Molecular genetics has revolutionized our ability to establish relationships between biological systems. Once the sequence of a gene is known, its homology with other genes can readily be established, and its predicted protein product can often be classified within a family with common biochemical activities. In signal transduction, the cast of characters includes membrane receptors, nucleotide binding proteins, protein kinases and phosphatases, and factors that regulate transcription. These components play important roles in cell differentiation and development. The first indication of this came from studies of genes that can be mutated to cause cancer(’). Over and over again, when the product of an oncogene was identified it turned out to be a signal transduction component. Recent studies indicate that genes associated with normal embryonic development encode the same sets of signal transduction components, and it is now generally accepted that cell growth and differentiation is controled by an interplay between hormonal factors, surface receptors, and intracellular signal transduction pathways that regulate gene expression. The involvement of signal transduction components in cell differentiation and growth control is true for bacterial as well as eukaryotic species. Recent advances have established B. subtilis as one of the best model systems for understanding the role of signal transduction in development. B. subtilis may provide the first instance where cell differentiation can be described in purely molecular terms. An understanding in depth, of the underlying mechanism has only been achieved, however, within the past year. The most recent finding has been the identification of a new receptor kinase, designated ComP(*). Over the past few decades a complex genetic picture of endospore formation and late growth development in B. subtilis has been established. This huge collection of information on the behavior of hundreds of defined mutant variants is suddenly becoming explicable in molecular terms. An excellent overview of this field has been provided in a collection of reviews issuing from the 10th International Spores Conference(3). B. subtifis normally lives in the soil where nutrients tend to be limited and conditions tend to fluctuate widely. Growth in this environment is generally limited by nutrient availability. Under these conditions some
cells divide with an asymmetrically placed septum, the larger daughter then proceeding to engulf its smaller sibling, which then becomes essentially the nucleus of a new cell that goes on to become a heat resistant spore. Spore formation is only one of the differentiated states within the B. subtifis repertoir. There is, for instance, an intermediate state called ‘competence’. Competent cells begin to differentiate by becoming smaller and less dense. They then enter a pre-sexual phase where they can take up exogenous DNA, i.e. they become transformation competent. A molecular genetic analysis of differentiation in B. subtifis has identified a network of interacting signal transduction proteins. There are, to date, four receptor/kinase proteins (ComP, SpoIIJ, DegS, and PhoR), that interact with five response regulators (ComA, SpoOA, SpoOF, DegU, and PhoP). The regulators are phosphoactivated switching elements that function in conjunction with the kinases to control gene exp r e ~ s i o n ( ~An ) . additional level of regulation is provided by sigma factors that act as variable promoter recognition subunits of RNA polymerase holoenzyme(5). These families of proteins (receptor/ kinases, response regulators, activator/repressors, and RNA polymerase variants) function by interacting with environmental signals, with one another, and finally with enhancer and promoter elements in the genome. Since the regulatory components modulate the expression of their own genes, there can be a programmed progression from one regulatory state to another. Only rapidly growing vegetative cells and spores appear to represent metastable states where environmental signals (nutrient deprivation or nutrient supply) are needed to trigger further developmental transitions. The receptor kinase, ComP, promotes the establishment of competence. The stimulus that interacts with the membrane receptor domain of ComP remains to be determined. One possibility is that ComP senses the level of a crucial nutrient. Alternatively, the protein may function as a pheromone receptor, providing an input for information from neighboring cells. This possibility is particularly intriguing in view of the fact that the transmembrane portions of the ComP protein appear to have a topology analogous to fungal sex factor receptors such as the Ste2 and Ste3 proteins in Saccharomyces cerevisiae(@. The crosstalk that has generally been observed between different kinases and response reg~lators(’9~) may function in B. subtilis to produce specific patterns of gene expression at specific times during development. The ComP kinase regulates at least two response regulators, ComA and S ~ O O A ( ~ComA ). plays an important role in the development of competence, while SpoOA facilitates the transition from vegetative growth to any of several differentiated states including both competence and sporulation. SpoOA is regulated by other kinases besides ComP. The most important among these is the SpoIIJ kinase(’.’’). Strains lacking both ComP and SpoIIJ are much more deficient in
sporulation than strains lacking either ComP or SpoIIJ alone(2). On the other hand, SpoIIJ does not seem to play a direct role in the regulation of ComA. SpoIIJ’s effects on competence appear to be primarily routed through SpoOA. Low level SpoOA activation favors competence by allowing some expression of a transcriptional regulator called AbrB that facilitates the expression of competence genes. High levels of SpoOA activation repress ubrB to levels that are insufficient for the expression of competence genes. Because AbrB also acts to repress the synthesis of a sigma factor, SpoOH, that is essential for sporulation, the overall effect is to push cells beyond competence into the sporulation differentiation pathway. Another transcriptional regulator, called Sin, also plays an important role in this transition, acting as a positive regulator for competence, and as a negative regulator of sporulation. Sin also plays a critical role in the development of motility, which in B. subtifis represents another differentiated phenotype, distinct from competence and sporulation. The regulatory network that mediates development in B. subtilis gives some indication of the enormous complexity we should expect to encounter in analogous systems in eukaryotic species. Undoubtedly, the few signal transduction components that have been identified among developmental genes in organisms such as Cuenorhabditis and Drosophila represent the tip of an enormous iceberg. Understanding cell differentiation at a molecular level will necessitate new formalisms for dealing with highly interconnected parallel signal transduction pathways. Research on relatively simple sys-
tems such as B. subtilis will undoubtedly lead the way in the formulation of this new science.
References 1 BISHOP.J. M. (1987). The molecular genetics of cancer. Science235.305-311. 2 WEINRAUCH, Y., PENCHEV, R.,DLJBNAU, E.. SMITH,I. A N D DUBNAU. D. (1990). A Bacillus subtilis regulatory gene product for genetic competence and sporulation resembles sensor protein members of the bacterial two-component signal-transduction systems. Genes Dev., (in press). 3 SMITH.I.. SLEPECKY, R. A. AND SETLOW.P. (1989). Regulation ofprocuryoric developmenr: srrucrural and functional analysis of bacterial sporularion and germinarion. American Society of Microbiology. Washington. D.C. 4 STOCK, J. B.. STOCK, A . M. A N D MOTTONEN. J. M. (1990). Signal transduction in bacteria. Nature 344, 395-400. 5 LOSICK. R. A N D PERO,J. (1981). Cascades of sigma factors. Cell 25,582-584. 6 CROSS,F.. HARTWELL, L. H., JACKSON. C. A N D KONOPKA. J. B. (1988). Conjugation in Saccharomyces cerevisiae. Ann. Rev. Cell Biol. 4. 429-457. 7 NINFA,A. J . , NINFA, E. G.. LUPAS.A , . STOCK.A , . MACASANIK. B. AND STOCK,J. (1988). Crosstalk between bacterial chemotaxis signal transduction proteins and the regulators of transcription of the Ntr regulon: evidence that nitrogen assimilation and chemotaxis are controlled by a common phosphotransfer mechanism. Proc. Narl. Acad. Sci. U.S.A. 85, 5492-5496. 8 ICO. M. M., NINFA,A. J.. STOCK,J. B. A N D SILHAVY, T. J . (1989). Phosphorylation and dephosphorylation of a bacterial activator by a transmembrane receptor. Genes Dev. 3, 598-605. 9 ANTONIEWSKI, C . , SAVELLI, B . AND STRACIER. P. (1990). The S p O / l J gene, which regulates early developmental steps in Bucillus suhtilis, belongs to a class of environmentally responsive genes. J. Bacteriol. 172, 86-93. 10 PERECO.M.. COLE,S. P.. BURBUI-YS. D.. TRACH.K. A N D HOCH,J. A . (1999). Characterization of the gene for a protein kinase which phosphorylates the sporulation-regulatory proteins SpoOA and SpoOF of Bacillus subrih. J . Bacteriol. 171, 6187-6196.
Jeffry B. Stock is at the Lewis Thomas Laboratory, Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014, USA.
cells divide with an asymmetrically placed septum, the larger daughter then proceeding to engulf its smaller sibling, which then becomes essentially the nucleus of a new cell that goes on to become a heat resistant spore. Spore formation is only one of the differentiated states within the B. subtifis repertoir. There is, for instance, an intermediate state called ‘competence’. Competent cells begin to differentiate by becoming smaller and less dense. They then enter a pre-sexual phase where they can take up exogenous DNA, i.e. they become transformation competent. A molecular genetic analysis of differentiation in B. subtifis has identified a network of interacting signal transduction proteins. There are, to date, four receptor/kinase proteins (ComP, SpoIIJ, DegS, and PhoR), that interact with five response regulators (ComA, SpoOA, SpoOF, DegU, and PhoP). The regulators are phosphoactivated switching elements that function in conjunction with the kinases to control gene exp r e ~ s i o n ( ~An ) . additional level of regulation is provided by sigma factors that act as variable promoter recognition subunits of RNA polymerase holoenzyme(5). These families of proteins (receptor/ kinases, response regulators, activator/repressors, and RNA polymerase variants) function by interacting with environmental signals, with one another, and finally with enhancer and promoter elements in the genome. Since the regulatory components modulate the expression of their own genes, there can be a programmed progression from one regulatory state to another. Only rapidly growing vegetative cells and spores appear to represent metastable states where environmental signals (nutrient deprivation or nutrient supply) are needed to trigger further developmental transitions. The receptor kinase, ComP, promotes the establishment of competence. The stimulus that interacts with the membrane receptor domain of ComP remains to be determined. One possibility is that ComP senses the level of a crucial nutrient. Alternatively, the protein may function as a pheromone receptor, providing an input for information from neighboring cells. This possibility is particularly intriguing in view of the fact that the transmembrane portions of the ComP protein appear to have a topology analogous to fungal sex factor receptors such as the Ste2 and Ste3 proteins in Saccharomyces cerevisiae(@. The crosstalk that has generally been observed between different kinases and response reg~lators(’9~) may function in B. subtilis to produce specific patterns of gene expression at specific times during development. The ComP kinase regulates at least two response regulators, ComA and S ~ O O A ( ~ComA ). plays an important role in the development of competence, while SpoOA facilitates the transition from vegetative growth to any of several differentiated states including both competence and sporulation. SpoOA is regulated by other kinases besides ComP. The most important among these is the SpoIIJ kinase(’.’’). Strains lacking both ComP and SpoIIJ are much more deficient in
sporulation than strains lacking either ComP or SpoIIJ alone(2). On the other hand, SpoIIJ does not seem to play a direct role in the regulation of ComA. SpoIIJ’s effects on competence appear to be primarily routed through SpoOA. Low level SpoOA activation favors competence by allowing some expression of a transcriptional regulator called AbrB that facilitates the expression of competence genes. High levels of SpoOA activation repress ubrB to levels that are insufficient for the expression of competence genes. Because AbrB also acts to repress the synthesis of a sigma factor, SpoOH, that is essential for sporulation, the overall effect is to push cells beyond competence into the sporulation differentiation pathway. Another transcriptional regulator, called Sin, also plays an important role in this transition, acting as a positive regulator for competence, and as a negative regulator of sporulation. Sin also plays a critical role in the development of motility, which in B. subtifis represents another differentiated phenotype, distinct from competence and sporulation. The regulatory network that mediates development in B. subtilis gives some indication of the enormous complexity we should expect to encounter in analogous systems in eukaryotic species. Undoubtedly, the few signal transduction components that have been identified among developmental genes in organisms such as Cuenorhabditis and Drosophila represent the tip of an enormous iceberg. Understanding cell differentiation at a molecular level will necessitate new formalisms for dealing with highly interconnected parallel signal transduction pathways. Research on relatively simple sys-
tems such as B. subtilis will undoubtedly lead the way in the formulation of this new science.
References 1 BISHOP.J. M. (1987). The molecular genetics of cancer. Science235.305-311. 2 WEINRAUCH, Y., PENCHEV, R.,DLJBNAU, E.. SMITH,I. A N D DUBNAU. D. (1990). A Bacillus subtilis regulatory gene product for genetic competence and sporulation resembles sensor protein members of the bacterial two-component signal-transduction systems. Genes Dev., (in press). 3 SMITH.I.. SLEPECKY, R. A. AND SETLOW.P. (1989). Regulation ofprocuryoric developmenr: srrucrural and functional analysis of bacterial sporularion and germinarion. American Society of Microbiology. Washington. D.C. 4 STOCK, J. B.. STOCK, A . M. A N D MOTTONEN. J. M. (1990). Signal transduction in bacteria. Nature 344, 395-400. 5 LOSICK. R. A N D PERO,J. (1981). Cascades of sigma factors. Cell 25,582-584. 6 CROSS,F.. HARTWELL, L. H., JACKSON. C. A N D KONOPKA. J. B. (1988). Conjugation in Saccharomyces cerevisiae. Ann. Rev. Cell Biol. 4. 429-457. 7 NINFA,A. J . , NINFA, E. G.. LUPAS.A , . STOCK.A , . MACASANIK. B. AND STOCK,J. (1988). Crosstalk between bacterial chemotaxis signal transduction proteins and the regulators of transcription of the Ntr regulon: evidence that nitrogen assimilation and chemotaxis are controlled by a common phosphotransfer mechanism. Proc. Narl. Acad. Sci. U.S.A. 85, 5492-5496. 8 ICO. M. M., NINFA,A. J.. STOCK,J. B. A N D SILHAVY, T. J . (1989). Phosphorylation and dephosphorylation of a bacterial activator by a transmembrane receptor. Genes Dev. 3, 598-605. 9 ANTONIEWSKI, C . , SAVELLI, B . AND STRACIER. P. (1990). The S p O / l J gene, which regulates early developmental steps in Bucillus suhtilis, belongs to a class of environmentally responsive genes. J. Bacteriol. 172, 86-93. 10 PERECO.M.. COLE,S. P.. BURBUI-YS. D.. TRACH.K. A N D HOCH,J. A . (1999). Characterization of the gene for a protein kinase which phosphorylates the sporulation-regulatory proteins SpoOA and SpoOF of Bacillus subrih. J . Bacteriol. 171, 6187-6196.
Jeffry B. Stock is at the Lewis Thomas Laboratory, Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014, USA.
