[]~EVIEWS
I n response to nutrient limitation, the Gram-positive bacterium Bacillus subtilis initiates a developmental program that culminates in the formation and release of a highly resistant, dormant endospore >3, Development of the endospore takes place in a sporangium consisting of two cellular compartments known as the mother cell and the forespore (Fig. 1). Gene expression in the two compartments of the sporangium is differentially regulated, with each compartment expressing its own set of developmental genes. The two separate programs of gene expression are not completely independent, however. Recent genetic, biochemical and molecular evidence indicates that there is a mode of communication between the mother cell and forespore that serves to coordinate gene expression in the two cells. (See Box 1 for a summary of the range of experimental approaches available for studying developmentally regulated gene expression in B. subtilis.)
Cellular differentiation and gene regulation during sporulation
Compartmentalizedgene expression during sp0rulati0n in Bacillus subtilis BARBARAKUNKEL Two important features of endospore development in Bacillus subtilis -the compartmentalization of mother cell gene expression and the coordination of mother cell gene expression with forespore development - are governed by the highly regulated expression of the sigK gene, which encodes the mother cell.specific RNApolymerase efactor eK. Compartmentalized expression of sigK is associated both with a chromosomal DNA rearrangement and with the restriction of sigK transcription to the mother cell A third mode of sigK regulation, which occurs at the level of activation of the sigK gene product by proteolytic processin& serves to couple gene expression between the mother cell andforespore compartments of the developing sporangiunt
Once triggered by nutrient limitation, sporulation in B. subtilis proceeds through a series of seven welldefined morphological stages that result in lysis of the sporangium and release of the mature endospore (Fig. 2) >3. One of the key features of this develop- of the mature spore, the cot genesS, which encode mental process is the formation of an asymmetrically the polypeptide components of the spore coat, and positioned septum (at stage II) which divides the the ssp genes 6, which encode a family of small, acidsporulating cell into two compartments, the mother soluble proteins located in the core of the spore. cell and the forespore. The two compartments presum- Genetic and molecular studies on the regulation of ably inherit identical chromosomes from the last round many spo, ger, cot and ssp genes indicate that of vegetative DNA replication, but then follow endospore development is correlated with a highly divergent pathways of cellular differentiation. The ordered program of gene expression in which sets of forespore develops into the mature spore, whereas coordinately regulated genes arc activated in a sequenthe mother cell contributes to the development o f t h e tial and compartment-specific manner throughout the spore and is then discarded when maturation of differentiation cycleT. the endospore is complete. Thus, the forespore can be This pattern of temporally and spatially regulated thought of as a germ-line cell, as it is the spore that gene expression is regulated primarily at the level of ultimately germinates and gives rise to subsequent transcription, and is governed, in part, by the sequenprogeny. The mother cell, on the other hand, is a tial appearance of different RNA polymerase terminally differentiating cell; both it and its chromo- (s factors~ 9; ~ factors are subunits of eubacterial RNA some are discarded by lysis when maturation of the polymerases that bind to core RNA polymerase and endospore is complete. confer on the holoenzyme the ability to utilize a speEndospore development requires the expression of cific class of promoters. In general, promoters rccog more than 80 different developmental genes3. Those nized by a given form of RNA polymerase hoh)enzyme genes that are specifically required for endospore formaII II tion are called spo genes~. For the most part, spo genes have been identified and defined by mutations that impair spore formation, but have little or no effect on vegetative growth. Such .si0o mutations generally block development at a definite stage in sporulation and are classified accordingly. Other genes that are involved in endospore development inFIGH clude the ger genes 4, whose Electron micrograph of a sporulating B. subtilis cell at an intermediate stage of sporulation. products are required for the The sporangium consists of two cellular compartments: a larger mother cell and a smaller forespore. Scale bar, 0.2 gm. Photograph kindly provided by A. Ryter {Pasteur Institute. Paris! normal germination properties TIG MAY1991 VOI.. 7 -','O. 5 g 19~)1 Elsex icl S c i e n c e Public, hers Lid (I :K/ 0 1 ( ~ - 9479 91 $02.00
m
[]~EVIEWS
Box 1. Studying developmentally regulated gene expression in Ig su&tilis A combination of molecular genetic and biochemical ~ otis has been used to study gene expression during endospore development in B. subt///s. Central to these studies has ~ n the cloning of developmer~l genes and the use of these cloned DNAs to monitor the expression of individual genes and to identify genetic and biochemical amenable to molecular genetic analysis, as DNA that is introducx'd into tim cell via DNA-mediated transformation is integrated into the genome by homologous recombination. Thus, genes can be mutated or otherwise manipulated in vitro and then ~ to the chromosome for a n a ~ of their activity in vf~. Gene fusions to the reporter gene lacZ have been extensively in the analysis of the temporal and spatial expression patterns of a ~ number of developmental genes, and for determining how the activation of such genes d e ~ on the products of other developmental genes~a-14,*6-18,a0;~,33,M. Such studies have made it possible to begin to the of gene expression during sporulation in terms of a regulatory network 7, have ted to the of regulatory genes (e.g. spollID 28), and have provided genetic evidence for the coupling of ~ r cell and forespore gene expression16a 7. Mutations that disrupt the dependence of ~er cell g~ne expression on fore.spore development have also been identified through the use of lacZ fusions3~, The ability to introduce genes that have been mutated or otherwise modified in vitro back into the chromosome by homologous recombination has made it possible to study the function of developmental genes in v/vo. Null alleles of genes can be made. for example, by transforming strains with nonrepticative plasmids that contain
internal restriction fragments from the gene of Lnterest20,24 or, alternatively, by directly s u b ~ t i n g the wild-type gene, by marker replacement, with deletion or insertion mutations constructed in v/fro5 20 24 3334 Genes that have been modified in vitro can also be introduced into the chromosome at unlinked sites (through the use of product in the sigK DNA rearrangement. Introduction of a cloned copy of the rearranged sigK gene into the chromosome of spolVCA mutant cells restored the capacity of the mutant cetb to spomlate, thus indicating that the sporVCA germ product has no essential function in sporulation other than to catalyse the DNA rearrangement 26. Site-directed mutagenesis is also a powerfid tool for directly analysing regulatory mechanisms governing gene expression. Replacement of the interrupted ~K-coding sequence in the Chromosome of vegetative bacteria with a cloned copy of the intact s/gK gene revealed that the sigK DNA rearrangement is not required for the spatial regutruncated version lacking the pro-amino acid coding ~ q u e ~ e was used to demonstrate that the dependence of aK-directed gene expression in the mother cell on forespore development is mediated at the level of pro-~ K processing37. Biochemical factors that govern the regulation of gene for transcription studies in vitro. Such studies have led to the identification of developmentally important transcription factors including 0
I n response to nutrient limitation, the Gram-positive bacterium Bacillus subtilis initiates a developmental program that culminates in the formation and release of a highly resistant, dormant endospore >3, Development of the endospore takes place in a sporangium consisting of two cellular compartments known as the mother cell and the forespore (Fig. 1). Gene expression in the two compartments of the sporangium is differentially regulated, with each compartment expressing its own set of developmental genes. The two separate programs of gene expression are not completely independent, however. Recent genetic, biochemical and molecular evidence indicates that there is a mode of communication between the mother cell and forespore that serves to coordinate gene expression in the two cells. (See Box 1 for a summary of the range of experimental approaches available for studying developmentally regulated gene expression in B. subtilis.)
Cellular differentiation and gene regulation during sporulation
Compartmentalizedgene expression during sp0rulati0n in Bacillus subtilis BARBARAKUNKEL Two important features of endospore development in Bacillus subtilis -the compartmentalization of mother cell gene expression and the coordination of mother cell gene expression with forespore development - are governed by the highly regulated expression of the sigK gene, which encodes the mother cell.specific RNApolymerase efactor eK. Compartmentalized expression of sigK is associated both with a chromosomal DNA rearrangement and with the restriction of sigK transcription to the mother cell A third mode of sigK regulation, which occurs at the level of activation of the sigK gene product by proteolytic processin& serves to couple gene expression between the mother cell andforespore compartments of the developing sporangiunt
Once triggered by nutrient limitation, sporulation in B. subtilis proceeds through a series of seven welldefined morphological stages that result in lysis of the sporangium and release of the mature endospore (Fig. 2) >3. One of the key features of this develop- of the mature spore, the cot genesS, which encode mental process is the formation of an asymmetrically the polypeptide components of the spore coat, and positioned septum (at stage II) which divides the the ssp genes 6, which encode a family of small, acidsporulating cell into two compartments, the mother soluble proteins located in the core of the spore. cell and the forespore. The two compartments presum- Genetic and molecular studies on the regulation of ably inherit identical chromosomes from the last round many spo, ger, cot and ssp genes indicate that of vegetative DNA replication, but then follow endospore development is correlated with a highly divergent pathways of cellular differentiation. The ordered program of gene expression in which sets of forespore develops into the mature spore, whereas coordinately regulated genes arc activated in a sequenthe mother cell contributes to the development o f t h e tial and compartment-specific manner throughout the spore and is then discarded when maturation of differentiation cycleT. the endospore is complete. Thus, the forespore can be This pattern of temporally and spatially regulated thought of as a germ-line cell, as it is the spore that gene expression is regulated primarily at the level of ultimately germinates and gives rise to subsequent transcription, and is governed, in part, by the sequenprogeny. The mother cell, on the other hand, is a tial appearance of different RNA polymerase terminally differentiating cell; both it and its chromo- (s factors~ 9; ~ factors are subunits of eubacterial RNA some are discarded by lysis when maturation of the polymerases that bind to core RNA polymerase and endospore is complete. confer on the holoenzyme the ability to utilize a speEndospore development requires the expression of cific class of promoters. In general, promoters rccog more than 80 different developmental genes3. Those nized by a given form of RNA polymerase hoh)enzyme genes that are specifically required for endospore formaII II tion are called spo genes~. For the most part, spo genes have been identified and defined by mutations that impair spore formation, but have little or no effect on vegetative growth. Such .si0o mutations generally block development at a definite stage in sporulation and are classified accordingly. Other genes that are involved in endospore development inFIGH clude the ger genes 4, whose Electron micrograph of a sporulating B. subtilis cell at an intermediate stage of sporulation. products are required for the The sporangium consists of two cellular compartments: a larger mother cell and a smaller forespore. Scale bar, 0.2 gm. Photograph kindly provided by A. Ryter {Pasteur Institute. Paris! normal germination properties TIG MAY1991 VOI.. 7 -','O. 5 g 19~)1 Elsex icl S c i e n c e Public, hers Lid (I :K/ 0 1 ( ~ - 9479 91 $02.00
m
[]~EVIEWS
Box 1. Studying developmentally regulated gene expression in Ig su&tilis A combination of molecular genetic and biochemical ~ otis has been used to study gene expression during endospore development in B. subt///s. Central to these studies has ~ n the cloning of developmer~l genes and the use of these cloned DNAs to monitor the expression of individual genes and to identify genetic and biochemical amenable to molecular genetic analysis, as DNA that is introducx'd into tim cell via DNA-mediated transformation is integrated into the genome by homologous recombination. Thus, genes can be mutated or otherwise manipulated in vitro and then ~ to the chromosome for a n a ~ of their activity in vf~. Gene fusions to the reporter gene lacZ have been extensively in the analysis of the temporal and spatial expression patterns of a ~ number of developmental genes, and for determining how the activation of such genes d e ~ on the products of other developmental genes~a-14,*6-18,a0;~,33,M. Such studies have made it possible to begin to the of gene expression during sporulation in terms of a regulatory network 7, have ted to the of regulatory genes (e.g. spollID 28), and have provided genetic evidence for the coupling of ~ r cell and forespore gene expression16a 7. Mutations that disrupt the dependence of ~er cell g~ne expression on fore.spore development have also been identified through the use of lacZ fusions3~, The ability to introduce genes that have been mutated or otherwise modified in vitro back into the chromosome by homologous recombination has made it possible to study the function of developmental genes in v/vo. Null alleles of genes can be made. for example, by transforming strains with nonrepticative plasmids that contain
internal restriction fragments from the gene of Lnterest20,24 or, alternatively, by directly s u b ~ t i n g the wild-type gene, by marker replacement, with deletion or insertion mutations constructed in v/fro5 20 24 3334 Genes that have been modified in vitro can also be introduced into the chromosome at unlinked sites (through the use of product in the sigK DNA rearrangement. Introduction of a cloned copy of the rearranged sigK gene into the chromosome of spolVCA mutant cells restored the capacity of the mutant cetb to spomlate, thus indicating that the sporVCA germ product has no essential function in sporulation other than to catalyse the DNA rearrangement 26. Site-directed mutagenesis is also a powerfid tool for directly analysing regulatory mechanisms governing gene expression. Replacement of the interrupted ~K-coding sequence in the Chromosome of vegetative bacteria with a cloned copy of the intact s/gK gene revealed that the sigK DNA rearrangement is not required for the spatial regutruncated version lacking the pro-amino acid coding ~ q u e ~ e was used to demonstrate that the dependence of aK-directed gene expression in the mother cell on forespore development is mediated at the level of pro-~ K processing37. Biochemical factors that govern the regulation of gene for transcription studies in vitro. Such studies have led to the identification of developmentally important transcription factors including 0
