School of Dentistry and Molecular Biology
Institute, University of California, Los Angeles, Los Angeles,
California 90095-1668
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INTRODUCTION |
Myxococcus xanthus is a
gliding bacterium that exhibits complex social behavior
(10). Upon starvation, cells aggregate to form fruiting
bodies (10). This cellular aggregation process requires
intercellular signaling, coordinated and directed cell movement, and
temporal and spatial gene expression (20, 22, 24, 45).
Previous studies showed that M. xanthus has dual motility
systems (17, 18): (i) system A (adventurous) is required for
the movement of single cells or small groups of cells; (ii) system S
(social) is required for the coordinated movement of large cell groups.
Social motility has been shown to be important for fruiting body
formation (17, 18, 30). The cellular aggregation process
also requires directed cell movement (45, 47). A group of
frz genes, homologous to the chemotaxis genes of
Escherichia coli and Salmonella typhimurium, were
found to be important for directing M. xanthus cells into
aggregation centers (32, 45, 47). To further understand the
mechanism(s) of cellular aggregation, we isolated and characterized
additional mutants that are defective in cellular aggregation. This
paper reports two of these mutants, both of which have a mutated gene
homologous to the dnaK gene of E. coli. The same
gene has also been identified by Patricia Hartzell's group at the
University of Idaho (58).
E. coli dnaK encodes a heat shock protein of the hsp70
family (3). It is regulated by and required for heat shock
(5, 13, 56). Many DnaK homologs have also been identified in
various other bacteria (27, 37, 40, 44, 55, 57, 59, 66). DnaK generally forms a multisubunit complex with DnaJ and GrpE. All
three genes are regulated by a heat-inducible alternative sigma factor
(sigma-32) (8, 14, 38, 43, 48, 54, 60, 61, 65). The
DnaK-DnaJ-GrpE complex serves as a molecular chaperone which is
involved in cellular motility, cell division, protein folding and
secretion, RNA and DNA synthesis, and the regulation of heat shock
responses (9, 12, 14, 15, 26, 43, 48, 54, 60, 61, 65). Here
we report that this M. xanthus DnaK homolog is required for
social motility and cellular aggregation of M. xanthus and
that the expression of this gene is not regulated by heat shock or by
nutrient conditions.
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MATERIALS AND METHODS |
Bacterial strains and culture conditions.
The bacterial
strains used in this study are listed in Table
1. E. coli strains were grown
and maintained in LB medium (35). M. xanthus was
grown and maintained at 32°C in CYE medium (6). Other
media used in this study include morpholinepropanesulfonic acid (MOPS)
medium (10 mM MOPS [pH 7.6], 8 mM MgSO4), A-1 minimum medium (4), and CF medium (16).
Phenotypic characterization of the nonfruiting mutants.
The
following experiments were performed to characterize the mutants. For
fruiting body formation, cells at about 5 × 108
cells/ml were placed on MOPS or CF plates (1.5% agar) and incubated at
32°C for 2 to 3 days. For the examination of developmental spores,
M. xanthus cells were spotted onto CF plates and incubated at 32°C for 7 days. Spore formation was then examined by light microscopy. The spores are refractile spherical cells which are resistant to 1% sodium dodecyl sulfate. For swarming, 20 µl of cells
at about 5 × 108 cells/ml was spotted on the center
of a swarming plate (CYE with 0.3% agar) and incubated at 32°C for 3 to 4 days (46, 49). Cell motility was assayed by time-lapse
videomicroscopy as described by Shi and Zusman (49). Social
motility was further studied by microscopic observation of colony edges
and by a cellular agglutination assay. The agglutination assay was
performed by the method described by Wu et al. (62). The
assays for chemotaxis and methylation of FrzCD were performed by
methods described previously (34, 46).
Heat shock and labeling conditions.
A procedure similar to
that described by Nelson and Killeen was used (39). Cells
grown at 24°C in A-1 medium (4) to exponential phase were
harvested, washed twice, and then resuspended at 0.1 unit of optical
density at 600 nm (OD600) in A-1 medium without methionine.
The cells were equilibrated at 24°C for 1 h and pulse-labeled with [35S]methionine (~10 µCi per ml) for 15 min at
40 or 24°C. The cells were immediately put on ice and mixed with cold
methionine at 10 µg per ml, harvested at 4°C, and washed twice with
15 mM sodium phosphate buffer (pH 7.0). Whole cell lysates were
prepared (34, 46) and subsequently analyzed by
electrophoresis on a sodium dodecyl sulfate-10% polyacrylamide gel
(42).
Molecular techniques.
Either P1::Tn5lac
(21) or P4::Tn5kan903 (courtesy of
Bryan Julien, Stanford University) was used for transposon mutagenesis as described previously (25). Myxophage Mx4 was used for
generalized transduction (41). EcoRI-digested
pUC18 was used to clone the mutations. M. xanthus genomic
DNA was isolated as described previously (2, 64), digested
with EcoRI, and ligated to the vector. Tn5
insertion was used as a selectable marker (it confers kanamycin resistance) for the cloning. The mutated genes were sequenced by using
primers complementary to the known DNA sequences at the ends of the
Tn5 constructs. DNA sequencing was performed by the automated DNA sequencing facility at the University of California, Davis. Sequence analysis was performed with BLAST and BCM Search Launcher (1, 52). 
-Galactosidase activities were assayed as described by Kroos et al. (24).
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RESULTS |
Isolation and phenotypic characterization of two nonfruiting
mutants.
A genetic screening was carried out to identify genes
involved in cellular aggregation and development of M. xanthus. Strain DZF1 is wild type with regard to fruiting body
formation but contains a leaky sglA gene, a gene involved in
social gliding motility (6). The strain was used for initial
transposon mutagenesis because it forms fewer cell clumps. Using
P4::Tn5kan903 and P1::Tn5lac, we isolated more than 10,000 Tn5 insertional mutants. These
mutants were streaked on CF plates and examined for cellular
aggregation and fruiting body development. About 200 mutants with
various degrees of defects in fruiting body formation were identified (11a). The linkage between the fruiting defects and
Tn5 insertions was confirmed by introducing the
Tn5 mutations back to DZF1 and to DZ2 by Mx4-mediated
generalized transduction. SW107 (Tn5lac insertion) and SW164
(Tn5kan903 insertion) are two such mutants identified in
this screening (Table 1). SW300 and SW301 are the two corresponding
mutants in the DZ2 background, produced by Mx4 generalized transduction
(Table 1). These two mutants are reported here because they exhibited
identical phenotypes and were later found to harbor mutations in the
same gene, sglK.
Figure 1 shows the phenotypes of the
mutant SW301 in comparison with the wild-type strain DZ2. SW300 showed
the same phenotypes as SW301 (data not shown). After 2 days of
incubation on CF plates, wild-type DZ2 cells formed visible fruiting
bodies (Fig. 1a), in which portions of cells eventually developed into
myxospores after 5 to 7 days. The mutants did not form any cellular
aggregates even after 5 days (Fig. 1b), and only a small portion of the
mutant cells developed into spores (<5% of the wild-type parent DZ2). We also tested the swarming ability of the mutants and found that they
were defective in swarming on CYE plates with 0.3% agar (Fig. 1d).
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FIG. 1.
Phenotypic characterization of wild-type and mutant
M. xanthus strains, performed as described in Materials and
Methods. Wild-type DZ2 forms fruiting bodies on CF agar after 2 days of
incubation (a); SW301 does not form any fruiting bodies even after 5 days (b). On CYE plus 0.3% agar, DZ2 forms a swarming colony about 4.0 cm in diameter after 5 days (c); the colony formed by SW301 is 1.5 cm
in diameter (d). On 1.5% agar, advancing colony edges of DZ2 contain
both single cells and large cell groups (e), while those of SW301
contain only single cells and small cell groups (f). SW300 shows the
same phenotype as SW301.
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The two nonfruiting mutants are normal in directed cell movement
but defective in social motility.
The swarming and fruiting
defects of the mutants may result from defects either in directed cell
movement or in cellular motility. Previous studies showed that
wild-type M. xanthus exhibits directed cell movements in
response to various chemicals (31, 47). These chemotactic
movements were found to be associated with cellular reversal frequency
and the modification of FrzCD, a methyl-accepting chemotaxis protein
(31, 47). The methylation of FrzCD was found to be
modulated over the course of development (33). Moreover, such changes in FrzCD methylation play important roles in directed cell
movement during fruiting body formation (33, 47, 53). SW300
and SW301 were tested with the various assays mentioned above and found
to respond to various chemicals as did the wild-type strain. For
example, they increased their cellular reversal frequency from once
every 4 to 6 min to about once every minute in response to 0.1%
isoamyl alcohol. Furthermore, the methylation patterns of the mutants
are similar to that of the wild type in response to fresh CYE,
starvation, and 0.1% isoamyl alcohol (Fig.
2). These data indicate that both SW300
and SW301 are normal for directed cell movements under the conditions
examined.
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FIG. 2.
Modification of FrzCD in wild-type and mutant strains.
The methylation/demethylation pattern of FrzCD was assayed by Western
blotting as previous described (34, 46). The arrowhead
indicates the position of methylated FrzCD. Wild-type and mutant cells
were treated with fresh CYE (chemoattractants) or 0.1% isoamyl alcohol
(chemorepellents) for 1 h or starved in MOPS buffer for various
lengths of time with shaking and then collected for FrzCD methylation
assay. Lanes 1 and 6, DZ2 and SW301 in fresh CYE medium, respectively;
lanes 2 and 7, DZ2 and SW301 in MOPS plus 0.1% isoamyl alcohol; lanes
3 to 5, DZ2 cells starved in MOPS buffer for 1, 8, and 24 h; lanes
8 to 10, SW301 cells starved in MOPS buffer for 1, 8, and 24 h.
SW300 shows the same methylation/demethylation patterns of FrzCD as
SW301 and DZ2.
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Using videomicroscopy, we also found that single cells of SW300 and
SW301 are as motile as wild-type cells. The mutant cells are also
proficient in swarming on 1.5% agar plates. Therefore, the mutants are
normal in A motility. As shown in Fig. 1f, the edges of the mutant
colonies only have single cells or small cell groups, indicating that
the mutant cells are defective in S motility. Moreover, unlike the
wild-type DZ2 cells, mutant cells grown in liquid culture do not form
cell clumps, which is another indication of defective S motility. It is
known that defects in S motility may result in reduced cellular
cohesion, which can be measured by an agglutination assay (50,
62). Figure 3 shows that both mutants are defective in agglutination, indicating that these strains
are defective in cellular cohesion. The defects of the mutants in
fruiting body formation and in swarming on 0.3% agar plates are also
consistent with defects in S motility (18, 23, 49).
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FIG. 3.
Agglutination assay. Cells were grown in CYE at 32°C
overnight to an OD600 about 0.5 and allowed to agglutinate
at room temperature, and the OD600 was measured every 10 min. The relative absorbance was calculated by dividing the absorbance
at a given time by the initial absorbance. The experiment was repeated
twice with similar results. DZ2 is the wild-type strain, whereas SW300
and SW301 are the two sglK mutants in the DZ2 background.
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SW300 and SW301 harbor mutations in the same gene, which is highly
homologous to the gene for heat shock protein DnaK.
Using the
Tn5 insertion as a selectable marker, the mutated genes in
SW300 and SW301 were cloned and partially sequenced. Sequence analysis
indicates that both SW300 and SW301 have Tn5 inserted in a
region encoding homologs of two heat shock proteins of E. coli, GrpE and DnaK. A GenBank search indicated that the DNA
sequence is identical to the M. xanthus sequence of
grpS and sglK recently submitted by Hartzell's
group at the University of Idaho (accession no. U83800). About 5 kb of
DNA in this genetic locus, a region which contains an operon consisting
of the grpE and dnaK homologs, has been sequenced
by Hartzell's group. Based on the DNA sequence, we found that both
SW300 and SW301 have the Tn5 insertions at the N terminus of
the sglK gene (Fig. 4). The
phenotypes of SW300 and SW301 are also largely consistent with what
they have described (28, 29, 58).
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FIG. 4.
Organization of grpS and sglK and
insertion of Tn5 in SW300 and SW301. A 3.68-kb
SacI-ApaI fragment is depicted. The open reading
frames of grpS and sglK are marked. The arrows
indicate the directions of the two open reading frames, which are 8 bp
apart. The predicted GrpS and SglK peptide sequences exhibit 26 and
58.5% identity with E. coli GrpE and DnaK, respectively.
grpS and sglK were also isolated by Hartzell's
group (GenBank accession no. U83800). The insertions in SW300 and SW301
are indicated. The circle indicates the insertion of
Tn5kan903 in SW301, whereas the square indicates the
insertion of Tn5lac in SW300.
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The DnaK homolog of M. xanthus is not regulated by
temperature or by growth conditions.
E. coli dnaK mutants
are temperature sensitive in growth and in cell division (5, 14,
15). We therefore examined the growth of the mutants on CYE
plates and their cell morphology under a microscope. At 40°C, the
absolute temperature maximum for M. xanthus growth
(19), both the mutant and the wild-type strains showed
scattered growth with no obvious defects in cell morphology. At all
other temperatures examined (15, 24, 32, and 38°C), normal growth and
cell morphology were observed for the mutants and the wild type alike.
Thus, it is unlikely that sglK in M. xanthus is
the functional equivalent of dnaK in E. coli.
It is known that the expression of DnaK in E. coli is
regulated by heat shock. Since SW300 contains a promoterless
lacZ gene inserted in the same orientation as the
sglK gene, we were able to assay its expression via the
level of -galactosidase. We found that the expression of the
M. xanthus dnaK homolog remained the same at different
temperatures (Fig. 5a), indicating that
the sglK gene may not be regulated by heat shock. In
addition, sglK appears to be constantly expressed during
vegetative growth (data not show) and shows little fluctuation under
developmental starvation (Fig. 5b).
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FIG. 5.
Expression of the sglK gene assayed by
-galactosidase activity from an sglK-lacZ fusion.
-Galactosidase activity was determined in strain SW300 as described
by Kroos et al. (24) and is presented in Miller units (MU)
(35). (a) Effect of temperature on the expression of
sglK. SW300 cells grown at 32°C were shifted to various
temperatures for either 5 min (solid bars) or 30 min (open bars) and
then collected for -galactosidase assay. (b) Effect of starvation on
expression of the sglK gene. SW300 cells grown in CYE medium
were resuspended in starvation medium (MOPS buffer). Samples were
collected for -galactosidase assay at different time points during
starvation. The data shown are averages of duplicate samples.
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It was somewhat surprising that sglK, a homolog of E. coli dnaK, seems not to be regulated by heat shock. Pulse-labeling
experiments were therefore performed to evaluate the expression of heat
shock proteins in both the mutant and the wild-type strains. As shown in Fig. 6, when cells were shifted from
24 to 40°C, a number of heat shock proteins were expressed in both
the wild-type and the mutant strains. The mutants have almost the same
expression patterns of heat shock proteins as the wild type. There is
no band missing in the mutants around the calculated molecular mass of
SglK (65.3 kDa). Therefore, unlike E. coli DnaK, SglK is not
a major heat shock protein in M. xanthus.
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FIG. 6.
Production of heat shock proteins by M. xanthus. Cells grown at 24°C were labeled as described in
Materials and Methods for 15 min with [35S]methionine and
then either shifted to 40°C for heat shock treatment (lanes 1, 3, and
5) or kept at 24°C (lanes 2, 4, and 6). Whole-cell lysates from the
same amount of cells were loaded on each lane. DZF1 lysates were loaded
on lanes 1 and 2, SW107 lysates were loaded on lanes 3 and 4, and SW164
lysates were loaded on lanes 5 and 6. Molecular mass standards are
indicated in kilodaltons on the left.
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DISCUSSION |
When M. xanthus cells are under the stress of
starvation, they undergo a developmental program in which cells
aggregate to form fruiting bodies and eventually differentiate into
myxospores (10). Identification of the molecular components
involved in this cellular process would facilitate the elucidation of
the underlying mechanisms of such a complex developmental process. Even
though many developmental genes of M. xanthus have been
identified (11, 51), their physiological functions are for
the most part not directly related to one another. Therefore, more
components involved in this cellular process are yet to be identified.
In this study, two mutants defective in fruiting body formation were
isolated through a genetic screening. Both mutants exhibited wild-type
behavior in single-cell motility and directed cell movement. The
mutants also showed modification of FrzCD similar to that of the
wild-type strain during starvation and in response to various chemicals
(Fig. 2). Nevertheless, they showed little or no aggregation under
developmental conditions (Fig. 1b). It was then discovered that the
mutants are defective in social motility (Fig. 1d and f), which could
possibly result in the defects in aggregation and fruiting body
development because of the impaired cell-cell interaction and
communication. Cloning and sequencing analysis indicated that both
mutations occurred in the same gene, encoding a DnaK homolog of the
hsp70 family.
As a stress protein, DnaK has been found to be required for various
stress responses in other bacteria, including responses to heat shock,
oxidative damage, and starvation (9, 12, 14, 15, 26, 43, 48, 54,
60, 61, 65). Considering that the developmental process of
M. xanthus is in essence a well-orchestrated stress response
to starvation, it is perhaps not too surprising that a DnaK homolog is
involved in this process. It should be noted, however, that the
expression of dnaK genes in other bacteria is up-regulated
by heat shock or starvation (8, 14, 38, 43, 48, 54, 60, 61,
65), whereas this M. xanthus dnaK homolog
(sglK) is apparently not regulated by heat shock or
starvation. Instead, it is constitutively expressed during both
vegetative growth and starvation (Fig. 5).
In E. coli, DnaK forms a protein complex with DnaJ and GrpE,
and such a complex has been found to be involved in a variety of
cellular processes, including cell motility, cell division, cell
growth, phage infection, protein transport, and heat shock response
(9, 12, 14, 15, 26, 43, 48, 54, 60, 61, 65). In contrast,
M. xanthus sglK seems to be involved only in some specific
cellular functions: the sglK mutants show no defect in cell
division, cell growth, single-cell motility, heat shock response, and
Mx4 phage infection, but they are defective in social motility and
fruiting body formation.
It would be interesting to find out how sglK may affect
these social processes of M. xanthus. The dnaK
mutation causes reduced expression of the flagellar genes in E. coli (48). It is possible that SglK is involved in the
regulation of the synthesis of social motility appendages in M. xanthus. The lack of such appendages may lead to defects in social
motility. Since sglK encodes a homolog of a molecular
chaperone, it is also conceivable that SglK in M. xanthus is
directly involved in the assembly of some apparatus required for social
motility. Alternatively, SglK could be involved indirectly in motility
by affecting the function of proteins in the regulatory hierarchy of
the social motility genes. Our preliminary results indeed indicate that
sglK may negatively regulate the expression of a DnaK-like
protein other than itself (data not shown). It should be noted,
however, that sglK mutants still possess A motility (Fig.
1f). Whatever structure or appendage is affected by sglK
mutations, either directly or indirectly, it must be unique to S
motility. It is not yet known whether this DnaK homolog is directly
required for fruiting body formation or whether it is the defects in
social motility which then lead to the nonfruiting phenotype. The
expression of sglK is not regulated by starvation, indicating that it is more likely involved in a general housekeeping function rather than specifically in fruiting body development. Future
experiments will focus on the studies of cellular surface structures of
sglK mutants and on further understanding the involvement of
SglK in social motility and development of M. xanthus.
We thank D. R. Zusman, Y. W. Han, Y. Li, P. Hartzell,
and P. Youderian for very helpful discussions. We also thank Bryan
Julien for kindly providing Tn5kan903.
This work was supported by NIH grant GM54666 to W. Shi and training
grant 5-T32-AI-07323 to Z. Yang.
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Abstract
Introduction
