.
-mercaptoethanol, 5% (w/v) CHAPS, and 2% (v/v)
ampholine (pH 3-10)). The whole cell extract was incubated for 1 h at 25 °C and centrifuged at 100,000 × g for 20 min. The supernatant was mixed with an equal volume of R buffer (8 M urea, 20 mM dithiothreitol, 0.5%
CHAPS, 0.5% ampholine (pH 3-10)) and subjected to rehydration of
Immobiline DryStrip (Amersham Biosciences; pH 3-10, 13 cm). Following
isoelectric focusing, the proteins were separated by SDS-PAGE (13%)
and detected by fluorography. To compare protein patterns during
development among various M. xanthus strains, proteins from
the same cell number spotted on the TM plate were applied to each gel.
Image analysis and quantification of protein spots were achieved with
Melanie II 2-D PAGE software (Bio-Rad).
-32P]ATP by T4
polynucleotide kinase and used to screen a gene library containing
1.0-1.5-kb HincII fragments of M. xanthus DNA in
pUC19. A positive clone designated pSP11 and containing a 1.1-kb
HincII fragment was obtained and sequenced.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (150K):
[in a new window]
Fig. 1.
Patterns of protein
synthesis in vegetative cells of M. xanthus DZF1
(A) and in developing cells (after 12 h
post-starvation) of DZF1 (B),
fruA::Tc (C), and
csgA731 (D) strains. Cells were
labeled with [35S]methionine, and whole cell proteins
were analyzed by two-dimensional GE followed by fluorography.
Windows a, b, and c are focused in
Fig. 2. Protein spots with numbers 1-14 were analyzed in
detail. The positions of molecular mass standards are given on the
right of each panel; from the top, 200, 116, 97, 66, 45, 31, 21.5, 14.5, and 6.5 kDa. The positions of isoelectric point
standards are given on the bottom of each panel;
from the left, 4.55, 5.20, 5.85, 6.55, 6.85, 7.35, 8.15, and 8.45. The
results of a detailed analysis of this figure are described in the
supplemental materials.
View larger version (124K):
[in a new window]
Fig. 2.
Time courses of morphogenesis and synthetic
rates of 14 proteins during development of M. xanthus
wild-type, fruA::Tc, and
csgA731 strains. A, morphogenesis of
M. xanthus DZF1, fruA::Tc, and csgA731
strains during development. Development of three strains was induced by
spotting vegetative cells on TM agar plates. The spots were
photographed through a dissecting microscope at the indicated times.
B-D, time courses of synthetic patterns of the spot 1-11
proteins as shown in window a (the spot 1-3 proteins)
(B), window b (the spot 4-9 proteins)
(C), and window c (the spot 10 and 11 proteins)
(D) during development of the indicated M. xanthus strains. The locations of windows a,
b, and c are indicated in Fig. 1. Graphs
1-14 indicate changes in the synthetic rates of the spot 1-14
proteins, respectively, in the three M. xanthus strains. The
density of each spot in the two-dimensional gels during development of
the three strains was estimated by Melanie II software and
plotted as percentage of spot volume of each spot. Symbols for each of
M. xanthus strains are as follows: circle, DZF1;
square, fruA::Tc; triangle,
csgA731. The x axes indicate time in hours, and
the y axes indicate percentages of spot volume. The values
at 0 h in graphs 3 and 10 indicate the
percentage of spot volume of vegetative cells.
View larger version (15K):
[in a new window]
Fig. 3.
Collision-induced dissociation MS/MS spectrum
of a doubly charged ion of a tryptic peptide (m/z
864.76) derived from the spot 13 protein. The amino acid
sequence AA(L/I)G(L/I)ENNT(L/I)SSVK was determined by interpretation of
the y-type product ion series as shown.
Amino acid sequences and relative molecular masses of tryptic peptides
derived from the spot 3, 10, 11, and 13 proteins
View larger version (10K):
[in a new window]
Fig. 4.
The amino acid sequence of the spot 11 protein, DofA. The amino acid sequence deduced from the DNA
sequence of the dofA gene is shown. The amino acid sequences
of two tryptic peptides determined by tandem mass spectrometry are
underlined.
5 Mx8 attB::pMFA05 (fruA+),
displayed a protein synthesis pattern similar to that of the wild-type
strain during development (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity from a
specific Tn5 lac fusion is not expressed in the A- to
E-signal mutants during development, the Tn5 lac fusion is
referred to as A- to E-signal-dependent, respectively. The
expression of at least 10 Tn5 lac fusions were
reported to be dependent on the csgA gene (11).
4273, regulated by the tps (encoding protein S) promoter,
was shown previously to be dependent on FruA function (19), consistent
with the present results. Among six
fruA-dependent proteins, only the production of
the spot 8 protein was dependent on CsgA function. The regulation of
synthesis of the selected 14 proteins by fruA and
csgA genes during development of M. xanthus was
summarized in Table II. Although both
fruA and csgA genes regulate the early
development of M. xanthus, the fruA and
csgA mutations differently regulate expression of some
specific developmental genes.
Regulation of the spot 1-14 protein synthesis by fruA and csgA genes
during development of M. xanthus
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Fig. 5.
A model for the control of the developmental
gene expression regulated by FruA and CsgA in M. xanthus. The fruA transcription requires
both A and E signals (20) and is initiated at 6 h post-starvation
(19). FruA, a response regulator in the His-Asp signal transduction
system, is proposed to be activated by phosphorylation with a yet
unidentified HPK X (20). We propose that FruA may be phosphorylated in
two steps: the C-signal (a product of the csgA
gene)-independent manner followed by the C-signal-dependent
manner. In the early stages of development, FruA is weakly
phosphorylated by HPK X in a C-signal-independent manner, resulting in
the production of weakly phosphorylated FruA, which is enough for the
expression of
fruA-dependent/csgA-independent
genes. As development proceeds, the concentration of C-signal
increases, which is proposed to be an activator for HPK X (20). This
results in highly phosphorylated FruA, allowing the expression of
fruA-dependent/csgA-dependent
genes. The csgA expression is fully activated by its own
product (21, 35-37).
Production of the spot 14 protein started during the early stage of
development and was maintained until the later stages of development in
the fruA and csgA mutants, whereas it was
repressed at the later stages of development in the wild-type strain.
In the fruA mutant, the expression of three Tn5
lac fusions, 4408, 4521, and 4455, known to be induced
during early stages of development, was found to be still inducible but
was no longer repressed at a later stage of development (19).
The present results strongly suggest that EF-Tu and Dru syntheses are regulated by the M. xanthus stringent response. The stringent response is an important signal for entry into the developmental process, and an M. xanthus relA mutant has been shown to be unable to develop (31, 32). The socE mutation bypassed the csgA requirement for development (39). The socE gene was normally expressed during vegetative growth, whereas socE expression was repressed by the stringent response when M. xanthus cells started the developmental process. Depletion of the socE gene was toxic for vegetative growth since socE depletion induces the stringent response even in the presence of amino acids. The socE expression could not be recovered during development. In contrast, EF-Tu and Dru expression was recovered during development. The recovery in EF-Tu and Dru synthesis may not be attributable to the end of the stringent response since (p)ppGpp was shown to be maintained at high levels during development (39). The timing of their recovery was different, and the fruA and csgA mutations differently affected their expression during development. These results suggest that mechanisms for the recovery of EF-Tu and Dru expression from the stringent response are different.
Whereas the Tn5 lac fusion experiments determined the
amounts of accumulated -galactosidase, the pulse-labeling
experiments used in the present work determined the synthetic rates of
various proteins. Hence, the pulse-labeling experiments present an
advantage for the analysis of the timing and regulation of gene
expression during M. xanthus development. In addition,
pulse-labeling experiments are essential for the identification of
proteins with high turnover rates. The spot 14 protein may be an
example of such high turnover proteins. The spot 14 protein exhibited a
high synthetic rate during the early stages of development, whereas it
could not be detected by CBB staining after 12 h, suggesting its
high turnover. Many of the 14 proteins described in the present work
can be used as developmental markers for the analysis of fruiting body
formation in M. xanthus.
We succeeded in cloning a novel gene dofA, whose expression
was dependent on FruA function but not on CsgA function. Further analysis of dofA will facilitate the elucidation of the
function of FruA, a key factor for fruiting body formation of M. xanthus. Such work is currently in progress in our laboratory.
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful to Monsanto's Cereon Microbiol Sequence Data Base for allowing us to access the data base. Nucleotide sequence of AB073986 was downloaded from Monsanto's Cereon Microbial Sequence Data Base. Monsanto has granted permission to deposit this sequence in GenBankTM as part of the publication process.
| |
FOOTNOTES |
|---|
* This work was supported in part by a grant from the Ministry of Education, Science, Sports and Culture of Japan and by the Foundation of Medicine and Dentistry of New Jersey.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.
The on-line version of this article (available at
http://www.jbc.org) contains the results of a detailed analysis of Fig. 1.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB073986 and AB073987.
To whom correspondence may be addressed. Tel.:
81-426-77-2568; Fax: 81-426-77-2559; E-mail:
komano-teruya@c.metro-u.ac.jp.
** To whom correspondence may be addressed: Dept. of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854. Tel.: 732-235-4161; Fax: 732-235-4783, E-mail: sinouye@waksman.rutgers.edu.
Published, JBC Papers in Press, May 7, 2002, DOI 10.1074/jbc.M111214200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: GE, immobilized pH gradient/sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CBB, Coomassie brilliant blue; EF-Tu, elongation factor Tu; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; HPK, histidine protein kinase; MS/MS, tandem mass spectrometry.
| |
REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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