Myxococcus xanthus sasN Encodes a Regulator That Prevents Developmental Gene Expression during Growth

Di Xu; Chun Yang; Heidi B Kaplan

doi:10.1128/jb.180.23.6215-6223.1998

. 1998 Dec;180(23):6215–6223. doi: 10.1128/jb.180.23.6215-6223.1998

Myxococcus xanthus sasN Encodes a Regulator That Prevents Developmental Gene Expression during Growth

Di Xu ¹, Chun Yang ¹, Heidi B Kaplan ^1,^*

PMCID: PMC107706 PMID: 9829930

Abstract

Myxococcus xanthus multicellular fruiting body development is initiated by nutrient limitation at high cell density. Five clustered point mutations (sasB5, -14, -15, -16, and -17) can bypass the starvation and high-cell-density requirements for expression of the 4521 developmental reporter gene. These mutants express 4521 at high levels during growth and development in an asgB background, which is defective in generation of the cell density signal, A signal. A 1.3-kb region of the sasB locus cloned from the wild-type chromosome restored the SasB⁺ phenotype to the five mutants. DNA sequence analysis of the 1.3-kb region predicted an open reading frame, designated SasN. The N terminus of SasN appears to contain a strongly hydrophobic region and a leucine zipper motif. SasN showed no significant sequence similarities to known proteins. A strain containing a newly constructed sasN-null mutation and Ω4521 Tn5lac in an otherwise wild-type background expressed 4521 at a high level during growth and development. A similar sasN-null mutant formed abnormal fruiting bodies and sporulated at about 10% the level of wild type. These data indicate that the wild-type sasN gene product is necessary for normal M. xanthus fruiting body development and functions as a critical regulator that prevents 4521 expression during growth.

Myxococcus xanthus fruiting body development is a highly coordinated process. To initiate the developmental program, the cells must sense nutrient limitation at high cell density (20, 28). If both conditions are met, about 100,000 rod-shaped vegetative cells aggregate to form a fruiting body, within which individual cells differentiate into spherical and refractile myxospores. Myxospores remain dormant and environmentally resistant until conditions permit germination (6, 7).

Nutrient limitation is sensed by M. xanthus cells, at least in part, by a rise in intracellular guanosine penta- and tetraphosphate ([p]ppGpp) (16, 43). Cell density appears to be sensed through an extracellular signal, termed A signal (28). The A signal has been identified as a mixture of specific amino acids that appear to be generated by M. xanthus autoproteolysis of surface proteins within the first hours after the onset of starvation (27). When the collective concentration of A signal in a community of starving cells reaches above 10 μM, cells initiate development by aggregating (28).

The expression of certain developmental genes requires independent input from both starvation and A signal (3, 24). The best-characterized gene of this class is designated 4521, in reference to the Ω4521 Tn5lac insertion. In wild-type strains, 4521 expression begins to increase at 1.5 to 2 h after the onset of development and increases greater than 10-fold by 6 to 8 h (19, 26). The expression of 4521 is cell density dependent (28). Its expression is low in starved cells at densities less than 3 × 10⁸ cells per ml and rises dramatically at densities above this level. The addition of exogenous A signal to low-density starved cells can rescue 4521 expression to near-maximal levels (28). When asg mutants, which are deficient in A-signal generation (19, 26), are starved at high density, 4521 is not expressed. Three unlinked asg genes (asgA, asgB, and asgC) have been identified previously (14, 25, 30). Their DNA sequences indicate that they encode regulatory proteins required for A-signal production (4, 35, 36). The expression of 4521 can be restored to asg mutants by the addition of exogenous A signal (27) or by the presence of asg suppressor mutations, designated sas (for suppressor of asg) (19).

The 4521 promoter has been determined by DNA sequence analysis and mutagenesis to be a member of the sigma-54-dependent family of promoters (23). Related studies have shown that a regulatory region of at most 146 bp upstream of the transcription start site is required for wild-type 4521 expression during growth and development (11). Transcription initiation of the 4521 promoter, similar to that of other sigma-54-dependent promoters, is expected to require an NtrC-like activator to bind upstream of the transcription start site (42).

To study the mechanism by which starvation and A-signal sensing are integrated, six mutants that expressed 4521 at a high level during growth and development were isolated in an asg background after UV mutagenesis (19). One of these mutations, sasB7, did not cluster with the others (sasB5, -14, -15, -16, and -17) (19). The sasB7 mutation is a gain-of-function mutation that maps approximately 4 kb away from the others, within the sasS gene, which encodes a histidine protein kinase component (50) of a putative two-component signal transduction system (49).

We report here the cloning, sequencing, and analysis of the wild-type sasN gene (previously referred to as sasB5 [50]) and the five clustered mutations (sasB5, -14, -15, -16, and -17) that map to it. We have determined that the wild-type sasN gene product is necessary for normal M. xanthus fruiting body formation and sporulation and that SasN functions as a critical regulator that prevents 4521 gene expression during growth.

MATERIALS AND METHODS

Plasmids, bacteria, and growth conditions.

The plasmids and bacterial strains used in this study are listed in Table 1. Strains such as DK6623 contain three genetic elements. The Ω4521 Tn5lac (Tc^r) insertion allows the expression of the 4521 reporter gene to be monitored (24, 26). The asgB480 mutation reduces A-signal production and thus blocks 4521 expression. The presence of sasB5, an asgB480 suppressor mutation, restores 4521 expression (19). M. xanthus strains were grown with vigorous shaking at 32°C in CTT liquid medium (1% Casitone [Difco], 10 mM Tris-HCl [pH 7.6], 1 mM K₂HPO₄-KH₂PO₄ [pH 7.7], 8 mM MgSO₄; pH 7.6) or on CTT agar (CTT liquid with 1.5% Bacto Agar). Kanamycin (40 μg/ml), tetracycline (12.5 μg/ml), or trimethoprim (250 μg/ml) was added when appropriate. The growing cells were used in the mid-exponential phase (80 to 160 Klett units, or 4 × 10⁸ to 8 × 10⁸ cells/ml). Escherichia coli was grown in Luria-Bertani (LB) broth (37) or on LB agar (LB broth plus 1.5% agar) containing ampicillin (50 μg/ml), kanamycin (40 μg/ml), or trimethoprim (100 μg/ml) when necessary.

TABLE 1.

Plasmid and strain list

Plasmid or strain	Relevant characteristic(s)	Source, reference, or derivation
Plasmids
pWM341	Kan^r	31
pBluescript SK(+)	Amp^r	Stratagene
pBGS18	Kan^r	44
pGEM3+	Amp^r	Promega
pHBK429	Kan^r Tn5 IS50L	12
pHBK274	Kan^rlacZ Mx8 phage attP	5.5-kb XhoI attP fragment from pLJS49 (40) ligated into the DraI-SphI site of pDAH274 (38)
pYC274	Kan^rattP	50
pYC1007	Kan^r SasB⁺	22-kb insert containing a portion of the wild-type sasB locus, resulting from in situ cloning of HK1406 chromosomal DNA
pYC1008	Kan^r	2.7-kb MluI fragment of pYC1007 ligated into the pBGS18 SmaI site
pYC1009	Kan^r	4.2-kb MluI fragment of pYC1007 ligated into the pBGS18 SmaI site
pYC1010	Kan^r	6.4-kb MluI fragment of pYC1007 ligated into the pBGS18 SmaI site
pYC1012	Kan^rattP	4.2-kb HindIII-EcoRI fragment of pYC1009 ligated into pYC274
pYC1209	Kan^r; internal fragment of sasS
pDX45-2	Amp^r	4.2-kb XbaI-XhoI fragment from pYC1009 ligated into HincII site of pGEM3
pDX45-2Tp	Tp^r	Dihydrofolate reductase gene from R-338 (47) ligated into the pDX45-2 EcoRI site
pDX45-2-12	Amp^r	Derived from pDX45-2; 1,409 bp deleted from the XbaI site
pDX45-2-16	Amp^r	Derived from pDX45-2; 1,766 bp deleted from the XbaI site
pDX45-2-24	Amp^r	Derived from pDX45-2; 2,614 bp deleted from the XbaI site
pDX45-2-25	Amp^r	Derived from pDX45-2; 2,812 bp deleted from the XbaI site
pDX77	Amp^r Kan^rattP	3.0-kb SmaI attP region from pHBK274 ligated into BamHI-HindIII site of pWM341
pDX76	Amp^r Kan^rattP	3.8-kb SmaI fragment containing attP-aph from pDX77 ligated into the pDX45-2-24 EcoRI site
pDX78	Amp^r Kan^rattP	3.8-kb SmaI fragment containing attP-aph from pDX77 ligated into the pDX45-2-12 EcoRI site
pDX80	Amp^r Kan^rattP	3.8-kb SmaI fragment containing attP-aph from pDX77 ligated into the pDX45-2-16 EcoRI site
pDX91	Amp^r Kan^rattP	3.8-kb SmaI fragment containing attP-aph from pDX77 ligated into the pDX45-2-25 EcoRI site
pSK500-4	Amp^r	2-kb XhoI-SacI fragment from pYC1009 ligated into the XhoI-SacI site of pBluescript SK
pDX61	Kan^raph	1.5-kb SalI-XhoI fragment of pWM341 ligated into the sasN SacI site in pSK500-4
pDX45-2-18	Amp^r	Derived from pDX45-2; 2,077 bp deleted from the XbaI site
pDX63.1	Amp^r Kan^r ΔsasN::aph	2.7-kb SalI-XhoI sasN-aph fragment of pDX61 ligated into SmaI-BamHI-digested pDX45-2-18
pDX63.4	Kan^r Tp^r ΔsasN::aph	1.1-kb fragment of dihydrofolate reductase gene ligated into the EcoRI site of pDX63.1
pDX56	Kan^r; internal sasN fragment	0.68-kb SacI-SalI fragment of pYC1009 ligated into PstI, EcoRI sites of pBGS18
pDX58	Tp^rsasN-lacZ	3.6-kb SalI-XhoI lacZ fragment from pHBK274 ligated to XbaI-NotI site of pDX45-2Tp
pDX45-2-6	Amp^r	Derived from pDX45-2; 845 bp deleted from the XbaI site
pDX66	Amp^r Kan^r	1.5-kb HindIII-SalI fragment pWM341 containing the aph gene ligated into the HindIII-SacII site of pDX45-2-6
pDX98	Amp^r Kan^r	4.1-kb insert containing the sasN sasB14 allele resulting from in situ cloning of DK6631
pDX99	Amp^r Kan^r	4.1-kb insert containing the sasN sasB15 allele resulting from in situ cloning of DK6632
pDX100	Amp^r Kan^r	4.1-kb insert containing the sasN sasB16 allele resulting from in situ cloning of DK6633
pDX101	Amp^r Kan^r	4.1-kb insert containing the sasN sasB17 allele resulting from in situ cloning of DK6634
pDX102	Amp^r Kan^r	4.1-kb insert containing the sasN sasB5 allele resulting from in situ cloning of DK6623
Strains
E. coli DH5α	supE44 ΔlacU169 (φ80lacZΔM15 hsdR17 recA1 endA1 gyrA96 thi-1 relA1)	15
M. xanthus
DK101	sglA1	17
DK1622	Wild type	18
DK6620	Tn5lac Ω4521 (Tc^r) sglA1	19
DK6623	Tn5lac Ω4521 (Tc^r) sglA1 asgB480 sasB5	19
DK6631	Tn5lac Ω4521 (Tc^r) sglA1 asgB480 sasB14	19
DK6632	Tn5lac Ω4521 (Tc^r) sglA1 asgB480 sasB15	19
DK6633	Tn5lac Ω4521 (Tc^r) sglA1 asgB480 sasB16	19
DK6634	Tn5lac Ω4521 (Tc^r) sglA1 asgB480 sasB17	19
DK6662	Tn5 Ω6658 (Tc^r) sglA1	19
HK1406	Tn5 Ω6658 (Tc^r)::pHBK429 sglA1	Electroporation of pHBK249 into DK6662
HK1210	ΔsasN::aph sglA1	Electroporation of pDX63.4 into DK101
HK1211	ΔsasN::aph	Electroporation of pDX63.4 into DK1622
HK1212	Tn5lac Ω4521 (Tc^r) sglA1 sasN disrupted by an internal fragment	Electroporation of pDX56 into DK6620
HK1217	sasN::lacZ sglA1 Kan^r	Electroporation of pDX58 into DK101
HK1218	Tn5lac Ω4521 (Tc^r) sglA1 asgB480 sasB5 sasS disrupted by an internal fragment	Electroporation of pYC1209 into DK6623
HK1503	Tn5lac Ω4521 (Tc^r) sglA1 sasS disrupted by an internal fragment	50

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General molecular biological methods.

Cloning and manipulations of plasmid DNA were performed according to standard protocols (37). Plasmid DNA for sequencing was prepared by using a QIAprep spin kit (Qiagen). DNA sequencing was performed at the DNA core facility of the Department of Microbiology and Molecular Genetics, University of Texas-Houston Medical School, with an ABI 373A DNA sequencer (Perkin-Elmer, Applied Biosystems Division).

Methods for genetic and developmental analysis of M. xanthus.

The following methods for M. xanthus manipulation used in this study have been previously described elsewhere: electroporation of plasmid DNA into M. xanthus (21), M. xanthus chromosomal DNA preparation (1), M. xanthus development on TPM starvation agar (24) and in submerged culture (25), viable myxospore assay (25), and β-galactosidase assays (24). The alterations to the chromosome for each M. xanthus strain constructed in this study were confirmed by Southern blot analysis (37).

In situ cloning of the wild-type sasB locus.

A 22-kb fragment of the wild-type sasB locus was cloned from strain DK6662 by the in situ cloning strategy (8). Strain DK6662 contains the Ω6658 Tn5-132 (Tc^r) insertion linked to the wild-type sasB locus (see Fig. 1A) (19). Briefly, the Kan^r plasmid pHBK429 (12), which contains the IS50 region of Tn5, was electroporated into DK6662 cells. The plasmid integrated into the chromosome by single-crossover homologous recombination with either the IS50R or the IS50L of the Ω6658 Tn5-132 (Tc^r) insertion to give Kan^r Tc^r recombinants. One Kan^r Tc^r recombinant, HK1406, was determined by Southern analysis to contain pHBK429 oriented in its chromosome such that digestion with EcoRI and recirculation would yield a plasmid containing a 22-kb region of the sasB locus. Such a plasmid, pYC1007, was prepared and used to transform E. coli DH5α cells. The physical distance between the Ω6658 Tn5-132 insertion and the five sasN mutations was estimated by cotransduction frequency to be about 8 kb (9, 19). Thus, it was possible that the 22-kb region might contain the wild-type alleles of these five mutations.

FIG. 1 — Genetic analysis of the *sasB* locus. (A) Physical map of the *M. xanthus sasB* locus in strain DK6662 containing the linked Tn5 Ω6658. The black line represents the *M. xanthus* DNA, and the white box represents Tn5 DNA. (B) Restoration of the SasB⁺ phenotype to the *sasB5* mutant DK6623 by subclones of the wild-type *sasB* locus integrated into the chromosomal *sasB* locus. The physical map of the *sasB* DNA cloned in pYC1007 is shown. The bars beneath the physical map indicate the fragments present in the subclones. The percentage of SasB⁺ recombinants is calculated as the number of SasB⁺ recombinants divided by the total number of Kan^r recombinants tested for the SasB phenotype, multiplied by 100. The number of recombinants tested in each experiment is more than 1,000. (C) Restoration of the SasB⁺ phenotype to the *sasB5* mutant DK6623 by subclones of the wild-type *sasB* locus integrated into the chromosomal Mx8 phage attachment site. The physical map of the *sasB* DNA cloned in pYC1012 is shown. Various restriction enzyme recognition sites used in generating subclones are shown. The exonuclease III digestions were generated starting at the *orf3* end of this region. The putative ORFs are represented by open boxes with horizontal arrows that indicate the predicted direction of transcription. The numbers of recombinants tested are the same, and the percentages of SasB⁺ recombinants are calculated, as described for panel B. The asterisks indicate that the plasmids were introduced into the *sasB* mutant strains, DK6631, DK6632, DK6633, and DK6634, and that the same results were observed.

Genetic complementation.

The Kan^r plasmids pYC1007, containing the complete 22-kb region linked to Ω6658 Tn5-132, and pYC1008, pYC1009, and pYC1010, containing three subclones, were tested for their ability to restore a SasB⁺ phenotype to the sasB5 mutant, DK6623, when integrated by a single-crossover recombination event into the sasB locus. Briefly, these plasmids were electroporated into DK6623 cells, plated onto CTT plates containing kanamycin, and overlaid with 0.04% 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-Gal), a chromogenic substrate of β-galactosidase. The SasB⁺ Lac⁻ Kan^r recombinant colonies, which expressed Ω4521 Tn5lac at a low wild-type level during growth, were tan and easy to differentiate from the parent-like SasB⁻ Lac⁺ Kan^r recombinant colonies, which expressed Ω4521 Tn5lac at a high level during growth and were blue.

Genetic complementation was used to determine the minimum region of the wild-type sasB locus that could restore a SasB⁺ phenotype to the five clustered sasB mutants, when placed in trans at the myxophage Mx8 attachment site, attB (40). The analysis was identical to that described above except that the DNA fragments to be tested were cloned into a vector, pDX77, which contains the attP region of the myxophage Mx8 and a kanamycin resistance gene. The resulting plasmids preferentially integrated into the attB site on the chromosome, generating Kan^r Tc^r recombinants.

DNA sequence determination and computer analysis.

A 4.2-kb MluI fragment, which complemented the sasB5, -14, -15, -16, and -17 mutations, was sequenced after being subcloned into pGEM3 to create plasmid pDX45-2. Nested deletion plasmids were generated with exonuclease III after the digestion of pDX45-2 with KpnI and XbaI by using the Erase-A-Base kit (Promega). Twenty plasmids of the appropriate lengths were sequenced with the T7 primer. To acquire the sequence of the second strand, a set of subclones was constructed by using pDX45-2 and its deletion derivatives and sequenced. The DNA sequence was edited and assembled by using the Genetics Computer Group’s sequence software package, version 9.1 (5). The Codonpreference program predicted the open reading frames (ORFs) based on the G-C codon bias of the third position (2) in this high-G+C-content (67.5 mol%) organism (32). Motif and hydrophobicity analyses were performed by the Internet services PrositeScan and TMpred at the Swiss Institute for Experimental Cancer Research (http://ulrec3.unil.ch/software).

Construction of sasN-null mutants.

The sasN insertion-deletion null mutation introduced into the wild-type strains DK101 and DK1622 was constructed and used to study the involvement of sasN in M. xanthus fruiting body development on starvation agar and in submerged culture, respectively. In each strain, the Tn5 kanamycin resistance gene, aph, replaced a 713-bp SacI-SmaI internal fragment of the sasN gene. The pDX63.4 plasmid generated for this strain construction contains aph flanked on one side by the 1.8-kb region upstream of the sasN SacI site and on the other side by the 0.8-kb region downstream of the sasN SmaI site. The strains were constructed by double-crossover recombination of pDX63.4 into the wild-type sasB locus, resulting in recombinants HK1210 in the DK101 background and HK1211 in the DK1622 background. A trimethoprim resistance gene located in the vector region of pDX63.4 is lost in the double-crossover recombination event; thus, the sasN-null mutants were identified among the Kan^r recombinants by their Tp^s phenotypes.

An internal-disruption mutation was generated by a single-crossover recombination event that incorporated a plasmid, pDX56, containing a fragment internal to sasN, into the M. xanthus chromosomal sasN gene (12). This created two truncated forms of the sasN gene separated by vector sequences; the 3′ end of one gene is absent, and the 5′ end of the other copy is absent. Plasmid pDX56, which contains a 680-bp internal SacI-to-SalI fragment of sasN in pBGS18, was introduced into DK6620 by electroporation. The null mutant HK1212 was selected for its Kan^r phenotype, and 4521 expression was monitored.

Construction of the sasN-lacZ fusion.

To generate a strain containing a sasN-lacZ transcriptional fusion, plasmid pDX58 was constructed and electroporated into DK101, creating HK1217. Plasmid pDX58 contains M. xanthus DNA from 128 bp upstream of the first predicted sasN translation start site through 780 bp downstream of the site, followed by the promoterless lacZ gene. When introduced by a single-crossover recombination event into the M. xanthus chromosome, the lacZ gene is located at the SasN amino acid residue 260 and the native sasN promoter controls its expression. Vector sequences separate lacZ from the other sasN gene, which is controlled by what is predicted to be a complete promoter region, beginning at the end of the upstream sasR gene.

Cloning and identification of the mutant alleles.

The sasB5, -14, -15, -16, and -17 mutant alleles were cloned from their corresponding mutant strains DK6623, DK6631, DK6632, DK6633, and DK6634, respectively, by a modification of the in situ cloning method (8), following integration of plasmid pDX66 into each mutant sasB locus. Briefly, plasmid pDX66, containing the kanamycin resistance gene, aph, and a 1-kb DNA fragment located 720 bp downstream of the sasN termination codon, was electroporated into the five strains. When this plasmid integrated by a single-crossover recombination event into the sasB locus, it formed a tandem duplication of the M. xanthus DNA present on pDX66 and introduced a selectable marker and an origin of replication adjacent to the mutated sasB alleles. The chromosomal DNA from one representative Kan^r Tc^r recombinant derived from each of the five sasB mutants was isolated, digested with MluI, ligated with T4 DNA ligase, and used to transform E. coli DH5α cells. The resulting plasmids were designated pDX102, pDX98, pDX99, pDX100, and pDX101, respectively. Each plasmid contains the pDX66 vector and a different intact sasN mutant allele. Subsequently, the internal 1.5-kb AgeI fragments containing the sasN mutant alleles were subcloned into the SmaI site of pBluescript SK(+) (Stratagene). Both strands of the AgeI fragments were sequenced, and the mutations were identified.

Nucleotide sequence accession number.

The nucleotide sequences of sasN and the surrounding region including orf2 and orf3 have been assigned GenBank accession no. AF076221.

RESULTS

Cloning the wild-type region that complements the sasB5, -14, -15, -16, and -17 mutations.

Five clustered point mutations (sasB5, -14, -15, -16, and -17) bypass the starvation and high-cell-density requirements for expression of the 4521 developmental reporter gene. As a result, strains containing these mutations express the 4521 gene at high levels during growth and development (19). A region of the wild-type M. xanthus chromosome adjacent to a Tn5 marker linked to these mutations (Fig. 1A) was cloned by the in situ cloning method developed by R. E. Gill et al. (8). This 22-kb fragment of the wild-type sasB region was able to restore the SasB⁺ phenotype to DK6623, the sasB5 mutant, when present as a tandem duplication in the sasB locus (Fig. 1B). Ninety percent of the selected recombinants were SasB⁺ (tan colonies when overlaid with X-Gal), and 10% of the selected recombinants were SasB⁻ (blue colonies when overlaid with X-Gal, identical to the DK6623 parent). An internal 4.2-kb MluI subclone of the 22-kb fragment in pYC1009 was also shown to restore the SasB⁺ phenotype to DK6623, at a similar percentage, when integrated at the sasB locus (Fig. 1B). Restoration of the SasB⁺ phenotype at about 90% suggests that these fragments contain a complete wild-type transcription unit. In tandem duplications, restoration of the wild-type phenotype at less than 100% is typical and is most likely a result of apparent gene conversion (45). Gene conversion in M. xanthus is documented to commonly occur at 10 to 25% in these types of crosses (41, 45).

To ensure that the 4.2-kb fragment contained a complete transcription unit, its ability to restore the SasB⁺ phenotype from a trans location in the Mx8 phage attachment site, attB, was tested. A plasmid containing a complete transcription unit should restore the wild-type phenotype at approximately 100% because gene conversion should not occur in this type of cross. Plasmid pYC1012 containing the 4.2-kb wild-type fragment was able to restore the SasB⁺ phenotype to the sasB5 mutant when integrated at attB (Fig. 1C); more than 99% of the selected recombinants were SasB⁺. These data indicate that the 4.2-kb fragment contains a functionally complete wild-type transcription unit and that the wild-type sasB⁺ allele is dominant to the sasB mutant alleles when they are present in a heterozygotic merodiploid.

Subclones of the 4.2-kb fragment integrated at the attB site were also tested in DK6623 (Fig. 1C). The 1.3-kb fragment of pDX76 was the smallest fragment tested which complemented the sasB5 mutant, indicating that it contained the minimum wild-type transcription unit. In addition, this 1.3-kb fragment of pDX76 restored the SasB⁺ phenotype to strains DK6631, DK6632, DK6633, and DK6634, containing the sasB14, -15, -15, and -17 mutant alleles, respectively, indicating that all of these mutations clustered in this region.

Sequence analysis of a 4.2-kb region of the sasB locus.

The complete DNA sequence of both strands of the 4,205-bp MluI fragment was determined. Three ORFs, designated ORF1, ORF2, and ORF3, were predicted based on the high G+C contents of the third codon positions in orf1, orf2, and orf3 (89.3, 90.6, and 92.3%, respectively), which is indicative of an ORF in high-G+C-content organisms (2) such as M. xanthus (67.5 mol% G+C) (32). The typical codon usage pattern previously observed for M. xanthus (39) was also observed for these genes.

The orf1 gene was designated sasN, based on the following information. First, the DNA sequence analysis shows that a complete ORF maps to the 1.3-kb MluI fragment (Fig. 2). Second, all of the five mutant alleles map within orf1 (Fig. 1C). Third, of the two tested derivatives of pDX45-2 unable to restore the SasB⁺ phenotype to DK6623, one lacked 95 bp of the 5′ end of the predicted orf1 coding region and the other lacked 175 bp of the 3′ end of the predicted orf1 coding region (Fig. 1C). Additional complementation experiments indicated that sasN comprises its own transcription unit, independent of the genes upstream and downstream (data not shown).

The sasN DNA and deduced amino acid sequences with the first of three possible ATG start codons are shown in Fig. 2. Possible ATG start codons are present at 129, 174, and 219 bp downstream of the termination codon of a gene designated sasR, which encodes a putative cognate response regulator of the SasS sensor kinase (49). Since no obvious consensus ribosome binding site could be identified within 6 to 12 bp upstream of any of the predicted start codons, the start site will be identified by further analysis. With the first ATG start codon, the sasN gene is predicted to encode a protein of 363 amino acids with a calculated molecular mass of 39.6 kDa. SasN showed no significant sequence similarities to known proteins.

Hydrophobicity analysis of the predicted 363-amino-acid SasN protein reveals a strongly hydrophobic region (residues 15 to 36) in the N terminus. Adjacent to the hydrophobic region is a leucine zipper motif (residues 56 to 77), containing four leucine residues separated by seven amino acids, often referred to as heptad repeats. Leucine zipper motifs are mediators of dimerization in eukaryotic (29) and prokaryotic (10) cytoplasmic proteins. Thus, the presence of the leucine zipper motif suggests that SasN may function as a homo- or a heterodimer. Many leucine zipper proteins are regulators that bind DNA directly with highly positively charged regions (29). The region of the predicted SasN protein after the leucine zipper, although highly positively charged (2 H, 6 K, 39 R/286 amino acids), is also highly negatively charged (24 D, 29 E/286 amino acids). The charged amino acids are dispersed, with no area being particularly positively charged. Further analysis will be useful to determine if SasN is multimeric and if it associates with other regulatory proteins, membrane fractions, and/or DNA.

The predicted translation start site of orf2 maps 694 bp downstream of the predicted sasN termination codon, and it appears to be transcribed in the same direction. It is expected to encode a protein of 341 amino acids with a calculated molecular mass of 37.5 kDa. The orf2 gene has been identified as a positive regulator of 4521 expression in an independent mutagenesis experiment (13). The orf3 gene, located downstream of orf2, is predicted to be transcribed in the opposite direction and to encode a protein of 222 amino acids with a calculated molecular mass of 23.6 kDa. The deduced amino acid sequences of these predicted proteins are not significantly similar to those of any proteins in the databases.

The sasN gene encodes a negative regulator of 4521 expression.

To determine the effect of a sasN-null mutation on 4521 gene expression in an otherwise wild-type background containing Tn5lac Ω4521, an internal-disruption sasN mutation was constructed and introduced into strain DK6620. The presence of the sasN-null mutation in this strain increases 4521 expression dramatically and dwarfs the parent strain’s roughly 10-fold increase in activity during growth and development (Fig. 3). In growing cells (time zero of development), the β-galactosidase specific activity of the sasN-null mutant is about 800 U, whereas the activity in the parent strain is about 20 U. This difference in β-galactosidase specific activity between growing cells of the sasN-null mutant and the parent strain indicates that SasN functions as a negative regulator of 4521 expression during growth. Furthermore, it indicates that all of the elements necessary for 4521 expression are present in wild-type cells during growth and points to SasN as the critical regulator that prevents 4521 expression. After 10 h of development, the β-galactosidase specific activity of the sasN-null mutant reaches about 2,000 U, whereas the activity in the parent strain is about 200 U. These data suggest that SasN may also function as a negative regulator of 4521 expression during development.

FIG. 3 — The effect of a *sasN*-null mutation on the expression of *4521*. β-Galactosidase specific activities of Tn5lac Ω4521 in the *sasN*⁺ strain DK6620 (○) and an isogenic *sasN* null mutant strain, HK1212 (•), are shown. The specific β-galactosidase activity was determined during growth and at different times during development on starvation agar. Each strain was tested in at least three independent experiments, and the averages of two recombinants are shown. ONP, o-nitrophenol.

The same 4521 expression pattern observed in this sasN-null mutant, HK1212, was observed for other sasN-null alleles that were generated by insertion-deletion or transposon insertion in an otherwise wild-type background (data not shown) and for the original suppressor alleles, which are in an asg background (19). These data reveal that sasN mutations confer the same phenotype regardless of whether a wild-type or a mutant asg allele is present.

sasN is necessary for normal fruiting body formation and sporulation.

To determine if a wild-type sasN gene was required for normal fruiting body development, a sasN-null mutation was introduced in two wild-type backgrounds and each strain was tested for the ability to form fruiting bodies and to sporulate. In contrast to the condensed and darkened fruiting bodies formed by wild-type DK101 cells after 2 days, the isogenic sasN-null mutant HK1210 formed loose asymmetric aggregates that did not compact or darken after prolonged incubation (Fig. 4A and B). In submerged culture, wild-type DK1622 cells formed tight and refractile fruiting bodies, whereas the sasN-null mutant HK1211 cells formed many loose, small, and flat aggregates (Fig. 4C and D). In addition, the sporulation efficiency of the sasN-null mutant HK1211 was about 10% that of wild type. A level of 7.5 × 10⁵ to 1.3 × 10⁶ spores/ml was determined for HK1211, compared to 1.0 × 10⁷ spores/ml for DK1622 after 7 days of development. The sasN-null mutants generated either by internal disruption or by transposon insertion showed the same abnormalities and deficiencies in development (data not shown). These data indicate that sasN is required for normal M. xanthus fruiting body formation and sporulation.

FIG. 4 — Developmental morphologies of *sasN*-null mutants. (A and B) Fruiting body development on TPM starvation agar is shown. Concentrated cells at a density of 4 × 10⁹ cells per ml in TPM buffer were inoculated onto TPM agar at 20 μl per spot and incubated at 32°C for 2 days. (A) DK101 wild-type cells; (B) HK1210 *sasN* internal-disruption null mutant. (C and D) Fruiting body development in submerged culture is shown. Cells (25 μl at a density of 4 × 10⁹ cells per ml) in MC7 buffer were inoculated into 1.7-cm-diameter wells containing 375 μl of MC7 starvation buffer and incubated at 32°C for 7 days. (C) DK1622 wild-type cells; (D) HK1211 *sasN* internal-disruption null mutant. Bar, 0.1 mm.

sasN is expressed during growth.

The high 4521 expression level in the sasN mutant suggests that SasN is a critical negative regulator that prevents 4521 expression during growth. To determine whether the sasN gene was expressed during growth, a strain containing a sasN-lacZ transcriptional fusion was generated. Strain HK1217 contains a tandem duplication of a sasN-lacZ transcriptional fusion and the wild-type sasN gene in an otherwise wild-type DK101 background. During growth, the β-galactosidase specific activity was 120 U. This activity can be compared to the basal level of 3 to 20 U observed during growth for most developmental genes. These data indicate that sasN is expressed at a moderately high level during growth and support the idea that the SasN functions to prevent developmental gene expression during growth. The sasN gene is also expressed during development, suggesting a potential role of SasN in development (49).

Expression of 4521 in a sasN sasS mutant is similar to that in the sasS parent strain.

SasS is a sensor histidine kinase of a putative two-component signal transduction system (49, 50) and functions as a positive regulator that is absolutely required for 4521 expression. sasS-null mutants express 4521 at a very low basal level (2 U) during growth and development (Fig. 5) (50). SasS has been proposed to sense the accumulation of A signal during early development (50).

FIG. 5 — Expression of *4521* in a *sasN sasS* mutant during growth and early development. β-Galactosidase specific activity profiles for strains HK1218 (*sasN sasS*), HK1503 (*sasN⁺ sasS*), and DK6623 (*sasN sasS⁺*) are shown. The specific β-galactosidase activity was determined during growth and development on starvation agar for each strain in at least three independent experiments, and results of a representative experiment are shown. ONP, o-nitrophenol.

To examine the possible functional relationship between SasN and SasS in regulating 4521, null mutations of each gene were placed in the same strain, HK1218. The HK1218 4521 expression was measured and compared to expression in the parent strains. The results (Fig. 5) show that the expression of 4521 in this sasN sasS mutant background was as low as that of the sasS-null mutant.

Cloning and identification of the sasB5, -14, -15, -16, and -17 mutant alleles.

The five clustered sasB mutant alleles, sasB5, -14, -15, -16, and -17, were cloned, sequenced, and identified. The mutation sasB16 is a C-to-T transition at nucleotide 267, which changes Q47 to an amber termination codon. Mutations sasB5, -14, -15, and -17 are identical C-to-A transversions at nucleotide 966 which change T280 to P. Mutation sasB15 also carries a G-to-A transition at nucleotide 729 which changes D201 to N. It is likely that sasB5, -14, -15, and -17 are siblings, because all of the mutations were isolated in the same screen (19).

Preliminary analysis of the SasN structure based on the DNA sequence suggests that the defect caused by the T280P substitution changes the structure and the charge distribution in a region in which three of the six amino acids are acidic. The fact that the strains containing the sasB16 nonsense mutation, the sasB5, -14, -15, and -17 missense alleles, and the sasN-null alleles constructed in this study have the same effect on 4521 expression suggests that the missense mutations generate nonfunctional or unstable proteins.

DISCUSSION

The M. xanthus sasN gene was identified and determined in this report to encode a negative regulator of the developmental reporter 4521 and to be required for wild-type fruiting body formation and sporulation. In the absence of SasN, as a result of either the original point mutations (19) or the null mutations constructed in this study, 4521 expression is extremely high during both growth and development (Fig. 3). These data indicate that all of the elements necessary for 4521 expression are present during growth and that SasN serves a critical role as a regulator that prevents 4521 expression during growth.

The mechanism of SasN function has yet to be determined. The two most likely options for SasN negative control of the sigma-54-dependent promoter 4521 are that SasN is a direct negative regulator, a repressor of the 4521 promoter, or that it is an indirect negative regulator, an inhibitor of an activator of 4521 expression. Biochemical evidence is necessary to definitively determine how SasN functions. The sasN DNA sequence and its predicted amino acid sequence have provided only limited insight into the mechanism of SasN action. However, the examination of 4521 expression in strains containing mutations in either or both of the genes encoding SasN or the 4521 activator, the SasS sensor kinase, has been more enlightening. The expression of 4521 during growth and development in the sasS mutant, in which the activated cognate response regulator of SasS should not accumulate, remains at a very low basal level (Fig. 5). The expression of 4521 in a mutant containing both sasS and sasN mutations shows the same low basal level (Fig. 5). Data such as these are typical of sigma-54-dependent promoters and have been used to argue that these promoters have no need for repressors. Specifically, in the absence of the activator the sigma-54–RNA polymerase holoenzyme can form a stable closed complex with the promoter but is unable to undergo isomerization to form an open complex that is transcriptionally competent (42). To our knowledge, all previously identified negative regulators of sigma-54-dependent promoters function indirectly by controlling the activity of activators. Thus, if SasN controls 4521 expression as a repressor, it would be the first identified repressor controlling a sigma-54 promoter. In addition, this low basal expression level in the absence of both the positive and the negative regulators suggests that there are no other independent regulators controlling 4521 expression.

The very high 4521 expression level during growth in the sasN mutant implies that there is a high level of the activated positive regulator. If SasN is a repressor, one would predict that the level of the activated positive regulator is normally high during growth and that SasN directly blocks activation of 4521 transcription. This block would have to be relieved soon after starvation to permit 4521 developmental expression. If SasN is an inhibitor of an activator, one would predict that the sasN mutation would cause an increase in the normally low level of the activated positive regulator. The potential targets of SasN inhibition are both elements of the two-component SasS-SasR system, which are the known positive regulators of 4521 expression (49, 50). It is possible that SasN inhibits SasS autohistidine kinase activity or stimulates SasS phosphatase activity. If SasR is the SasN target, SasR∼P would be stabilized as a result of the sasN mutation. A SasN-SasS interaction that stimulates dephosphorylation of SasR∼P would be similar to the nitrogen assimilation pathway in the enteric bacteria that controls transcription of many promoters (33), including the best-studied sigma-54-dependent promoter, glnA (22, 34).

SasN is an intriguing regulator that controls developmental gene expression during the transition from growth to development. This activity is reminiscent of the Bacillus subtilis transition state regulators AbrB, Hpr, and Sin (46). However, SasN is also required for M. xanthus fruiting body formation and sporulation, a characteristic that differentiates it from these B. subtilis regulators that do not confer a Spo⁻ phenotype. It is likely that SasN plays additional roles in M. xanthus development, such as regulating other developmentally expressed genes. Understanding the mechanism of SasN action and the elements that control SasN activity and/or expression should help to clarify how M. xanthus cells limit the critical transition from growth to development to the appropriate conditions by integrating information on their nutrient status and cell density.

ACKNOWLEDGMENTS

We thank T. Hoover for helpful discussions, K. Borkovich for use of her microscope, and J. Eraso for critical reading of the manuscript.

This investigation was supported by Public Health Service grant GM47444 to H.B.K. from the National Institutes of Health.

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[B1] 1.Avery L, Kaiser D. In situ transposon replacement and isolation of a spontaneous tandem duplication. Mol Gen Genet. 1983;191:99–109. doi: 10.1007/BF00330896. [DOI] [PubMed] [Google Scholar]

[B2] 2.Bibb M J, Findlay P R, Johnson M W. The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences. Gene. 1984;30:157–166. doi: 10.1016/0378-1119(84)90116-1. [DOI] [PubMed] [Google Scholar]

[B3] 3.Bowden M G, Kaplan H B. The Myxococcus xanthus developmentally expressed asgB-dependent genes can be targets of the A signal-generating or A signal-responding pathway. J Bacteriol. 1996;178:6628–6631. doi: 10.1128/jb.178.22.6628-6631.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4.Davis J M, Mayor J, Plamann L. A missense mutation in rpoD results in an A signaling defect in Myxococcus xanthus. Mol Microbiol. 1995;18:943–952. doi: 10.1111/j.1365-2958.1995.18050943.x. [DOI] [PubMed] [Google Scholar]

[B5] 5.Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984;12:387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6.Dworkin M. Recent advances in the social and developmental biology of the myxobacteria. Microbiol Rev. 1996;60:70–102. doi: 10.1128/mr.60.1.70-102.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7.Dworkin M, Kaiser D, editors. Myxobacteria II. Washington, D.C: American Society for Microbiology; 1993. [Google Scholar]

[B8] 8.Gill R E, Cull M G, Fly S. Genetic identification and cloning of a gene required for developmental cell interactions in Myxococcus xanthus. J Bacteriol. 1988;170:5279–5288. doi: 10.1128/jb.170.11.5279-5288.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9.Gill R E, Shimkets L J. Genetic approaches for analysis of myxobacterial behavior. In: Dworkin M, Kaiser D, editors. Myxobacteria II. Washington, D.C: American Society for Microbiology; 1993. pp. 129–155. [Google Scholar]

[B10] 10.Giraldo R, Nieto C, Fernandez-Tresguerres M E, Diaz R. Bacterial zipper. Nature. 1989;342:866. doi: 10.1038/342866a0. [DOI] [PubMed] [Google Scholar]

[B11] 11.Gulati P, Xu D, Kaplan H B. Identification of the minimum regulatory region of a Myxococcus xanthus A-signal-dependent developmental gene. J Bacteriol. 1995;177:4645–4651. doi: 10.1128/jb.177.16.4645-4651.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12.Guo D, Bowden M G, Pershad R, Kaplan H B. The Myxococcus xanthus rfbABC operon encodes an ABC transporter homolog required for O-antigen biosynthesis and multicellular development. J Bacteriol. 1996;178:1631–1639. doi: 10.1128/jb.178.6.1631-1639.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13.Guo, D., and H. B. Kaplan. Unpublished data.

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PERMALINK

Myxococcus xanthus sasN Encodes a Regulator That Prevents Developmental Gene Expression during Growth

Di Xu

Chun Yang

Heidi B Kaplan

Abstract