INTRODUCTION

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The gram-negative soil bacterium Myxococcus xanthus undergoes multicellular development upon nutrient deprivation. The developmental process requires the coordinated effort of approximately 10⁵ cells and culminates in the formation of the multicellular fruiting body (3). Within the fruiting body, rod-shaped vegetative cells differentiate into environmentally resistant, metabolically quiescent myxospores.

The transition from a colony of vegetatively growing rods to a spore-filled fruiting body requires cell-cell communication to coordinate changes in cell behavior and movement. These changes are facilitated by the production of several cell-derived extracellular signals (2, 6, 21). Production and reception of these signals result in a signal transduction cascade that directs the coordinated expression of specific developmentally regulated genes. Thus far, three signaling systems, the A-, C-, and E-signaling pathways, have been described in detail for M. xanthus (2, 6). To gain a better understanding of the relationship between the various signaling systems and gene expression, a battery of developmentally regulated Tn5lac fusions has been used to examine the dependence of gene expression on each signaling system (14-16, 20). Such analyses have allowed the identification of genetic regulatory circuits used by M. xanthus to control developmental-gene expression. Additionally, this type of genetic approach has allowed the placement of additional genes, such as those responsible for the reception and transduction of the various signals, onto the genetic regulatory circuits.

A-signaling mutants are blocked early in development (about 1 to 2 h poststarvation), and as such, expression of most developmentally regulated Tn5lacZ fusions is impaired. A-signal is a mixture of amino acids and peptides, believed to be produced by extracellular proteolysis, that acts as a quorum sensor (21). Defective C-signaling results in a developmental block at approximately 6 to 8 h post-starvation initiation, and activation of Tn5lac fusions expressed after this time point is diminished (15). C-signal activity requires the presence of a cell surface-associated protein, CsgA, which shares a high degree of sequence similarity and identity with members of the short-chain alcohol dehydrogenase family (22). CsgA is encoded by the csgA gene (7), and csgA mutants fail to aggregate or to sporulate properly (30). Furthermore, csgA mutants do not form the synchronous cell waves, or ripples, that are characteristic of developing cells. Little is known about how C-signal is transmitted. Transduction of C-signal does require FruA, a two-component response regulator (4). The phenotype of cells carrying a fruA mutation is similar to that of csgA mutant cells (32). Thus far, FruA is the only proposed component of the C-signal transduction pathway.

Previous work on early developmental-gene expression has identified a bifurcation early in the M. xanthus developmental pathway. One branch, designated the population starvation branch (31), requires the A-signal quorum sensor for expression of its genes. The genes on the second branch, designated the cellular starvation branch (31), require only the starvation initiation signal (p)ppGpp for expression. By studying mutants, it has been determined that both branches are required for fruiting-body development and sporulation, indicating that the branches eventually converge. Mutations in specific genes in either branch lead to developmental arrest. One such gene, which is on the cellular starvation branch, is sdeK (5, 16, 17), a proposed histidine kinase-encoding gene. Here we provide direct evidence that sdeK encodes a histidine kinase, and we further define the role that sdeK plays in developmental signal transduction. We also propose that SdeK may be the link between the cellular and population pathways and that the SdeK signal transduction pathway works in concert with the C-signal transduction pathway to regulate gene expression through the aggregation stage in M. xanthus development.

	MATERIALS AND METHODS

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Bacterial strains, transductions, and plasmids. A complete list of strains and plasmids used in this study is shown in Table 1. Plasmids were propagated in Escherichia coli DH5 (8). M. xanthus DK1622, which is wild type for both fruiting-body formation and sporulation, was chosen as the wild-type strain for this study (12). Strain MS1512 (DK1622 sdeK1) was constructed as described previously for the DK101 derivative MS1503 (5). Myxophages Mx4 ts18 ts27 hrm and Mx8 clp2, which have been described previously (1, 24), were used to transduce the Tn5lacZ transcriptional fusions employed in this study from their parental strains (16) into MS1512. Myxophage Mx8 was used to transduce csgA::Tn5-132 (18) from DK5216 into MS1523. Plasmids pJEF39 and pJEF40 were introduced into MS1512 via electroporation as described previously (28). The resulting kanamycin-resistant transformants were screened by Southern blot analysis (29) for confirmation of a single insertion of the appropriate plasmid at the correct chromosomal location, within the sdeK locus.

Media for growth, transductions, and development. The M. xanthus strains were grown with vigorous shaking at 32°C in CTT broth (1% Casitone [Difco Laboratories], 10 mM Tris-HCl [pH 7.6], 1 mM KH₂PO₄, 8 mM MgSO₄) or on CTT plates containing 1.5% agar (Difco). CTT plates were supplemented with 40 µg of kanamycin monosulfate (Sigma) per ml or 12.5 µg of oxytetracycline (Sigma) per ml as required. Myxospores and transduced cells were suspended in CTT soft agar (CTT broth containing 0.7% agar) prior to being plated. E. coli DH5 was grown with shaking at 37°C in Luria broth or on Luria-Bertani agar medium as described previously (29). Luria broth and Luria-Bertani agar medium were supplemented with 40 µg of kanamycin monosulfate or 50 µg of ampicillin (Sigma) per ml as needed. Stocks of the generalized transducing phages Mx4 and Mx8 were prepared on donor cells grown with vigorous shaking at 32°C in CYE broth (1% Casitone, 0.5% yeast extract [Difco Laboratories], 8 mM MgSO₄). M. xanthus development was conducted at 32°C on TPM agar medium, which is composed of TPM buffer (10 mM Tris-HCl [pH 7.6], 1 mM KH₂PO₄, 8 mM MgSO₄) and 1.5% agar.

Development, sporulation efficiencies, rippling assays, and -galactosidase expression. Vegetatively growing M. xanthus cells were harvested during mid-exponential phase by centrifugation (7,700 × g for 5 min) and plated for development as described previously (16). Progress of fruiting-body development was observed visually by using a dissecting microscope (Wild-Heerburg). Determination of sporulation efficiencies was conducted as described previously (34).

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Developmental rescue experiments. Wild-type DK1622 cells and the isogenic sdeK1 derivatives were prepared for developmental assay as described above. Cells suspended in TPM medium were mixed in equal proportions, applied to TPM agar in 20-µl aliquots, and incubated at 32°C for 5 days.

A- and C-factor assays. A-factor assays were conducted, as described previously, by the in situ method (16). One unit of A-factor is defined as the amount required to produce 1 U of -galactosidase activity above background (21). C-factor assays were performed by a developmental mixing protocol. Cells defective for C-signal production (DK2630) were mixed with an equal proportion of DK1512 (sdeK1) cells, and the cell mixture was plated for development as described above. Germinated spores (100-colony sample size) were then scored for kanamycin resistance, to determine lineage, by patching colonies onto CTT plates containing kanamycin.

Overproduction and purification of His-SdeK. His-SdeK was overproduced using the plasmid pTrcHisB (Invitrogen). The putative sdeK open reading frame (ORF) was cloned into pTrcHisB as a 1.6-kb BamHI-HindIII fragment from psdeK1 to form psdeK2 (Table 1). An additional 33 bases at the 5' end of the putative sdeK ORF were included in the clone because of restriction site availability. As a result, SdeK contained an additional 11 amino acids at its N terminus. The overexpressed SdeK contained an N-terminal polyhistidine tag. E. coli cells containing the plasmid psdeK2 were grown as described above and induced to express His-SdeK by the addition of 1 mM isopropyl--D-thiogalactopyranoside (IPTG) when culture turbidity reached an optical density at 600 nm of 0.3. Protein was overproduced for 3 h, at which point the cells were pelleted (1,000 × g for 10 min) and stored at 20°C.

Phosphorylation assays. Frozen aliquots of His-SdeK or His-SdeKH286A protein were thawed to room temperature and diluted 10-fold in 20 mM Tris (pH 8.7). The protein was incubated 1 h on ice to allow refolding. The phosphorylation assays were performed with a 30-µl reaction volume containing 50 mM Tris (pH 7.6), 50 mM KCl, 10 mM MgCl₂, 1 mM DTT, and 0.1 mM EDTA. Refolded protein was added to a final concentration of 0.5 µM. The reaction was initiated by the addition of 30 µCi of [-³²P]ATP (6,000 Ci/mmol; NEN Life Science Products, Inc.). The reaction mixture was incubated at room temperature for 10 min, and the reaction was stopped by addition of 0.2 volume of 5× protein loading buffer (250 mM Tris [pH 6.8], 10% glycerol, 0.02% bromophenol blue, 1% sodium dodecyl sulfate [SDS], 150 mM -mercaptoethanol). The samples were heated for 3 min at 55°C and subsequently loaded onto an SDS-12% polyacrylamide gel. Electrophoresis was performed at a constant 200 V for approximately 1 h. The gel was subsequently soaked in approximately 15 ml of SDS running buffer (100 mM Tris base, 800 mM glycine, 35 mM SDS) twice, for 20 min each time, to remove any unincorporated [-³²P]ATP. Labeled protein was detected with a PhosphorImager (Storm 840; Molecular Dynamics).

Site-directed mutagenesis. The plasmid psdeK2 (Table 1) was used as the template for mutagenesis. The single-stranded primers used to generate His-SdeKH286A and SdeKH286A within psdeK2.5 and pJEF40, respectively, were primer 1 (5'-GGCGTGCTGGGCGCGGACCTGGGCAATCC-3') and primer 2 (5'-GGATTGCCCAGGTCCGCGCCCAGCACGCC-3') (Fisher). Mutagenesis was carried out by the method described by the manufacturer of the ExSite kit (Stratagene).

	RESULTS

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SdeK is a histidine kinase. As reported previously, the ORF contains the 4408 Tn5lacZ insertion, which disrupts a gene that encodes a histidine kinase, as determined by sequence analysis (5). The defining characteristic of histidine kinases is an autophosphorylation activity in which ATP is used to phosphorylate a conserved histidine residue (9, 27). To determine whether SdeK possesses this activity, an N-terminal polyhistidine-tagged SdeK fusion protein (His-SdeK) was constructed and purified from E. coli DH5. When overexpressed in E. coli, the His-SdeK fusion protein formed inclusion bodies, which were then purified, solubilized, and refolded as described in Materials and Methods. The His-SdeK purification scheme yielded a protein preparation of approximately 70 to 80% purity, as determined by Coomassie staining of the polyacrylamide gel after electrophoresis (data not shown). The soluble, refolded protein preparation was then assayed for autophosphorylation in the presence of [-³²P]ATP. A parallel assay was performed on MBP-EnvZ (a gift from M. Igo) as a positive control. Figure 1 shows that both the His-SdeK fusion protein (lane 2) and the MBP-EnvZ control (lane 1) were labeled in the presence of [-³²P]ATP.

View larger version (46K): [in this window] [in a new window]   FIG. 1.   His-SdeK is a histidine kinase. Phosphorylation reactions with MBP-EnvZ (lane 1), His-SdeK (lane 2), and His-SdeKH286A (lane 3) are described in Materials and Methods. H268 is required for phosphorylation of His-SdeK (lane 3). Two sets of MBP-EnvZ and His-SdeK phosphorylation reactions were performed; one product set was incubated for 10 min at 100°C prior to electrophoresis (lanes 4 and 5). MBP-EnvZ (lane 4) and His-SdeK (lane 5) were stable to 10 min of heat treatment at 100°C (compare lanes 1 and 2 with lanes 4 and 5, respectively). Phosphoproteins were visualized by phosphorimaging. Stability of the phosphorylated forms of His-SdeK and MBP-EnvZ to acid and to base treatment was assayed (lanes 6 through 9). After phosphorylation and electrophoresis, two identical gels were treated with 0.2 M HCl (lanes 6 and 7) or 1.0 M NaOH (lanes 8 and 9).

Phosphorylated His-SdeK displays lability characteristics indicative of a histidine phosphate label. The stability of the phosphorylated His-SdeK fusion protein was assayed at a high temperature and under acidic and basic conditions to determine whether the phosphorylated His-SdeK protein was phosphorylated on a histidine residue. Phosphohistidyl residues are heat and acid labile but base stable, while phosphoester groups, such as phosphoserines and phosphotyrosines, are heat and acid stable but base labile (1a, 27). Heating of phosphorylated His-SdeK at 100°C for 10 min resulted in a significant decrease in phosphorylation activity, as determined by phosphorimaging (Fig. 1, lane 5). The loss of phosphorylated protein was not due to heat-induced protein degradation (data not shown). Furthermore, after incubation of an SDS-polyacrylamide gel containing phosphorylated His-SdeK for 30 min at 50°C in pH 4 buffer, phosphorylated protein was no longer detectable by phosphorimaging (Fig. 1, lane 7). Coomassie staining of the gel indicated that the loss of labeled protein did not result from protein degradation. Incubation in basic buffer (see Materials and Methods) yielded no change in intensity of the labeled His-SdeK (Fig. 1, lane 9). A phosphorylated MBP-EnvZ control displayed similar characteristics in each experiment (Fig. 1, lanes 4, 6, and 8).

H286 in SdeK is required for activity. It was determined, based on protein sequence alignment, that the putative site of autophosphorylation of SdeK is H286 (5). To test whether this histidine is required for autophosphorylation of His-SdeK, the residue was changed to an alanine by site-directed mutagenesis (see Materials and Methods). The gene encoding the SdeK mutant protein in which an alanine is substituted for the histidine at position 286 (SdeKH286A) is carried on the plasmid psdeK2.5 (Table 1). Sequencing of psdeK2.5 verified that no additional mutations were introduced during the site-directed mutagenesis. The corresponding fusion protein, His-SdeKH286A, was then expressed, purified, and assayed for activity in parallel with the wild-type His-SdeK fusion. Equal amounts of wild-type and mutant protein were assayed in each reaction. The His-SdeKH286A fusion protein exhibited no detectable autophosphorylation activity (Fig. 1, compare lanes 2 and 3). Thus, H286 is essential for His-SdeK activity.

View larger version (154K): [in this window] [in a new window]   FIG. 2.   The putative phosphorylation site, H286, is required for in vivo activity of SdeK. M. xanthus strains DK1622 (wild type [WT]), MS1512 (sdeK1), MS1527(sdeK1 sdeK+), and MS1526 (sdeK1 sdeKH286A) were developed for 3 days on TPM agar. Photographs were taken 72 h after development initiation. Magnification, ×96.

Is sdeK required for extracellular signaling? If the defects in fruiting-body formation and sporulation in the sdeK mutant strain are caused by a deficiency in extracellular signal production, then codevelopment with wild-type cells should rescue the developmental phenotype. Isogenic wild-type DK1622 cells and DK4300 (sdeK::Tn5lac) cells were mixed at a 1:1 ratio and allowed to codevelop on TPM agar. After 3 days, spores were harvested and assayed for viability. To determine whether any of the spores were derived from DK4300 cells, 100 colonies were scored on CTT plates containing kanamycin. None of the 100 colonies scored after codevelopment was kanamycin resistant, indicating that there was no rescue of the DK4300 sporulation defect by codevelopment with wild-type cells.

The sdeK mutant produces both A- and C-signal. While the above-described experiments demonstrated that the phenotype of an sdeK-null mutant cannot be rescued extracellularly, the experiments did not specifically address whether sdeK1 cells are defective in the production of specific extracellular signals. Therefore, we assayed the ability of the sdeK1 mutant to produce A- and C-signal. A-signal production by wild-type, asgA476, and sdeK1 cells was assayed directly by measuring the amount of A-factor released into the cell suspension (20, 28). Conditioned medium was assayed for A-factor activity by using the A-factor-dependent 4521 Tn5lacZ gene fusion as a reporter as previously described (20, 28). The results, shown in Table 2, indicated that the levels of A-factor produced by sdeK1 cells were equivalent to those produced by wild-type cells, thereby demonstrating that sdeK1 cells are capable of producing A-factor.

SdeK is required for activation of developmental-gene expression. To further define the role played by SdeK in M. xanthus development and to determine the timing of its action, expression of several Tn5lac reporter fusions in the sdeK1 mutant background was studied. Nine developmentally regulated Tn5lacZ fusions were transduced into the sdeK1 strain. All transductants were verified for single-copy insertion at the appropriate location by Southern analysis prior to use. Isogenic wild-type and sdeK1 strains carrying each of the nine Tn5lac reporter fusions individually were assayed for developmental expression of each fusion. Two of the fusions, 4455 and 4469, like sdeK, lie on the cellular starvation pathway branch and require only the starvation initiation signal for expression. They are expressed independently of A- and C-signal. The 4455 fusion, which is activated between 1 and 2 h poststarvation, is expressed at wild-type levels in the sdeK1 background (data not shown), while 4469 expression, which is activated at approximately 4 h poststarvation, is increased about twofold over wild-type levels (Fig. 3A).

View larger version (49K): [in this window] [in a new window]   FIG. 3.   Effects of sdeK1-null genotype on expression of Tn5lac fusions 4469 (A-signal-independent fusion) (A), 4403 (C-signal-dependent fusion) (B), 4406 (C-signal-dependent fusion) (C), 4435 (C-signal-dependent fusion) (D), 4400 (partially C-signal dependent fusion) (E), and 4414 (partially C-signal dependent fusion) (F). Tn5lacZ fusions were transduced into sdeK1 strain MS1512 as described in Materials and Methods. Cells were plated for development, and mean -galactosidase specific activities were determined from experiments done in triplicate at various time points as described in Materials and Methods. Error bars represent standard deviations of the means. Each triplicate experiment was conducted two or three times, and the data presented are from one representative experiment. Wild type, ; sdeK1, ; csgA, . ONP, o-nitrophenol.

	DISCUSSION

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The sdeK gene was originally defined by the 4408 Tn5lacZ transcriptional fusion. We previously reported that sdeK expression is dependent on (p)ppGpp and becomes activated when cells begin to enter stationary phase (5), which resulted in its classification as a starvation-dependent gene. Although sdeK is expressed immediately upon starvation, disruption of sdeK results in both a block in M. xanthus development approximately 6 to 8 h poststarvation at the early aggregation stage and a strong sporulation defect (5, 15). Analysis of the sdeK sequence led to the conclusion that sdeK encodes a histidine kinase. Here we have presented biochemical and genetic data demonstrating that SdeK is a histidine kinase and have further characterized the role that SdeK plays in M. xanthus development.

SdeK shares a high degree of sequence similarity and identity with the PhoR family of histidine kinases (5). The feature that best defines a histidine kinase is its ability to autophosphorylate on a conserved histidine residue (9, 27). We constructed an SdeK fusion protein with a polyhistidine tag at its N terminus (His-SdeK) and found that it was phosphorylated when incubated with [-³²P]ATP. Based on sequence analysis of SdeK, H286 was identified as the putative phosphorylation site. When this residue was changed to an alanine by site-directed mutagenesis, the protein was no longer phosphorylated in vitro, nor was it able to complement the sdeK1 phenotype. Thus, H286 is required for in vitro phosphorylation of His-SdeK and in vivo function of SdeK. Phosphorylated His-Sdek also shows the heat and acid lability and base stability indicative of histidine kinases. Analysis of these data has led us to conclude that SdeK is a histidine kinase.

SdeK and C-signal both exert control over a similar set of genes. The fact that M. xanthus development does not progress beyond the aggregation stage (6 to 8 h poststarvation) when sdeK is disrupted indicates that SdeK plays a critical role in development. We demonstrated that sdeK cells are able to produce both A- and C-signal to wild-type levels and cannot be rescued extracellularly when codeveloped with wild-type cells. These data indicate that SdeK is not involved in the production of extracellular signals.

What role does the SdeK signal transduction pathway play in development? We have previously proposed that M. xanthus cells monitor their nutritional status by monitoring their translation efficiency via relA and the stringent response (31). Our current model for the regulation of early developmental-gene expression is depicted in Fig. 4. Upon activation of the developmental program, a bifurcation in the pathway occurs. One branch, designated the population-sensing branch, leads to A-signal production and reception; in addition, this branch controls production and reception of C-signal, a second extracellular signaling system required for aggregation (13, 23). The second branch acts independently of any extracellular signals and only requires starvation for activation of gene expression (31). This branch has been designated the cellular starvation branch. Genes under the control of this branch include 4469 and sdeK (31). Based on the fusion data presented in this paper, we confirm that these two branches converge at approximately 6 to 8 h post-development initiation (15). The question now becomes the following: what is the mechanism by which SdeK and its signal transduction pathway interact with the C-signaling pathway to alter gene expression at this point in development?

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Model for early developmental-gene expression. Development is initiated by amino acid limitation, which activates the stringent response mediated by the ribosome-associated protein RelA. RelA activation induces an increase in the level of (p)ppGpp, which activates the cellular starvation pathway and, in conjunction with AsgA and AsgB, leads to the production of A-signal (upward-pointing, broadly dashed arrow on left) and activation of the population-sensing pathway. C-signal, a second extracellular signal required for regulation of coordinated cell movement and gene expression, lies on the population-sensing branch and is depicted by the upward-pointing, broadly dashed arrow on the right. Genes expressed along the two different branches are designated by thinner, more narrowly dashed arrows at the time of activation. The two branches merge at approximately 6 h, at which point both C-signal (downward-pointing, broadly dashed arrow on right) and SdeK become necessary for gene expression. The model also shows the inhibitory effect (bent tipless arrow) of SdeK on the expression of 4469.