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Journal of Bacteriology, August 1999, p. 4969-4977, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Sigma Factor Displacement from RNA Polymerase during Bacillus subtilis Sporulation

Jingliang Ju, Theresa Mitchell, Howard Peters III,dagger and W. G. Haldenwang*

Department of Microbiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7758

Received 14 October 1998/Accepted 2 June 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

As Bacillus subtilis proceeds through sporulation, the principal vegetative cell sigma  subunit (sigma A) persists in the cell but is replaced in the extractable RNA polymerase (RNAP) by sporulation-specific sigma  factors. To explore how this holoenzyme changeover might occur, velocity centrifugation techniques were used in conjunction with Western blot analyses to monitor the associations of RNAP with sigma A and two mother cell sigma  factors, sigma E and sigma K, which successively replace sigma A on RNAP. Although the relative abundance of sigma A with respect to RNAP remained virtually unchanged during sporulation, the percentage of the detectable sigma A which cosedimented with RNAP fell from approximately 50% at the onset of sporulation (T0) to 2 to 8% by 3 h into the process (T3). In a strain that failed to synthesize sigma E, the first of the mother cell-specific sigma  factors, approximately 40% of the sigma A remained associated with RNAP at T3. The level of sigma A-RNAP cosedimentation dropped to less than 10% in a strain which synthesized a sigma E variant (sigma ECR119) that could bind to RNAP but was unable to direct sigma E-dependent transcription. The E-sigma E-to-E-sigma K changeover was characterized by both the displacement of sigma E from RNAP and the disappearance of sigma E from the cell. Analyses of extracts from wild-type and mutant B. subtilis showed that the sigma K protein is required for the displacement of sigma E from RNAP and also confirmed that sigma K is needed for the loss of the sigma E protein. The results indicate that the successive appearance of mother cell sigma  factors, but not necessarily their activities, is an important element in the displacement of preexisting sigma  factors from RNAP. It suggests that competition for RNAP by consecutive sporulation sigma  factors may be an important feature of the holoenzyme changeovers that occur during sporulation.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The pivotal event establishing sporulation-specific gene expression in Bacillus subtilis is the reprogramming of the bacterium's RNA polymerase (RNAP) (52). This occurs when the principal promoter recognition subunit (sigma A) of the vegetative cell RNAP is replaced by analogous sporulation-specific subunits (sigma E, sigma F, sigma G, and sigma K) (18, 52). These alternative sigma  factors control both the timing and localization of spore gene expression.

Early in sporulation, B. subtilis partitions itself into two unequal compartments with unique developmental fates. The smaller, forespore compartment is eventually engulfed by the larger, mother cell compartment, which nurtures the forespore as it develops into a mature endospore. Each of the sporulation-specific sigma  factors is active in only one of these two compartments. Mother cell gene expression is controlled by the sequential appearance of sigma E followed by sigma K, while forespore-specific genes are activated first by sigma F and then by sigma G (7, 8, 10, 18, 20, 30, 37, 41, 42, 44, 52, 60). The genes encoding each of the early sporulation sigma  factors (sigma E and sigma F) are expressed at the onset of sporulation (31, 36, 56); however, neither of these factors is active until later in development, when the two separate compartments are formed. sigma E and sigma F are each kept silent by a unique means. sigma F is bound to an inactivating anti-sigma F protein (SpoIIAB), while sigma E is formed as an inactive proprotein, pro-sigma E (9, 13, 36, 43, 51). Pro-sigma E becomes active only after 27 amino acids are cleaved from its amino terminus (36). The septation event initiates a process that leads to the release of sigma F from its antagonist in the forespore (1, 3, 12). sigma F then directs the transcription of a gene, spoIIR, whose product in turn triggers pro-sigma E processing in the mother cell (23, 29, 39). Once active in their particular compartments, sigma E and sigma F induce expression of the genes for the sigma factors which will ultimately replace them (sigma K and sigma G, respectively) (14, 34, 35, 44, 53). sigma K and sigma G, like their predecessors, are initially inactive. sigma K is formed as a proprotein which is processed and activated in response to a signal from the developing forespore (7, 8, 41). sigma G's activity is restricted by SpoIIAB, the same anti-sigma protein that inhibited sigma F (16, 30). Turnover of SpoIIAB in the forespore is thought to be an important factor in activating sigma G (30, 33).

Although much is known about the mechanisms by which the sporulation-specific sigma  factors are expressed and activated, the process by which they replace sigma A on RNAP is still unclear. sigma A persists in sporulating B. subtilis (54), and yet by 2 to 3 h after the onset of sporulation, the RNAP extracted from these bacteria is virtually devoid of sigma A (19, 38). Early studies of sigma A's displacement from RNAP revealed that treatment of a sporulating B. subtilis culture with the protein synthesis inhibitor chloramphenicol allowed sigma A to again become extractable as an RNAP subunit (47). This result was interpreted as evidence for the existence of a short-lived protein inhibitor of sigma A that appears in sporulating B. subtilis to block sigma A's binding to RNAP.

Anti-sigma factor proteins are now known to restrict the ability of several B. subtilis factors to form RNAP holoenzymes. In addition to sigma F and sigma G (9, 13, 16, 30, 33), the general stress response (sigma B) and motility (sigma D) sigma  factors of B. subtilis are controlled by inhibitory binding proteins (4, 6, 11). It is plausible that a similar protein could be involved in restricting sigma A's access to RNAP in sporulating B. subtilis. Alternatively, the presence of the sporulation-specific sigma  factors themselves could be the basis for sigma A's exclusion from RNAP. If the sporulation sigma  factors are effective competitors for a limited pool of core RNAP, their mere presence might be sufficient to deny sigma A access.

Using velocity sedimentation techniques to separate RNAP from unassociated sigma  factors and Western blot analyses to locate RNAP, sigma A, sigma E, and sigma K in these gradients, we revisited the problem of sigma A release during sporulation. The pattern of sigma  factor association with RNAP in wild-type and mutant B. subtilis was found to be consistent with a model in which competition for RNAP by sporulation sigma  factors is necessary for E-sigma A decrease and the changeover from E-sigma E to E-sigma K that occurs later in development. If unknown sporulation factors facilitate this process, they do not appear to be adequate for separating the preexisting sigma  factors from RNAP in the absence of the sigma  proteins that will replace them.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Plasmids and bacterial strain constructions. The B. subtilis strains and plasmids used in this study are listed in Table 1. pGEM3C500 is the Escherichia coli vector pGEM-3Zf(+)cat (58) containing a 500-bp PstI DNA fragment from the interior of the spoIIGA coding sequence (25). Transformation of this plasmid into B. subtilis, followed by selection for chloramphenicol acetyltransferase, generates transformants (SE500) in which the plasmid has inserted into spoIIGA and separated sigE (spoIIGB) from its promoter element. pBZ1 was obtained from Lee Kroos (Michigan State University). It carries the sigKCR109 allele cloned downstream of Pspac. An EcoRI/HindIII fragment from pBZ1, carrying Pspac::sigKCR109, was cloned into EcoRI- and HindIII-cut pUS19 (5) to create pUS109CR. B. subtilis BK410 (35) has a deletion of the 3' end of sigK (spoIIIC94). Transformation of pUS109CR into BK410 and selection for the vector-encoded Spcr results in single-site recombinants in which the vector has integrated within the 5' end of sigK. This creates a single intact sigK gene, controlled by its normal promoter, and an inactive, truncated sigK gene downstream of the vector sequence. Pspac. Spo- clones would have the sigK109CR mutation included in the intact gene. BK410-1 was one such clone. S410 is SMY transformed with chromosomal DNA from BK410-1 and with selection for the sigK109CR-linked Spcr. pUS117CR is a 1.1-kbp DNA fragment carrying the sigE117CR allele cut from pJ89CR117 (27) with PstI and cloned into PstI-cut pUS19. SE117CR is SMY transformed with pUS117CR, with selection for the vector-encoded Spcr, followed by screening for the Spo- phenotype of the sigE117CR allele. SF9030 is SMY transformed with chromosomal DNA from EUR9030 (30) and selection for the spoIIAC-disrupting erm cassette. The SigE- SigF- strain (SEF500) was generated by transforming SE500 (SigE-) with this same EUR9030 DNA.

                              
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  		TABLE 1.   B. subtilis strains and plasmids used in this study

Induction of sporulation. B. subtilis, grown overnight in Luria broth, was diluted 1/20 into Difco Sporulation medium (DSM) and incubated at 37°C. The onset of sporulation (T0) was taken as the time that the culture stopped exponential growth.

Velocity gradient analysis. Cells were harvested into equal volumes of crushed ice at various times after the onset of sporulation, washed in 1 M NaCl, concentrated 20-fold, and disrupted in a resuspension buffer (10 mM Tris [pH 8.0], 1 mM EDTA, 50 mM NaCl, 10 mM MgCl2, 0.3 g of phenylmethylsulfonyl fluoride per liter, 3 mM dithiothreitol) by passage (three times) through a French press at 12,000 lb/in2. Cell debris was removed by a low-speed centrifugation (10,000 rpm, 45 min, SS-34 rotor [Sorvall]). Supernatant samples (0.5 ml) were applied onto 11-ml linear glycerol gradients (15 to 30% glycerol in resuspension buffer) and centrifuged for 22 to 24 h at 37,000 rpm and 4°C in a Sorvall TH641 rotor. After centrifugation, 0.5-ml samples were collected from the bottom of the tube. Two volumes of ethanol were added to this sample. This precipitated 80 to 90% of the protein, without apparent preference for any of the proteins under investigation. The precipitated material was resuspended in sample buffer and analyzed by Western blotting. When Triton was used in the analysis, it was added to both the sample buffer (1.0%) and the glycerol gradient (0.1%).

Western blot analysis. Fractions from the velocity gradient were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on gels containing 10% acrylamide. Subsequent steps were as done previously (11). The anti-beta ' and anti-sigma A mouse monoclonal antibodies were obtained as previously described, using B. subtilis core RNAP and sigma A-His6 as the inoculated antigens (11). The anti-sigma E monoclonal antibody has been described previously (55). A sample of rabbit polyclonal antibody against sigma K (60) was provided by L. Kroos. Bound antibody was detected with alkaline phosphatase-conjugated goat anti-mouse or goat anti-rabbit antibodies, as appropriate. Western blot data were quantitated with an AlphaImager 2000 (Alpha Innoteck Corp., San Leandro, Calif.) and its associated software.

General methods. DNA manipulations were performed by standard protocols. Transformation of naturally competent B. subtilis cells was carried out as described by Yasbin et al. (57).


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Patterns of association of sigma A with RNAP. RNAP purified from sporulating B. subtilis contains little sigma A; nevertheless, sigma A can be immunoprecipitated from crude extracts of such cells (54). In an attempt to better define the status of sigma A in sporulating B. subtilis, we prepared an anti-sigma A monoclonal antibody and used it as a probe to monitor sigma A's abundance and RNAP association in B. subtilis extracts.

In an initial experiment, we examined the sigma A levels in sporulating B. subtilis and compared this value to the sigma A level which existed previously. Extracts were prepared from B. subtilis that had been harvested at the onset of sporulation and at 1.5-h intervals thereafter and analyzed by Western blotting as described in Materials and Methods. As noted by others (40, 54), the ratio of sigma A to the core RNAP beta ' subunit was found to be essentially unchanged as the cells proceeded through 4.5 h of sporulation (T4.5) (Table 2). Thus, a significant drop in the sigma A/core RNAP ratio is not responsible for sigma A's disappearance from the sporulating cell's extractable RNAP.

                              
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  		TABLE 2.   Relative sigma A abundance

Velocity centrifugation techniques can readily separate RNAP holoenzymes (approximately 5 × 105 Da) from free sigma  subunits (typically 2.5 × 104 to 5 × 104 Da) and had been used by others to monitor the association of mutant sigma  factors with RNAP (49). We applied this technique, in conjunction with Western blot analyses, to address the question of sigma  factor-RNAP associations during sporulation. We first asked whether the RNAP-sigma factor fractionation profile seen following velocity centrifugation would resemble the sporulation-specific changes in RNAP holoenzyme composition that had been documented previously.

The most obvious change in the RNAP profile of sporulating B. subtilis occurs at approximately T2 to T3. At this time, E-sigma A becomes rare and E-sigma E, a holoenzyme carrying the first of the mother cell-specific sigma  factors, becomes evident. The loss of sigma A from RNAP and the synthesis of sigma E do not occur if sporulation is blocked by mutation in the spo0A gene (19). In such cells, E-sigma A remains extractable under culture conditions that induce sigma A displacement in wild-type B. subtilis. Crude extracts were prepared from wild-type and spo0A mutant cells and subjected to centrifugation through 15 to 30% glycerol gradients. The gradients were then fractionated and analyzed by Western blotting with monoclonal antibodies against sigma A, sigma E, and the core RNAP beta ' subunit as probes. At the onset of sporulation, approximately half of the sigma A present in the wild-type B. subtilis extract cosedimented with the RNAP marker (beta ') (Fig. 1A, T0). By 3 h into the process (Fig. 1A, T3), most of the sigma A no longer moved with the RNAP marker and instead was found higher in the gradient. A similar analysis of the spo0A extract (Fig. 1B) revealed a persistent association of sigma A with RNAP throughout this same period.


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  		FIG. 1.   Fractionation of extracts from sporulating and Spo0A- B. subtilis. Wild-type B. subtilis SMY (A) and its congenic Spo0A- variant S0A514 (B) were grown in DSM. Samples were harvested at the end of exponential growth (T0) or at 1.5-h intervals thereafter (T1.5, T3, and T4.5), the cells were disrupted, and the resulting crude extracts were fractionated by centrifugation through a linear gradient of 15 to 30% glycerol, as described in Materials and Methods. Fractions were collected and analyzed by Western blotting with anti-beta ', anti-sigma A, and anti-sigma E antibodies as probes. Fraction 1 represents the bottom of the centrifuge tube. The two bands detected by the anti-sigma E antibody in panel A at T1.5 represent sigma E and its slightly larger precursor (pro-sigma E).

The Western blot data for the T3 extracts were quantitated by densitometry to estimate the relative abundance of each protein in the separate fractions (Table 3). In both the wild-type and Spo0A mutant extract gradients, fractions 4 to 10 contained approximately 85% of the RNAP beta ' marker. These same fractions contained approximately 50% of the Spo0A extract's sigma A but only 2% of the sigma A that was present in the wild-type extract. In order to verify that the estimates of sigma A in the various fractions were based on measurements that were in a linear response range, fractions that corresponded to the RNAP-containing fractions and those that did not contain RNAP were pooled, serially diluted, and analyzed by Western blotting for RNAP and sigma  factor contents. The amount of the sigma A component cosedimenting with the RNAP marker was consistently less than 10% of the sigma A that was detectable in cells that had progressed beyond the initial stages of sporulation, while it remained at 50% or greater in Spo0A- extracts (data not shown). The experiment also revealed that the amount of extract that we analyzed, although optimal for the sigma  factor signal, had begun to saturate the beta ' signal in the peak beta ' fractions. Thus, the fractions indicated as the principal RNAP-containing fractions contain even more of the RNAP than depicted in the figures or tables.

                              
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  		TABLE 3.   Partitioning of sigma A and sigma E at T3

The Western blot analyses of the wild-type B. subtilis strain also revealed some interesting aspects of the distributions of pro-sigma E and sigma E in these extracts (Fig. 1A). Pro-sigma E and sigma E were detectable in the T1.5 extract. Virtually all of the sigma E, and a portion of the pro-sigma E, cosedimented with the RNAP marker, while most of the pro-sigma E moved to a position below RNAP in the gradient. The "pro" sequence of sigma E is believed to tether pro-sigma E to the B. subtilis cytoplasmic membrane (22, 23, 28). It is likely that the fast-sedimenting forms of pro-sigma E represent molecules of pro-sigma E that are associated with membrane components. In Triton-treated extracts, pro-sigma E no longer sediments to the bottom of the gradient but is found either in the RNAP fractions or higher in the gradient, while the sedimentation pattern of sigma E is unaltered (data not shown). At T3, only mature sigma E was evident in the gradient fractions. In this and the earlier (T1.5) sample, almost all of the detectable sigma E was restricted to the RNAP-containing fractions, while by T4.5 most of the sigma E was no longer associated with RNAP but sedimented as if it was free within the extract (Fig. 1A). Apparently, sigma E readily associates with RNAP early in sporulation, but between T3 and T4.5, its ability to remain bound to RNAP declines.

We conclude from these experiments that the velocity gradient technique allows us to monitor sigma -RNAP associations during sporulation, that sigma A displacement from RNAP is largely complete by T3, and that sigma E, like sigma A, becomes less likely to be RNAP bound as sporulation progresses.

Effect of sigma factor mutations on E-sigma A persistence. sigma A dissociates from RNAP in sporulating B. subtilis but continues to form an RNAP holoenzyme in Spo0A-deficient cells. Among the genes whose expression depends on Spo0A are those which encode the first of the mother cell- and forespore-specific sigma factors, sigma E and sigma F, respectively (2, 45, 46, 56). To determine whether either of these early-sporulation sigma factors is required, directly or indirectly, for the displacement of sigma A from RNAP, we repeated the fractionation analysis with extracts that had been prepared from B. subtilis strains lacking one or both of these transcription factors. Representative Western blot analyses of extracts fractionated by velocity gradient centrifugation are illustrated in Fig. 2. The extracts were prepared from sporulating cultures of the mutant strains that had been harvested at T3. The distribution of the sigma  factors in these gradients is quantitated in Table 3. The loss of sigma E, sigma F, or both proteins allowed a substantial retention of the sigma A in the RNAP fractions (Fig. 2). In each of the three mutant strains, approximately 30% of the extract's sigma A sedimented in the peak RNAP fractions (Table 3).


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  		FIG. 2.   Fractionation of SigE- and SigF- B. subtilis. Wild-type B. subtilis (SMY) (A) and its congenic SigF- (SF9030) (B), and SigE- (SEF500) (C), and SigE- SigF- (SEF500) (D) variants were grown to T3 in DSM and analyzed as described for Fig. 1. The protein band reacting with the anti-sigma E antibody is sigma E in wild-type B. subtilis (A) and pro-sigma E in the SigF- strain (B).

sigma A is believed to be dispersed at equivalent concentrations in the mother cell and forespore (40). It is therefore reasonable to assume that any factor that causes a significant displacement of sigma A from RNAP would have to be active in the large mother cell compartment, where the bulk of the sigma A should lie. The loss of either the forespore-specific sigma F or the mother cell-specific sigma E caused a similar retention of sigma A in the RNAP fraction. This suggests that the mother cell factor responsible for displacing sigma A requires sigma F for its appearance. sigma F-dependent transcription in the forespore is needed for pro-sigma E processing (29, 39). Thus, mature sigma E could be the missing factor necessary for the bulk of the sigma A displacement. A need for mature sigma E would indicate either that processed sigma E, but not its proprotein, can compete with sigma A for RNAP or that a sigma E-dependent gene product is the actual effector of sigma A displacement.

To distinguish between these possibilities, we next analyzed the sigma A-RNAP association profile in a B. subtilis strain whose only source of sigma E is an allele (sigECR117) which encodes a product that can bind to RNAP but fails to recognize sigma E-dependent promoters (27). If competition between sigma E and sigma A for RNAP is needed for sigma A's dissociation from RNAP, then sigma A should still be displaced in the sigECR117 mutant strain. Alternatively, if a sigma E-dependent gene product is responsible, the sigECR117 allele should be equivalent to a sigE null mutation and allow E-sigma A to persist. When the sigma A pattern in cells harvested at T3 of sporulation was analyzed, only 7.5% of the sigma A remained associated with the RNAP during the centrifugation analysis (Table 3). It therefore appears that the presence of the mature sigma E protein itself is more important than sigma E's transcriptional activity for sigma A displacement from RNAP.

Effect of chloramphenicol on sigma E persistence and sigma A RNAP displacement. We next revisited the observation that E-sigma A could be more readily isolated from sporulating B. subtilis if the culture was pretreated with chloramphenicol (47). In a previous study of the effects of antibiotics on sigma E processing and turnover, we found that sigma E, once formed, persists in chloramphenicol-treated cells (26). We therefore looked at the effect of chloramphenicol treatment on the sigma A association with RNAP at different times in sporulation. Chloramphenicol treatment could influence the cosedimentation of sigma A with RNAP, but the time at which treatment was applied had a significant effect on the outcome. When chloramphenicol was added to the culture before significant sigma E accumulation and sigma A displacement, at T1.5, sigma E synthesis was compromised, and cells harvested 0.5 h later, at T2, did not show as great a degree of sigma A displacement as an untreated culture at T2 (Fig. 3). However, when a culture was treated with chloramphenicol after sigma E had accumulated and sigma A was largely displaced, at T3, sigma E persisted, and there was no obvious difference at T3.5 in the amount of sigma A that cosedimented with the RNAP in either the treated or untreated cultures (Fig. 3). Thus, in our hands, chloramphenicol treatment did not return displaced sigma A to RNAP but could block its initial displacement. The data sustain a correlation between the presence of sigma E and the decrease in E-sigma A.


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  		FIG. 3.   Effect of chloramphenicol treatment on E-sigma A persistence. Wild-type B. subtilis (SMY) was grown in DSM. At 1.5 and 3 h after the end of exponential growth, chloramphenicol was added to portions of the culture, which were harvested 0.5 h later. T2.0 + CM and T2.0 represent portions of the culture harvested at 2 h with and without chloramphenicol treatment, respectively. T3.5 + CM and T3.5 are similar cultures harvested at T3.5. The culture samples were analyzed as described for Fig. 1. The anti-sigma E antibody detected pro-sigma E and sigma E at T2 and only sigma E at T3.5.

Replacement of E-sigma E by E-sigma K. Sigma factor substitution is an ongoing process during sporulation (52). In the mother cell compartment, sigma E, which becomes the principal sigma  factor on RNAP by T3, is itself supplanted on RNAP after T3 (Fig. 1A). There is an aspect to the E-sigma E loss that is distinct from E-sigma A replacement. sigma A persists in the cell even though E-sigma A declines. In contrast, both E-sigma E and sigma E protein levels fall after sigma E's period of activity (55, 60, 61). The previously documented decline of sigma E, as well as its dissociation from RNAP, can be noted in Fig. 1A (T4.5). By T4.5 the bulk of the remaining sigma E has become displaced from the RNAP-containing fractions and sediments higher in the gradient. Presumably, this slower-migrating species represents free sigma E. Zhang and Kroos have found that sigma E is negatively regulated by sigma K (60, 61). They showed that sigma K reduced sigma E-dependent transcription and, more recently, the synthesis of sigma E itself (60, 61). We therefore examined the effects of a sigma K loss on the sigma E profile in our systems. Our data (Fig. 4) confirm the finding of Zhang and Kroos that sigma E persists in the absence of sigma K and, in addition, show that sigma E continues to cofractionate with RNAP in the sigma K-deficient strain (e.g., compare Fig. 1A, T4.5, with Fig. 4, T4.5). sigma K itself, or a sigma K-dependent process, thus affects both sigma E levels and sigma E's ability to associate with RNAP.


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  		FIG. 4.   Fractionation of crude extracts from SigK- B. subtilis. SigK- B. subtilis (SK1027) was grown in DSM, and samples were harvested at the end of growth (T0) and at 1.5-h intervals thereafter (T1.5, T3.0, and T4.5). The samples were analyzed as described for Fig. 1. The anti-sigma E antibody detects pro-sigma E and sigma E at T1.5 and T3 and primarily sigma E at T4.5.

In an attempt to distinguish between sigma K, as competitor of sigma E, or an unknown gene product under sigma K's control as an effector of sigma E displacement and loss, we examined the sigma E profile in a B. subtilis mutant whose sigma K (sigma KCR109) could bind RNAP but could not recognize sigma K-dependent promoters (61). There is a limitation to this experiment which was not encountered in the similar experiment with the sigma ECR119 mutant. sigE is expressed from a sigma A-dependent promoter (32), and therefore, sigma E synthesis is unaffected by the activity of the sigma  factor product that is made. sigK, in contrast, is transcribed first by E-sigma E and then by E-sigma K (34, 35). Given that the sigma KCR109 protein is ineffective as a sigma  factor, the sigma K protein levels in this strain remain dependent on E-sigma E. Western blot analyses of extracts prepared from sporulating wild-type and sigKCR109 mutant strains revealed that the mutant strain formed approximately 25% of the sigma K protein that was found in wild-type B. subtilis (data not shown). Perhaps due to this lower sigma K level and a continued dependence on E-sigma E for sigma K synthesis, sigma E persisted in the sigma KCR109 strain but nevertheless became increasingly displaced from RNAP as the mutant sigma K protein accumulated (Fig. 5). Quantitation of the antibody reactions in Fig. 5 revealed that the sigma E abundance was essentially unchanged between T4.5 and T6 (i.e., the sigma E antibody signal at T4.5 was 94.3% of the signal at T6.0 [62.5 × 103 and 66.3 × 103 pixels, respectively]); however, the percentage of unbound sigma E rose from 14.5 to 30%. During this same period, the abundance of sigma KCR109 increased 2.4-fold, with virtually all of the sigma K being in the RNAP-containing fractions. The disproportionate association of sigma K with RNAP, relative to that of sigma E, and the coincidence of an approximately twofold increase in sigma K abundance with a twofold increase in free sigma E suggest that sigma K can compete with sigma E and displace it from RNAP in these extracts.


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  		FIG. 5.   Fractionation of crude extracts from sigK109CR B. subtilis. B. subtilis S410 (sigK109CR) was grown in DSM. Samples harvested at 4.5 h (T4.5) and 6 h (T6) after the end of growth were analyzed as described for Fig. 1 with antibodies against beta ', sigma A, sigma E, and sigma K as probes. The anti-sigma E antibody detected sigma E. The anti-sigma K antibody detected sigma K and its slower-migrating precursor, pro-sigma K.

The anti-sigma K antibody detects both sigma K and its larger precursor form (pro-sigma K). Pro-sigma K, like pro-sigma E, is believed to be associated with cell membrane components (59). This likely accounts for pro-sigma K's distribution at the bottom, and throughout, the gradient (Fig. 5). The distribution of pro-sigma K in these gradients differs somewhat from the profile which we had observed for pro-sigma E in similar gradients. Pro-sigma E was more highly enriched in the RNAP-containing fractions and appeared to either cosediment with RNAP or migrate as faster-sedimenting, and presumably membrane-bound, forms (Fig. 1A [T1.5], 2B, 3, and 4 [T1.5]). The pro-sigma K, in contrast, was spread more diffusely throughout the gradient, with less of it in the RNAP fractions and a portion of it near the top of the gradient, where it is free from high-molecular-weight associations (Fig. 5). Pro-sigma E thus appears more likely than pro-sigma K to be associated with RNAP. Whether this is due to inherent differences between the two proproteins in their capacity for RNAP binding or to an effect of other components in the extract at the time of their synthesis (e.g., sigma E present to compete with pro-sigma K) is not clear.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

As B. subtilis proceeds into sporulation, its extractable RNAP contains decreasing amounts of the principal vegetative cell sigma  factor, sigma A, and increasing amounts of sporulation-specific sigma  factors (18, 52). Early in sporulation (T2 to T3), the principal RNAP holoenzyme species that can be isolated contains sigma E (19), the first sporulation-specific sigma  factor that is present in the large mother cell compartment (10). After T4, the level of sigma E falls and the second mother cell holoenzyme, E-sigma K, appears and continues the next stage of transcription in that compartment (55, 60). In the present study we have examined some of the properties of the E-sigma Aright-arrowE-sigma Eright-arrowE-sigma K progression. We found, as have others (40), that sigma A and RNAP levels remain relatively constant until at least T4.5 of sporulation. Nevertheless, by 3 h into this process, the percentage of sigma A that cosediments with RNAP falls from 50% to less than 10% (Fig. 1A). In contrast, sigma A association with RNAP remains at 50% in a B. subtilis strain which is unable to sporulate due to a mutation in the sporulation-essential spo0A gene (Fig. 1B). Thus, as initially reported more than 20 years ago, sigma A persists in sporulating B. subtilis but becomes displaced from RNAP by sporulation-specific factors (47, 54).

Although our gradient analyses showed a progressive loss of E-sigma A as B. subtilis proceeds into sporulation, a recent study, using a histidine tag to withdraw RNAP from B. subtilis extracts, found E-sigma A to persist throughout sporulation (17). We are unable to explain the differences in our findings. The absence of sigma A has long been a hallmark of RNAP from sporulating B. subtilis (19, 38, 47, 54). Given that sigma A persists during sporulation, it is possible that some feature of the Ni2+ resin extraction technique allowed it to reassociate with RNAP during the purification.

Based on immunofluorescence experiments, sigma A is believed to be in roughly equal concentrations in the mother cell and forespore (40). The mother cell compartment of a typical B. subtilis cell is at least five times the size of the forespore. Therefore, whatever is responsible for sigma A displacement during sporulation needs to be active in the large mother cell, where the bulk of the RNAP and sigma A is likely to be found. Our data implicate sigma E, the first of the mother cell-specific sigma  factors, as having a direct role in sigma A displacement. Mutations which block sigma E synthesis alter the sigma A retention on RNAP from the few percent found in wild-type B. subtilis to a level almost as great as that seen in the spo0A mutant. sigma E's role in sigma A dissociation from RNAP appears to be related to the presence of the sigma E protein rather than its transcriptional activity. A mutant sigma E (sigma ECR119), which can bind to RNAP but cannot direct RNAP to sigma E promoters, reduced the amount of sigma A that cosedimented with RNAP from the 30% observed in the absence of sigma E to below 10%. The notion that sigma E is preferentially bound to RNAP in early sporulating cells is supported by the distribution of sigma E in the gradient analyses. Until sigma K appears, virtually all of the detectable sigma E in the crude extracts cosediments with RNAP (Fig. 1A, 2, 3, and 4), while a significant portion of the sigma A in the extracts is unassociated. Either a sporulation-specific factor, which does not depend on sigma E for its expression, weakens sigma A's ability to compete with sigma E or sigma E is an inherently more potent RNAP binding protein than is sigma A. The latter possibility is attractive. sigma A, but not sigma E, is released from RNAP when these holoenzymes are bound to phosphocellulose (19, 48). This implies that sigma E binds more tightly to RNAP than does sigma A. In addition, the synthesis of sporulation sigma  factors in vegetatively growing B. subtilis induces the expression of genes whose transcription depends on them (see, e.g., references 24, 44, 46, 50, and 51). Thus, sporulation sigma  factors can gain access to RNAP to at least some degree in the presence of sigma A without the aid of additional sporulation factors.

In wild-type strains, active sigma E initiates the synthesis of sigma K, the second of the two mother cell sigma  factors. In such strains, there is a displacement of sigma E from RNAP and a drop in sigma E protein levels. Zhang and Kroos have documented that the decrease in sigma E levels is dependent on active sigma K (60, 61). Our current data confirm the need for sigma K in sigma E disappearance and also show that the presence of sigma K protein alters the profile of sigma E's association with RNAP. Before the appearance of sigma K, or in mutant B. subtilis that cannot synthesize sigma K, most of the sigma E present in crude extracts cosediments with RNAP (Fig. 1A [T3] and 4); however, after sigma K's appearance, a significant portion of the sigma E becomes displaced from RNAP (Fig. 1A [T4.5] and 5). Coincident with the sigma E displacement, virtually all of the mature sigma K is found in the RNAP-containing fractions (Fig. 5). Thus, sigma K effectively competes with sigma E for RNAP, with the result that E-sigma K becomes a preferred holoenzyme species.

Competition among sigma  factors has been implicated as an element of postexponential gene expression in both E. coli and B. subtilis. The stationary-phase sigma  factor of E. coli, sigma S, appears to compete with this organism's housekeeping sigma 70 during the transition to stationary phase (15). Overproducing either sigma 70 or sigma S was found to shift the pattern of transcription in stationary-phase E. coli in favor of those promoters recognized by the sigma  factor that was overexpressed (15). More germane to our present study, Hicks and Grossman have found that altering sigma A levels in B. subtilis affects gene expression by the sporulation-essential, transition state sigma  factor sigma H (21). Enhanced or restricted sigma A production during growth was found to decrease or increase, respectively, sigma H-dependent gene expression. Overproduction of sigma A also delayed the production of heat-resistant spores, an outcome that might be expected if, as Hicks and Grossman suggested, sporulation-specific sigma  factors had to compete with this sigma A pool for a limited population of RNAP core (21).

Recently, Lord et al. analyzed the replacement of sigma A by the forespore-specific sigma  factor sigma F (40). They noted, as did we, that the intracellular concentrations of core RNAP and sigma A were virtually unchanged during the first 3 h of sporulation. In addition, they determined that by the time the first sporulation sigma  factors are activated (i.e., after septation), the concentration of sigma A and sigma F exceeds the concentration of RNAP and that competition for core RNAP must be occurring (40). Although the simplest model for sigma  factor substitution would have sigma F as a more effective competitor for RNAP than sigma A, Lord et al. found that sigma A's affinity for RNAP was 25-fold greater than that of sigma F (40). Based on this disparity, they proposed that an anti-sigma A factor is synthesized or activated during sporulation to allow sigma F to successfully compete for RNAP. If this or a similar hypothetical factor also participates in the exchange of sigma E for sigma A, our data argue that its appearance depends on Spo0A, but not active sigma E or sigma F, and that it cannot in itself remove sigma A from RNAP but would instead enhance the competitiveness of the sporulation sigma  factors to displace sigma A. In vitro studies of the affinities of purified sigma A, pro-sigma E, and sigma E for RNAP, as well as transcription competition analyses with purified proteins, will be useful in determining whether sigma E has the affinity for RNAP that would allow it to directly supplant sigma A on RNAP.


    ACKNOWLEDGMENTS

This work was supported by NSF grant MCB-9727927.

We thank Lee Kroos for stimulating discussions, unpublished results, bacterial strains, and anti-sigma K antibody. We also thank Alan Grossman, Charles Moran, and Patrick Stragier for providing strains and/or plasmids.


    FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78284-7758. Phone: (210) 567-3957. Fax: (210) 567-6612. E-mail: Haldenwang{at}UTHSCSA.EDU.

dagger Present address: Gene Regulation and Chromosome Biology Laboratory, ABL Basic Research Program, NCI-FCRDC, Frederick, MD 21702.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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