A negative regulator linking chromosome segregation to developmental transcription in Bacillus subtilis

Authors

  • Marguerite A. Cervin,

    1. Departments of, Microbiology and Immunology,
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  • George B. Spiegelman,

    1. Departments of, Microbiology and Immunology,
    2. Medical Genetics, University of British Columbia, 6174 University Boulevard, Vancouver, BC, V6T 1Z3, Canada.,
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  • Brian Raether,

    1. Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, NX-1, La Jolla, CA 92037, USA.
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  • Kari Ohlsen,

    1. Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, NX-1, La Jolla, CA 92037, USA.
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  • Marta Perego,

    1. Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, NX-1, La Jolla, CA 92037, USA.
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  • James A. Hoch

    1. Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, NX-1, La Jolla, CA 92037, USA.
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James A. Hoch E-mail hoch@scripps.edu; Tel. (619) 784 7905; Fax (619) 784 7966.

Abstract

The Spo0JA and Spo0JB proteins of Bacillus subtilis are similar to the ParA and ParB plasmid-partitioning proteins, respectively, and mutation of spo0JB prevents the expression of stage II genes of sporulation. This phenotype is a consequence of Spo0JA activity in the absence of Spo0JB, and its basis was unknown. In the studies reported here, Spo0JA was found specifically to dissociate transcription initiation complexes formed in vitro by the phosphorylated sporulation transcription factor Spo0A and RNA polymerase with the spoIIG promoter. This repressor-like activity is likely to be the basis for preventing the onset of differentiation in vivo. Spo0JB is known to neutralize Spo0JA activity in vivo and also to interact with a mitotic-like apparatus responsible for chromosome partitioning. These data suggest that Spo0JA and Spo0JB form a regulatory link between chromosome partition and development gene expression.

Introduction

A central determinant governing entry into sporulation by Bacillus subtilis is the phosphorylation state of the transcription factor Spo0A (Hoch, 1993). Among its several sporulation-essential functions, phosphorylated Spo0A (Spo0A∼P) activates the transcription of stage II operons including spoIIA, spoIIE and spoIIG (Satola et al., 1991; 1992; Trach et al., 1991; Bird et al., 1992; 1993; 1996; York et al., 1992; Baldus et al., 1994). The spoIIA and spoIIG operons encode the sporulation-specific sigma factors σF and σE, which function in the forespore and mother cell compartments of the developing spore respectively (Errington, 1993; Stragier and Losick, 1996). Spo0A phosphorylation is accomplished by the phosphorelay signal transduction system (Burbulys et al., 1991). The phosphorelay is a signal integration circuit that regulates the state of Spo0A phosphorylation through the summation of phosphate input by the protein kinases of the phosphorelay and phosphate removal by protein phosphatases, which may act either on Spo0A∼P directly or on the phosphorelay intermediate Spo0F∼P (Ohlsen et al., 1994; Perego et al., 1994; 1996). While it is conceivable that all of the signals influencing the decision to sporulate are transduced by the phosphorylation–dephosphorylation reactions and integrated by the phosphorelay, it has never been proven that elevated levels of Spo0A∼P per se are sufficient to initiate developmental transcription of stage II genes.

The spo0J stage 0 sporulation locus, defined by a single allele spo0J93, has been known for some time to be required for stage II gene expression, but its function has been obscure (Mysliwiec et al., 1991). When sequence analysis of the chromosome origin region became available, the spo0J locus was revealed to consist of two genes with homology to the parA, parB or korB, incC genes of plasmids (Ogasawara and Yoshikawa, 1992). This homology information led to the obvious conclusion that the spo0J locus may have a role in chromosome segregation and suggested that the physical act of segregating the chromosome into a forespore compartment may directly affect transcription of subsequent genes in the sporulation process (Hoch, 1993). Subsequent studies showed that the ParA equivalent, Spo0JA, also known as Soj, was the cause of the sporulation-negative phenotype when the ParB equivalent, Spo0JB, also known as Spo0J, was defective (Ireton et al., 1994). The sporulation-negative phenotype caused by deletion of the spo0JB gene could be relieved by the deletion of spo0JA (Ireton et al., 1994). This important finding indicates that the ParA homologue Spo0JA acts as a negative regulator of sporulation in the absence of the ParB homologue Spo0JB. Furthermore, these proteins may influence normal chromosome segregation, as spo0JB mutants produced a low frequency of anucleate daughter cells (Ireton et al., 1994).

Important clues as to the roles of the proteins of the spo0J locus were obtained by Sharpe and Errington (1995). These investigators found that a mutant lacking both spo0JA and spo0JB genes had normal septation rates and cell length distributions and normal time of formation and positioning of the sporulation-specific asymmetric septum, despite its defect in partitioning. However, the kinetics of spore formation were retarded, and the specificity of DNA transfer to the prespore was relaxed. It was proposed that the Spo0J (Spo0JB) protein was responsible for a centromere-like function in the cell, which was, in turn, responsible for the directionality of chromosome transfer to the poles of the cell. Recent studies in several laboratories have revealed that chromosome separation occurs by a mitotic-like apparatus and that the Spo0JB protein, like the ParB protein of Caulobacter crescentus, associates with this apparatus and co-segregates with the origin of chromosome replication (Glaser et al., 1997; Lin et al., 1997; Mohl and Gober, 1997; Webb et al., 1997). The roles played by these proteins in mitotic segregation are not understood. Although Spo0JB and C. crescentus ParB have similar physical segregation behaviour, only the latter is essential for growth and, by implication, Spo0JB is not an essential component of the mitotic apparatus. C. crescentus ParA does not co-segregate with the mitotic-like apparatus but appears to be essential for growth (Mohl and Gober, 1997). In contrast, deletion of spo0JA in a spo0JB +B. subtilis strain yields a strain that sporulates normally and does not have a partition defect (Sharpe and Errington, 1996). These data are not consistent with Spo0JA playing an essential role in chromosome segregation. Moreover, the data indicate that Spo0JA is capable of blocking the initiation of sporulation in a mutant strain lacking Spo0JB.

In the experiments described here, the mechanism of inhibition of sporulation by Spo0JA was investigated. Spo0JA was found specifically to dissociate transcription initiation complexes formed by Spo0A∼P and RNA polymerase at the promoter region of the spoIIG operon. We discuss the results in view of the genetically demonstrated neutralization of this repressor-like activity of Spo0JA by Spo0JB and suggest a model of how the mitotic apparatus couples the regulation of stage II gene expression to chromosome replication and segregation.

Results

The spo0JB mutation does not affect phosphorelay gene transcription

As transcription of stage II genes is positively regulated by Spo0A∼P and these genes are not expressed in a spo0JB mutant, it was proposed that Spo0JA might act by inhibiting the phosphorylation of Spo0A (Ireton et al., 1994). In order to test this proposal, the expression of the genes for three of the component proteins of the phosphorelay was examined. β-Galactosidase fusions to kinA, spo0F and spo0A were studied in wild type and a spo0JB mutant as a function of growth and sporulation. The results were the same for all; no significant difference in timing or expression level was found for any of these genes. Figure 1 shows the results for the induction of the spo0A gene. As Spo0A∼P is required for the induction of both spo0F and spo0A, it seems unlikely that Spo0A∼P levels were affected by the spo0JB mutation (Strauch et al., 1993). As a further test of this notion, the repression of abrB was followed. Spo0A∼P is known to be a direct repressor of abrB transcription (Strauch et al., 1990). The spo0JB mutant was not significantly different from the wild-type strain in the kinetics of abrB repression as a function of growth (Fig. 1). These data make it unlikely that Spo0JA acts to inhibit the synthesis of phosphorelay proteins or to inhibit the flow of phosphate to Spo0A.

Figure 1.

. β-Galactosidase analyses of transcription from the spo0A (A) and abrB (B) promoters using lacZ fusions in a wild-type and spo0JB deletion strains. (○) data from the wild-type strain; (□) data from the spo0JB mutant. Time is in hours, where 0 is the time when the culture shifts from exponential growth to stationary phase.

Spo0JA inhibits transcription from stage II gene promoters

If Spo0JA did not regulate the level of Spo0A∼P, the possibility existed that it was a direct repressor of stage II gene transcription. The effect of Spo0JA on in vitro transcription was tested using the Spo0A∼P-activated spoIIG and spoIIE promoters, the Spo0A∼P-repressed abrB promoter and the A2 promoter from phage φ29 that is not affected by Spo0A∼P. Increasing concentrations of Spo0JA inhibited transcription from the spoIIG promoter, while transcription from the A2 promoter was not affected significantly (Fig. 2). The highest concentration of Spo0JA reproducibly inhibited the Spo0A∼P stimulation of spoIIG transcription by over 70%. Transcription from the Spo0A∼P-dependent spoIIE promoter was inhibited to the same extent as that from the spoIIG promoter. Thus, purified Spo0JA did not act as a general transcription inhibitor but was specific for Spo0A∼P-dependent promoters. Furthermore, in vitro transcription repression of the abrB promoter by Spo0A∼P was not relieved by the addition of Spo0JA (data not shown), suggesting that Spo0JA does not act as a general antagonist of Spo0A∼P. These in vitro properties of Spo0JA on transcription exactly mimic the phenotypic effects of a spo0JB mutation on these genes in vivo.

Figure 2.

. Effect of Spo0JA on in vitro transcription. Transcription assays were carried out as described in Experimental procedures. The templates used were: the spoIIG promoter (▪); the φ29 A2 promoter (□). The percentage transcription inhibition was calculated by determining the Cerenkov radiation in gel slices containing the transcripts and expressing it as a percentage of the no Spo0JA added control.

The step in transcription initiation affected by Spo0JA was examined using the carboxyl-terminal domain of Spo0A (termed Spo0A-C). The carboxyl-terminal domain and phosphorylated Spo0A act identically in transcription assays, and the use of this domain eliminated the presence of phosphorelay proteins in the assay as well as negating the possibility that Spo0JA acts as a phosphatase. This portion of Spo0A (representing amino acids 149–267) activates transcription of the spoIIG promoter in vitro, as shown in Fig. 3, lane 1 (Grimsley et al., 1994). However, significant inhibition of spoIIG transcription occurred when Spo0JA was added before initiation and either before (Fig. 3, lane 2, 89% inhibition) or after (Fig. 3, lane 4, 80% inhibition) the formation of Spo0A-C–RNAP–DNA complexes. Interestingly, maximal inhibition of transcription (Fig. 3, lane 3, 95% inhibition) occurred when Spo0JA and Spo0A-C were preincubated together before adding RNAP, template DNA and initiating nucleotides, raising the possibility that Spo0JA interacts with the C-terminal domain of Spo0A. When Spo0JA was added after preincubation of RNAP–Spo0A-C–DNA with ATP and GTP (which permits the synthesis of an 11-mer RNA), a minimal effect on transcription was observed (Fig. 3, lane 5, 12% inhibition). These results show that Spo0JA effectively inhibits Spo0A-C-dependent transcription at the spoIIG promoter at any step before initiation. Furthermore, as Spo0JA inhibits Spo0A-C, it is unlikely that Spo0JA inhibition of Spo0A∼P is caused by a Spo0A∼P phosphatase activity or that Spo0JA interacts with the amino-terminus domain of Spo0A∼P to effect inhibition of transcription.

Figure 3.

. Spo0JA inhibition of transcription initiation complex formation. Single-round transcription assays were carried out essentially as described in Experimental procedures except that Spo0A-C (500 nM) was used instead of Spo0A∼P and components were added in successive stages. The components in the first stage were incubated at 37°C for 2 min before the addition of the second-stage components. After 2 additional min at 37°C, further initiation was blocked, and initiated complexes were allowed to elongate by the addition of heparin plus UTP and CTP. After 5 min, the products of the reaction were separated by electrophoresis through a polyacrylamide gel containing 7 M urea and the level of transcripts was assessed by exposing the gel to X-ray film overnight. Spo0JA was used at a concentration of 10 μM.

The target of Spo0JA repression

The mechanism of Spo0JA transcription repression was investigated by both DNase I protection and gel mobility shift assays. Using the spoIIG promoter, we did not observe any Spo0JA-dependent protection from, or hypersensitivity to, DNase I with either Spo0JA alone or in combination with Spo0A∼P and/or RNAP. These results suggest that, although Spo0JA repressed transcription, it did not form stable complexes with the spoIIG promoter, making it unlikely that the Spo0JA inhibition of transcription resulted from exclusion of the polymerase from the promoter. Therefore, it seemed likely that the target of Spo0JA inhibition was Spo0A∼P–RNAP complexes with the spoIIG promoter, and gel mobility shift assays were then used to test this possibility.

In the gel mobility shift assays (Fig. 4A and B), we used either a weak competitor, which does not dissociate protein–DNA complexes (calf thymus DNA), or a stringent competitor, which invades and dissociates all but very stable complexes (heparin). Neither Spo0A∼P, Spo0A-C, Spo0JA nor Spo0A∼P plus Spo0JA formed complexes with the spoIIG promoter that survived challenge with calf thymus DNA (Fig. 4A, lanes 1–4). RNAP alone bound to the spoIIG promoter, resulting in the formation of two complexes, I and II, and the binding was unaffected by the presence of Spo0A∼P (Fig. 4A, lanes 5 and 6; Bird et al., 1996). Other analyses have shown that, in the upper band (complex II), the DNA is wrapped around the RNAP, while in the lower band (complex I), the DNA is not wrapped around the RNAP (G. B. Spiegelman, unpublished observations). Spo0JA did not alter the complexes formed by RNAP (Fig. 4, lane 7), showing that it did not affect RNAP binding.

Figure 4.

. Spo0JA dissociates the Spo0A∼P–RNAP–spoIIG promoter complex. A. Reactions were carried out as described in Experimental procedures and stopped by the addition of either calf thymus DNA (CT) or heparin. Spo0JA was present at a concentration of 10 μM (lanes 3, 4, 11 and 13). ATP and GTP were at 0.4 mM. When present, Spo0A∼P and Spo0A-C were used at concentrations of 200 and 100 nM respectively. RNA polymerase was added at 100 nM final concentration. B. Gel shift assays were performed as described in (A), except that all reactions were stopped by the addition of heparin. Lanes 1 and 2 did not contain any Spo0JA. Lanes 3–8 contained 2.5, 3.3, 5.0, 6.7, 7.5 and 10 μM Spo0JA respectively. Lanes 9–11 contained 3.3, 6.7, and 10 μM Spo0JA respectively.

The addition of ATP and GTP to a reaction containing DNA, Spo0A∼P and RNAP yielded a third complex, which could be resolved by electrophoresis (complex III; Fig. 4A, lane 8). Complex III, unlike complexes I and II, was resistant to the addition of heparin, as would be expected if it contained an RNAP that had initiated RNA synthesis (4Fig. 4A; compare lane 6 with lane 9, and lane 8 with lane 10; Bird et al., 1996). Complex III was also formed by the addition of ATP plus GTP to reactions containing RNAP and Spo0A-C (Fig. 4A, lane 12). Spo0JA inhibited the formation of the initiated complex (complex III; Fig. 4A, lanes 11 and 13), even though it did not affect the binding of RNAP to the promoter region (complexes I and II; Fig. 4A, lane 7). Increasing concentrations of Spo0JA added before initiation prevented formation of the initiated complex over a narrow concentration range (Fig. 4B, lanes 1–8). In contrast, the addition of Spo0JA after initiation of RNA synthesis had little effect (Fig. 4B, lanes 9–11). A trivial explanation for these results that Spo0JA sequestered or destroyed the ATP was unlikely, as Spo0JA had no ATPase activity (K. L. Ohlsen, unpublished results), and ATP is present at a 40-fold molar excess over Spo0JA. Thus, Spo0JA appeared specifically to dissociate complexes of RNA polymerase, Spo0A∼P and the spoIIG promoter DNA.

Spo0JA binds single-stranded spoIIG promoter DNA

Numerous attempts to footprint Spo0JA on the spoIIG promoter were unsuccessful and, as shown in Fig. 4, Spo0JA did not form stable complexes with double-stranded DNA. Our recent finding that Spo0A∼P–RNAP–spoIIG complexes contain denatured DNA (Rowe-Magnus and Spiegelman, 1998) raised the possibility that Spo0JA might bind specifically to single-stranded spoIIG promoter DNA. Figure 5 shows the results of the mobility shift assay that also mapped the binding site for Spo0JA on the spoIIG promoter. The promoter DNA fragment was labelled with [γ32-P]-ATP and polynucleotide kinase at a BamHI site 135 bp downstream of the transcription start site, +1. This fragment was cleaved with restriction enzymes having sites at −100 (HindIII), −43 (AseI) or −22 (AluI) (Fig. 5A). The cleaved fragments were denatured, incubated with Spo0JA and subjected to gel mobility shift assay. Fragments generated with HindIII or AseI retained the ability to be bound by Spo0JA, but binding to the AluI fragment was reduced dramatically (Fig. 5B). These results indicate that Spo0JA does not bind ssDNA non-specifically and binds to the spoIIG promoter in the region between −43 and −22, suggesting that this binding ability is likely to contribute to its repressor-like activity on this promoter. Further experiments are required to define the minimum binding site and to determine whether Spo0JA also binds to the other DNA strand in this region.

Figure 5.

. Spo0JA binds to single-stranded DNA. A. Nucleotide sequence of the spoIIG promoter. The region between +1 and −100 is indicated. Solid bars define the region of binding of the Spo0A protein as determined by D. Rowe-Magnus and G. B. Spiegelman (manuscript submitted). The −10 and −35 regions are in bold characters, and the restriction enzymes used are shown. The abnormally long spacing between the −10 and −35 regions is characteristic of some spoII promoters (Satola et al., 1991). B. The gel mobility shift assay was carried out as described in Experimental procedures, except that the [γ-32P]-ATP-labelled PvuII fragment of pUCIIGtrpA was digested with either HindIII, AseI or AluI and then purified by electroelution. The templates were boiled for 3 min and fast-cooled on ice before use in the assay. The assay was carried out in a final volume of 20 μl, either with a Spo0JA input of 300 ng (+) or without Spo0JA (−).

Spo0JB regulation of Spo0JA activity

At this point, it seemed clear that Spo0JA prevented spoIIG gene transcription by dissociating the Spo0A∼P plus RNAP complexes. Results from genetic experiments (Ireton et al., 1994) indicated that the activity of Spo0JA was counteracted by Spo0JB. The question of the mechanism by which Spo0JB prevented Spo0JA repression remained unanswered. Because ParA and ParB of P1 prophage are thought to interact, the simplest mechanism would be physical interaction between Spo0JA and Spo0JB. However, purified Spo0JA and Spo0JB showed no tendency to form complexes in any of the in vitro tests that we tried. Therefore, we devised a test for complexes preformed in vivo.

A multicopy plasmid was prepared containing spo0JA and spo0JB with a four-histidine carboxyl-terminal extension on the gene product of spo0JB. Similarly, a multicopy plasmid with only the promoter region and the spo0JA gene with a carboxyl-terminal histidine extension was prepared. Extracts prepared from strains bearing these plasmids were subjected to metal chelate chromatography, and the bound proteins were eluted. Analysis of these eluted fractions on polyacrylamide gels is shown in Fig. 6. The eluate from the strain bearing a histidine-modified Spo0JA showed a predominant stained protein band at the Spo0JA position and several larger minor bands (Fig. 6, lane A-1). Analyses of this eluate by Western analysis with antibody to Spo0JA revealed that two of the minor bands with slightly higher molecular weight reacted with the antibody (Fig. 6, lane B-1). Protein sequence analyses showed that these higher molecular weight bands were authentic Spo0JA that migrates anomalously (B. Raether, unpublished data). Antibody to Spo0JB was found to react with a band in the eluate, but it migrated at the Spo0JA position, suggesting that the antibody cross-reacted with Spo0JA rather than indicating that the fraction contained Spo0JB (Fig. 6, lane C-1). Analyses of the eluate from the strain bearing both Spo0JA and Spo0JB in multicopy and a histidine extension on Spo0JB only gave a single stained band at the molecular weight position of Spo0JB (Fig. 6, lane A-2). No reactivity in this fraction was found using antibody to Spo0JA (Fig. 6, lane B-2), and antibody to Spo0JB reacted with a band at the position of Spo0JB (Fig. 6, lane C-2). Therefore, Spo0JA did not co-purify with Spo0JB or vice versa.

Figure 6.

. Spo0JA and Spo0JB purification from B. subtilis cultures. The Spo0JA protein (lane 1) was purified from a B. subtilis strain carrying plasmid pMB380.2 that expresses a Spo0JA protein with a 6x-histidine tag extension at the carboxy-terminal end. The Spo0JB protein (lane 2), modified to contain four histidine residues at the carboxy-terminal end, was purified from a strain carrying plasmid pMB381, which also expresses an unmodified Spo0JA protein. A. Coomassie blue-stained SDS–polyacrylamide gel. B. Western blot probed with an antibody raised against Spo0JA. C. Western blot probed with an antibody raised against Spo0JB.

Sporulation studies of the strains used in the antibody experiments showed that the histidine extensions on Spo0JA and Spo0JB did not affect their activities in vivo. The control strain bearing only the vector sporulated at a frequency of 37%, whereas the presence of a multicopy spo0JA reduced the frequency to 9%. This is a lesser reduction than has been seen in a spo0JB mutant, but the strain used here still contained an intact chromosomal copy of spo0JB. Adding the spo0JB gene to this plasmid, albeit with a histidine extension, raised the sporulation frequency to 23%, which is close to the 27% observed for a multicopy plasmid with the modified spo0JB gene alone. Furthermore, the spo0JA and spo0JB genes carrying the His-tag coding extensions were introduced in single copy in the B. subtilis chromosome. The resulting strains had phenotypes consistent with the Spo0JA–His-tag and Spo0JB–His-tag products acting as wild-type proteins. The data are consistent with the histidine extensions on Spo0JA and Spo0JB having no effect on their functions.

Discussion

The results presented here establish that elevated levels of phosphorylated Spo0A transcription factor are necessary but not sufficient for the initiation of stage II gene transcription in sporulation. The Spo0JA repression of spoII gene expression prevents transcription in what appear to be normal cellular levels of Spo0A∼P. There may well be other control circuits that regulate at this level and that are independent of the Spo0A∼P concentration. We have found that the Spo0JA inhibition of spoIIG transcription in vitro may be competed by additional Spo0A∼P, which may explain why some sof mutants of Spo0A are able to restore sporulation in a spo0JB mutant (M. A. Cervin and G. B. Spiegelman, unpublished data). Such mutations alter Spo0A, making it subject to phosphorylation by other kinases and thereby allowing bypass of the phosphorelay and its control circuits.

To our knowledge, Spo0JA is the first transcription repressor described that specifically dissociates an activator–RNAP promoter–complex. As Spo0A∼P is required for transcription from spoIIA and spoIIE promoters as well as from the spoIIG promoter, Spo0JA probably inhibits expression from all of these promoters by a common mechanism. It seems likely that Spo0JA binding to the −43 to −22 region of the spoIIG promoter is involved in the mechanism of dissociation of Spo0A∼P–RNAP–promoter complexes. As Spo0JA had no effect on the electrophoretic mobility of the native DNA fragment containing the spoIIG promoter, it must not induce denaturation on its own. However, its binding may shift the equilibrium between the native and denatured state in a region that could be destabilized by the binding of Spo0A∼P plus RNAP. The data in Fig. 4 indicate that both Spo0A∼P and RNAP are necessary for that binding, as Spo0JA also had no effect on the binding of RNAP alone. A simple model to explain the effect of Spo0JA on the complexes detected by mobility shift assays would be that some sort of destabilization of the DNA duplex, induced by the binding of Spo0A∼P, serves as a nucleation site for Spo0JA binding and subsequent denaturation of the DNA strands, and that the strand denaturation is incompatible with RNAP binding. As RNAP binding is rapid and reversible, the promoter complexes would break up unless RNA synthesis has been initiated. Additional experiments to trap and characterize intermediates in the dissociation are in progress.

The roles of the ParA proteins and their regulatory interactions with ParB proteins are not well understood. The ParA of P1 prophage plasmid is essential for partitioning and is also known to be involved in the feedback regulation of the par operon expression (Davis et al., 1992). Experiments with P1 have shown that Par protein levels must be kept low and in the correct ratio to obtain efficient partitioning (Davis et al., 1996). Overexpression of ParA or ParB in C. crescentus causes cell division defects and filamentation (Mohl and Gober, 1997), and the ParA protein is essential for viability. Spo0JA overexpression in a Spo0JB + background causes a sporulation deficiency in B. subtilis similar to that seen in a spo0JB mutant. Thus, the evidence is consistent with the idea that the ratio of Spo0JA to Spo0JB proteins is an essential factor for correct functioning. This ratio could be crucial if Spo0JA and Spo0JB form a complex, and the non-complexed proteins each have distinct regulatory roles. Evidence for a complex in this class of proteins was best demonstrated with ParA and ParB of P1, as ParB stimulates the ATPase activity of ParA in vitro and the ParA-mediated autoregulation of the par operon in vivo (Friedman and Austin, 1988; Davis et al., 1992). Identical results have been obtained with the SopA–SopB homologues of ParA–ParB from the F plasmid (Mori et al., 1989; Watanabe et al., 1992). We found no evidence that Spo0JA and Spo0JB interacted by co-purification studies from gently prepared extracts using His-tagged proteins and metal chelate chromatography. It remains possible that the two proteins interact in vivo, but the interaction is too weak to detect. Purified Spo0JA had no detectable ATPase activity with or without Spo0JB, precluding experiments of the type used to detect interaction of the P1 or F Par proteins. Furthermore, neither Spo0JA nor Spo0JB appear to regulate transcription of the spo0J locus (K. Ohlsen & J. A. Hoch, unpublished), which points out another difference between these proteins and the ParA–ParB proteins of P1 prophage or the equivalent proteins of F plasmid.

Despite the lack of evidence for physical interaction, there seems little doubt that Spo0JB regulates the activity of Spo0JA in some manner. Loss of Spo0JB results in blocked stage II gene transcription and, as we show here, this could be attributed to the negative effect of Spo0JA on transcription. As Spo0JB is associated with the mitotic apparatus, it is not unreasonable to imagine that Spo0JB regulates Spo0JA to couple stage II gene transcription to chromosome replication and/or segregation. For example, in growing cells, Spo0JB may be sequestered by the mitotic apparatus, leaving Spo0JA free to inhibit stage II gene expression. As growth slows and cells prepare for sporulation, there may be a release or modification in Spo0JB that activates it to inhibit the activity of Spo0JA, thereby releasing stage II genes from repression. Alternatively, Spo0JB might be indirectly responsible for Spo0JA regulation by controlling the expression or activity of another protein with inhibitory properties. This model would explain how the cell prevents spurious initiation of development and asymmetric septum formation in cells with a partially replicated chromosome even if the Spo0A transcription factor is fully phosphorylated and active (Mandelstam et al., 1971). It also explains why DNA synthesis inhibitors block sporulation at stage 0 (Shibano et al., 1978; Ireton and Grossman, 1992; 1994).

The regulation mechanism suggested for Spo0JA–Spo0JB could have global significance, as ParA–ParB homologues are present in a wide range of bacteria (Fraser et al., 1995; Bult et al., 1996; Hilbert et al., 1996). Thus, it is possible that the Spo0JA homologues in various organisms play a similar role as negative regulators of transcription, possibly timing stationary phase events to cell division or chromosome partitioning.

Experimental procedures

Genetic nomenclature

The partition genes in this manuscript have been named spo0JA and spo0JB to reflect their relationship to parA and parB, respectively, of the plasmid partition systems. Initially, these genes were non-descriptively designated orf253 and orf282, and one or more were known to be part of the spo0J locus. Subsequently, they were designated soj (suppressor of OJ) and spo0J. This nomenclature, however, is confusing with regard to the homologous ParA and ParB family of proteins and genetically misleading. spo0JA is, in fact, a sporulation gene because, as a negative regulator, it has a sporulation phenotype when overexpressed, its gene product regulates transcription of sporulation genes and, furthermore, it is not a true suppressor.

Bacterial strains and growth conditions

The strains used in this study are derivatives of JH642 (trpC 2, phe-1). A spo0JB deletion strain (JH15016) was obtained by replacing a NdeI–Pml I fragment with the Kmr cassette gene from pJM114 (Perego, 1993). The spo0A–lacZ construct (pJB0Alac) was obtained by cloning a blunted ClaI–HpaI fragment carrying the spo0A promoter in the blunted BamHI site of pDH32 (Perego, 1993). Transformation of pJB0Alac into JH642 and JH15016 gave strains JH15012 and JH15022 respectively. The abrB–lacZ fusion construct pJM5139 has been described by Perego et al. (1988). JH642 and JH15016 carrying pJM5139 are named JH12601 and JH11451. Cultures for β-galactosidase assays were grown in Schaeffer's sporulation medium (Schaeffer et al., 1965) and assayed as described previously (Ferrari et al., 1988). The units of activity were calculated according to Miller (1972).

Transcription assays

The spoIIG promoter used in the transcription assays was a 600 bp fragment liberated by PvuII digestion of plasmid pUCIIGtrpA (Bird et al., 1996). This fragment carries the spoIIG promoter from −110 to +135, cloned between HindIII and BamHI sites upstream of the trpA transcription terminator. The PvuII fragment, which has vector sequences on either side of the cloned fragment, was isolated as described previously (Bird et al., 1996). The A2 promoter was contained on a PvuII restriction endonuclease fragment from plasmid pUCA2trpA. This plasmid is identical to pUCIIGtrpA, except that the HindIII to BamHI portion containing the spoIIG promoter has been replaced by a 200 bp fragment containing a TaqI fragment from plasmid p238-5 (Dobinson and Spiegelman, 1987), which contains the A2 promoter from phage φ29. The abrB promoter was isolated as an EcoRI fragment from plasmid pJM5134 as described previously (Greene and Spiegelman, 1996). The spoIIE promoter was amplified from strain JH642 and cloned into the vector component of pUCIIGtrpA, replacing the spoIIG sequences. The PvuII fragment from this vector was used in transcription. Assays were carried out in a 10 μl final volume. For the spoIIG promoter, the template DNA (4 nM) was incubated with Spo0A∼P (200 nM), RNA polymerase (25 nM), ATP (0.4 mM), [α-32P]-GTP (5 μM, 20 Ci mmol−1) and the indicated concentrations of Spo0JA for 2 min at 37°C. Complexes were then challenged with heparin plus nucleotides and allowed to elongate for 5 min at 37°C. Final concentrations were: heparin, 10 μg ml−1; UTP, 0.4 mM; CTP, 0.4 mM. In the case of the A2 promoter, the initial incubation contained template (4 nM) and RNAP (25 nM). After 2 min, Spo0JA and ATP plus GTP were added. After a further 2 min, complexes were challenged with the heparin, CTP and UTP mixture as above. Elongation reactions were stopped by the addition of 10 μl of 7 M urea, 0.1% xylene cyanol, 0.1% bromophenol blue in 0.5 × TBE (Sambrook et al., 1982). The transcripts were separated on an 8% polyacrylamide gel containing 7 M urea in 0.5 × TBE (Sambrook et al., 1982) and exposed to X-ray film. Bacillus subtilis RNAP was purified as described previously (Dobinson and Spiegelman, 1987) and was generously donated by D. Rowe-Magnus, University of British Columbia.

Purification of Spo0JA and Spo0JB

A Spo0JA expression plasmid was constructed in the pET20b vector (Novagen) that promotes the expression of proteins fused to six histidine residues at the carboxyl-terminal end. The Spo0JA coding sequence was amplified by polymerase chain reaction (PCR) using genomic DNA from strain JH642 as template and cloned into the NdeI–XhoI sites of pET20b, giving plasmid pET20.0JA. The fusion protein was produced in E. coli strain BL21 (DE3) (Novagen). Cells were grown in 5 l of LB with 100 μg ml−1 ampicillin at 37°C. When the OD600 reached 0.7, IPTG was added to a final concentration of 1 mM, and the culture was grown for an additional 2.5 h at 30°C. Cells were harvested, resuspended in buffer A [50 mM potassium phosphate, pH 8.0, 300 mM NaCl, 1 mM β-mercaptoethanol, 0.5 mM phenylmethylsulphonyl fluoride (PMSF)] containing 20 mM imidazole, sonicated and centrifuged for 30 min at 26 000 × g. The supernatant was mixed with 10 ml of a 50% slurry of Ni-NTA agarose (Qiagen) for 1 h at 4°C and then loaded into a 8 × 3 cm column. The column was washed with buffer A containing first 20 mM and then 50 mM imidazole. The Spo0JA protein was eluted with 50 ml of buffer A containing 100 mM imidazole. The protein was dialysed in buffer A, and the concentration was determined using the Bradford Assay (Bradford, 1976). Purified protein was more than 90% pure as judged by SDS–PAGE.

The Spo0JA and Spo0JB proteins were purified from B. subtilis JH642 carrying multicopy plasmids derived from pBS19 (Perego and Hoch, 1987). Plasmid pMB380.2 contains the entire promoter region and the Spo0JA coding sequence modified to contain six codons for histidine at the 3′ end of the gene. Plasmid pMB381 contains the entire spo0J locus, and it promotes the expression of an intact Spo0JA protein and a modified Spo0JB protein carrying four histidine residues at the carboxy-terminal end. Cells were grown in Schaeffer sporulation medium containing Cm at 5 μg ml−1. Cell growth was monitored at OD525, and cells were harvested 2 h after the transition state (T2). Purification of the proteins was carried out essentially as described previously for the purification of Spo0JA from E. coli with the exception that Spo0JB eluted with 50 mM imidazole.

Spo0A-C was purified to homogeneity and kindly provided by J. Nerem and T. Msadek from the Hoch Laboratory at The Scripps Research Institute.

Gel mobility shift assays

The HindIII/BamHI fragment of pUCIIGtrpA was labelled at the BamHI end with [γ-32P]-ATP (Bird et al., 1996) and approximately 1 × 104 Cerenkov counts were used per reaction in a 10 μl volume. The components indicated were mixed on ice in a reaction buffer (10 mM HEPES, pH 8.0, 10 mM MgAc, 0.1 mM DTT, 80 mM KAc, 0.1 mg ml−1 BSA) and then incubated at 37°C for 3 min. Reactions were stopped by the addition of 3 μl of a stop buffer (20% glycerol in the reaction buffer) containing either 300 μg ml−1 sonicated calf thymus DNA or 100 μg ml−1 heparin. Samples were loaded onto an 8% polyacrylamide gel (40% acrylamide:1.38% bisacrylamide) in 0.8 × TAE (Sambrook et al., 1982) containing 2% glycerol. Samples were electrophoresed at 12 V cm−1 for 1.5 h, and then the gel was dried and exposed to Kodak XAR X-ray film overnight at 80°C.

Antibodies and Western blotting

Polyclonal antibodies were raised against the Spo0JA protein purified from E. coli as described in this paper and against the Spo0JB protein purified from E. coli as described by Glaser et al. (1997). Western blotting was carried out as described previously (Sambrook et al., 1982) using the ECL system (Amersham Life Science) for detection.

Acknowledgements

G.B.S. and M.A.C. were supported by grants from the Natural Science and Engineering Research Council of Canada (5-86783 and 5-81866). Research at The Scripps Research Institute was supported by grant GM19416 from the National Institute of General Medical Sciences, National Institutes of Health, United States Public Health Service.

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