Suppressors of a spo0A missense mutation and their effects on sporulation in Bacillus subtilis

Biochimie (1992) 74, 679--688 © Soci6t6 fran~aise de biochimie et biologie mol6culaire / Elsevier, Paris

679

Suppressors of a spoOA missense mutation and their effects on sporulation in Bacillus subtilis A D G r o s s m a n , T Lewis, N L e v i n , R DeVivo Department of Biology, Building 56-510, Massachusetts Institute of Technology, Cambridge, MA 02139, USA (Received 20 January 1992; accepted 20 February 1992)

Summary. m The spoOA gene product of Bacillus subtilis is a transcriptional regulator that is required for the initiation of sporulation. It has not been possible to isolate mutations that suppress the sporulation defect caused by spoOA null mutations. We describe the isolation and characterization of mutations that suppress the severe sporulation defect caused by a spoOA missense mutation (spoOA9V). Two suppressor mutations, spa2 and spa4, have been characterized in combination with, and separated from, the spoOA9V mutation. Both were located in the carboxyl half of Spo0A, in the putative DNA binding, transcriptional activation region. spa2 was in codon 174, causing a leucine to arginine change (spoOA174LR), and spa4 was in codon 162 (of 267), causing a histidine to arginine change (spoOA162HR). spa2 and spa4 significantly restored sporulation to the spoOA9V mutant, however, the appearance of heat resistant spores was delayed relative to wild-type. When separated from spooA9V, that is, as single mutations in spoOA, spa4 caused a delay in sporulation, while spa2 allowed apparently normal sporulation. The spa mutations caused interesting phenotypes when combined with other early sporulation mutations, spa2 suppressed the sporulation defect caused by spoOEll. This was most easily seen in spoOEll abrB double mutants, which had a much more severe sporulation defect than the spooEII single mutant. That is, spoOEll and abrB mutations caused a synthetic (synergistic) sporulation phenotype. Both the spa2 spoOA9V and the spa4 spoOA9V alleles greatly enhanced the sporulation defect caused by mutations in spoilJ, spoOJ and spoOK. The significance of these synthetic sporulation defects is discussed.

synthetic sporulation mutations I spoOF. I abrB I spoOK I spoOJ I spollJ I response regulator / histidine protein kinase Introduction

The initiation of sporulation in Bacillus subtilis is controlled in part by the spoOA gene product, spoOA is required for the expression of genes that are normally induced in response to nutrient depletion. Null mutations completely block sporulation and cause defects in many of the other processes associated with the end of normal growth, including defects in the development of genetic competence and the production of extracellular proteases and antibiotics [1-4]. Spo0A is a DNA binding protein that controls expression of genes that are involved in the transition from growth to stationary phase [5, 6]. It belongs to a family of prokaryotic regulatory proteins that are homologous in their amino termini and are involved in signal transduction and regulation of gene expression in response to changes in external conditions [7-11]. The activity of these regulatory proteins (called response regulators) is modulated by phosphorylation of an aspartate residue in the conserved amino terminus. The response regulator is one compo-

nent of the so-called 'two component regulat3ry systems'. The other component is a histidine protein kinase (also called the sensor component), which autophosphorylates on a histidine residue and transfers the phosphate to the cognate regulator. The family of histidine protein kinases share homglogy in their carboxy termini [7,11]. In contrast to the other well-characterized response regulators, the activation of Spo0A involves a multicomponent phospho-relay [12]. The spoOF and spoOB gene products are needed for the initiation of sporulation and are part of the phospho-relay that leads to the activation of Spo0A [ 12]. SpoOF is similar to the amino terminus of Spo0A a,d other response regulators [13], and is probably directly phosphorylated in a phospho-transfer reaction involving one or more histidine protein kinases [ 12,14]. The phosphate from SpoOF is transferred to Spo0B, which then transfers it to Spo0A [12]. Once activated, Spo0A~P acts as both a repressor and an activator of transcription, apparently depending on the location of the target site [5, 6].

680

spoOA mutations (rvtA, sol, surOB) have been isolated that bypass the normal phosphorylation pathway. These altered function mutations were isolated as extragenic suppressors of spoOF or spoOB mutations [15-19], and may make Spo0A activation independent of phosphorylation. It is more likely that they make Spo0A a direct substrate for one or more of the histidine protein kinases because at least some of the sol mutants still depend on kinases for sporulation [19]. Other mutations that might make Spo0A activation independent of phosphorylation have been described recently [20]. Additional altered function mutations in spoOA have been isolated that modify control of sporulation initiation. The col mutants (control of initiation) sporulate under conditions that strongly inhibit sporulation of wild-type bacteria [21 ]. In contrast to the sof and rvtA mutations, col mutations do not suppress the sporulation defect caused by mutations in spoOB and spoOF [211, indicating that they do not bypass the phospho-relay. The col mutations do suppress the sporulation defect caused by the spoOEll mutation [21]. spoOE encodes an inhibitor of sporulation. Null mutations lead to enhanced sporulation while overproduction of the wild-type protein inhibits sporulation [221. spoOEll is a nonsense mutation that produces a truncated protein that inhibits sporulation [22, 23]. The fact that some altered function mutations in spoOA suppress spoOEll, but do not bypass the need for spoOB and .spoOF is consistent with the notion that Spo0E normally acts to inhibit a late step in the phospho-relay, perhaps controlling the transfer of phosphate from Spo0B~P to Spo0A (discussed in [241). Null mutations in spoOA cause the most pleiotropic phenotype of all the sporulation mutations. Much of that pleiotropy is due to increased expression of abrB in spoOA mutants [25]. abrB encodes a DNA binding protein [6, 26, 27] that represses expression of many genes, including spoOE [22], spoVG [26, 28], and genes involved in protease and antibiotic production [26, 29, 30]. Normally, Spo0A represses expression of abrB, and as cells enter stationary phase and Spo0A is activated, less AbrB is made, leading to increased expression of genes repressed by AbrB. Null mutations in abrB cause increased expression of many genes and suppress many of the phenotypes caused by spoOA mutations, but not the sporulation defect [31-36]. Partial loss of function mutations in spoOA (spoOA9V, and spoOA153) have been described that cause a defect in sporulation, but that do not cause the pleiotropic phenotypes of null mutants [2]. Because of this difference in phenotype, spoOA9V, and spoOA153 were thought to define a different gene,

called spoOC, that was very near spoOA. It has since been shown that spoOA9V and spoOA153 are different missense mutations in codon 257, the l lth codon from the stop codon [8, 9]. These mutations seem primarily to affect the ability of Spo0A to function as an activator of transcription, and have less effect on its function as a repressor of abrB [37]. In this report we describe the isolation and characterization of suppressor mutations that allow the spoOA9V mutant to sporulate. Two suppressors, spa2 and spa4, have been characterized both in combination with and separated from the spoOA9V mutation. Both were in spoOA and caused sporulation phenotypes ih addition to their effect on spoOA9V. The spa2 mutation allowed apparently normal sporulation, but suppressed the sporulation defect caused by the spoOEll mutation, spa4 caused a delay in sporulation. Both the spa2 spoOA9V and the spa4 spoOA9V double-mutant alleles greatly enhanced the sporula[ion defect caused by mutations in spollJ, spoOJ, and

spoOK. Materials

and methods

Strains, media, and transformation.

Bacillus subtilis strains used are listed in table I. All are derived from the wild type strain JH642 and contain the trpC2 and pheAl mutations. Standard E coil strains were used for cloning as we described previously [381. LB medium [391 was used for routine growth of E coli and B subtilis. DS medium [40] was used as the nutrient sporulation medium. Media were solidified for plates with 15 g of agar (Difco) per !. Antibiotics were used as described previously 1381.

Clal [

H~al

B~III

I t

~150bPI

EcoRl I

Sacl ] Bcll I Hincll I

II

spoOA

19V

pJF1361;pNL34; pDVll; pDV14

I I

I [

I

I

pJFI599

pNLI cat

~ I

pNL41

I I

cat

I

!

pNI~A I

pDV10-1

I

I pDV171-i8,20 I Fig 1. Map of the spoOAregion and plasmids used. Plasmids containing, DNA from the spoOA region are shown and described further in Materials and methods. The approximate location of the spoOA9V mutation, causing an alanine to valine ch~ge in codon 257 (spoOA257AV),is indicated.

681 Table !. Strains used. All strains are derived from JH642 and contain the trpC2 and pheA1 mutations.

Strain

Genotype

Source or comments

JI-I642 JH647 JH695 AG234

trpC2 pheA1 spoOE11 spoOA9V spoOA9V sigB::cat dall

AG475

AspoOA::cat

AG522 AG567

spollJ::Tngl 7 spoOA9V-cat

AG571

spoOA*-cat

AG588

spa2 spoOA9V-cat

AG590

spa4 spoOA9V-cat

AG610

spoOEll abrB::Tn917

AG670

spa4 spoOA9V

AG674

(spa4 spoOA9V)::pJF1361

AG678

spa4-cat (spoOA9V+)

AG680

spa2 spoOA9V

AG686

spa2-cat (spoOA9V+)

AG701

(spa2 spooA9V)::pJF1361

A(3708

spa2 spoOA9VspoliJ::Tn917

AG709

spa4 spoOA9VspollJ::Tn917

AG903 AG 1080

spoOEl l abrB::Tn917 spoOA::pDV11 (spa2) spooK418::Tn9171ac

J Hoch [22, 231 [8,91 JH695 transformed to Cmr with sigB::cat dall DNA (cat and dal are =50% linked) JH642 transformed with linearized pNIAI [38, 47] JH695 transformed to Cm r with pNL53A; cat =95% linked to spoOA9V JH642 transformed to Cmr with pNL53A; cat =95% linked to spoOAgV EMS treatment of AG567; original isolate of spa2 EMS treatment of AG567; original isolate of spa4 JH647 transformed with abrB::Tn917 DNA [26, 481 AG234 transformed to dal+spa4 CmS with DNA from AG590 AG670 transformed with pJF1361. Used to clone spa4. AG670 transformed to Cm~ with pNL53A AG234 transformed to dal+spa2 Cm s with DNA from AG588 AG680 transformed to Cmr with pNL53A AG680 transformed with pJF 1361. Used to clone spa2 AG680 transformed with DNA from spollJ::Tngl 7 AG670 transformed with DNA from spoilJ: :Tngl 7 AG610 transformed with pDVI 1 (spa2)

AG 1081

spoOJ::Tn917~HU26 !

AG 1083 AGI084

spa4 spoOA9V spoOK::Tn9171ac spa4 spoOA9VspoOJ::Tngl7

AG 1086

spa4-cat spoOK::Tn9171ac

AG 1087

spa4-cat spooJ::Tn917

AG 1088 AG 1089

spa2 spooA9V spoOK::Tn9171ac spa2 spoOA9VspoOJ::Tn917

AG 1091

spa2-cat spoOK::Tn9171ac

AG ! 092

spa2-cat spoOJ::Tngl 7

JH642 transformed with spoOK::Tn9171ac DNA [38] JH642 transformed with spoOJ::Tn917 DNA [471 AG670 transformed with spoOK::Tn9171ac DNA AG670 transformed with spoOJ::Tn917 DNA AG678 transformed with spoOK::Tn9171ac DNA AG678 transformed with spoOJ::Tn917 DNA AG680 transformed with spoOK::Tn9171ac DNA AG680 transformed with spoOJ::Tn917 DNA AG686 transformed with spoOK::Tn9171ac DNA AG686 transformed with spoOJ::Tn917 DNA

682

E coil and B subtilis cells were made competent and transformed by standard procedures [41, 42]. Cloning spoOA (pNLI ). Due to the initial unavailability of clones of spoOA, we isolated a clone (pNLI, fig 1) using colony hybridization and a synthetic oligonucleotide probe based on the published DNA sequence [8, 9]. B subtilis chromosomal DNA was cut with Hpal and Bcll and fragments of approximately 1-2 kb were gel-purified and cloned into pJHl01 [43] that had been cut with Nrul and BamHI. These enzymes were used so that an intact spoOA structural gene would be separated from its promoter and cloned into the tet gene of pJH101 (similar to pBR322) in the orientation opposite that of transcription. Clones containing spoOA were identified by standard colony hybridization procedures [42] using the synthetic oligonucleotide 5'-CATCTTCCTGCCCAAAG-3' (made by the Microchemistry laboratory of the Harvard University Biological Laboratories), which was complementary to nucleotides 261-277 of the spoOA structural gene.

Plasmids. Plasmids containing parts of the spoOA region are shown in figure 1. pJF1361 and pJF1599 were obtained from J Hoch [8]. pJF1361 contains B subtilis DNA from the Clal site upstream to the EcoRl site in the 3' end of spoOA cloned into the integrative vector pJHl01 [43] between the ClaI and EcoRI sites. pJF1599 contains B subtilis DNA from the EcoRI site in the 3' end of spoOA to the Hincll site downstream, cloned between the EcoRI and Smal sites of pUC9. pNL 1 was constructed as described above. pDVI0-1 was derived from pJFI361 by deleting from Bglll to EcoRI. pDVII and pDVI4 contain the spa2 and spa4 alleles of spoOA, respectively. They were made by integrating pJF1361 into spoOA (spa2 spoOA9V and spa4 spoOA9V) and cutting the plasmid back out such that the spa mutations were contained on the plasmid. Briefly, chromosomal DNA was made from strains AG701 [(spa2 spoOA9V)::pJF1361 'Spo +'] and AG674 [(spa4 spoOAgV)::pJFi361 'Spo÷'], digested with EcoRI, ligated at dilute DNA concentration to promote intramolecular ligation, and transformed into E coli, selecting for resistance to arnpiciUin. The resulting plasmids, pDVII and pDVI4, contained spa2 and spa4, respectively. This was shown by integrating these plasmids into JH695 (spoOA9V) and recovering 'SPO+' (spa spoOAgV) transformants. (In contrast to spoOA null mutants, spoOA9V mutants are easily transformed.) pDVI7, pDVI8, and pDV20 contain wild type, spa2, and spa4 DNA from the Bglll to EcoRI sites in spoOA, respectively, and were used for DNA sequencing. They were derived from pJF1361, pDVil, and pDVI4 by deleting from Bgill to the BamHI site in the vector. pNLA 1 contains the AspoOA::cat deletion-insertion mutation that was used to make strain AG475. pNL41 was constructed in two steps. First, the spoOA region from pJF1361 was subcloned into pUCI8, a vector that does not have a cat gene. pJF1361 was cut with EcoRI and BamHI to release the --1.05 kb Clal to EcoRI ~FoOAfragment as well as part of the backbone of pJHl01, and ligated into EcoRI to BamHl of pUCI8 to produce pNL34. Second, the cat cassette (SmaI to BamHI) from pMlll01 [44] was cloned between HpaI and BgiIl of spoOA, generating a 247-bp deletion that removed 222 bp of the spoOA coding region (fig 1). The AspoOA::cat mutation

was recombined into the chromosome by transforming wildtype cells with linearized pNL41 and selecting fo~ Cm r transformants. The AspoOA::cat mutants had the typical spoOA phenotypes: they were SPO-, defective in antibiotic and protease production, and sensitive to polymyxin. The structure of one transformant was verified by Southern hybridization (NL and ADG, unpublished results). pNL53A contains the cat cassette from pMl1101 [44] cloned into the Sacl site, --350 bp downstream of spoOA, in pJF1599. It was used to construct strain AG567 and AG571 (table I). The cat gene downstream of spoOA had no effect on sporulation. Sporulation of strain AG571 (spoOA+-cat) was indistinguishable from that of strain JH642 (spoOA+), and the sporulation phenotype of AG567 (spoOA9V-cat) was indistinguishable from that of JH695 (spoOA9V).

Mutagenesis. Strain AG567 (spoOA9V-cat) was grown in nutrient sporulation medium (DS medium) to mid-log; cells were centrifuged, resuspended in 1/7 volume of Spizizen salts [45] and treated with 4% ethy|methanesulfonate (EMS) for 15 rain. Cells were centrifuged, washed and resuspended in prewarmed DS medium. The mutagenized culture was split into subcultures to ensure isolation of independent mutations. 24 h after the end of vegetative growth, samples were heated to 80°C for 15 rain to kill cells that had not sporulated. Portions of the heat-treated cultures were then spread onto DS agar plates to obtain single colonies. Samples tested 48 h after the end of vegetative growth contained approximately the same number of spores as those at 24 h, indicating that there was not a large class of suppressor mutations causing an extended delay in spomlation. The EMS mutagenesis was shown to be effective in increasing the frequency of mutational events. Mutagenesis enhanced the frequency of streptomycin resistant (100 ~tg/ml) mutants by a factor of -- 1000.

DNA sequencing. spoOA÷, spa2, and spa4 were sequenced from pDVI7, pDVI 8, and pDV20, respectively. Double-stranded DNA was sequenced using the USB Sequenase Kit and standard protocols. Sequencing was done with two primers purchased from New England Biolabs: the pBR322 EcoRl site 15-mer clockwise primer, ~ d ~he pBR322 BamHl site 16-met counterclockwise primer.

Spore assays. Cells were grown in DS medium at 37°C and the percent spores were determined as the ratio of heat resistant (80°C for 15 min) colony forming units to total colony forming units on LB plates, times 100% at the indicated times (usually > 20 h after the end of exponential growth).

Results

Attempts to isolate second site suppressor mutations o f a spoOA null mutation were unsuccessful. We constructed a stable, well defined spoOA null m u tation, AspoOA::cat (Materials and methods), and used strains c a r r y i n g this mutation to select for Spo+ revertants. E v e n after h e a v y mutagenesis, we were unable

683 to isolate Spo + revertants from the spoOA null mutant. In addition, we were unable to isolate Spo+ revertants from a AspoOA::cat abrB double mutant, abrB mutations suppress some of the phenotypes associated with the spoOA mutant, but not the sporulation defect. An alternative approach was to look for suppressors of spoOA missense mutations. Two alleles of spoOA, spoOA9V and spoOA153, cause a less severe phenotype than spoOA null mutations. While null mutants are pleiotropic and defective in many of the processes associated with stationary phase, spoOA9V and spoOA153 mutants seem to be defective only in sporulation. Both spoOA9V and spoOA153 are in the 257th codon, 11 codons upstream from the stop codon. The spoOA9V mutation results in a change from alanine to vafine, whereas spoOAI53 causes a change to glutamic acid [8, 9]. We have isolated and characterized two second site suppressors of the spoOA9V mutation. (We have been unable to obtain the spoOA153 mutation.) Both suppressor mutations were in spoOA and allowed the spoOA9V mutant to sporulate at a significant frequency. We first describe the isolation of suppressors of spoOA9V (spa mutations), their genetic and molecular characterization, and then the phenotypic characterization of the mutants. Isolation of similar suppressors of spoOA9V has recently been described [37].

Isolation of suppressors of spoOA9V. A strain was constructed to facilitate characterization of suppressors of spoOA9V. AG567 contained the cat gene (conferring resistance to chloramphenicol) very close to the spoOA9V mutation, but 3' to the spoOA coding sequence (see Materials and methods). Linkage be'~ween spoOA9V and cat allowed these genes to be co-transformed at a high frequency (.--90--95%). This provided a way to determine quickly if the spoOAgV mvtation was still present in the revertants, and simultaneously, if the suppressor mutation was linked to the cat marker. The frequency with which the Spo- phenotype caused by spoOA9V could be transferred into a wild type strain by selecting for chloramphenicol resistance (Cmr) would indicate if the suppressor mutation was in the spoOA region of the chromosome. Eleven Spo + revertants were isolated from the spoOA9V-cat strain (AG567) following EMS mutagenesis. After mutagenesis, cells were grown, subjected to sporulation conditions, and heat resistant spores were selected (80°C, 15 min). Samples were then spread on DS agar plates to allow spores to germinate and grow into colonies. Only cells which contained a mutation that suppressed the sporulation defect caused by spoOA9V, or a back.mutation, would survive the

heat treatment. Spo + revertants (or pseudo-revertants) were present at a frequency of --lOS. Quantitative sporulation assays indicated that all of the revertants produced >10S-fold more spores per ml than the spoOA9V parent (table II). Among the eleven Spo+ revertants, two (AG588 and AG590) had distinct colony morphologies different from wild type, indicating that these were most likely pseudo-revertants. Colony morphologies of the other nine revertants were indistinguishable from the isogenic wild type. We describe the genetic, moIecular, and phenotypic characterization of the two pseudo-revertants, spa2 and spa4.

Recovery of spoOA9V-catfrom two of the revertants The two revertants with distinct colony morphologies contained second-site suppressors that were closely linked to, but separable from, the original spoOA9V mutation. Chromosomal DNA was prepared from the Spo* revertants and used to transform wildtype cells (JI-I642) to chloramphenicol resistance. Transformants were examined for their sporulation phenotype. When the donor DNA came from the original spoOA9V-cat parent (AG567), =95% of the Cmr transformants were Spo-. When donor DNA from two of the revertants (AG588 and AG590) was used in a similar transformation into wild type cells (JH642), Spo- transformants were also obtained, indicating that these revertants did not contain a back mutation. However, the frequency of Cm r ~ransformants that were Spo- was only =2% (compared to the expected 90-95% if the suppressor mutations were unlinked to spoOA), indicating that in both cases the suppressor mutation was tightly linked to cat, and perhaps in spoOA. Of the remaining rcvertants tested, all seemed to have back mutations or suppressor mutations that we could not separate from the spoOA9V mutation. To facilitate characterization, the two second site suppressor mutations (spa2 in AG588 and spa4 in AG590) were introduced by transformation into unmutagenized cells (containing the spoOA9V mutation). The spa mutations were transferred into the recipient AG234 (spoOA9V, sigB::cat, dal) by congression with the unlinked dat marker. Selection was for the the ability to grow without D-alanine (dal+) and the transformants were tested for the acquisition of the spa mutation as judged by their ability to sporulate. Approximately 1-2% of the dal+ transformants were 'Spo +' with DNA from AG588 (spa2) and AG590 (spa4). Of these, approximately 6-7% were sensitive to chloramphenicol, indicating cotransformation of sigB+ with dal + and separation of cat from spoOA9V in the donor DNA. One Cm s transformant from each cross (AG670 (spa4, spoOA9IO and AG680 (spa2, spoOA9V)) was chosen for further characterization.

684

Both spa mutations are in spoOA. The linkage of spa2 and spa4 to cat indicated that these mutations could be in spoOA. The location of the spa mutations was further defined by marker rescue type experiments with clones containing different parts of spoOA. If a plasmid carries a wild-type copy of the suppressor locus, then integration of a plasmid into the spa spoOA9V double mutant should, at some frequency, 'rescue' the spa mutation and result in a Spo- (spoOA9V) phenotype. The plasmids used for these rescue experiments end at the EcoRI site in spoOA and will not rescue the spoOA9V mutation, which is 3' to EcoRI (fig 1). Both spa2 and spa 4 appeared to map between the Bglll and EcoRI sites in spoOA. Integration of pJF1361, which contains DNA from Clal to EcoRl (fig 1), into AG670 (spa4, spoOA9V) and AG680 (spa2, ~poOA9V) resulted in approximately 50 and 30% Spo- transformants respectively. The Spo- phenotype arises when the integration of pJF1361 occurs between the spa mutation and spoOA9V, generating a truncated copy of spoOA containing the spa mutation and an intact copy of spoOA containing spa+ and spoOA9V. Transformation of the same two strains with pDV10-1, which contains DNA from ClaI to BgllI of spoOA, did not result in any Spo- transformants (<0.3%). Together these results indicated that both spa2 and spa4 were located between the Bgill and EcoR! sites in spoOA.

Cloning and sequencing spa2 and spa4. spa2 and spa4 were cloned by integrating pJF1361 into spoOA and cutting the plasmid back out such that the spa mutations were contained on the plasmid (see Materials and methods). The resulting plasmids, pDVII and pDVI4, contained spa2 and spa4, respectively.

spoOA+, spa2 and spa4 were sequenced from plasmids derived from pJF1361, p D V l l , and pDV14. Deletions were made in these three plasmids between the Bglll site in spoOA and the BamHl site in the vector backbone. The resulting plasmids, pDVI7 (spoOA+), pDVI8 (spa2), and pDV20 (spa4) were used in double stranded DNA sequencing reactions (see Materials and methods). Only one base change was found in each of the spa clones. The spa2 mutation was a CTC to CGC transversion at codon 174, causing a leucine to arginine change (spoOA174LR), and the spa4 mutation was a CAT to CGT transition at codon 162, causing a histidine to arginine change (spoOA162HR). These changes are in the carboxyl half of the protein, in the putative DNA binding or transcriptional activation region.

Sporulation of spoOA9V is significantly restored by the spa mutations. Both spa2 and spa4 significantly suppressed the sporulation defect caused by spoOA9V (table II). The spoOA9V mutation essentially causes a complete block in sporulation (<10 spores/ml). The spa2 spoOA9V double mutant sporulated at frequencies between 2 and 30%, while the spa4 spoOA9V double mutant sporulated a bit less well, typically between 0.2 and 4%. While the level of suppression was significant, sporulation was somewhat less efficient than in wild-type cells. In addition, sporulation of both spa spoOA9V strains was delayed relative to wild-type (table II).

spa4 spoOA9V+caused a delay in sporulation. In order to test the effects of the spa2 and spa4 mutations on sporulation in the absence of the spoOA9V mutation, we converted each spa spoOA9V double mutant to spoOA9V+ by introduction of pNL53A. pNL53A contains DNA from the extreme 3' end of spoOA. The EcoRI site in spoOA conveniently separates the spoOA9V mutation from the spa mutations. Thus, transformation of strains containing the spoOA9V mutation with pNL53A should result in some fraction of spoOAgV* transformants. Strains JH695 (spoOA9V), AG670 (spa4 spoOAgV), and AG680 (spa2 spoOA9V) were transformed with pNL53A, that had been linearized, selecting for Cmr. In all three cases, two classes of transformants were observed based on different colony morphologies on sporulation plates. Approximately 15% of the Cmr transformants of JH695 were Spo+, indicating conversion to spoOA9V+. Similarly, two classes of transformants were obtained when pNL53A was introduced into AG670 and AG680. Most transformants had the Table 11. Sporulation of spa2 and spa4 revertants. Data shown are from a single experiment. Similar results were obtained in at least four separate experiments. Typically, at T~0 and I"_,2o AG670 gave --=102--103 and ~-106--107 spores/ml; AG680 gave ---102--103 and --107-108 spores/mi; AG678 gave -106--107 and --10s spores/ml; and AG686 gave ~-10s and ~-10s spores/ml, respectively.

Strain

Relevantgenotype

AG571 AG567 AG670 AG680 AG678 AG686

spo+ spoOA9V spa4 spoOA9V spa2 spoOA9V spa4 spa2

Percent spores 1"1o Tz6 23 <0.00001 0.0002 0.0005 0.2 77

70 <0.00001 0.4 2 100 100

685 morphology of the parent strain (spa spoOA9V), while 20-30% had a different morphology.

colony

It seemed likely that the transformants with colony morphologies different from the parent had been converted to spoOA9V+. One transformant with the 'different' morphology from each strain was tested for sporulation in DS medium. Strains AG678 (spa4, spoOA9V+) and AG686 (spa2, spoOA9V+) produced approximately the same number of spores as wild type (JH642) when sporulation was measured > 20 h after the initiation of sporulation (table II), indicating that these strains in fact had been converted to spoOAgV+. However, when sporulation was measured =8-12 h after the initiation of sporulation, the spa4 mutation (but not spa2) caused a reproducible decrease in the number of spores (table II). Thus, sporulation was delayed in the spa4 mutant.

spa2 partly suppressed the sporulation defect caused by spoOEll and spoOEll abrB. Several different mutations in spoOA suppress the sporulation defects caused by other spoO mutations [15-19]. Most of these were isolated as suppressors of spoOF or spoOB mutations, and some are able to suppress the sporulation defects caused by mutations in spoOB spoOE spoOF spoOJ spoOK and/or spollJ (kinA). We tested the effects of the spa alleles of spoOA on the sporulation phenotypes of some of these spo mutants. The sporulation defect caused by the spoOEll mutation was partly suppressed by spa2. This was most easily seen in spoOEll abrB double mutants (table III). Preliminary experiments indicated approximately 5-10-fold suppression of spoOEll by spa2. Since this effect was not very large and was difficult to reproduce, we tested the ability of spa2 to suppress Table I!I. Effects of spa2 and abrB mutations on sporulation of the spoOEll mutant. Data shown are from a single experiment. Heat resistant spores were determined approximately 20 h after the initiation of spomlation in DS medium. Spores per ml are presented, rather than percent spores, due to the decrease in viable cells in spoOEll abrB at late times after To. Viable cell counts were approximately the same for all strains at least until =Tt. Typically, the spoOEll mutant gave =105-106 spores/ml, while the spoOEll abrB double mutant gave =103-104 spores/ml.

Strain

Relevantgenotype

Spores/ml

JH642 JH647 AG610 AG703

spo+ spoOEll spoOEll,abrB spoOEll,abrB, spa2

6 x

10s 3 X 105 6 x 103 1 x 107

the more severe sporulation defect in spoOEll abrB double mutants. We had noticed that abrB mutations dramatically enhanced the sporulation defect caused by the spoOEII mutation (table III). spoOEI1 produces a truncated protein (nonsense fragment) that blocks sporulation [22, 23], and abrB mutations cause increased expression of spoOE [22]. The more severe sporulation defect in the spoOEll abrB double mutant was almost certainly due to overexpression of the nonsense fragment of SpoOE that blocks sporulation. spa2 partly suppressed the sporulation defect of the spoOEll abrB double mutant (table III). In contrast to the effect of spa2 on sporulation of the spoOEll mutants, none of the spa alleles suppressed the sporulation ~.~efect of a spoOB mutant. That is, none of the four sFoOA alleles containing spa mutations (spa2, spa4, spa2 spoOA9V, and spa4 spoOA9V) was able to suppress the sporulation defect of a spoOB mutant. These results indicate that the spa mutations did not bypass the normal phosphorylation pathway needed to activate SpoOA. This is in contrast to the sof and twtA alleles of spoOA which suppress the sporulation defects caused by spoOB, spoOF, and spoOEll mutations. (soft and rvtAll also suppress the sporulatiora defect of spoOEll abrB double mutants (K Hicks ~tnd ADG, unpublished results).)

The spa2 spoOA9Vand spa4 spoOA9Valleles caused a synthetic sporulation defect when combined with some early sporulation mutations. We tested the effects of spa2, spa2 spoOA9V, spa4, and spa4 spoOAgV on the ability of other early spo mutants to sporulate. Both the spa2 spoOA9V and the spa4 spoOAgV alleles caused synthetic sporulation phenotypes in combination with a null mutation in spollJ (table IV). That is, the defect caused by the combination of the spollJ mutation with the spa spoOA9V allele was more severe than expected from Table IV. Effects of spa2 spoOA9V and spa4 spoOAgV mutations on sporulation of a spoliJ mutant. Data shown are from a single experiment. Heat resistant spores were determined approximately 20 h after the initiation of sporulation in DS medium.

Strain

Relevantgenotype

JH642 AG522 AG670 AG709 AG680 AG708

spo÷ spolIJ::Tn917 spa4 spoOA9V spa4 spoOA9V,spollJ spa2 spoOA9V spa2 spoOA9EspolIJ

Percent spores 89 12 0.7 0.001 4.3 0.007

686 the individual phenotypes. An even more severe sporulation defect was seen when spa2 spoOA9V and spa4 spoOA9V were combined with mutations in spoOK and spoOJ (table V).

Discussion We have isolated and characterized two different suppressors of the spoOA9V mutation. Both suppressar mutations were in spoOA: spa2 at codon 174, causing a leucine to arginine change (spoOA174LR), and spa4 at codon 162 (of 267), causing a histidine to arginine change (spoOA162HR). Both mutations significantly restored sporulation of the spoOA9V mutant, although the appearance of heat resistant spores was delayed relative to wild-type. When separated from spoOA9V, that is, as single mutations in spoOA, spa4 caused a delay in sporulation, while spa2 allowed apparently normal sporulation. Similar suppressors of spoOA9V (called suv) have recently been described [37]. The suv4 mutation is the same as spa4, and suv3 is in the same codon as spa2, except that it causes a leucine to phenylalanine change [37]. The effects of the suv mutations on timing of sporulation were not reported. Neither of the spa mutations was able to suppress the sporulation defects caused by mutations in spoOB or spoOF, indicating that they did not bypass the normal phosphorylation pathway needed to activate Spo0A. spa2 was able to partly suppress the sporulation defect caused by the spoOEll mutation. This was most easily seen in spoOOEll abrB double mutants, which had a much more severe sporulation defect than the spoOEll mutant alone. This synthetic sporulation defect caused by spoOEll and abrB is probably due to overexpression of the spoOEll nonsense fragment in abrB mutants [22, 23]. The effect of the spa2 spoOA9V and spa4 spoOA9V alleles of spoOA on sporulation of spoilJ, spoOK, and spoOJ mutants was remarkable. Both spoOA alleles caused synthetic sporulation defects in combination with each of these three spo mutations. The spoIIJ, spoOK, and spoOJ mutations are all likely to cause defects in the phospho-relay needed to activate Spo0A. spollJ (kinA) encodes a histidine protein kinase [ 14, 46] that can phosphorylate SpoOF in vitro [ 12, 14], apparently initiating the phospho-relay [ 12]. spolIJ mutants are also suppressed by some of the sol mutations [19]. spoOK and spoOJ mutants are suppressed by some of the mutations in spoOA [17, 18] (K Ireton, AD Grossman, unpublished results), and they can be suppressed by overproducing KinA (SpoIIJ) [38] (K Ireton, AD Grossman, unpublished results). The spa spoOA9V alleles seem to encode a partly defective Spo0A, as evidenced by delayed or reduced sporulation. It could be that these mutant

Table V. Effects of spa mutations on sporulation of spoOK

and spoOJ mutants. Data shown are from a single experi-

ment. Heat resistant spores were determined approximately 20 h after the initiation of sporulation in DS medium.

Strain

Relevantgenotype

JH642 AG 1080 AG 1081 AG670 AG 1083 AG 1084 AG678 AG 1086 AG 1087 AG680 AG 1088 AGI089 AG686 AG 1091 AG 1092

spo+ (spa+) spoOK::Tn9171ac spoOJ::TnOl 7 spa4 spooA9V spa4 spooA9V,spoOK spa4 spoOA9V,spoOJ spa4 spa4, spoOK spa4, spoOJ spa2 spooA9V spa2 spooA9V,spoOK spa2 spoOA9V,spoOJ spa2 spa2, spoOK spa2, spoOJ

Percentspores 64 5.6 7.4 2.8 0.0003 0.00002 78 0.8

12 27 0.002 0.00003 92

21 24

spoOA gene products are partly defective in activation by the phospho-relay, or in their ability to activate transcription, and when combined with other mutations that decrease the efficiency of the phospho-re!ay, cause a synthetic phenotype. Alternatively, the synthetic phenotype could be due to defects in different aspects of Spo0A activation. There seem to be alternative ways to activate Spo0A. spoOA mutants are more pleiotropic than spo08 or spoOF mutants, indicating a pathway separate from the phospho-relay for activating Spo0A. In addition, some of the sof mutations, which bypass the need for spoOB and spoOF, still require kinases for activation, indicating at least one other mechanism for activating the mutant Spo0A [19]. While it is clear that the phospho-relay is normally required for sporulation, it is possible that after the initial activation of Spo0A by the phospho-relay, another pathway is also involved in maintaining the activated form. Mutations that alter this additional pathway might not cause a significant phenotype if the phospho-relay is functioning normally. However, if the phospho-relay is slightly impaired, then a mutation that alters the additional pathway might cause a much more severe sporulation defect. It seems possible that the spa spoOA9V mutant is defective in activation by a pathway different from the phospho-relay, and the synthetic sporulation phenotype is caused by the combination of defects in two partly redundant pathways of Spo0A activation. Of course, further charac-

687 terization o f spoOA m u t a t i o n s and genes involved in the initiation o f sporulation will help determine the m e c h a n i s m s and p a t h w a y s used to activate Spo0A.

Acknowledgments We are grateful to K Siranosian and D Rudner for technical assistance; K lreton for comments and suggestions on the manuscript. This work was started in the laboratory of R Losick at Harvard University, and we are grateful for his support and encouragement. We thank J Hoch for providing pJF1361 and pJF1599. ADG is a Lucille P Markey Scholar in Biomedical Sciences and this work was supported in part by a grant from the Luciile P Markey Charitable Trust. Support in part also came from Public Health Services grant GM41934 to ADG from the NIH, and BRSG 2 S07 RR07047-23 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, NIH.

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