SdeK Is Required for Early Fruiting Body Development in Myxococcus xanthus

JOURNAL OF BACTERIOLOGY,0021-9193/98/$04.0010

Sept. 1998, p. 4628–4637 Vol. 180, No. 17

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

SdeK Is Required for Early Fruiting Body Developmentin Myxococcus xanthus

ANTHONY G. GARZA,1 JEFFREY S. POLLACK,1 BARUCH Z. HARRIS,1 ALBERT LEE,1

INGRID M. KESELER,2† ELLEN F. LICKING,2 AND MITCHELL SINGER1*

Section of Microbiology, University of California, Davis, Davis, California 95616,1 andDepartment of Biochemistry, Stanford Medical School, Stanford, California 943062

Received 25 November 1997/Accepted 16 June 1998

Myxococcus xanthus cells carrying the V4408 Tn5lac insertion at the sde locus show defects in fruiting bodydevelopment and sporulation. Our analysis of sde expression patterns showed that this locus is induced earlyin the developmental program (0 to 2 h) and that expression increases approximately fivefold after 12 h ofdevelopment. Further studies showed that expression of sde is induced as growing cells enter stationary phase,suggesting that activation of the sde locus is not limited to the developmental process. Because the peak levelsof sde expression in both an sde1 and an sde mutant background were similar, we conclude that the sde locusis not autoregulated. Characterization of the sde locus by DNA sequence analysis indicated that the V4408insertion occurred within the sdeK gene. Primer extension analyses localized the 5* end of sde transcript to aguanine nucleotide 307 bp upstream of the proposed start for the SdeK coding sequence. The DNA sequencein the 212 and 224 regions upstream of the sde transcriptional start site shows similarity to the s54 family ofpromoters. The results of complementation studies suggest that the defects in development and sporulationcaused by the V4408 insertion are due to an inactivation of sdeK. The predicted amino acid sequence of SdeKwas found to have similarity to the sequences of the histidine protein kinases of two-component regulatorysystems. Based on our results, we propose that SdeK may be part of a signal transduction pathway requiredfor the activation and propagation of the early developmental program.

In response to starvation, cells of the gram-negative soil bac-terium Myxococcus xanthus initiate a developmental programthat culminates in the formation of a dome-shaped structurecalled a “fruiting body.” Construction of the multicellular fruit-ing body requires nutrient deprivation, the coordinated effortof approximately 105 cells, and a solid surface. Within the fruit-ing body, individual cells differentiate into environmentally re-sistant and metabolically quiescent spores (for reviews, seereferences 17, 18, and 47).

Fruiting body development proceeds through a series ofsteps that require cell-cell signaling. At least five extracellularsignals (designated A-signal through E-signal) are needed forconstruction of a spore-filled fruiting body (6, 10, 28, 29, 40).Several studies have examined the effects of extracellular sig-naling mutations on expression of developmentally regulatedTn5lac reporter gene fusions (22–24, 27). The results of thesestudies suggest that each extracellular signal coordinates thetemporal expression of a unique set of developmentally regu-lated genes.

The genes tagged by Tn5lac fusions have been positioned onthe M. xanthus developmental pathway based on their tempo-ral regulation by extracellular signaling events (23). In the earlyportion of the developmental pathway (between 0 and 6 h),there are two distinct classes of development genes: thosewhich require the extracellular A-signal for full expression, andthose which do not. Thus, there appears to be a bifurcation ofthe early developmental pathway into an A-signal-dependentbranch and an A-signal-independent branch.

Characterization of Tn5lac insertion mutants showed thatthe A-signal-independent fusion V4408 inhibits constructionof normal-looking fruiting bodies (24, 25). This result suggeststhat the V4408 Tn5lac insertion defines a locus that is requiredfor fruiting body formation and that the A-signal-independentpathway is essential for propagating the developmental pro-gram. Consistent with this proposal is the finding that theV4408 insertion reduces expression of a second developmen-tally regulated locus called “devRS” (25). Recent studies haveshown that a wild-type copy of the devRS locus is needed forprogression through the later stages of the developmental pro-cess (24, 25, 53).

We are interested in the regulation of gene expression in theA-signal-independent pathway and how the products of thesegenes propagate the developmental program. In this paper, wedescribe our analysis of the locus defined by V4408 Tn5lacinsertion, designated sde (for starvation-induced, developmentessential). To begin to understand how the sde genes are reg-ulated, we examined the patterns of sde expression in devel-oping cells and in vegetatively growing cells and localized thetranscriptional start site for the sde operon. DNA flankingV4408 was cloned and characterized by DNA sequence anal-ysis and insertion mutagenesis to determine which gene in thesde locus is required for fruiting body development.

MATERIALS AND METHODS

Bacterial strains and plasmids. A complete list of the strains and plasmidsused in this study is shown in Table 1. Plasmids were propagated in Escherichiacoli DH5a (11) or JM101 (34). DK101 is wild type for fruiting body developmentand sporulation, and it was chosen as the parent of all strains used in this studybecause it carries the sglA1 mutation (14). The sglA1 allele allows for dispersedgrowth in liquid cultures. Previous work by Kroos et al. (24) demonstrated thatthe developmental defects produced by the V4408 insertion are identical in bothan sglA1 background and an sglA1 background, indicating that these defects arecaused by the V4408 insertion itself and are independent of the sglA1 allele.

Strain MS1503 is a derivative of DK101 that carries a 700-bp deletion in thesde locus. The deletion removes 550 bp from the 59 end of the predicted SdeK

* Corresponding author. Mailing address: Section of Microbiology,One Shields Ave., University of California, Davis, Davis, CA 95616.Phone: (530) 752-9005. Fax: (530) 752-9014. E-mail: [email protected].

† Present address: Stanford Human Genome Center, Department ofGenetics, Stanford Medical School, Stanford, CA 94306

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coding sequence and an additional 150 bp of upstream DNA. The 700-bp dele-tion in MS1503 was constructed by a two-step gene replacement procedure witha derivative of plasmid pBJ114 (16), called pJEF9. Plasmid pJEF9 carries thegalK gene, which can be used as a counterselection for the loss of an integratedplasmid, and a 1.9-kb internal EcoRI-HindIII fragment from the sde locus. This1.9-kb insert was generated by deleting 700 bp of DNA from the internal portionof a 2.6-kb fragment of the sde locus. Plasmid pJEF9 was introduced into DK101cells as described below, and Kanr transformants that contain a single copy ofpJEF9 integrated by homologous recombination at the sde locus were identifiedby Southern blot analysis (43). The single integration event produced a tandemduplication with one copy of sdeK intact and one copy of sdeK with its 59 enddeleted. After identification of Kanr transformants with a single plasmid inser-tion, cells were inoculated into flasks containing 5 ml of CTT broth (componentsgiven below) and incubated at 32°C overnight with vigorous agitation. Cells wereharvested from overnight cultures by centrifugation, resuspended in 500 ml ofTPM buffer (components given below), and aliquoted onto CTT plates supple-mented with 2% galactose. In the presence of galactose, expression of the galKgene from plasmid pJEF9 is lethal to M. xanthus, and Galr colonies can arisefrom cells which have undergone a plasmid excision event. To identify isolatesthat had excised the chromosomal copy of plasmid pJEF9, the Galr colonies werereplica plated onto CTT in the presence and absence of kanamycin. Coloniesthat were only able to grow in the absence of kanamycin (Kans) were analyzedfurther. Excision of plasmid pJEF9 can yield a strain with a wild-type copy ofsdeK or a strain with a deletion at the 59 end of sdeK. Chromosomal DNA wasisolated from Kans colonies (43) and used for Southern blot analysis (43) toidentify strains carrying the deletion.

Media for growth and development. M. xanthus strains were grown at 32°C inCTT broth containing 1% Casitone (Difco Laboratories), 10.0 mM Tris hydro-chloride (pH 8.0), 1 mM KH2PO4, and 8 mM MgSO4 or on plates containingCTT broth and 1.5% Difco Bacto-Agar. CTT broth and CTT plates were sup-plemented with 40 mg of kanamycin sulfate per ml (Sigma) or 2% galactose(Sigma) as needed. CTT soft agar is CTT broth containing 0.7% Difco Bacto-Agar. E. coli DH5a and JM101 were grown at 37°C in Luria-Bertani broth (LB)containing 1% tryptone (Difco), 0.5% yeast extract (Difco), and 0.5% NaCl or onplates containing LB and 1.5% Difco Bacto-Agar. LB and LB plates weresupplemented with 50 mg of ampicillin per ml (Sigma) or 40 mg of kanamycinsulfate per ml (Sigma) as needed. Fruiting body development was carried out at32°C on TPM plates containing TPM buffer (10.0 mM Tris hydrochloride [pH8.0], 1 mM KH2PO4, and 8 mM MgSO4) and 1.5% Difco Bacto-Agar.

b-Galactosidase, developmental, and sporulation assays. To harvest vegeta-tively growing cells, M. xanthus strains were inoculated into a flask containing 10ml of CTT broth, and the culture was placed at 32°C with vigorous swirling. Atvarious times after the culture reached a density of 2 3 108 cells per ml, 500-ml

aliquots were removed and quick-frozen in liquid nitrogen. Developmental cellswere harvested from TPM agar plates as described previously (24), and b-galac-tosidase assays were performed with quick-frozen cell extracts (20). b-Galacto-sidase specific activity is defined as nanomoles of o-nitrophenol (ONP) producedminute21 milligram of protein21.

Fruiting body development on TPM agar plates was monitored visually with adissecting microscope (Wild-Heerbrug). Two methods were used to examinesporulation efficiency. First, the number of ovoid-shaped, phase-bright sporesproduced by developing cells was determined with a Petroff-Hausser countingchamber and phase-contrast microscopy. Second, the viability of these spores wasdetermined as described previously (53). Due to day-to-day variation in theabsolute spore number, spore assays are reported as percentage of that of thewild type for each experiment. One hundred percent wild-type sporulation wasbetween 2 3 106 and 2 3 107 spores/ml, which corresponds to 1 to 10% sporu-lation efficiency based on the number of input cells.

Analysis of RNA. To isolate RNA during growth, M. xanthus cells were inoc-ulated into CTT broth, and the culture was placed at 32°C with vigorous shaking.Cells were harvested at various times after the culture reached a density of 2 3108 cells per ml. To isolate developmental RNA, approximately 5 3 1010 cellswere harvested from TPM agar plates as previously described (20). After har-vesting, vegetatively growing cells and developing cells were quick-frozen inliquid nitrogen as described above for the b-galactosidase assays. RNA wasisolated from quick-frozen cells by the hot phenol method (43). Total cellularRNA was used in slot blot hybridizations as described by Kaplan et al. (20). Twoprobes were used for the slot blot hybridization experiments: an ApaI fragmentcontaining 300 bp of DNA immediately upstream of the V4408 insertion site andan NcoI-PstI fragment containing 1.2 kb of DNA downstream of the V4408insertion site. Similar results were found with both probes. The specificity ofthese probes was confirmed by using yeast mRNA, which yielded no detectablesignal. Primer extension analyses were carried out as described previously (15)with the modifications of Mirel and Chamberlin (36). The primers used for theseanalyses were 59-GCCACCCTAACCCCGCAGCA-39 and 59-ACACTCCCTTCATACAGACGCAG-39. Both primers were synthesized by Operon Technolo-gies, Inc., (Alameda, Calif.).

Plasmid transfer to M. xanthus. Plasmids containing DNA fragments from thesde locus were electroporated into DK101 or MS1503 cells by the technique ofPlamann et al. (41). Southern blot analysis (43) was used to identify electropo-rants that contain a single copy of the appropriate plasmid integrated by homol-ogous recombination into the sde locus or a single copy of the appropriateplasmid integrated by site-specific recombination into the chromosomal Mx8phage attachment site (attB). Kanr electroporants carrying a single plasmidinsertion were scored for development and sporulation efficiency as needed.

TABLE 1. Bacterial strains and plasmids used in this study

Strain orplasmid Relevant characteristic Source or

reference

StrainsE. coli

DH5a supE44 DlacU169 f80DlacZM15 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 11JM101 D(lac pro) thi supE F9 traD36 proAB lacIq DlacZM15 34

M. xanthusDK101 sglA1 (wild-type development) 14DK4408 sglA1 sdeK::Tn5lac V4408 24MS1503 sglA1 DsdeK This studyMS1506 sglA1 DsdeK attB::pJEF4 This studyMS1510 sglA1 sde::pELF1 This studyMS2010 sglA1 trcF::pBAR101 This study

PlasmidspBJ114 Kanr galK 16pBGS18 Kanr 49pBluescript SKII Ampr StratagenepPLH343 Kanr 19pBAR101 Kanr, pBGS18 containing 1 kb of trcF on a StuI-SalI fragment This studypIMK50 Ampr Kanr, pBluescript SKII containing 9.5 kb of V4408 Tn5lac and 12.5 kb of upstream DNA on a XhoI fragment This studypIMK51 Ampr, pBluescript SKII containing 50 bp of V4408 Tn5lac and 6 kb of upstream DNA on a BamHI fragment This studypELF1 Kanr, pBGS18 containing 50 bp of V4408 Tn5lac and 6 kb of upstream DNA on a BamHI fragment This studypJEF1 Kanr, pBGS18 containing sdeK and 6 kb of upstream DNA on a BamHI-PstI fragment This studypJEF2 Kanr, pBGS18 containing the internal sdeK 1.2-kb NcoI-PstI fragment This studypJEF2.5 Kanr, pBGS18 containing a 25-kb NcoI (within sdeK)-EcoRI fragment This studypJEF3 Kanr, pPLH343 containing sdeK and 1 kb of upstream DNA on a StuI-PstI fragment This studypJEF4 Kanr, pPLH343 containing sdeK and 1 kb of upstream DNA on a StuI-HindIII fragment This studypJEF9 Kanr, pBJ114 containing a 1.9-kb fragment of the sde locus (with a 700-bp ApaI internal deletion) This study

VOL. 180, 1998 sde OPERON 4629

Cloning the sde locus. To isolate chromosomal DNA upstream (with respect tolacZ transcription) of the V4408 Tn5lac fusion and insertion, genomic DNA wasprepared from strain DK4408 (43), digested with XhoI, and ligated into XhoI-digested plasmid pBluescript SKII. The DNA ligation mixture was electropo-rated into E. coli cells as described previously (43), and Kanr colonies wereidentified on LB plates containing kanamycin. Plasmid DNA was isolated fromovernight cultures grown in kanamycin-supplemented LB plates as described bySambrook et al. (43). One clone, plasmid pIMK50, that contains a 22-kb insertwas identified by digestion of the plasmid DNA with the appropriate restrictionenzymes. Subsequently, a BamHI fragment from pIMK50 with 50 bp of the 59end of V4408 Tn5lac and 6 kb of chromosomal DNA upstream of the insertionwas identified by restriction analysis, purified, and ligated into BamHI-digestedpBluescript SKII to yield plasmid pIMK51. To confirm that the approximately 6kb of DNA cloned into pIMK51 is identical to DNA located upstream of thechromosomal V4408 insertion, this 6-kb BamHI fragment was used as a probefor Southern blot analysis (43). Chromosomal DNA prepared from strainDK4408 was digested with SmaI, SalI, XhoI, EcoRI, or PstI, separated on an0.8% agarose gel, and blotted onto a nylon membrane. The 6-kb BamHI probemade from pIMK51 showed a hybridization pattern identical to that obtainedwhen the same blot was probed with the 59 end of Tn5lac. The restriction mapgenerated from this Southern blot analysis was similar to the one constructedpreviously by Kroos et al. (24) for DNA upstream of the V4408 insertion.

An in situ cloning technique was used to isolate chromosomal DNA down-stream (with respect to lacZ transcription) of the V4408 Tn5lac fusion andinsertion as described by Gill et al. (9) and Stephens and Kaiser (50). Kanr

transformants of strain DK101 containing a single insertion of plasmid pELF1 orpJEF2 in the sde locus were isolated as described above. After identification ofKanr transformants with the single insertion event, chromosomal DNA wasisolated and digested with PstI or EcoRI for pELF1 and pJEF2, respectively.Each chromosomal digest was then self-ligated and electroporated into E. coliDH5a (43). Electroporated cells were plated on LB plates containing kanamycinand incubated at 37°C overnight. Plasmid DNA was isolated and digested withthe appropriate restriction enzymes to confirm the composition of the clones.One clone (plasmid pJEF1) carries a 7.7-kb insert with 6 kb of chromosomalDNA upstream of the V4408 insertion site and 1.7 kb of chromosomal DNAdownstream of the V4408 insertion site. The second clone (plasmid pJEF2.5)carries a 25-kb insert, with 1.2 kb of sdeK DNA and approximately 24 kb ofdownstream DNA. Neither plasmid contains Tn5lac DNA.

DNA sequence analysis. DNA was sequenced by the dideoxynucleotide chain-termination method (44) with the Sequi-Therm cycle sequencing kit (EpicentreTechnologies, Madison, Wis.) and custom-designed oligonucleotide primers syn-thesized by Operon Technologies, Inc. The nucleotide sequence of both strandsof a 6.3-kb region in the sde locus was determined in this manner. The DNAsequence was analyzed with ABI prism software and assembled with Deneba

Sequencher software. Protein sequences were analyzed with the TMpred pro-gram to search for hydrophobicity and potential membrane-spanning domains.

RESULTSPhenotypes of the V4408 mutant. Fruiting body develop-

ment in M. xanthus is accompanied by a series of morpholog-ical and behavioral changes that can be easily observed (Fig. 1).These changes include cellular aggregation, mound formation,and spore maturation. Kroos et al. (24, 25) reported that theV4408 Tn5lac insertion in the sde locus blocks fruiting bodydevelopment and sporulation. To further examine these be-havioral and morphological defects caused by V4408, cellsfrom isogenic strains DK101 and DK4408 (DK101::Tn5lacV4408) were spotted on TPM agar for developmental assays. Acomparison of the developmental phenotypes for each strain isshown in Fig. 1. Aggregation of DK4408 cells began at aboutthe same time (8 h) as aggregation of wild-type DK101 cells.However, the aggregates of DK101 cells formed mound-shaped structures after 24 h of development, while the aggre-gates of DK4408 cells failed to compact into mounds through-out the 48 h that development was observed. Between 12 and24 h of development, the DK4408 aggregates dissociated intofeatureless, translucent mats of cells, whereas the mounds ofDK101 cells developed into darkened, spore-filled fruiting bodies.

Two methods were used to assay the sporulation efficiency ofDK4408 and DK101 cells developing on TPM agar plates for 5days. To determine the number of heat-resistant and sonica-tion-resistant ovoid spores produced by developing cells, weused a Petroff-Hausser counting chamber and phase-contrastmicroscopy. To examine the viability of these spores, we placedthem on CTT agar and counted the number of colonies thatarose. The results from the sporulation assays are summarizedin Table 2. The two techniques yielded similar results; thenumber of spores produced by DK4408 cells was about 0.8% ofthat produced by DK101 cells and the number of viable spores

FIG. 1. Behavior of M. xanthus cells during development on TPM agar plates. The developmental progress of wild-type DK101 cells, DK4408 cells, MS1503 cells,and MS1506 cells is shown. Cells were spotted on TPM starvation agar and monitored visually as described in Materials and Methods. Photographs were taken at thetimes indicated.

4630 GARZA ET AL. J. BACTERIOL.

from DK4408 cells was about 0.1% of that produced by DK101cells.

Expression of sde during growth and development. The V4408Tn5lac insertion in DK4408 places lacZ under transcriptionalcontrol of a promoter in the sde locus. Using this lacZ reporter-gene fusion, we examined sde expression in DK4408 cells de-veloping on TPM agar (Fig. 2A). b-Galactosidase specific ac-tivity began to increase within 2 h after starvation-initiatedfruiting body development. Peak b-galactosidase specific activ-ity was observed around 12 h of development, and these levelsremained relatively unchanged over the next 36 h. The level ofb-galactosidase specific activity after 12 h of development isapproximately fivefold higher than that at the onset of thedevelopmental program. To examine expression of sde duringvegetative growth, we assayed b-galactosidase specific activityin DK4408 cells placed in CTT liquid media. Figure 2B showsthat b-galactosidase specific activity started to increase duringthe transition from exponential growth to stationary phase andthat b-galactosidase specific activity continued to increase wellinto stationary phase. The peak b-galactosidase specific activityobserved in stationary phase is approximately threefold higherthan the peak observed in exponential phase. These findingsshow that expression of sde is induced in both developing cellsand in vegetative cells entering stationary phase.

To determine whether the increases in b-galactosidase spe-cific activity observed during growth and development ofDK4408 cells occur at the level of sde mRNA accumulation,RNA slot blot hybridization analysis was used. Total RNA waspurified from DK101 cells during development on TPM agarand growth in CTT broth. The RNA was transferred to nylonmembranes and probed with a 300-bp ApaI fragment contain-ing DNA immediately upstream of the V4408 insertion site(Fig. 3), and the relative levels of sde mRNA were quantified.The results shown in Fig. 2C indicate that the relative increasesin sde mRNA during growth and development are similar torelative increases in b-galactosidase specific activity. Further-more, these findings show that sde expression does not changein an sde1 or sde mutant background, suggesting that this locusis not autoregulated.

Cloning the sde locus. In a previous study, Kroos et al. (24)mapped several restriction sites in DNA upstream (with re-spect to lacZ transcription) of the V4408 insertion. The restric-

tion enzyme XhoI cuts once within V4408 Tn5lac and once ashort distance upstream of the insertion, generating a 22-kbchromosomal fragment with 9.5 kb of V4408 Tn5lac and ap-proximately 12.5 kb of upstream DNA (Fig. 3). The fact thatthe 9.5 kb of V4408 Tn5lac on this XhoI fragment contains thegene that confers kanamycin resistance (nptII) allowed us toidentify a plasmid (pIMK50) carrying the 22-kb XhoI insert. A6-kb BamHI fragment from plasmid pIMK50 was subcloned

FIG. 2. Patterns of sde expression. The mean b-galactosidase specific activ-ities found in DK4408 cells and the mean sde mRNA levels found in DK101 cellswere determined from three independent experiments. Error bars are the stan-dard deviations of the mean. The open squares represent b-galactosidase specificactivity, and solid diamonds represent cell density. The mean b-galactosidasespecific activities found at the indicated times during development on TPM agar(A) and during vegetative growth in CTT broth (B) are shown. The mean foldincrease in b-galactosidase specific activity (■) and the mean fold increase in sdemRNA (o) are compared for 0 to 12 h of development on TPM agar plates and0 to 53 h of growth in CTT liquid (C).

TABLE 2. Developmental phenotypes ofwild-type and mutant strainsa

Strain Presence of fruitingbody developmentb

% of wild-typesporesc

% of wild-typeviable sporesd

DK101 1 100.0 6 7.7 100.0 6 19.5DK4408 2 0.8 6 0.3 0.1 6 0.1MS1503 2 0.1 6 0.1 0.2 6 0.1MS1506 1 117.1 6 9.7 90.0 6 39.2MS2010 1 NDe 100f

a Cells were placed on TPM agar and allowed to develop for 5 days. Devel-opment was monitored visually as described in Materials and Methods. Directspore counts and spore viability assays were used to determine the sporulationefficiency of a strain. Spore assays were performed three times for each strainexcept where indicated. The mean values (6 standard deviations) for the sporeassays are shown as a percentage of that of DK101 (wild type). The number ofspores produced by wild-type cells ranged from 2 3 106 to 2 3 107.

b 1, fruiting bodies detected visually; 2, fruiting bodies not detected visually.c Values were determined by counting the number of heat- and sonication-

resistant spores on a Petroff-Hausser chamber by phase-contrast microscopy.d Values were determined by transferring heat- and sonication-resistant spores

to CTT agar plates, incubating the plates for 5 days, and counting the number ofcolonies that arose from the spores.

e ND, not determined.f This value was derived from one spore assay.


into pBluescript SKII and into pBGS18 to yield plasmidspIMK51 and pELF1, respectively. This 6-kb BamHI fragmentwas subsequently used as a probe for Southern blot analysis(data not shown), and DNA sequence analysis was used to con-firm that it contained 50 bp from the 59 end of V4408 Tn5lacand 6 kb of chromosomal DNA upstream of this insertion.

An in situ cloning technique was used to isolate chromo-somal DNA downstream of V4408 Tn5lac. Plasmid pELF1 wasintroduced into strain DK101 by electroporation, and Kanr

colonies were isolated for further analysis. One Kanr isolate(MS1510) was shown by Southern blot analysis to carry a singlecopy of plasmid pELF1 integrated in the chromosome by ho-mologous recombination (data not shown). The findings fromthe Southern blot analysis indicated that the restriction enzymePstI cuts once within the multicloning site of pELF1 and once1.7 kb downstream of the plasmid insertion site. Thus, by di-gesting chromosomal DNA prepared from MS1510 with PstIand ligating the pool of PstI fragments, we generated a plasmid(pJEF1) that carries 6 kb of chromosomal DNA upstream ofthe V4408 insertion site, 1.7 kb of chromosomal DNA down-stream of the V4408 insertion site, and no V4408 Tn5lac DNA(Fig. 3). The composition of pJEF1 was confirmed by restric-tion analysis and by DNA sequencing. To obtain an additional24 kb of DNA downstream of the PstI site, a similar procedure

was used (see Materials and Methods). The resulting clonecontaining this additional DNA was designated pJEF2.5.

DNA sequence analysis of the sde locus. A 6.3-kb region ofthe native sde locus from the upstream EagI site to the down-stream HindIII site was characterized by DNA sequence anal-ysis (Fig. 3 [GenBank accession no. AF031084]). This region ofDNA covers 4.1 kb upstream and 2.2 kb downstream of theV4408 insertion site. Two open reading frames (ORFs), initial-ly designated ORF1 and ORF2, were identified. Both ORFs havea strong bias toward G 1 C nucleotides in the third position ofthe codons (92.7% for ORF1 and 89.2% for ORF2), which istypical for genes in M. xanthus and other high-G 1 C organ-isms (3, 47).

The 3.6-kb ORF1 is located upstream of the V4408 insertionsite, and it is predicted to be transcribed in the opposite ori-entation relative to lacZ from the V4408 Tn5lac fusion. Thissuggests that ORF1 is not part of the operon defined by V4408.The deduced amino acid sequence of the ORF1 product shows44% identity and 64% similarity to the E. coli transcriptionrepair coupling factor, TrcF (45, 46). In E. coli, TrcF is in-volved in strand-specific repair of DNA lesions that blocktranscription by RNA polymerase. We have renamed ORF1“trcF” based on this strong sequence similarity.

The 1.6-kb ORF2 is predicted to be transcribed in the same

FIG. 3. Physical map of the V4408 insertion region. The broadened black line indicates the region that has been characterized by DNA sequence analysis. Boxesshow the locations of the indicated ORF. The arrow inside each box shows the predicted orientation of transcription of the ORF. The location of the V4408 insertion(triangle) was determined by DNA sequencing and Southern blot analysis as described in Materials and Methods. An enlargement of Tn5lac is shown above the triangle.Plasmids containing the indicated DNA fragments of the sde locus are shown below the map of the V4408 insertion region. The parentheses indicate the extent of thedeleted region in plasmid pJEF9, and the parallel slashes indicate contiguous DNA sequence that extends to the EcoRI site, approximately 24 kb downstream fromthe end of the sdeK ORF. The 300-bp ApaI fragment and the 1.1-kb NcoI-PstI used for slot blot hybridization analysis of sde mRNA are represented by stippled boxes.


orientation relative to lacZ from the V4408 Tn5lac fusion. Inaddition, the DNA sequence analysis revealed that the V4408insertion site is in the 59 end of ORF2, indicating that ORF2 ispart of the operon defined by V4408. The C-terminal 228residues of the putative ORF2 product show strong similarityand identity to the transmitter domain in histidine kinase pro-teins. Histidine kinase proteins function as sensors in two-component signal transduction systems, and a conserved histi-dine residue, which is often found in the transmitter domain ofthese proteins, is phosphorylated in response to a particularintracellular or extracellular signal. Four highly conserved re-gions have been identified in the transmitter domain of thesesensor proteins (38, 51). Region H contains the histidine res-idue that serves as the site of autophosphorylation, and regionG is thought to function as a nucleotide binding site (38, 51).A specific function has not yet been assigned to region N orregion D/F. The sequence alignments shown in Fig. 4 indicatethat ORF 2 (designated SdeK for starvation-inducible, devel-opment-essential kinase) contains all four of these conservedregions.

The deduced amino acid sequence in the N-terminal portionof SdeK yielded no significant similarities to protein sequencesin the GenBank database. However, previous analyses haveshown that the amino acid sequences of the N-terminal regionof histidine kinases are not well conserved (38). This lack of

conservation is consistent with the proposed function of theN-terminal region as input domains that change the activity ofeach protein in response to a specific signal. Further analysis ofthe N-terminal region of SdeK revealed no hydrophobic mem-brane-spanning regions, suggesting that SdeK may localize tothe cytoplasmic compartment of M. xanthus cells. Thus, SdeKmay belong to the family of cytoplasmic histidine kinase pro-teins with an N-terminal input domain followed by a C-termi-nal transmitter domain. Some of the members of this family ofproteins are DegS from Bacillus subtilis (13, 26), KinA fromBacillus subtilis (2, 39), NifL from Azotobacter vinelandii (4,42), and NtrB from E. coli (35).

The 59 end of the sdeK ORF is defined by a TAG stop codon(1169, Fig. 5A). Because the N-terminal domain is not con-served in histidine kinases, the sequence analysis does not aidin assigning the start of SdeK translation. The 59 end of thesdeK ORF contains two potential ribosome binding sites fortranslation initiation of the SdeK protein. The first putativeribosome binding site is separated from a downstream GTGcodon (1307, Fig. 5A) by 3 nucleotides, whereas the secondputative site is separated by 8 nucleotides from a CTG codon(1238, Fig. 5A). Because GTG is more commonly used as atranslation initiation codon than CTG (7), we propose that thedownstream site at 1307 is the probable start of the SdeKprotein coding sequence.

FIG. 4. Alignment of the SdeK C terminus with conserved regions in the transmitter domain of known histidine kinase proteins. Conserved sequences of FixL fromRhizobium meliloti (1, 5), KinA from Bacillus subtilis (2, 39), KinB from B. subtilis (54), NtrB from E. coli (35), PhoR from E. coli (30), and SdeK from M. xanthus areincluded. The four highly conserved regions (H-Box, N-Box, D/F-Box, and G-Box) in histidine kinase proteins are shown (38, 51). Amino acid residues found in 10to 100% of the histidine kinase proteins are presented above the sequence alignment as presented by Stock et al. (51). Black boxes indicate that a residue is absolutelyconserved among all histidine kinases. The dark gray boxes indicate that a residue is found in 75 to 98% of histidine kinases. The light gray boxes indicate that a residueis found in 10 to 74% of histidine kinases.


Localizing the 5* end of the sde developmental transcript.The DNA sequence of the sde locus strongly suggests that thesdeK gene is the first gene in the sde transcriptional unit. Toidentify the 59 end of the sde developmental transcript, primerextension analysis was performed with 12-h (the time at whichpeak levels of sde mRNA were detected) developmental RNAand a primer (ORF12) that is complementary to the regionaround the putative start codon for SdeK (Fig. 5A). Figure 5Bshows that the 59 end of the sde mRNA mapped to a guaninenucleotide 307 bp upstream of the putative start of the SdeKcoding region. In a similar primer extension experiment with asecond primer (ORF12-2), the same 59 end was detected forsde mRNA (data not shown). Analysis of this 307-bp regionupstream of sdeK revealed no obvious ORF, suggesting thatsdeK is the first gene in the sde transcript.

Examination of the 59 region preceding the putative tran-scriptional start site for the sde operon revealed a sequencewith similarities to the s54 family of promoters (52) (Fig. 5C).The similarity is strongest in the 224 region, with 5 out of 7nucleotides identical to the s54 consensus sequence. The sim-ilarity is less conserved around the 212 region, with only 2 outof 5 matches to the consensus. However, the highly conserveddinucleotides GG in the 224 region and GC in the 212 regionare found. No similarities to other families of bacterial pro-moters were observed.

Identifying the gene in the sde locus that is required fordevelopment and sporulation. The DNA sequence analysisshowed that the V4408 Tn5lac insertion is located within thesdeK coding sequence. Because sdeK and trcF appear to be indiverging operons, this finding implies that the developmentalblock caused by V4408 does not result from inactivation of thetrcF gene. The most plausible explanation for the V4408 phe-

notype is an inactivation of sdeK function or a polar effect ona gene or genes further downstream of sdeK.

To confirm that inactivation of trcF is not responsible for theV4408 phenotype, we constructed strain MS2010 and exam-ined its ability to form fruiting bodies and to sporulate (Table2). MS2010 is a derivative of strain DK101 containing an in-sertion of plasmid pBAR101 in the chromosome. The insertionin MS2010 produces two incomplete copies of the trcF gene.When placed on TPM agar plates, cells of MS2010 formednormal-looking fruiting bodies after 24 h of development, andthe number of viable spores produced by these fruiting bodieswas similar to the number of viable spores produced by wild-type DK101 fruiting bodies. These findings indicate that inac-tivation of the trcF gene is not responsible for the developmen-tal arrest observed for the V4408 insertion mutant.

To determine whether the developmental defects caused bythe V4408 insertion are due to an inactivation of the sdeK gene,we constructed a strain, designated MS1503, that carries adeletion in sdeK (DsdeK). This deletion removes 550 bp fromthe 59 end of the putative SdeK coding region, including theputative translation initiation sites, as well as an additional 150bp of upstream DNA. When a fragment of DNA located down-stream of the 700-bp deletion was used as a probe for slot blothybridization analysis, no sde mRNA was detected in theMS1503 mutant (data not shown), indicating that the deletionhas a polar effect on downstream transcription. To examine thedevelopmental defects caused by DsdeK, MS1503 cells were as-sayed for development and compared to DK101 and DK4408

FIG. 5. (A) DNA sequence of the region upstream of the V4408 Tn5lacinsertion. The black triangle shows the location of the V4408 insertion. The bentarrow above the 11 shows the 59 end of the predicted sde transcript, and the 212and 224 regions are indicated. The stop codon (TAG) that defines the 59 end ofthe sdeK ORF (1169) is shown in lowercase. Potential ribosome binding sitesand translational start codons for the SdeK protein are in boldface. Nucleotidesmarked with an asterisk are complementary to the 39 end of M. xanthus 16SrRNA (37). The dashed arrow above the GTG at position 1307 is the proposedstart of the SdeK coding sequence. The underlined regions show the sequencesthat are complementary to the two primers (ORF12 and ORF12-2) used toidentify the 59 end of the sde transcript. (B) Mapping the 59 end of the sdedevelopmental transcript by primer extension analysis. A, C, G, and T show theDNA sequencing ladders. PE, primer extension with total RNA prepared fromDK101 cells developing on TPM agar for 12 h and primer ORF12 (Materials andMethods). (C) Comparison of the sde promoter region to the s54 consensussequence and three M. xanthus s54 promoters: mbhA (21), 4521 (21), and pilA(55). The proposed start of transcription is designated by the 11.


cells (Fig. 1). Cells from all three strains began to aggregate atabout the same time, but DK4408 cells and MS1503 cells failedto compact into mounds after 48 h of development. In addi-tion, the number of spores and the number of viable sporesproduced by strain MS1503 were about 1,000-fold lower thanthose in wild-type strain DK101 and similar to those in strainDK4408 (Table 2). Thus, the defects in development andsporulation observed by the DsdeK strain are similar to thedefects caused by the V4408 insertion.

To demonstrate that the loss of sdeK function was respon-sible for the developmental phenotypes observed for strainMS1503, we attempted to complement the mutation with aplasmid, pJEF4, that contains a wild-type copy of sdeK, as wellas the upstream DNA sequences required to drive expressionof this gene. In addition, plasmid pJEF4 carries the Mx8attPsite for integration at the chromosomal Mx8 phage attachmentsite, attB. This plasmid was introduced into strain MS1503, giv-ing rise to strain MS1506. MS1506 cells were assayed for de-velopment and compared to DK101 and MS1503 cells to de-termine whether a wild-type copy of the sdeK gene can rescuethe defects caused by the DsdeK mutation (Fig. 1). MS1506cells aggregated at approximately the same time as wild-typeDK101 cells. However, these aggregates took 48 h to fullycompact into darkened fruiting bodies, while DK101 cells tookonly 24 h. When we examined the sporulation efficiency ofstrain MS1506 after 5 days, we found that it was similar to thatof strain DK101 (Table 2). These findings show that the defectsin development and sporulation caused by the DsdeK mutationcan be complemented by providing a wild-type copy of thesdeK gene at an ectopic site, although development appears tobe slightly delayed. Our results suggest that the developmentalblock produced by the V4408 insertion is due to an inactivationof the sdeK gene and not to a polar effect on a gene down-stream of sdeK.

DISCUSSION

Tn5lac transcriptional fusions were used by Kroos and Kai-ser (22) to identify approximately 30 developmentally regu-lated loci in the M. xanthus chromosome. Expression of eightof the Tn5lac fusions is induced within a few hours after star-vation initiates the developmental program (24). Among theseeight early fusions, V4408 Tn5lac is unique because the inser-tion itself produces a developmental block (24, 25). Thus, theV4408 insertion defines a genetic locus that is absolutely re-quired for progression through the developmental cycle. Wehave designated this locus sde, and in this report we haveexamined its regulation and its potential function in develop-ment.

Expression of sde is induced when cells initiate developmentand when cells enter stationary phase. Expression from theV4408 Tn5lac fusion is induced early in the M. xanthus devel-opment program (0 to 2 h), well in advance of the first visiblesigns of cellular aggregation. Peak expression of sdeK was notobserved until approximately 12 h into development, when theaggregates of cells are compacting into mounds. After 12 h ofdevelopment, the level of sde expression was found to be aboutfivefold higher than at the onset of development. Further anal-ysis of V4408 expression showed that induction of sde occurs asgrowing cells begin to enter stationary phase. Expression of sdecontinued to increase well into stationary phase, giving a peakinduction of about threefold. These results show that inductionof sde is not limited to the M. xanthus developmental program.Our finding that levels of expression of sde are similar in bothan sdeK1 and an sdeK mutant background suggests that sdeK isnot autoregulated.

What is the event that leads to activation of the sde locus?The results of our expression studies suggest that sde may beinduced when nutrient levels are insufficient to maintain sus-tained growth. Previous findings are consistent with this hy-pothesis. For example, Singer and Kaiser (48) and Harris et al.(12) showed that expression of V4408 Tn5lac is dependent onhigh levels of the intracellular starvation signal (p)ppGpp. Fur-thermore, the conditions that activated expression of sde in ourstudies correlate with the conditions previously shown to gen-erate relatively high levels of (p)ppGpp (31–33). Thus, the sdelocus may be expressed either directly or indirectly in responseto the starvation signal (p)ppGpp.

The 5* end of the sde transcript is located 307 bp upstreamof the putative start codon for SdeK. The results of primerextension analyses with developmental RNA showed that the59 end of the sde transcript is located 307 bp upstream of theputative SdeK coding region. Because this 307-bp region doesnot appear to contain a bona fide ORF, we believe that sdeK isthe first gene in the sde operon. This finding suggests that sdemRNA has a relatively long leader sequence. A similar resultwas observed by Fisseha et al. (8), who proposed that theC-signal-dependent V4403 mRNA has a 142-bp leader se-quence. The sde leader could potentially be involved in regu-lation of sde expression at either the transcriptional or trans-lational level. Alternatively, it could be involved in regulatingthe stability of sde mRNA.

The DNA sequence upstream of the transcriptional start forsde has similarity to the s54 family of promoters. This class ofpromoters has sequence identity in the 212 and 224 regions,with the GC dinucleotide in the 212 region and the GG dinu-cleotide in the 224 region being highly conserved (52). Theputative promoter for sde contains these two conserved dinu-cleotides, and it has strong identity to the consensus sequencein the 224 region (5 out of 7 nucleotides). Recent work byKeseler and Kaiser (21) suggests that two developmentallyregulated genes, mbhA and 4521, have s54-like promoters.Based on their findings, they proposed that development inM. xanthus could be regulated by a cascade or series of positiveactivators all utilizing the same s54 RNA polymerase holoen-zyme (21). Although the temporal regulation of these threegenes during development is similar, each has unique require-ments for full expression. For example, mbhA and 4521 requirethe extracellular A-signal for full expression, while sde does not(27, 29). Furthermore, mbhA requires a solid surface for ex-pression, while 4521 and sde do not (21). Thus, the contrastbetween levels of expression of these genes may reflect thedifferences in their utilization of upstream activator proteins.

The product of the sdeK gene is required for fruiting bodydevelopment and sporulation. The results of the DNA sequenceanalysis with cloned fragments of the sde locus localized thesite of the V4408 insertion to the sdeK ORF, 96 bp downstreamof the putative translation start codon of the SdeK protein. Asecond ORF, designated trcF, was identified upstream of thesdeK gene in what appears to be a diverging operon. An inser-tion within the trcF ORF has no detectable effect on develop-ment or sporulation, indicating that the product of this gene isnot required for fruiting body formation.

Cells carrying the V4408 insertion or the 700-bp deletion insdeK form loose aggregates, but these aggregates fail to com-pact into visible mound-shaped structures. Furthermore, thesemutations in sdeK reduce the number of spores and the num-ber of viable spores from about 100- to 1,000-fold. Thus, bothmutations cause similar defects in development and in sporu-lation. When a wild-type copy of the sdeK gene is provided atan ectopic site, the developmental defects caused by the DsdeKmutation could be complemented, although fruiting body for-


mation was slightly delayed. Because other studies have foundthat gene expression from the attB site can be substantiallyreduced compared to expression from native sites (8, 21), wepropose that this delay may be due to lower levels of sdeKexpression from attB. These findings suggest that the defects indevelopment and sporulation observed for the V4408 insertionmutant are due to an inactivation of the sdeK gene, implyingthat the product of sdeK is required for normal fruiting bodyformation.

The C terminus of SdeK has similarity to the transmitterdomain of histidine kinase proteins. When we searched theGenBank database for similarities to the putative product ofthe sdeK gene, we found that the C-terminal 228 amino acidsof SdeK have similarity to the transmitter domain of histidinekinase proteins of two-component regulatory systems. In thetwo-component paradigm, the histidine kinase componentsenses a specific intracellular or extracellular signal. In re-sponse to this change, it autophosphorylates on a conservedhistidine residue within the transmitter domain. The phospho-ryl group can then be transferred to an aspartate residue withinthe receiver domain of the cognate response regulator compo-nent, modulating its activity. Typically, the response regulatoris a DNA-binding protein that alters gene expression. Thus,two-component regulatory systems allow cells to monitor en-vironmental and physiological changes and then respond ac-cordingly.

The finding that SdeK may be a cytoplasmic histidine kinasehas several implications. First, it suggests that SdeK may bepart of a signal transduction pathway. Disruption of the sdeKgene blocks development prior to mound formation, indicatingthat this signal transduction pathway is needed relatively earlyin the developmental program. Second, the activity of SdeKmay be modulated by an intracellular signal or by associationwith a membrane-bound component which senses some extra-cellular change. Third, SdeK may propagate the input signal byaltering the activity of a response regulator, allowing this pro-tein to change developmental gene expression or some othercellular process. Because a wild-type copy of the sde locus isneeded for developmental expression of the devRS genes (25),it is tempting to speculate that one potential target of the SdeKsignal transduction pathway is the devRS operon. Further un-derstanding of the function of the proposed SdeK signalingpathway requires that we identify the input signal for SdeK, itsresponse regulator partner, and the target or targets of theresponse regulator.

ACKNOWLEDGMENTSWe thank Peggy Baer and Jodi Nunnari for helpful discussions and

for critical reading of the manuscript. In addition, we thank StaciaHoover and Dean Lavell for technical assistance with sequencing andfor loaning computer software.

This work was supported (in part) by a National Institutes of Healthpostdoctoral fellowship (GM19080) to A.G.G. and a National Insti-tutes of Health grant (GM54592) to M.S.

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SdeK Is Required for Early Fruiting Body Development in Myxococcus xanthus

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