SCOTT PANZER,' RICHARD LOSICK,1* DONGXU SUN,2 AND PETER SETLOW2. Department of .... NOTES. 563. 5 am). (n a). 0. _0_. >m..0. -cx va a. 0. O en. °0 x. >.0. nE i' 8. 0s o 0. -a .... Mason, J. M., R. H. Hackett, and P. Setlow. 1988.
Vol. 171, No. 1
JOURNAL OF BACTERIOLOGY, Jan. 1989, p. 561-564
0021-9193/891010561-04$02.00/0 Copyright © 1989, American Society for Microbiology
Evidence for an Additional Temporal Class of Gene Expression in the Forespore Compartment of Sporulating Bacillus subtilis SCOTT PANZER,' RICHARD LOSICK,1* DONGXU SUN,2 AND PETER SETLOW2 Department of Cellular and Developmental Biology, The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138,1 and Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 060322 Received 22 August 1988/Accepted 30 September 1988
We present evidence indicating that the previously studied, sporulation-induced gene 0.3 kb, which encodes a stable RNA present at late developmental stages, is transcribed in the forespore chamber of sporulating cells of Bacillus subtilis. Compartmentalized gene expression was demonstrated on the basis of subcellular fractionation experiments in which severalfold-higher levels of 0.3 kb-directed I-galactosidase specific activity were observed in forespore extracts than in extracts from the mother cell and dependence studies in which 0.3 kb transcription was found to be blocked in mutants bearing mutations in spoIIIA, spolIIE, and spoIIIG, genes which are known to govern forespore gene expression. Also, 0.3 kb transcription could be switched on during growth in cells in which transcription of the forespore regulatory gene spoIlIG was engineered to be activated in response to the lac inducer IPTG (isopropyl-j3-D-thiogalactopyranoside). Although it is transcribed in the forespore, 0.3 kb is switched on at a later developmental stage than other previously studied foresporeexpressed genes, and hence it appears to be representative of an additional temporal class of compartmentalized gene expression.
appearance of optically refractile prespores) of sporulation and that is inferred to encode a small basic polypeptide of 6,750 daltons (11, 16). Because its transcript is conspicuous in total RNA from late sporulating cells, we were interested to know whether 0.3 kb is transcribed in the mother cell or in the forespore compartment. To study the compartmentalization of 0.3 kb expression, we took advantage of the existence of a previously constructed operon fusion of 0.3 kb to the lacZ gene of Escherichia coli (16) and of methods for the subcellular localization of P-galactosidase (1, 9, 17). The 0.3 kb-lacZ fusion was introduced into the prophage of the temperate B. subtilis bacteriophage SPi by a previously described recombinational transfer procedure (13, 19) to create an SP,::0.3 kb-lacZ transducing phage. Cells (25 ml) of strain 168 bearing the transducing phage were harvested at a late stage of sporulation (9 h after the end of the exponential phase of growth in Difco sporulation medium) and then frozen at -70°C for storage. To achieve differential breakage of the mother cell and forespore compartments, the cells were thawed, collected by centrifugation in a Beckman Microfuge washed in 50 mM Tris hydrochloride at pH 8.0, suspended in 1 ml of buffer A of Singh et al. (15) containing 800 jig of lysozyme per ml, and incubated at 37°C for 15 min. The lysozyme-treated cells were collected by microcentrifugation for 4 min and suspended in 0.5 ml of buffer A by vortexing vigorously for 30 s. This treatment was sufficient to cause extensive breakage of the mother cells. The remaining cells and debris were collected by centrifugation, and the supernatant fluid (the mother cell fraction) was recovered and placed on ice. The cell pellet was washed and suspended in 1 ml of buffer A and then sonicated for 10 s at 100 W, which disrupted the remaining mother cells. After a second cycle of centrifugation, washing, and sonication, the remaining material (largely forespores and cell debris) was collected by centrifugation, suspended in 0.2 ml of buffer A plus 0.2 ml of acid-washed glass beads (75 to 100 jim; Sigma Chemical Co.) and sonicated twice for 30-s intervals at 140 W. After the addition of 0.6 ml of buffer A, followed by vigorous
Endospores of the gram-positive bacterium Bacillus subtilis are produced within sporangia that consist of two compartments known as the mother cell and the forespore (reviewed in references 7 and 8). The mother cell and the forespore are believed to contain identical chromosomes, but they have different developmental fates (6). The forespore ultimately becomes the core of the spore and is responsible for the production of the small, acid-soluble proteins known as SASPs (3), which are packaged with the spore chromosome (14). The mother cell, on the other hand, governs the production of coat and other proteins that constitute the outer protective layers of the spore but it is ultimately discarded by lysis when spore formation is complete. Examples of genes expressed in the forespore compartment, in addition to the SASP-encoding ssp genes (9), are genes of the spoVA operon (1, 6), the germination gene gerA (A. Moir, personal communication), and the glucose dehydrogenase-encoding gene gdh (12). These genes appear to be switched on synchronously during hour 2 to 3 of sporulation, and they may constitute a regulon of forespore gene expression. Examples of genes that are known or inferred to be expressed in the mother cell are the sporulation genes spoIIIC (17), spoIVC (5), spoVB (6), and spoVJ (J. Errington, personal communication), the germination gene gerE, and the spore coat gene cotA (S. Panzer, undergraduate honors thesis, Harvard University, Cambridge, Mass., 1988). In contrast to the known forespore-expressed genes, mother cell-expressed genes are switched on in an ordered, temporal sequence that spans several developmental stages (5, 13, 17). Here we present evidence assigning the expression of a previously identified, sporulation-induced gene called 0.3 kb (11, 16) to the forespore chamber. Based on its time of induction, 0.3 kb is evidently representative of an additional temporal class of forespore gene expression. The 0.3 kb gene specifies a stable, 300-base RNA that appears at a late stage (hour 4 or about 30 min before the *
Corresponding author. 561
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TABLE 1. Compartmentalization of 0.3 kb expression'
Expt
,B-Galactosidase sp act (nmol of ONPG hydrolyzed/ min per mg of protein) in: Forespore Mother cell fraction fraction
Activity ratio cell) (forespore/mother
6.3 1 7.5 1.2 >20 2 4.0 6 9.5 m.. -cx0 >.0 a va °0 x
o. 0 (A I._a) =
O
en
(Ac
O-B 0 l80 oi'nE 0s
-a 5
Hours after the addition of IPTG FIG. 2. (a) Time course of 0.3 kb-directed ,-galactosidase synthesis in cells with and without induction of aoG synthesis. Wild-type B. subtilis 168 cells that were lysogenized with SPP::0.3 kb-lacZ and carried either plasmid pDG298 or pDG148 were grown in 2x YT medium (16 g of Bacto-Tryptone, 10 g of yeast extract, and 5 g of NaCl per liter) with kanamycin (10 pLg/ml). Plasmid pDG298 (D. Sun, P. Stragier, and P. Setlow, unpublished results) carries the spoIIIG gene downstream from, and under the control of, the spac promoter, while pDG148 (18) is the parental plasmid, which contains spac but lacks spolfIG. At an optical density of 0.3, IPTG was added to 5 mM to samples of the cultures, and the level of p-galactosidase was determined during subsequent incubation. Symbols: *, strain with pDG298 plus IPTG; O, strain with pDG298 without IPTG; A, strain with pDG148 with IPTG; A, strain with pDG148 without IPTG. (b) Time course of glucose dehydrogenase synthesis after induction of (G synthesis in vegetative cells. oG synthesis was induced in wild-type cells lysogenized with SPP::0.3 kb-lacZ and carrying plasmid pDG298 as described for panel a. Samples were taken for measurement of glucose dehydrogenase (0) and ,B-galactosidase (U). Enzyme assays were as previously described (9).
the fusion and subtracted this from the activity observed in the presence of the fusion. Transcription was significantly impaired in mutants bearing mutations in spoIIIA, spoIIIE, and spoIIIG (Fig. la to c), genes which are known to be required for transcription of ssp and certain other forespore genes but not for transcription of the mother cell-expressed spoIVC gene (1, 5, 9; D. Sun, P. Stragier, and P. Setlow, unpublished results). (Impaired expression in a spoIIIE mutant was also observed in earlier work [16] in which 0.3 kb-directed ,B-galactosidase synthesis was measured in cells bearing the fusion on a multicopy plasmid.) In contrast, little impairment of transcription (within the level of significance of our measurements) was observed in mutants bearing mutations in spoIIIC and spoIIID (Fig. ld and e), genes which are thought to be involved in mother cell gene expression (1, 5, 17). The dependence of 0.3 kb transcription on spoIIIG is of special significance because this stage III gene has been shown to encode a newly discovered RNA polymerase sigma factor called cfG that directs transcription of forespore genes (D. Sun, P. Stragier, and P. Setlow, unpublished results). Transcription of spoIIIG (also referred to as sigG) is itself limited to the forespore compartment, and hence the selective appearance of cG in the forespore accounts for the compartmentalization of expression of ssp and other genes whose promoters are utilized by aG RNA polymerase (C. Karmazyn-Campelli, C. Bonamy, B. Savelli, and P. Stragier, personal communication). As a direct test of the involvement of spoIIIG in the activation of 0.3 kb, we introduced the fusion into cells bearing plasmid pDG298 (D. Sun, P. Stragier, and P. Setlow, unpublished results), which carries a modified spoIIIG gene, engineered so as to bring its transcription under the control of the IPTG (isopropyl-p-Dthiogalactopyranoside)-inducible spac promoter (18). In such cells, transcription of the crG structural gene can be
induced under conditions of vegetative growth by the addition of the lac inducer IPTG. The 0.3 kb gene was switched on in these cells as a response to the addition of IPTG (Fig. 2a). However, the time of induction of 0.3 kb-directed P-galactosidase synthesis was delayed by about 1 h compared with the onset of glucose dehydrogenase synthesis (Fig. 2b) or sspA-lacZ expression (data not shown) under the same conditions. In sum, our results suggest that 0.3 kb is a foresporeexpressed gene, but its induction seems to occur at a later developmental stage (hour 4 of sporulation) than that of other previously studied forespore genes (hour 2 to 3). This conclusion is based on the following kinds of time course experiments in which 0.3 kb expression was monitored: (i) directly by the appearance of its 300-base RNA product (11); (ii) indirectly by measuring 0.3 kb-directed ,B-galactosidase synthesis in cells bearing the fusion on a multicopy plasmid (16); and (iii) indirectly by measuring fusion-directed galactosidase synthesis in cells bearing a single copy of 0.3 kb-lacZ in the chromosome (Fig. 1). Also, in preliminary work we have found that under sporulation conditions the time of induction of 0.3 kb-directed ,B-galactosidase synthesis in fusion-bearing cells is later by 1 to 2 h than the time of appearance in the same cells of glucose dehydrogenase, the production of the forespore-expressed gdh gene. Our results point to the possibility of greater heterogeneity in the timing of gene activation in the forespore than previously anticipated. In this regard, the forespore can be likened to the mother cell, for which a highly ordered sequence of gene activation has been documented. Whatever the basis for the delayed activation of 0.3 kb in the forespore, it may be noteworthy that its transcription in vegetative cells in response to IPTG-induced synthesis of arG is slower to ensue than transcription of sspA or gdh under the same circumstances. Possible interpretations of this 1-
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observation are that 0.3 kb induction depends on the attainment of a certain critical concentration of EoG, the induction of a regulatory gene under spoIIIG control, or both. Conceivably, one or more SASPs, some of which are DNAbinding proteins that package the forespore genome, contribute to the activation of 0.3 kb. We are grateful to L. Kroos for helpful advice and guidance. We thank P. Stragier for making his spoIIIG mutant and spac-spoIIIG fusion available before publication. This work was supported by Public Health Service grants GM18568 to R.L. and GM19698 to P.S. from the National Institutes of Health. LITERATURE CITED 1. Errington, J., and J. Mandelstam. 1986. Use of a lacZ fusion to determine the dependence pattern and the spore compartment expression of sporulation operon spoVA in spo mutants of Bacillus subtilis. J. Gen. Microbiol. 132:2977-2985. 2. Errington, J., and J. Mandelstam. 1986. Use of a lacZ fusion to determine the dependence pattern of sporulation operon spoIIA in wild-type Bacillus subtilis and in spo mutants. J. Gen. Microbiol. 132:2987-2989. 3. Fliss, E. R., M. J. Connors, C. A. Loshon, E. Curiel-Quesada, B. Setlow, and P. Setlow. 1985. Small, acid-soluble spore proteins of Bacillus: products of a sporulation-specific, multigene family, p. 60-66. In J. A. Hoch and P. Setlow (ed.), Molecular biology of microbial differentiation. American Society for Microbiology, Washington, D.C. 4. Kroos, L., A. Kuspa, and D. Kaiser. 1986. A global analysis of developmentally regulated genes in Myxococcus xanthus. Dev. Biol. 117:252-266. 5. Kunkel, B., K. Sandman, S. Panzer, P. Youngman, and R. Losick. 1988. Promoter for a sporulation gene in the spoIVC locus and its use in studies of temporal and spatial control of gene expression. J. Bacteriol. 170:3513-1322. 6. Lencastre, H., and P. Piggot. 1979. Identification and different sites of expression for spo loci by transformation of Bacillus subtilis. J. Gen. Microbiol. 114:377-389. 7. Losick, R., and P. Youngman. 1984. Endospore formation in Bacillus subtilis, p. 63-88. In R. Losick and L. Shapiro (ed.),
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