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Journal of Bacteriology, October 2006, p. 7267-7273, Vol. 188, No. 20
0021-9193/06/$08.00+0     doi:10.1128/JB.00744-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Blocking Chromosome Translocation during Sporulation of Bacillus subtilis Can Result in Prespore-Specific Activation of ^G That Is Independent of ^E and of Engulfment

Vasant K. Chary, Panagiotis Xenopoulos, and Patrick J. Piggot^*

Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Received 24 May 2006/ Accepted 1 August 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Formation of spores by Bacillus subtilis is characterized by cell compartment-specific gene expression directed by four RNA polymerase factors, which are activated in the order ^F-^E-^G-^K. Of these, ^G becomes active in the prespore upon completion of engulfment of the prespore by the mother cell. Transcription of the gene encoding ^G, spoIIIG, is directed in the prespore by RNA polymerase containing ^F but also requires the activity of ^E in the mother cell. When first formed, ^G is not active. Its activation requires expression of additional ^E-directed genes, including the genes required for completion of engulfment. Here we report conditions in which ^G becomes active in the prespore in the absence of ^E activity and of completion of engulfment. The conditions are (i) having an spoIIIE mutation, so that only the origin-proximal 30% of the chromosome is translocated into the prespore, and (ii) placing spoIIIG in an origin-proximal location on the chromosome. The main function of the ^E-directed regulation appears to be to coordinate ^G activation with the completion of engulfment, not to control the level of ^G activity. It seems plausible that the role of ^E in ^G activation is to reverse some inhibitory signal (or signals) in the engulfed prespore, a signal that is not present in the spoIIIE mutant background. It is not clear what the direct activator of ^G in the prespore is. Competition for core RNA polymerase between ^F and ^G is unlikely to be of major importance.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Formation of spores by Bacillus subtilis is a simple two-cell differentiation process that has become a paradigm for studying cell differentiation in prokaryotes. Central to this process is compartmentalization of gene expression between the two cell types. Sporulation involves asymmetrical division that yields two cells of different sizes, which have different developmental fates. The smaller cell, called the prespore (or forespore), ultimately develops into the mature, heat-resistant spore. The larger cell, called the mother cell, is necessary for spore formation but ultimately lyses. Expression of different genes in the two cells is governed by the activation of four RNA polymerase sigma factors: ^F and then ^G in the prespore and ^E and then ^K in the mother cell (for a review, see reference 14). Each of these sigma factors directs expression of distinct regulons, which have recently been delineated by microarray analysis; the smallest regulon, the regulon for ^F, contains about 50 genes, and the largest, the regulon for ^E, contains perhaps 200 genes (7, 42, 49).

The first sigma factor to become active, ^F, does so soon after formation of the spore septum, and its activation leads rapidly to the activation of ^E (12; for a review, see reference 14). Development continues with the mother cell engulfing the prespore. Upon completion of engulfment, the prespore is entirely within the mother cell and so is no longer in direct contact with the medium. At this time, ^G becomes active in the prespore, and it in turn triggers activation of ^K in the mother cell. Coordination of the cascade of sigma factor activation is ensured by intracellular controls and by intercellular communication between the two compartments (14, 25).

In this report, we focus on the activation of ^G, which is encoded by the spoIIIG locus (27, 47). The spoIIIG locus is first transcribed during sporulation by read-through from the spoIIG locus. However, the transcript is translated poorly, if at all, into ^G (27, 45); it is not needed for spore formation (45), and its role is obscure. Productive transcription of spoIIIG is initiated from a promoter immediately upstream of the gene. It is directed by RNA polymerase containing ^F and so is confined to the prespore (17). This transcription is delayed compared to that of other ^F-directed genes (19, 42), and it requires a ^E-directed signal from the mother cell (17, 29). It also requires expression of the ^F-directed spoIIQ locus (48); since the SpoIIQ protein is located in the membrane, its regulation of spoIIIG transcription is also thought to be indirect (34). Placing spoIIIG under a strong ^F-directed promoter (44) or mutating the spoIIIG promoter (10) can result in ^E-independent transcription of spoIIIG. However, this does not make activation of ^G independent of ^E, indicating that there are additional controls dependent on ^E; indeed, these controls appear to be more important for spore formation (10).

When first formed, ^G is not active. Its activation depends on completion of engulfment of the prespore (43), which requires the activity of several ^E-directed genes (1, 14). Activation also requires the ^E-directed expression of the spoIIIA operon, whose products are thought to transmit some signal from the mother cell to the prespore via the SpoIIIJ protein (8, 38). It is not clear if the SpoIIIA-mediated signal is distinct from the signal that engulfment is completed, as strains with spoIIIA mutations complete engulfment (30). A protein encoded in the spoIIIA operon, SpoIIIAH, interacts with the prespore protein SpoIIQ, suggesting that there is an additional regulatory mechanism that acts via SpoIIIA (2). However, it remains to be established whether SpoIIQ does indeed have a role in ^G activation in addition to its role in spoIIIG transcription (2, 48).

There are additional controls that prevent ^G from becoming active in the other cell type, the mother cell (5, 40). These controls have complicated analysis of the controls of prespore activation. For example, mutations that bypassed the need for spoIIIA in ^G activation resulted in deregulation of ^G expression in the mother cell but not in the prespore (5, 40). Such mutations overcame SpoIIAB-mediated inhibition of ^G in the mother cell, a regulatory system that may be quite separate from the SpoIIIA-SpoIIIJ system regulating ^G in the prespore.

Here we focused on ^G activation in the prespore. We found that it is possible to activate ^G in the prespore independent of ^E activity and of spoIIQ; in the conditions used, ^G became active after septum formation and before engulfment was initiated. These results suggest that ^E/SpoIIQ regulation normally ensures that ^G does not become active until engulfment is completed. Premature ^G activation does not curtail ^F activity, suggesting that competition for core polymerase is not a major factor in determining ^G activation or ^F inactivation.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Media. B. subtilis was grown in modified Schaeffer's sporulation medium (MSSM) or on Schaeffer's sporulation agar (31, 35). When required, the medium contained 5 µg chloramphenicol/ml, 1.5 µg erythromycin/ml, 3.5 µg neomycin/ml, 100 µg spectinomycin/ml, or 10 µg tetracycline/ml. Escherichia coli was grown on Luria-Bertani lysogeny broth agar containing 100 µg ampicillin/ml when required.

Strains. B. subtilis 168 strain BR151 (trpC2 metB10 lys-3) was used as the parent strain. The B. subtilis strains used are listed in Table 1. Strain PS1120 with spoIIIG inserted at amyE (45) was kindly provided by Peter Setlow (Connecticut Health Sciences Center); the amyE::spoIIIG construct was introduced by transformation into strains listed in Table 1, with selection for the linked cat marker. The spoIIIE36 mutation is spo-36 described by Hranueli et al. (15). Mutants with spoIIR inactivated were constructed by double crossover at the spoIIR locus in such a way that 342 bp at the 3' end of the gene was replaced with P_sspA-lacZ or P_sspA-gfp transcriptional fusions linked to an antibiotic resistance cassette. The fusions were designed so that their expression could not be driven by the spoIIR promoter or by the promoter for the resistance cassette. The P_spoIIR-cfp and P_spoIIR-lacZ fusions were inserted at the spoIIR locus by double crossover, disrupting the locus. The P_sspA-yfp fusion was present in a derivative of the pAMß1 replicon shuttle plasmid pJAR2 (3). Vectors with the gfp, cfp, and yfp genes were kindly provided by W. G. Miller (28), D. Z. Rudner (6), and P. J. Lewis (11), respectively. E. coli DH5 (Gibco-BRL) was used to maintain plasmids. Details of plasmid and strain construction are available on request.

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TABLE 1. B. subtilis strains used

Fluorescence microscopy. Cultures were grown in MSSM at 37°C. A 200-µl portion of culture was mixed with 0.2 µl of a 1-mg/ml stock solution of FM4-64 (Molecular Probes) in phosphate-buffered saline (Gibco-BRL). Samples were incubated at 37°C for 5 min, and 1 µl of an unfixed sample was transferred to a slide and visualized essentially as described by Pogliano et al. (33). Images were captured using a Leica DM IRE2 microscope with a TCS SL confocal system as described previously (5). Excitation for green fluorescent protein (GFP) and FM4-64 was at 488 nm; emission for GFP was captured at wavelengths between 500 and 550 nm, and emission for FM4-64 was captured at wavelengths between 600 and 730 nm. Excitation for cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) was at 458 and 514 nm, respectively, and emission was captured at wavelengths between 465 and 500 and between 525 and 550 nm, respectively. In general, there was 4x line averaging and 3x frame averaging.

Other methods. ß-Galactosidase activity was assayed essentially as described previously (3). Specific activity was expressed in nanomoles of o-nitrophenyl-ß-D-galactopyranoside hydrolyzed per minute per milligram (dry weight) of bacteria. Results of typical experiments are shown below; the activities are the means for duplicate cultures in an experiment. The methods used for transformation of B. subtilis and for sporulation by exhaustion in MSSM and other methods were essentially the methods described previously (31).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Rendering ^G activation in the prespore independent of ^E activity. Productive transcription of spoIIIG, the structural gene for ^G, is initiated from a ^F-directed promoter and takes place in the prespore (45). This ^F-directed transcription normally requires activation of ^E in the mother cell (17, 29). When the spoIIIG promoter was relocated to the origin-proximal amyE locus, its transcription, as assayed with a P_spoIIIG-lacZ transcriptional fusion, still required ^E activity (10, 17) (Fig. 1, compare strains SL5430 and SL10117). However, we found that when translocation of the origin-distal 70% of the chromosome into the prespore was blocked by a spoIIIE36 mutation, ^E activity was no longer required for the ^F-directed P_spoIIIG-lacZ transcription (Fig. 1, compare strains SL10086 and SL11106); increased ^F-directed transcription in a spoIIIE mutant background has been observed previously for ^F-directed genes (37, 39, 46). The transcription of spoIIIG remained absolutely dependent on ^F (data not shown).

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FIG. 1. The PspoIIIG promoter is expressed in the absence of E when it is located at the origin-proximal amyE locus in a spoIIIE36 mutant. Transcription directed by the PspoIIIG promoter was assessed by determining the ß-galactosidase activity in the following strains containing an amyE::PspoIIIG-lacZ fusion: SL5430 (spo+) (), SL10117 (spoIIGB::erm) (), SL10086 (spoIIIE36) (•), and SL11106 (spoIIIE36 spoIIGB::erm) ().

Activity of ^E is normally required for the activation of ^G, as well as for the transcription of spoIIIG. The observations described above for the transcription of spoIIIG encouraged us to test whether ^G became active in the absence of ^E when chromosome translocation was blocked by a spoIIIE36 mutation. To monitor ^G activity, a P_sspA-lacZ transcriptional fusion was introduced into the origin-proximal spoIIR locus. This insertion inactivated spoIIR and so prevented activation of ^E (18); loss of ^E activity in strains with this construct was confirmed by their failure to initiate engulfment and also by the absence of expression of a ^E-dependent P_spoIID-lacZ fusion when it was introduced by appropriate crosses (data not shown). The spoIIIE36 mutation prevented translocation of the origin-distal spoIIIG locus into the prespore and so prevented its ^F-directed transcription (52). Consistent with this result, no ^G activity was detected in a spoIIIE36 mutant, strain SL12918, in which spoIIIG is at its natural locus (Fig. 2). However, when the spoIIIG locus was inserted into the origin-proximal amyE locus, ^G activity was readily detected (Fig. 2, strain SL12916). The level of ^E-independent ^G activity in strain SL12916 was similar to the level of ^G activity of a spo⁺ strain, SL10369 (Fig. 2), although expression appeared to start slightly earlier, during spore formation. Thus, ^E activity was not required for either transcription of spoIIIG or ^G activation. However, there was still a requirement for ^F activity, as P_sspA-lacZ expression was blocked by inactivation of spoIIAC, the structural gene for ^F (Fig. 2, strain SL12938).

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FIG. 2. Activation of G independent of E in a spoIIIE36 mutant with spoIIIG located at amyE. The activity of G was assessed by determining the ß-galactosidase activity in the following strains: SL10369 (spo+ PsspA-lacZ@sspA) (•), SL12916 (spoIIIE36 amyE::spoIIIG spoIIR::PsspA-lacZ) (), SL12918 (spoIIIE36 spoIIR::PsspA-lacZ) (), SL12936 (spoIIIE36 amyE::spoIIIG spoIIR::PsspA-lacZ spoIIR@spoIIR) (), and SL12938 (spoIIIE36 amyE::spoIIIG spoIIR::PsspA-lacZ spoIIR@spoIIR spoIIAC::neo) ().

^E-independent activation of ^G is confined to the prespore. To test the location of the ^E-independent activation of ^G in an amyE::spoIIIG spoIIIE36 strain, SL12864 was constructed, in which a ^G-dependent P_sspA-gfp transcriptional fusion was inserted into the spoIIR locus. Because of the absence of ^E activity, this strain underwent sporulation division but did not initiate engulfment. Strain SL12864 expressed GFP, and expression was confined to the prespore (Fig. 3B and Table 2); similar results were obtained when spoIIGB, which is the structural gene for ^E, was also inactivated (data not shown). Mutants lacking ^E activity often form prespores at both ends of the sporulating organism (the abortively disporic phenotype [16, 30]). This phenotype was observed with SL12864, and in the majority of the disporic cells the GFP signal was detected in both prespores (Fig. 3B). The results confirmed that the ^E-independent ^G activity is confined to the prespore. In the spo⁺ strain, SL10969, the prespore-specific ^G activity was detected only in bacteria that had completed engulfment (Fig. 3A), whereas in SL12864 it was detected at the preengulfment stage (Fig. 3B).

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FIG. 3. Location of E-independent G activity. (A to C) Examples of bacteria stained with FM4-64 (red) and expressing GFP (green) under control of the G-directed sspA promoter. (A) SL10969 (spo+); (B) SL12864 (spoIIIE36 amyE::spoIIIG spoIIR) (the arrows indicate an abortively disporic cell); (C) SL12929 (spoIIIE36 amyE::spoIIIG spoIIR+) (the arrow indicates a partly engulfed prespore). (D to I) Strain SL12972 expressing CFP (blue) under control of the F-directed spoIIR promoter (D, F, G, and I) and expressing YFP (yellow) under control of the G-directed sspA promoter (E, F, H, and I). Bacteria were stained with FM4-64 (red). Panel F is an overlay of panels D and E and includes the FM4-64 image. Panel I is an overlay of panels G and H and includes the FM4-64 image. Bar = 3 µm for all images.

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TABLE 2. Location of fluorescent protein expressed from G- and F-directed promotersa

The efficiency of P_sspA-gfp expression in SL12864 was comparable to that in a spo⁺ strain (SL10969) in which the P_sspA-gfp fusion was inserted at sspA and to that of the spoIIIE36 strain SL12929, in which the activity of ^E was restored by complementing spoIIR (Table 2). These data are in agreement with the quantitative analysis of P_sspA-lacZ expression in the two genetic backgrounds, as mentioned above. In spo⁺ strains, ^G does not become active in the prespore until after completion of engulfment (32), whereas the ^E-independent activation of ^G occurs after the sporulation division and before initiation of engulfment (indeed, engulfment is not completed in strains that lack ^E activity). The mechanism of ^E-dependent activation of ^G includes, but is probably not limited to, expression of the spoIIIA locus (8, 38). Whatever the details, this mechanism is not required when the origin-distal 70% of the chromosome is trapped in the mother cell and spoIIIG is located in the prespore.

In order to simultaneously visualize both ^F and ^G activities, we constructed strain SL12972 with P_spoIIR-cfp (^F-directed) and P_sspA-yfp (^G-directed) transcriptional fusions. The P_spoIIR-cfp fusion was inserted into and disrupted spoIIR. The P_sspA-yfp fusion was present in an autonomously replicating plasmid; increasing the gene dosage of sspA had been shown previously to cause only a modest increase in its mRNA level (26). The strain containing the fusions also had a spoIIIE36 mutation and spoIIIG located at amyE. The activities of both ^F and ^G were confined to the prespores (Table 2, strain SL12972, and Fig. 3D to I). The two activities were generally detected in the same preengulfment prespores (Table 2) in both monosporic and disporic organisms.

Restoration of ^E activity to a spoIIIE36 amyE::spoIIIG mutant does not change the timing of ^G activation. During the normal course of spore formation, ^G does not become active until after completion of engulfment, and its activation depends on the activity of ^E. Above, we describe conditions in which ^G became active in the preengulfment prespore and in the absence of ^E. We were interested to see if restoration of ^E activity in these conditions affected the timing or location of ^G expression. In the strains used, ^E was not active because of the loss of spoIIR. In order to restore ^E activity, a functional copy of spoIIR was introduced into strain SL12864 by single (Campbell-like) crossover at the truncated spoIIR locus, yielding strain SL12929. The restoration of spoIIR indeed resulted in activation of ^E, as indicated by bacteria proceeding toward the completion of engulfment and by the expression of a ^E-dependent P_spoIID-lacZ fusion when it was introduced into the strain (data not shown). In strain SL12929, ^G activity was confined predominantly to the prespore. Importantly, it appeared after completion of the sporulation division septum and before the initiation of engulfment was discernible. The proportion of cells in which ^G activity was detected for strain SL12929 was similar to the proportion for strain SL12864, in which ^E was not active (Table 2 and Fig. 3C). In strain SL12929, substantial numbers of bacteria proceeded toward the completion of engulfment (Fig. 3C), consistent with the restoration of ^E activity in a spoIIIE36 mutant. The ^G activity in SL12929 was initially prespore specific, although during prolonged incubation there was a breakdown of compartmentalization accompanied by extensive cell lysis, as is typical of spoIIIE mutants in which the prespores are unstable and lyse (data not shown) (4, 22, 43). The effect of restoration of ^E activity on the time of ^G expression was also tested by integrating a functional copy of spoIIR into strain SL12916 by single crossover. The expression of the ^G-directed sspA-lacZ fusion in the resulting strain, SL12936, was very similar to the expression in strain SL12916, in which ^E was not active (Fig. 2). Restoration of ^E activity did not restore ^K activity to the spoIIIE36 mutant strains (data not shown).

Activation of ^G in the prespore does not markedly curtail ^F activity. Sigma factors ^F and ^G are very similar to each other (13). They have overlapping promoter specificities (13), and both are inhibited by the anti-sigma factor SpoIIAB (9, 20, 40). During normal spore formation, they are activated successively in the prespore, and there are indications that ^F activity is curtailed when ^G becomes active (23). Thus, ^F and ^G may be in direct competition with each other. One possibility is that ^G outcompetes ^F for core RNA polymerase and supplants it. Alternatively, ^F might outcompete ^G and prevent it from becoming active until some other factor removes ^F from the competition. The ^E-independent activation of ^G described above provided the possibility to test the effect of direct competition between ^F and ^G in the preengulfment prespore.

There was little difference in ^F activity between strains SL12857 and SL12861 (Fig. 4), which differ only in the presence of an expressed copy of spoIIIG located at amyE in strain SL12857. Thus, the activity of ^G in the preengulfment prespore does not seem to have an appreciable effect on ^F activity. The strains contained a P_spoIIR-lacZ fusion inserted at spoIIR to assay ^F activity; the insertion disrupted spoIIR and so prevented ^E activation.

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FIG. 4. Effect of G activity on F activity in the preengulfment prespore. The activity of F was assessed by determining the ß-galactosidase activity in the following strains: SL12857 (spoIIIE36 spoIIR::PspoIIR-lacZ) () and SL12861 (spoIIIE36 spoIIR::PspoIIR-lacZ amyE::spoIIIG) (•).

This result reinforces the conclusion from studies of a lonA mutant that competition between ^F and ^G for core polymerase may not be important in determining the activities of these sigma factors (23). We infer that competition between the sigma factors for core RNA polymerase does not explain the normal delay in activation of ^G until after the completion of engulfment that occurs in spo⁺ strains.

Expression of ^G activity in an amyE::spoIIIG spoIIIE36 mutant is independent of spoIIQ. The appearance of ^G activity ordinarily requires expression of the ^F-directed spoIIQ locus (48). This expression is prespore specific (24) and so might regulate ^G expression separately from the ^E-directed mother cell signal(s). Transcription of spoIIIG directed by ^F normally depends on expression of the spoIIQ locus, even when spoIIIG is relocated to amyE (47); it is possible that the SpoIIQ protein is required for activation of ^G, as well as for spoIIIG transcription (2). We wanted to test whether this dependence on spoIIQ was retained in a spoIIIE36 amyE::spoIIIG strain. For this purpose, the spoIIQ mutant SL13225 was constructed. Strain SL13225 displayed ß-galactosidase activity in sporulation conditions (Fig. 5) similar to that of the isogenic spoIIQ⁺ strain, SL12916 (Fig. 5), except that at later times (6 to 7 h after the end of exponential growth) strain SL13225 displayed somewhat more activity than the spoIIQ⁺ strain. Thus, inactivation of spoIIQ did not impair ^G activity in a spoIIIE36 amyE::spoIIIG strain, indicating that spoIIIG transcription and ^G activation did not require either SpoIIQ or ^E in this genetic background. Indeed, the only effect of spoIIQ inactivation was to increase ^G activity at later times.

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FIG. 5. Activation of G is independent of SpoIIQ in a spoIIIE36 amyE::spoIIIG background. The activity of G was assessed by determining the ß-galactosidase activity in the following strains: SL12916 (spoIIIE36 amyE::spoIIIG spoIIR::PsspA-lacZ) () and SL13225 (spoIIIE36 amyE::spoIIIG spoIIR::PsspA-lacZ spoIIQ::neo) (•).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Gene expression during formation of spores by B. subtilis is controlled by the successive activation of RNA polymerase sigma factors in the order ^F-^E-^G-^K through a complex pattern of intra- and intercellular signals, in which activation of the later sigma factors in the sequence depends on activation of the earlier factors (25, 32). Thus, the activation of ^G in the prespore, which occurs upon completion of engulfment, depends on prior activation of ^F in the prespore and prior activation of ^E in the mother cell. We report here conditions in which ^G is activated in the prespore before engulfment is completed and independent of ^E activity. To our knowledge, this is the first report of overlapping ^F and ^G activities in the preengulfment prespore. The premature prespore-specific ^G activity depends on the genetic background of the strains used. In the strains used the origin-distal 70% of the chromosome was retained in the mother cell, and spoIIIG, the structural gene for ^G, was inserted into an origin-proximal site, which was present in the prespore. The relocation of spoIIIG is necessary for its transcription to be directed by ^F, which is active only in the prespore. The premature prespore-specific ^G activity is seen in strains that lack ^E activity and is not affected by restoration of ^E activity. Ordinarily, ^E activity is required both for spoIIIG transcription and for ^G activation (14). Thus, ^E has lost both roles with respect to ^G. In the mutant background, ^G becomes active soon after septum formation, as does ^F, rather than after completion of engulfment. Although ^G is activated earlier, the level of ^G activity, as assayed with a P_sspA-lacZ transcriptional fusion, is similar to the level that occurs in a spo⁺ strain. From these results, we suggest that the role of the ^E-mediated control is to ensure that activation of ^G occurs after the completion of engulfment.

It seems plausible that ^E activity is ordinarily needed to reverse some inhibitory signal (or signals) in the prespore, which is not present in the mutant strains. One possibility is that expression of a ^F-directed gene in the origin-distal 70% of the chromosome provides an inhibitory signal whose functions include coordinating ^G activation with the completion of engulfment. Moving spoIIIG to an origin-proximal site is, in itself, insufficient to overcome this inhibitory signal. However, when the origin-distal region remains trapped in the mother cell in a spoIIIE mutant, there is no inhibitory signal in the prespore and ^G becomes active in the absence of ^E. With this said, no ^F-directed gene encoding such an inhibitory signal has been identified yet. Exclusion of the lonA gene from the prespore in the spoIIIE mutants may also facilitate premature ^G activation because ^G is particularly sensitive to the LonA protease (36, 39). Another possibility (which does not exclude the possibility described above) is that some structural component of the engulfing septum and/or an origin-distal portion of the chromosome is involved in ^G activation. A possible candidate is SpoIIIE. This large, 787-residue protein is already known to have several roles, and it might be part of the mechanism that ensures that ^G does not become active until completion of engulfment. It is located in the sporulation septum (51) and is required both for chromosome translocation (50) and for completion of engulfment (41). SpoIIIE forms a single focus in the engulfing septum until engulfment is completed, when it becomes dispersed (41). It may be that changes in SpoIIIE conformation, associated with completion of engulfment, are part of the pathway that relieves ^G inhibition. Certainly, loss of SpoIIIE function is pivotal to the system described here for obtaining ^E-independent prespore-specific ^G activity.

The same changes in genetic background also removed any requirement for SpoIIQ in ^G activation. SpoIIQ is made in the prespore. It is normally required for transcription of spoIIIG and some other ^F-directed genes, although it is unlikely to act directly on the regulated promoters (48). It may also be required for ^G activation and has been shown to interact with SpoIIIAH, whose expression is normally required for ^G activation (2). In some conditions it is required for the completion of engulfment (48). The mode of SpoIIQ action remains unclear, although the interaction with SpoIIIAH suggests that SpoIIQ might ordinarily be in the signal pathway from the mother cell that is activated on completion of engulfment. Reinforcing the evidence for such a pathway, the same changes in genetic background that remove any requirement for ^E in ^G activation also remove the need for SpoIIQ.

Once it becomes active, ^G can direct transcription of its own structural gene. This positive feedback loop provides a mechanism for the rapid accumulation of ^G when it is needed. However, inappropriate expression can be toxic (21), so the positive feedback loop needs to be tightly controlled. In this report we describe premature activation of ^G. Nevertheless, this activation occurs only during spore formation and only in the prespore. It is absolutely dependent on ^F activity, and a totally unregulated feedback loop is not established. We previously described a system that leads to mother cell-specific ^G activity, which is independent of ^F and the anti-sigma factor SpoIIAB (5). Mutations that render ^G insensitive to regulation by SpoIIAB also result in inappropriate activation of the feedback loop (40), as do mutations that inactivate lonA, which encodes a protease that can act on ^G (36). However, in none of these conditions is ^G activity constitutive. Thus, there are several mechanisms to prevent totally unregulated expression of the ^G-directed positive feedback loop. The LonA protease is thought to prevent nonspecific activation of ^G during stationary phase or during a stress response (36) and may help prevent activation in the mother cell (5). The SpoIIAB protein prevents activation in the mother cell (5, 40). The results reported here support the view that the ^E/SpoIIQ system prevents activation of ^G in the prespore before completion of engulfment. None of the mechanisms appears to have a role during vegetative growth, suggesting that there may be still other systems regulating ^G activity.

 
This work was supported by Public Health Service grant GM43577 to P.J.P. from the National Institutes of Health.

 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, 3400 North Broad Street, Philadelphia, PA 19140. Phone: (215) 707-7927. Fax: (215) 707-7788. E-mail: piggotp{at}temple.edu.

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Journal of Bacteriology, October 2006, p. 7267-7273, Vol. 188, No. 20
0021-9193/06/$08.00+0     doi:10.1128/JB.00744-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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