INTRODUCTION

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Myxococcus xanthus, a gram-negative, rod-shaped, soil bacterium, responds to starvation by differentiating metabolically dormant, spherical myxospores that are resistant to heat and desiccation (for a general review, see reference 9). These spores are contained within a macroscopic structure, the fruiting body, that is formed when the cells glide into and form aggregates of approximately 10⁵ cells. This macroscopic structure is thought to enhance the eventual dispersion of its spores. To achieve its selective advantage, fruiting body development is highly regulated. For example, aggregation and morphogenesis of a fruiting body must precede the physiological and morphological differentiation of cells into the dormant myxospores. Before development, cells assess population density and nutrient conditions (47). Once the process has begun, the population then guides itself through several developmental checkpoints by producing several extracellular signals, the best understood of which are the A-, C-, and E-signals (7, 26, 31, 45). Mutants defective in producing one of these signals fail to develop or sporulate; however, signal-deficient mutants can be induced to sporulate if they are developed in coculture with either a wild-type strain or a mutant from a different signal class (13). A-factor, one of the earliest signals, has been shown to be a mixture of amino acids that must be present at a particular concentration to serve as a quorum sensor (19, 34). The C-signal is a cell surface protein which requires cell alignment for its transmission and helps bring large numbers of cells into late, symmetric aggregates (48).

One genetic route to understanding the mechanisms that regulate development is to find genes that are necessary for, or are regulated by, the developmental process. Two known developmentally regulated genes have promoters that resemble those recognized by the minor sigma factor ⁵⁴. Those genes are mbhA (42) and the A-signal-dependent gene termed 4521 (24). Keseler and Kaiser have shown that single base changes in six particular residues in the region from 12 to 24 (which distinguishes ⁵⁴ from ⁷⁰ promoters) ahead of the 4521 RNA start site decreased expression of this gene by 90% and that deletions of base 19 decreased expression by 95% (24), as was expected of a ⁵⁴ promoter. Attempts to investigate the overall role of ⁵⁴ in development by deleting the gene rpoN have been hampered because this gene, which encodes the ⁵⁴ subunit in M. xanthus, is essential for viability (25). ⁵⁴ promoters are unique in that each requires a specific activator protein in addition to RNA polymerase holoenzyme binding at the promoter, and these activator proteins are often part of sensory circuits. The best-studied examples of such activators are NtrC (NRI), which regulates nitrogen assimilation genes among enteric bacteria, and NifA, which regulates the nitrogen fixation genes in Rhizobium, Klebsiella, and other bacteria (35, 41).

Unable to explore the effects of a deletion of rpoN on development in general, we have turned to its specific activator proteins. The domain structure of the well-documented activator proteins is conserved along genus lines (40) (Fig. 1). The N-terminal domain is usually where the activator protein itself is modified (often by phosphorylation), which can affect the cooperativity of binding to the DNA as well as the ATPase activity of the activator. Promoter binding is directed by the C-terminal domain of the protein, while the central region contains an ATP binding domain, which is also the most highly conserved of the three (40) (Fig. 1).

View larger version (4K): [in this window] [in a new window]   FIG. 1.   Common structure of 54 activator proteins. The N-terminal domain is the least conserved among genera and has a length which ranges from 12 to 400 amino acids. Some activators are phosphorylated on an aspartate residue in this domain (P). The C-terminal domain of 65 to 130 amino acids has a helix-turn-helix (HTH) motif characteristic of DNA binding proteins. The central domain of about 240 amino acids is the most highly conserved and has an ATP binding motif. Approximate locations of the PCR primers used to amplify the central domain are indicated by the converging arrows. Consensus data are from reference 40.

A PCR technique that specifically amplifies sequences corresponding to the central, strongly conserved, nucleotide binding domain of ⁵⁴ transcriptional activator proteins has been developed by Kaufman and Nixon. Using heterologous primers, they were able to amplify 14 NtrC-like PCR fragments of ~475 bp each from M. xanthus chromosomal DNA (23). We have used these fragments in a way calculated to cause internal disruptions in their corresponding genes. Each fragment was inserted into a plasmid incapable of replication within M. xanthus, and the resulting plasmids were introduced into M. xanthus. Antibiotic resistance encoded by the plasmid-borne gene should result only by incorporation of that plasmid directly into the gene homologous to the PCR fragment used. The result of the technique should be an altered chromosomal structure resulting from an insertional mutation at the chromosomal locus. We have tested this mutagenesis approach to selectively mutate potential ⁵⁴ activator proteins. From 14 individual NtrC-like DNA fragments of 475 bp each, we have attempted disruption of the homologous chromosomal regions. Mutants generated this way were compared with a wild-type strain to assess effects on development. Results of this initial sampling are presented here.

	MATERIALS AND METHODS

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Media for growth or development. Myxobacteria were propagated at 32°C in CTT broth (1% Bacto Casitone, 10 mM Tris-HCl [pH 8.0], 8 mM MgSO₄, 1 mM KPO₄ [pH 7.6]) or CTT agar (CTT broth plus 1.5% Bacto Agar) unless otherwise indicated. CTTYE (CTT plus 0.2% Bacto Yeast Extract) was used after electroporation of M. xanthus. Kanamycin (40 µg/ml) or oxytetracycline (12.5 µg/ml) was added where indicated below. A1 minimal medium has been described previously (3) and was solidified with either 1.5% Bacto Agar or 0.8% agarose. TPM buffer (10 mM Tris-HCl [pH 7.6], 8 mM MgSO₄, 1 mM KPO₄ [pH 7.6]) and TPM agar (TPM buffer plus 1.5% Bacto Agar) were used to induce development as previously described (28). Briefly, exponential cells were harvested by centrifugation and resuspended in TPM buffer to a concentration of 5 × 10⁹ cells/ml. Ten- or twenty-microliter droplets were then placed on the surfaces of TPM agar plates. Development was also induced in submerged culture (30); cells were developed in 24-well, polystyrene microtiter plates. -Galactosidase activity was assayed in cells harvested from either submerged culture or TPM agar plates (from five 20-µl droplets) as previously described (21). Escherichia coli cultures were grown in L broth or L agar (44) supplemented with 40 µg of kanamycin per ml where indicated below. Growth, in liquid, of M. xanthus was monitored by measuring turbidity in a Klett-Summerson photoelectric colorimeter equipped with a red filter.

PCR amplification of ⁵⁴ transcriptional activator sequences from M. xanthus. Highly degenerate primers were used to amplify the central domain of ⁵⁴ transcriptional activator genes by methods that were previously described (23).

Plasmid and strain construction. The M. xanthus wild-type strain DK 1622 was used as the starting point for strain construction, so that the products would be isogenic. All the strains and plasmids that were used are listed in Table 1. Thirteen 475-bp (nominal) PCR fragments were ligated into the EcoRI site in pBGS18 (49), generating plasmids pLAG1 through pLAG13. The plasmids were prepared and introduced into E. coli XL1 by either transformation or electroporation according to the methods of Sambrook et al. (44). After propagation in E. coli, these plasmids were extracted and electroporated into M. xanthus as described previously (22). That procedure was modified as follows. Eighty microliters of concentrated M. xanthus cell suspension (10,000 Klett units) was pipetted into a 0.2-cm-gap-size cuvette with 1 to 3 µl of plasmid DNA and electroporated at 1.3 kV, 400 W, and 25 mF, which gave time constants of 8.0 to 9.0 ms. Immediately following electroporation, 1 ml of CTTYE was added to the cuvette; the entire contents were then transferred to a flask containing 1.5 ml of CTTYE, and the mixture was incubated with shaking at 32°C for 5 h before being plated onto selective medium. Colonies were picked, and streaked onto CTT plus kanamycin agar. DNA was prepared from these bacteria (1) and restricted, and the digests were analyzed by Southern hybridization to confirm disruption of the target gene. Southern blots were probed both with the corresponding 475-bp fragment used to produce the gene disruption mutant and with a fragment from the kanamycin resistance gene of pBGS18.

Nutritional requirements. Strains were inoculated onto A1 minimal agar with kanamycin, grown for 3 to 5 days, and finally transferred to a plate containing A1 agarose plus kanamycin. To assess auxotrophy, the resulting biomass of each disruption strain was compared with that of its nondisrupted parent growing on the same plate.

Sporulation assay. The efficiency of sporulation was measured as described previously (27) with modification. After development on TPM agar (10-µl droplets of 5 × 10⁹ cells/ml) for 3 days, the cells from five droplets were harvested by scraping them into microcentrifuge tubes containing 1 ml of TPM buffer. The contents of each tube were then treated in a cup horn sonicator (Tekmar) for 5 min at maximum power with ice-water cooling to disrupt fruiting bodies and disperse spores. Residual vegetative cells were killed subsequently by heating the tubes at 50°C for 2 h. After serial dilution and plating of the suspensions, the sporulation efficiency was measured as the number of colonies present relative to the number of cells initially deposited on the TPM agar. To determine whether development could be rescued in a developmentally defective strain by coculture, the activator disruption strains were mixed in a 1:1 ratio with other strains. For these rescue experiments, five 20-µl droplets of the mixed cell suspensions were deposited onto TPM agar and the sporulation efficiency was measured and compared with that of the wild type. The amounts of A- and C-factor produced were determined by the quantitative assessment of how much an asgA or a csgA mutant could be induced to sporulate when it was in coculture with the testing strain compared to the level of sporulation of the wild type under the same conditions.

Introduction of reporter gene fusions into gene disruption mutants. Myxophage Mx4ts18ts27 hrm (Mx4) (5) was used to transduce Tn5-132lacZ promoter fusions of 4521 (from DK 6620) and mbhA (from DZF 4619) into each of the M. xanthus disruption strains. In some cases, the mbhA::Tn5-132lacZ fusion was transduced from DK 7870 into the disruption strains by Mx8clp2 (Mx8) (38). Some developmentally regulated Tn5-132lacZ fusions were transduced into DK 7823 with Mx4 (28). The Tn5-132lacZ fusion 4414 was transduced from DK 5511 by Mx8 into DK 7814 and DK 7837. Transductants were selected on CTT plus kanamycin plus oxytetracycline medium, and the structures of the transductants were confirmed by Southern blot analysis (28).

	RESULTS

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⁵⁴ transcriptional activator sequences from M. xanthus. With NtrC (NRI) protein as the prototype, the amino acid sequences of members of the family of known transcriptional activators for ⁵⁴ promoters have been divided into the three domains shown in Fig. 1. As mentioned above, the N-terminal domain is where the activator protein is often modified and the C-terminal helix-turn-helix domain of the protein is where binding to DNA is mediated. In collaboration with our laboratory, Kaufman and Nixon PCR amplified sequences corresponding to the consensus central ATP binding region from M. xanthus chromosomal DNA. Locations of the PCR primers used by Kaufman and Nixon are indicated in Fig. 1. From M. xanthus they obtained 14 unique NtrC-like fragments from a sample of 60 M13 clones (23). M. xanthus DNA appears to be several times more productive of unique fragments than the three other bacterial species surveyed: Rhizobium meliloti, Bacillus subtilis, and Synechococcus sp. strain PCC7002. The possible significance of this finding is taken up in Discussion.

Creation of activator-disruption mutant strains. To investigate the physiological functions of putative activators of ⁵⁴-dependent genes, each of the PCR-amplified fragments was integrated by homologous recombination into M. xanthus, as diagrammed in Fig. 2. M. xanthus prefers to integrate chimeric plasmids through sequence homology (11). The 475-bp DNA fragments, since they were recovered after PCR amplification with primers that targeted the central domain of the protein, were expected to be completely internal to their corresponding genes (see the PCR primer sites in Fig. 1). As illustrated in Fig. 2, when homologous recombination occurs between an internal fragment and the complete copy that resides in the chromosome, the expected result is two incomplete copies of the gene, one of which, called 5', is missing sequences upstream of the amplified segment and the other of which, called 3', is missing sequences downstream. Vector DNA should separate the two incomplete copies from each other; gene function in this case should be totally lost because both copies are defective. The plasmid vector, which bears a gene that encodes kanamycin resistance, is not capable of autonomous replication in M. xanthus. Accordingly, the primary way of obtaining a kanamycin-resistant transformant is by integration, by using the homologous recombination just described, thus creating an insertional mutation that is expected to be null.

View larger version (10K): [in this window] [in a new window]   FIG. 2.   Gene disruption in M. xanthus by homologous recombination. A plasmid that carries an internal fragment of the 54 activator target gene (shaded box) recognizes its homolog within the genome (black box). Because the PCR-amplified fragment is completely internal to the central domain (Fig. 1), that fragment can be considered to have the 5' end of the activator gene (5') and the 3' end of the gene (3') deleted. A single homologous crossover event should lead to a tandem duplication of the internal gene fragment and incorporation of the vector into the chromosomal gene, resulting in a knockout (or null) mutation in that gene. The vector bears the gene that encodes kanamycin resistance. E indicates sites of restriction enzyme cleavage used for Southern blot analysis.

View larger version (49K): [in this window] [in a new window]   FIG. 3.   Southern blots of the insertion strains. Approximately 1 µg of DNA was loaded in each lane. DNA preparations were restricted with SmaI. SmaI sites are indicated with an E in the diagram of Fig. 2. Probes are listed across the top. The first lane of each blot contains wild-type DNA, and the second lane contains DNA from the insertion mutant. The locations of standard size markers (with sizes in kilobases) are indicated. Each blot has been probed with the PCR fragment used to create that mutant. (B) DNAs from the wild type and each of the mutants were also probed with a portion of the kanamycin resistance gene from pBGS18. Standard size markers of 23, 9.4, 6.5, 4.4, 2.3, and 2.0 kb are shown.

Mutant characterization. The next phase of the investigation sought to find the physiological functions of the putative ⁵⁴ activators or, more properly, of their target genes. The strategic reason for placing physiology ahead of gene sequencing was to discover which putative activators, considering their number, have the most relevance to development. Accordingly, growth, swarming motility, sporulation, developmental gene expression, and capacity for fruiting body development were examined for each of the seven disruption strains. Temporarily, these strains are designated by the PCR fragment numbers assigned by Kaufman and Nixon (23), Mxa191 to Mxa296. Functional names will be assigned when the genes to which these fragments correspond have been isolated and characterized.

Growth, colony morphology, and swarming. The disruption strains were maintained in growth media containing kanamycin to select against loss by excision of the integrated plasmid by homologous recombination between the two copies of the tandemly duplicated gene. For purposes of retaining the duplication, the short length of the duplication and, consequently, less frequent recombination work to advantage in decreasing the frequency of excision. Kanamycin-resistant derivatives of our laboratory strains generally grow more slowly in the presence of this antibiotic than in its absence. DK 1622 Km^r, for example, has a doubling time of 5.5 to 6.5 h in kanamycin medium, 1 or 2 h longer than DK 1622 in drug-free medium. Only one activator-disruption strain showed a significant difference in doubling time from DK 1622 Km^r and all other mutants; Mxa191 doubled after 12 h. All the strains, including Mxa191, were able to grow on the completely defined minimal medium A1 (3), despite their having been selected on the nutritionally rich CTTYE agar, which could have supported growth of many kinds of auxotrophs. The colony morphology of the disruption strains on CTT agar appeared normal (i.e., like that of the reference strain, DK 1622, referred to as the wild type), except for Mxa287, whose colonies were smaller than wild-type colonies, less rough in appearance, and less cohesive as cell masses when they were mechanically disturbed. Their colony morphology resembled that of an asgA mutant, which fails to release A-factor (31). However, the colonies of Mxa287 are not identical to those of an asg mutant, since Mxa287 forms yellow colonies like those of the wild type whereas in general asg mutants form tan colonies (36).

Fruiting body development. Three of the eight disruption mutant strains showed normal fruiting body development during 3 days of incubation on TPM agar (Table 3). However, five were clearly abnormal under laboratory conditions. One of these strains, Mxa296, developed normally on TPM agar but failed to develop in submerged culture. Submerged culture was carried out in the wells of plastic culture dishes, and this type of culture requires that the cells can adhere to and create a biofilm on the polystyrene floor of a well in order to develop fruiting bodies (30). Although the basis for the surface specificity of agar versus that of polystyrene is unknown, the target activator in Mxa296 is clearly necessary for the ability of DK 1622 to initiate development under these particular conditions.

View larger version (175K): [in this window] [in a new window]   FIG. 4.   Development of strains on TPM agar at 24 h. (A) Wild type; (B) Mxa287; (C) Mxa213; (D) Mxa259. Arrowheads indicate incompletely closed structures. The scale bar in panel A measures 0.15 cm, and the scale is the same for all frames.

Sporulation. The capacity of starvation-induced cultures of each of the three aggregation-defective mutants to sporulate was assessed. Sporulation normally follows aggregation and occurs within the aggregate. The disruption mutants Mxa287, Mxa213, and Mxa259 formed no viable heat- and sonication-resistant spores even after 3 days of development on TPM agar (Table 4). Mxa259 forms darkened aggregates but no viable spores, showing that the darkening of fruiting bodies need not indicate the presence of mature spores. These nonsporulating mutants could also not be induced to form spores by coculturing them with an equal number of wild-type cells. The sporulation level of Mxa287, when it was cocultured with the wild type, remained below 0.1% of wild-type levels (Table 5). It did increase somewhat, but the increase in spore number for Mxa287 was about 3 orders of magnitude less than the rescue seen with signaling mutants such as asg or csg (results shown in Table 5). The failure of wild-type cells to rescue the sporulation defect of Mxa287 and of the other aggregation mutants (data not shown) shows that their primary defects are cell autonomous, not the production of an extracellular factor(s) released by wild-type cells that might be required for sporulation, such as A-factor, C-factor (13), or E-factor (7).

A- and C-factor production. The aggregation-defective mutant Mxa287 was also specifically tested for its capacity to produce A- and C-factors, because failure to produce either of these factors is known to lead to aggregation arrest and failure to sporulate (32, 48). The ambient levels of A-factor and C-factor were measured by mixing the aggregation mutant with either an A-signal-deficient (asgA) or a C-signal deficient (csgA) mutant, coculturing the mixtures under starvation conditions to induce development, and finally counting the number of spores formed by the asgA and csgA indicator strains, differentially marked by drug resistance. It was evident that the Mxa287 mutant strain made both the A- and C-factors since it induced the asgA and csgA signal-defective mutants to sporulate (Table 5). Mxa287 made roughly wild-type levels of both factors, as judged by the number of viable spores formed. Thus, Mxa287 produces A- and C-factors but fails to respond to the extracellular signals that are contributed by the wild-type during coculture. For these reasons, and others stated below, the putative activator targeted in mutant Mxa287 appears to be part of a signal reception rather than a signal production pathway.

Expression of developmentally regulated reporters. Tn5lacZ transcriptional fusions have been constructed as reporters for two particular ⁵⁴-transcribed genes, mbhA and 4521 (24, 25, 42). mbhA encodes a myxobacterial hemagglutinin. While these genes are developmentally expressed, neither is essential for development. If the activator proteins sampled in the current collection of disruption mutants include the specific activators for those particular target genes, then those reporters should not be expressed. The mbhA and 4521 operon fusions were transduced into all the activator mutants, and the specific activity of -galactosidase produced from the mbhA or 4521 reporter was measured after development had been induced. Activator mutant Mxa287 was unable to express the A-signal-dependent 4521 reporter, which is normally expressed at about 2 h into development, the normal time of A-signaling (28). The Mxa296 strain did not express that fusion when it developed in submerged culture but did express 4521 when it developed on TPM agar (Fig. 5A). This expression pattern of -galactosidase from Tn5lacZ 4521 parallels the ability of Mxa296 to aggregate on agar and its lack of ability to aggregate in submerged culture. However, Mxa296 was able to express 4521 in buffered suspension with either TPM buffer, which is used for TPM agar development, or MC7 buffer, which is used for submerged-culture development. These results also indicate that Mxa296 has a preaggregation defect that is expressed before the strain has developed for 2 to 3 h in submerged culture, the normal time for 4521 expression. This defect may be in the recognition of polystyrene as a proper surface for development; alternatively, polystyrene may be inhibitory to development in this mutant.

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Expression of -galactosidase from the Tn5lac 4521 reporter. (A) Strain Mxa296. The squares indicate -galactosidase specific activity from cells developed on TPM agar, and the circles indicate -galactosidase specific activity from cells in submerged culture. (B) Strain Mxa287. The squares reflect -galactosidase expression in a wild-type background, and the circles show expression in a Mxa287 mutant background. The inset shows 4521 expression in the Mxa287 mutant background plotted on a more sensitive scale. ONP, o-nitrophenyl.

View larger version (17K): [in this window] [in a new window]   FIG. 6.   Expression of developmentally regulated Tn5lac reporter fusions in Mxa287. The squares represent -galactosidase specific activity in a wild-type background, and the circles represent -galactosidase specific activity of the fusions in a Mxa287 mutant background. ONP, o-nitrophenyl.

	DISCUSSION

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Endospore formation in B. subtilis has been shown to be regulated by a cascade of different sigma factors, each of which is responsible for the expression of a particular set of genes in the differentiation of the spore from the mother cell (14). At least five sigma factors of the ⁷⁰ family have been found in M. xanthus, the deletion of some of which leads to developmental defects (18); however, none of these sigma factors, when they are deleted, cause the extreme developmental defects produced by deletion of ^H, ^F, ^E, ^G, or ^K (all members of the ⁷⁰ family), which terminates Bacillus spore formation (14). There is no evidence that these M. xanthus sigma factors are regulated as a cascade.

Sigma factors of the ⁵⁴ family are both structurally and functionally different from members of the larger ⁷⁰ family (39). ⁵⁴ promoters are often used for growth on certain alternate carbon sources among different Pseudomonas spp. (39), on alternate nitrogen sources in some enteric species (35, 41), or for nif genes in some bacteria capable of nitrogen fixation (39, 43). ⁵⁴ is cell cycle regulated, but is not vital, in the swarmer cells of Caulobacter crescentus, where expression of several genes of flagellum biosynthesis and assembly are timed by the cell cycle (4).

In contrast to its nonessential role in other bacteria, ⁵⁴'s role in the growth of M. xanthus has been found to be essential (25). Although the majority of developmentally regulated promoters recognized to date appear to be recognized by a member of the ⁷⁰ family (6), at least three promoters, mbha, pilA (55), and 4521, have ⁵⁴ hallmarks. Constrained by the requirement of ⁵⁴ for growth, to investigate the extent of ⁵⁴ use during development, we have created insertion mutations in genes expected to encode ⁵⁴ activator proteins. The strategy of Kaufman and Nixon (23) appears to identify multiple ⁵⁴ activator proteins in M. xanthus. Starting their search with Rhizobium, they revealed genes for possible activators in Bacillus as well as Myxococcus but none in Synechococcus. With an internal fragment of a presumptive activator gene, the chromosomal gene can be mutated by plasmid insertion. In this way it was possible for us to integrate 7 of 14 Mxa fragments. One other fragment, Mxa15, was represented by a Tn5 insertion in pilR, bringing the number of the 14 fragments that are available for analysis to 8. The remainder of this discussion examines the range of functions suggested in this initial sample of ⁵⁴ activators.

The Mxa296 mutant was able to develop fruiting bodies on TPM agar, where it showed a wild-type morphology, but it was unable to develop in submerged culture. Its activator-related defect is early in development, preceding 4521 expression at 2 h since the 4521 reporter is also not expressed in the insertion mutant in submerged culture but is on agar. Both starvation and A-signaling are required for 4521 expression (47). It is therefore possible that either the recognition of starvation or the response to A-signal depends on the Mxa296 activator. However, both starvation recognition and A-signaling can occur in suspension (33) and therefore are not dependent on attachment to a surface. Since the Mxa296 insertion mutant could express the 4521 reporter in suspension, it is possible that the polystyrene vessel used for submerged culture inhibited development of this mutant. Whether wild-type cells can adapt to a polystyrene surface while the Mxa296 activator mutant cannot is a possibility that will require further investigation.

One insertion mutant is affected in social gliding motility by the inactivation of pilR, and this mutant has developmental defects as a consequence. ⁵⁴ activators often function as response regulators for two-component regulatory systems (35, 46). PilR activates the transcription of pilA, which encodes the major pilin subunit of the polar type IV pili. pilS, which lies immediately upstream of pilR, is homologous to the sensor element of a pilS-pilR two-component system (54). However, pili are found on M. xanthus cells under all laboratory growth conditions so far tested (53), and so the environmental signal that activates the PilR response via PilS has yet to show itself physiologically. The pilR gene could not be disrupted by the insertion of Mxa15 into its target gene in this study. The failure to obtain the Mxa15 insertion mutant is most likely a consequence of the short length of homology of the PCR fragment, since a Tn5 insertion mutant of pilR is viable.

Three other mutants completely arrested aggregation but at distinctly different stages. With the timing of development of the wild type as the reference, Mxa287 halted early (2 to 4 hours), Mxa213 stopped midway in aggregation at about 5 h, and Mxa259 apparently halted near the end of aggregation.

The Mxa287 mutant arrests as an almost featureless mat of cells that resembles the morphology of mutants that do not produce A-factor, such as asgA mutants (31). The Mxa287 mutant also has a sporulation defect which is comparable to that of an asgA mutant. The mutant fails to express the A-signal-dependent 4521-lacZ fusion, while it does express the starvation-responsive but A-signal-independent reporters 4469, 4455, and 4411 (Fig. 6). These data indicate that Mxa287 fails to carry out A-signaling. Since, on the one hand, Mxa287 produces wild-type levels of A-factor as judged by its capacity to rescue the sporulation defect of an asgA mutant and, on the other hand, is not rescued by coculture with wild-type cells, the data point to a fault in the A-factor response pathway, as opposed to one in A-factor production.

The failure of this strain to express -galactosidase from the 4521-lacZ promoter fusion, which is known to be driven by a ⁵⁴ promoter (24) and possibly to have a binding site for such an activator protein (12), may indicate that the activator lacking in Mxa287 directly controls 4521 transcription. As stated above, starvation is a prerequisite for the activation of 4521 (47), but Mxa287 is not expected to be a constituent of the starvation recognition circuit since the mutant strain expresses the starvation-dependent, but A-independent, fusion 4469 at high levels (Fig. 6). Mxa287 also expresses the developmentally regulated gene fusions 4455 and 4411 (Fig. 6). These fusions are also considered A-signaling independent (2, 27); the pattern of reporter activity supports the specificity of the Mxa287 activator for A-signaling. In a recent report, Yang and Kaplan (56) cite unpublished experiments which are consistent with Mxa287 being an activator of 4521.

That the 4521 gene is not essential for development while the Mxa287 mutant fails to develop implies the existence of additional genes that are dependent on the Mxa287 putative activator, at least one of which is necessary for fruiting body development. The identities of these genes remain to be elucidated. The Mxa287 mutant transcribes the mbhA-lacZ promoter fusion at roughly normal levels (Fig. 6), suggesting a different activator for mbhA. It has been reported that asg mutants do not accumulate MbhA protein during development as judged by hemagglutination assays (36), suggesting that mbhA is A-signal dependent. In sum, Mxa287 appears to activate a particular set of A-signal-dependent genes, at least one of which is essential for development.

The mutants Mxa213 and Mxa259 arrest aggregation at stages later than that of the Mxa287 mutant (Fig. 4). Neither of them can sporulate, and neither can be induced to sporulate by exposure to wild-type cells, indicating that their defects are also not in the production of extracellular signals. The failure of Mxa213 and Mxa259 to express the 4414-lacZ fusion, which is known to be C-signal dependent (28, 50), may indicate defects in their responses to C-signal.

The activator insertion mutants are expected to be null, because they would produce only a 5' deletion and a 3' deletion version of the activator protein. However, the possibility that the 5' deletion allele retains activity might be considered. Several, but not all, ⁵⁴ activator proteins have been found to be active and to become independent of sensory input when they lack only their N-terminal regulatory regions (8, 10, 37). Depending on the extent of 5' deletion, recombination between the plasmid clone and the M. xanthus activator genes (Fig. 2) might generate a functional central ATP binding domain combined with a functional C-terminal DNA binding domain that would constitute an activator able to function without regulatory control. Regions within the central domain are needed for several functions of the activator protein: cooperative binding of the activator to the DNA, ATP binding, ATP hydrolysis, and binding of the protein to the RNA polymerase holoenzyme (51). The published reports of active 5' deletion proteins are all of engineered recombinant activator proteins for which care has been taken to retain their entire central domains with all of their functions intact. The PCR primers used by Kaufman and Nixon lie not at the borders of the central domain but fully within it, so it is unlikely that the 5' deletion recombinant allele retains activity. Nevertheless, as each activator protein is examined, this possibility should be kept in mind.

Fourteen different activator protein sequences were found in the first screening of M. xanthus (23). Eight of these were successfully inactivated in this study. The PCR primers were not designed for the high G+C content (70%) of M. xanthus DNA, and an activator was not found for mbhA, which is thought to have a ⁵⁴ promoter (42). It is possible that this first PCR screen has not saturated the genome. In conclusion, the multiplicity and phenotypic range of developmentally defective ⁵⁴ activator mutants clearly indicate that these presumed activators, and by implication ⁵⁴ itself, play multiple roles in the regulation of development of M. xanthus.