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A Love Affair with Bacillus subtilis

2014; Elsevier BV; Volume: 290; Issue: 5 Linguagem: Inglês

10.1074/jbc.x114.634808

1083-351X

Richard Losick,

RNA and protein synthesis mechanisms

My career in science was launched when I was an undergraduate at Princeton University and reinforced by graduate training at the Massachusetts Institute of Technology. However, it was only after I moved to Harvard University as a junior fellow that my affections were captured by a seemingly mundane soil bacterium. What Bacillus subtilis offered was endless fascinating biological problems (alternative sigma factors, sporulation, swarming, biofilm formation, stochastic cell fate switching) embedded in a uniquely powerful genetic system. Along the way, my career in science became inseparably interwoven with teaching and mentoring, which proved to be as rewarding as the thrill of discovery. My career in science was launched when I was an undergraduate at Princeton University and reinforced by graduate training at the Massachusetts Institute of Technology. However, it was only after I moved to Harvard University as a junior fellow that my affections were captured by a seemingly mundane soil bacterium. What Bacillus subtilis offered was endless fascinating biological problems (alternative sigma factors, sporulation, swarming, biofilm formation, stochastic cell fate switching) embedded in a uniquely powerful genetic system. Along the way, my career in science became inseparably interwoven with teaching and mentoring, which proved to be as rewarding as the thrill of discovery. I was a junior at Princeton University (1963–1964) when I walked into the Frick Chemical Laboratory office of a newly arrived professor of chemistry, Charles Gilvarg. I was a chemistry major and knew I wanted to be a scientist for as long as I could remember, but I understood little about what that meant. There was a lot of buzz about this brilliant biochemist from New York University, and I was hoping I could land a position on his research team. I remember how nervous I was for my interview, but fortunately, Gilvarg did accept me into his laboratory. He was an intimidating presence but, as I came to appreciate, a wise and dedicated mentor who launched me on my career in science. In what culminated in my senior thesis, I was charged with studying how α-acetylation affected the use of lysine oligopeptides by Escherichia coli. I learned that following α-acetylation, oligopeptides were poorly taken up by E. coli, derepressing the production of a reductase in the lysine biosynthetic pathway. This was by no means an earth-shattering discovery, but I was thrilled to have learned something that no one else on the planet had known before. It is a lesson I have tried to pass on to generations of undergraduates: few things in college are more rewarding than engaging in research and discovering something, however modest, that no one knew before. I learned another lesson when I submitted my senior thesis. Gilvarg went through it with a fine-toothed comb, sparing no opportunity to criticize unjustified conclusions. In fact, I was delighted and flattered that he cared so much about what I had done and written that he took the time to give me detailed feedback. Later, he submitted a paper based on my senior thesis to the Journal of Biological Chemistry, whose legendary editor John T. Edsall would later become my colleague at Harvard University. When a review arrived, Gilvarg wrote in the margin, “This shows the referee really read, understood and appreciated the paper.” It was the same lesson I had taken away from his critique of my thesis. “Effect of α-acetylation on utilization of lysine oligopeptides in Escherichia coli” was my first publication (1Losick R. Gilvarg C. Effect of α-acetylation on utilization of lysine oligopeptides in Escherichia coli.J. Biol. Chem. 1966; 241: 2340-2346Abstract Full Text PDF PubMed Google Scholar), and it appeared in the very journal that half a century later would invite me to write this Reflections article. It was not, however, with E. coli that I had a love affair, nor was it with Salmonella (Salmonella anatum) despite the intense relationship that S. anatum and I developed during my three years as a graduate student in Phil Robbins' laboratory at the Massachusetts Institute of Technology (MIT; Gilvarg had urged me to do my Ph.D. research with Robbins). It was an exciting time to be in the Robbins laboratory; a highlight of that period was the discovery of the C55 polyisoprenoid lipid carrier involved in O-antigen biosynthesis. (Interestingly, the same lipid carrier was simultaneously shown to be required for peptidoglycan biosynthesis by my future mentor and friend Jack Strominger.) My own project focused on an S. anatum phage (ϵ15) that uses O-antigen with α-glycosyl linkages as a receptor (2Losick R. Robbins P.W. Mechanism of ϵ15 conversion studied with a bacterial mutant.J. Mol. Biol. 1967; 30: 445-455Crossref PubMed Scopus (34) Google Scholar). Cleverly, upon infection and lysogeny, ϵ15 converts the O-antigen to β-glycosyl linkages, thereby blocking its brethren from infecting the same cell. I provided evidence that the phage replaces the α-polymerizing enzyme of its host with a phage-borne β-polymerase. It turned out that another, even cleverer phage (ϵ34) uses the β-linked O-antigen as a receptor, a cat-and-mouse tale that Robbins and I recounted in a Scientific American article (3Losick R. Robbins P.W. The receptor site for a bacterial virus.Sci. Am. 1969; 221: 120-124Crossref PubMed Scopus (11) Google Scholar). Robbins was an inspirational mentor who liked nothing more than doing experiments with his own hands, working side by side with members of his own laboratory (including, at the time, the future independent scientists Dennis Bray at the University of Cambridge, Andrew Wright at Tufts University Medical School, and Henry Wu at the Uniformed Services University of the Health Sciences). As the end of my Ph.D. studies approached, I decided I wanted to pursue postdoctoral research with Julius Adler at the University of Wisconsin. I was mesmerized by his experiments on chemotaxis and by the fact that it was possible to study behavior in E. coli. However, my intention to move to the University of Wisconsin was upended when Salvador Luria nominated me to be a junior fellow in the Harvard Society of Fellows. Luria was one of my heroes at MIT and an inspiring presence and teacher. (He invited graduate students to his home to discuss the writings of the French existentialist Albert Camus!) I think the fix was in as Senior Fellow Jim Watson had been Luria's first student and rightly or wrongly trusted his judgment. Watson told me that Jack Strominger was coming to Harvard and suggested that, given my experience with cell-surface biochemistry, I ask him for space in his new laboratory down the hall. Strominger agreed, and I soon found myself on the third floor of the Biological Laboratories, with Jim Watson and Wally Gilbert and their joint laboratory (with Klaus Weber) on one side and George Wald on the other. This was an extraordinary location and an intensely exciting time, as the third floor was home to nine individuals who had won or would later win a Nobel Prize. In addition to Wald, Watson, and Gilbert, future Nobel Prize-winning inhabitants of the third floor included the undergraduate Marty Chalfie; graduate students Bob Horvitz, Mario Capecchi, and Craig Mello; the postdoctoral fellow Rich Roberts; and Roger Kornberg doing a one-year stint as a junior fellow. (Another future Nobelist, Sid Altman, was on the fourth floor.) To briefly recount two stories from this era, Rich Roberts, then, like me, in the Strominger laboratory, set out to sequence a tRNA involved in bacterial cell wall synthesis, becoming one of the few individuals in the Boston area in the early 1970s who could sequence RNA. This made him attractive to Jim Watson, who invited Roberts to join him at the Cold Spring Harbor Laboratory (CSHL), where Watson was moving to become the director. At CSHL, Roberts made the startling finding that the 5′-ends of adenovirus (late) mRNAs were all the same; splicing had joined a common 5′-sequence to the coding sequence for different virion proteins (4Chow L.T. Gelinas R.E. Broker T.R. Roberts R.J. An amazing sequence arrangement at the 5′ ends of adenovirus 2 messenger RNA.Cell. 1977; 12: 1-8Abstract Full Text PDF PubMed Scopus (641) Google Scholar). This extraordinary discovery earned Roberts a Nobel Prize, and it all began with his attempting to sequence a bacterial tRNA! Fast forward to the late 1980s, when graduate student Craig Mello was studying extrachromosomal DNA arrays in Caenorhabditis elegans with assistant professors Victor Ambros and Dan Stinchcomb. At the time, Ambros was discovering the first microRNA (5Lee R.C. Feinbaum R.L. Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.Cell. 1993; 75: 843-854Abstract Full Text PDF PubMed Scopus (8535) Google Scholar), a remarkable irony given that microRNAs act in the same manner as the siRNAs that Mello (and Andy Fire) would later discover and develop into a Nobel Prize-winning tool for silencing genes in the nematode (6Fire A. Xu S.-Q. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature. 1998; 391: 806-811Crossref PubMed Scopus (10807) Google Scholar). I came to Harvard with the intention of studying the interaction of phage T4 with the E. coli membrane. I was even awarded a National Science Foundation grant to pursue this project, but geography and history altered everything! Down the hall, Dick Burgess and Andrew Travers had discovered that in E. coli, a subunit of RNA polymerase (called sigma) dictated promoter recognition. This suggested that there might be alternative sigma factors driving the expression of alternative gene sets (7Burgess R.R. Travers A.A. Dunn J.J. Bautz E.K. Factor stimulating transcription by RNA polymerase.Nature. 1969; 221: 43-46Crossref PubMed Scopus (625) Google Scholar). Meanwhile, my friend and dorm mate from Princeton and housemate in Cambridge, Linc Sonenshein, who was also a classmate at MIT, was studying Φe, a phage of the endospore-forming bacterium B. subtilis, in the Luria laboratory (Fig. 1). Piggybacking on the robust dormancy properties of the spore, Φe becomes trapped in the developing spore, shielding it from harsh environmental conditions, only to emerge when its unfortunate host attempts to return to vegetative growth upon germination. Sonenshein and I continued to interact frequently after my move to Harvard, regaling each other with stories about B. subtilis and RNA polymerase, respectively. Sonenshein was the matchmaker who started my romance with B. subtilis. Putting two and two together, Sonenshein and I postulated that the elaborate developmental process of endospore formation was an ideal candidate for a regulatory system that could be driven by alternative sigma factors. To begin to investigate this notion, we grew vegetative and sporulating cells of B. subtilis at MIT and took the centrifuge bottles with the cell pellets to Harvard's Biological Laboratories, where we would break the cells open and isolate RNA polymerase using the methods of Burgess et al. (7Burgess R.R. Travers A.A. Dunn J.J. Bautz E.K. Factor stimulating transcription by RNA polymerase.Nature. 1969; 221: 43-46Crossref PubMed Scopus (625) Google Scholar). (On one occasion, Matt Meselson saw us leaving the Biological Laboratories with the empty bottles and accused us of stealing Harvard equipment. We showed him Luria's name embossed on the metal bottles.) We were thrilled to be able to demonstrate that the template specificity of RNA polymerase is altered during the transition from vegetative growth to sporulation (8Losick R. Sonenshein A.L. Change in the template specificity of RNA polymerase during sporulation of Bacillus subtilis.Nature. 1969; 224: 35-37Crossref PubMed Scopus (0) Google Scholar). Sonenshein and I were right to focus on RNA polymerase, as became clear in the ensuing years, but our focus was premature. The discovery of alternative sigma factors had to await the invention of DNA cloning to provide templates for assaying promoter-specific transcription. Later, after joining the Harvard faculty, I was able to recruit outstanding graduate students, including Arno Greenleaf, Tom Linn, Rosalind Shorenstein, and Robert Tjian, who purified the housekeeping sigma factor σA from vegetative cells, found that σA was replaced by other RNA polymerase-binding proteins during sporulation, and demonstrated that sporulation involved an elaborate program of protein synthesis (9Linn T.G. Greenleaf A.L. Shorenstein R.G. Losick R. Loss of sigma activity of RNA polymerase of Bacillus subtilis during sporulation.Proc. Natl. Acad. Sci. U.S.A. 1973; 70: 1865-1869Crossref PubMed Google Scholar, 10Shorenstein R.G. Losick R. Purification and properties of the sigma subunit of ribonucleic acid polymerase from vegetative Bacillus subtilis.J. Biol. Chem. 1973; 248: 6163-6169Abstract Full Text PDF PubMed Google Scholar, 11Tjian R. Losick R. An immunological assay for the sigma subunit of RNA polymerase in extracts of vegetative and sporulating Bacillus subtilis.Proc. Natl. Acad. Sci. U.S.A. 1974; 71: 2872-2876Crossref PubMed Scopus (29) Google Scholar, 12Linn T. Losick R. The program of protein synthesis during sporulation in Bacillus subtilis.Cell. 1976; 8: 103-114Abstract Full Text PDF PubMed Google Scholar). The search for alternative sigma factors was successful because Watson's graduate student Jan Pero joined the project. (She also became my wife and life partner!) Jan had become an expert on gene regulation based on her studies of phage λ in the Watson laboratory. I told her about my collaboration with Sonenshein, whom we followed to the University of Paris-Sud in Orsay, France, where he was doing postdoctoral research with the pioneering sporulation geneticist Pierre Schaeffer (and where I met Patrick Stragier, who would later become a close collaborator) (Fig. 2). Upon our return to Cambridge, Pero decided to focus on the B. subtilis phage SP01, a close relative of Φe. Thanks to the work of Shunzo Okubo at the Osaka University Medical School, who kindly sent us key mutants from Japan, and of E. Peter Geiduschek at the University of Chicago and later at the University of California, San Diego, phage SP01 was known to exhibit a temporal pattern of gene expression under the control of phage regulatory genes (13Okubo S. Yanagida T. Fujita D.J. Olsson-Wilhelm B.M. The genetics of bacteriophage SPO1.Biken J. 1972; 2: 81-97Google Scholar, 14Fujita D.J. Ohlsson-Wilhelm B.M. Geiduschek E.P. Transcription during bacteriophage SPO1 development: mutations affecting the program of viral transcription.J. Mol. Biol. 1971; 57: 301-317Crossref PubMed Scopus (0) Google Scholar). With Jack Strominger championing my cause, I was at that point appointed an assistant professor in what was then the Department of Biology. Pero was joined on the SP01 project by my graduate students Tom Fox and Tjian (who had switched from sporulation to the phage). Pero and Fox discovered that the transcription of phage early genes was directed by the host's RNA polymerase containing the σA factor and that successive expression of phage middle and late genes was driven by alternative, phage-encoded sigma factors. One of the early genes (28) specified an alternative sigma factor specific for phage middle genes, two of which (33 and 34) reprogrammed core RNA polymerase to transcribe late genes. In one of the most breathtaking scientific periods of my career, Tjian and Pero reconstituted phage late transcription with the products of phage genes 33 and 34 (15Pero J. Tjian R. Nelson J. Losick R. In vitro transcription of a late class of phage SPO1 genes.Nature. 1975; 257: 248-251Crossref PubMed Google Scholar). Fox proved that RNA polymerase-associated phage proteins were the products of genes 28, 33, and 34 by use of nonsense suppression to generate gene products with altered isoelectric points (16Fox T.D. Losick R. Pero J. Regulatory gene 28 of bacteriophage SPO1 codes for a phage-induced subunit of RNA polymerase.J. Mol. Biol. 1976; 101: 427-433Crossref PubMed Google Scholar). These findings were the first direct demonstration in any organism of promoter-specific transcription directed by alternative sigma factors. Pero joined the faculty and continued the SP01 work in her own laboratory, where she discovered that phage promoters recognized by the phage-modified forms of RNA polymerase exhibited conserved sequences centered about 10 (−10) and 35 (−35) bp upstream of the transcription start site. These −10 and −35 sequences were distinct from the corresponding elements characteristic of promoters recognized by the σA-containing host RNA polymerase. This led to the proposal, which proved to be correct, that sigma factors work by directly contacting and recognizing cognate sequences in promoter −10 and −35 regions (17Losick R. Pero J. Cascades of sigma factors.Cell. 1981; 25: 582-584Abstract Full Text PDF PubMed Google Scholar). In parallel with the study of SP01, graduate student Steve Clark tackled another B. subtilis phage called PBS2. Instead of modifying the host RNA polymerase, PBS2 encodes its own multisubunit RNA polymerase (18Clark S. Losick R. Pero J. New RNA polymerase from Bacillus subtilis infected with phage PBS2.Nature. 1974; 252: 21-24Crossref PubMed Scopus (21) Google Scholar). The discovery of phage-encoded alternative sigma factors was a powerful incentive to return to the question of how B. subtilis turns on expression of different genes at different times during sporulation. The discovery of a family of stage-specific and compartment-specific sigma factors became the core of the genetic network controlling sporulation that my laboratory was to decipher. The development of recombinant DNA methods in the early to mid-1970s enabled my students Jacqueline Segall, Michelle Igo, and Frank Ollington and postdoctoral fellows Peter Zuber and Mike Stephens to clone DNA templates with individual bacterial promoters and use the cloned DNAs to study the regulation of individual genes (19Segall J. Losick R. Cloned Bacillus subtilis DNA containing a gene that is activated early during sporulation.Cell. 1977; 11: 751-761Abstract Full Text PDF PubMed Google Scholar, 20Ollington J.F. Haldenwang W.G. Huynh T.V. Losick R. Developmentally regulated transcription in a cloned segment of the Bacillus subtilis chromosome.J. Bacteriol. 1981; 147: 432-442Crossref PubMed Google Scholar, 21Ollington J.F. Losick R. A cloned gene that is turned on at an intermediate stage of spore formation in Bacillus subtilis.J. Bacteriol. 1981; 147: 443-451Crossref PubMed Google Scholar, 22Zuber P. Losick R. Use of a lacZ fusion to study the role of the spoO genes of Bacillus subtilis in developmental regulation.Cell. 1983; 35: 275-283Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 23Stephens M.A. Lang N. Sandman K. Losick R. A promoter whose utilization is temporally regulated during sporulation in Bacillus subtilis.J. Mol. Biol. 1984; 176: 333-348Crossref PubMed Scopus (12) Google Scholar). This made possible the discovery of the first alternative bacterial sigma factor (then called σ37 and later σB) in 1979 by my postdoctoral fellow Bill Haldenwang (24Haldenwang W.G. Losick R. A modified RNA polymerase transcribes a cloned gene under sporulation control in Bacillus subtilis.Nature. 1979; 282: 256-260Crossref PubMed Google Scholar, 25Haldenwang W.G. Losick R. Novel RNA polymerase sigma factor from Bacillus subtilis.Proc. Natl. Acad. Sci. U.S.A. 1980; 77: 7000-7004Crossref PubMed Google Scholar). The σB factor turned out not to be a sporulation sigma factor but rather a stress response sigma factor, as demonstrated in my laboratory by Craig Binnie, Mary Lampe, and Igo (26Binnie C. Lampe M. Losick R. Gene encoding the σ37 species of RNA polymerase sigma factor from Bacillus subtilis.Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 5943-5947Crossref PubMed Google Scholar, 27Igo M.M. Losick R. Regulation of a promoter that is utilized by minor forms of RNA polymerase holoenzyme in Bacillus subtilis.J. Mol. Biol. 1986; 191: 615-624Crossref PubMed Google Scholar, 28Igo M. Lampe M. Ray C. Schafer W. Moran Jr., C.P. Losick R. Genetic studies of a secondary RNA polymerase sigma factor in Bacillus subtilis.J. Bacteriol. 1987; 169: 3464-3469Crossref PubMed Google Scholar) and by Price and co-workers (29Duncan M.L. Kalman S.S. Thomas S.M. Price C.W. Gene encoding the 37,000-Dalton minor sigma factor of Bacillus subtilis RNA polymerase: isolation, nucleotide sequence, chromosomal locus and cryptic function.J. Bacteriol. 1987; 169: 771-778Crossref PubMed Google Scholar). Shortly after the discovery of σB in 1981, Haldenwang and Naomi Lang-Unnasch uncovered the first sporulation-specific sigma factor, then called σ29 and later called σE (30Haldenwang W.G. Lang N. Losick R. A sporulation-induced sigma-like regulatory protein from Bacillus subtilis.Cell. 1981; 23: 615-624Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 31Trempy J.E. Bonamy C. Szulmajster J. Haldenwang W.G. Bacillus subtilis σ factor σ29 is the product of the sporulation-essential gene spoIIG.Proc. Natl. Acad. Sci. U.S.A. 1985; 82: 4189-4192Crossref PubMed Google Scholar, 32Moran Jr., C.P. Lang N. Banner C.D. Haldenwang W.G. Losick R. Promoter for a developmentally regulated gene in Bacillus subtilis.Cell. 1981; 25: 783-791Abstract Full Text PDF PubMed Google Scholar). The ensuing decade saw the discovery of what turned out to be the four remaining sporulation sigma factors through work from my laboratory (33Kroos L. Kunkel B. Losick R. Switch protein alters the specificity of RNA polymerase containing a compartment-specific sigma factor.Science. 1989; 243: 526-5289Crossref PubMed Google Scholar) as well as from the laboratories of my former postdoctoral fellow Charles Moran, my collaborator Stragier, Peter Setlow, and Issar Smith (34Dubnau E. Weir J. Nair G. Carter 3rd, L. Moran Jr., C. Smith I. Bacillus sporulation gene spo0H codes for σ30 (σH).J. Bacteriol. 1988; 170: 1054-1062Crossref PubMed Google Scholar, 35Sun D.X. Stragier P. Setlow P. Identification of a new σ-factor involved in compartmentalized gene expression during sporulation of Bacillus subtilis.Genes Dev. 1989; 3: 141-149Crossref PubMed Google Scholar, 36Karmazyn-Campelli C. Bonamy C. Savelli B. Stragier P. Tandem genes encoding σ-factors for consecutive steps of development in Bacillus subtilis.Genes Dev. 1989; 3: 150-157Crossref PubMed Google Scholar). Thus, five sigma factors in total (σE, σF, σG, σH, and σK) were responsible for the program of gene expression that transformed a growing cell into a dormant spore (and with the surprise that the gene for σK was a composite of two partial coding sequences that were fused by excision of a large segment of the chromosome during sporulation (37Stragier P. Kunkel B. Kroos L. Losick R. Chromosomal rearrangement generating a composite gene for a developmental transcription factor.Science. 1989; 243: 507-512Crossref PubMed Google Scholar)). Just as the phage sigma factors fit neatly into a simple hierarchical regulatory cascade (σA → σSP01-28 → σSP01-33,34), so too the sporulation regulatory proteins fell into a linear dependent sequence (σH → σF → σE → σG → σK) but with an exciting spatial twist. Spore formation takes place not in a single cell, but rather in a sporangium. After a process of asymmetric division, the sporangium consists of two cellular compartments known as the forespore and the mother cell. Thus, we had to consider not only the time of appearance of each of the five sigma factors but also their location over the course of development. It turned out that σH is present in the predivisional sporangium, but the remaining four are compartment-specific transcription factors: σF and σG are active in the forespore, and σE and σK are active in the mother cell (as demonstrated by Adam Driks, Liz Harry, Peter Margolis, and Kit Pogliano) (38Margolis P. Driks A. Losick R. Establishment of cell type by compartmentalized activation of a transcription factor.Science. 1991; 254: 562-565Crossref PubMed Google Scholar, 39Driks A. Losick R. Compartmentalized expression of a gene under the control of sporulation transcription factor σE in Bacillus subtilis.Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 9934-9938Crossref PubMed Google Scholar, 40Harry E.J. Pogliano K. Losick R. Use of immunofluorescence to visualize cell-specific gene expression during sporulation in Bacillus subtilis.J. Bacteriol. 1995; 177: 3386-3393Crossref PubMed Google Scholar). Moreover, σE activity in the mother cell is dependent on σF in the forespore; σG activity in the forespore is tied to activation of σE in the mother cell; and activation of σK in the mother cell is dependent on σG. Stragier and I called this pattern of intercompartmental control “crisscross regulation” (Fig. 3) (41Losick R. Stragier P. Crisscross regulation of cell-type-specific gene expression during development in B. subtilis.Nature. 1992; 355: 601-604Crossref PubMed Scopus (0) Google Scholar). How did crisscross regulation work? It turned out that both σE and σK are initially produced as inactive proproteins (pro-σE and pro-σK) and that conversion to the active sigma factors is controlled by analogous (but, remarkably, non-homologous!) intercompartmental signal transduction pathways under the control of σF and σG, respectively (42LaBell T.L. Trempy J.E. Haldenwang W.G. Sporulation-specific σ factor σ29 of Bacillus subtilis is synthesized from a precursor protein.Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 1784-1788Crossref PubMed Google Scholar, 43Cutting S. Oke V. Driks A. Losick R. Lu S. Kroos L. A forespore checkpoint for mother cell gene expression during development in Bacillus subtilis.Cell. 1990; 62: 239-250Abstract Full Text PDF PubMed Google Scholar, 44Hofmeister A.E. Londoño-Vallejo A. Harry E. Stragier P. Losick R. Extracellular signal protein triggering the proteolytic activation of a developmental transcription factor in B. subtilis.Cell. 1995; 83: 219-226Abstract Full Text PDF PubMed Google Scholar). Astonishingly, the pro-σK-processing enzyme (SpoIVFB) would later turn out to be the founding member of a family of membrane-embedded metalloproteases that includes site-2 proteases (45Rudner D.Z. Fawcett P. Losick R. A family of membrane-embedded metalloproteases involved in regulated proteolysis of membrane-associated transcription factors.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 14765-14770Crossref PubMed Scopus (173) Google Scholar), which, as shown by Michael Brown and Joseph Goldstein at the University of Texas Southwestern Medical Center (46Sakai J. Duncan E.A. Rawson R.B. Hua X. Brown M.S. Goldstein J.L. Sterol-regulated release of SREBP-2 from cell membranes requires two sequential cleavages, one within a transmembrane segment.Cell. 1996; 85: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar), are proprotein-processing enzymes for mammalian transcription factors (sterol regulatory element-binding proteins) involved in cholesterol biosynthesis. The σK pathway was worked out by postdoctoral fellows Simon Cutting, Lee Kroos, Valerie Oke, Orna Resnekov, and David Rudner (43Cutting S. Oke V. Driks A. Losick R. Lu S. Kroos L. A forespore checkpoint for mother cell gene expression during development in Bacillus subtilis.Cell. 1990; 62: 239-250Abstract Full Text PDF PubMed Google Scholar, 47Cutting S. Driks A. Schmidt R. Kunkel B. Losick R. Forespore-specific transcription of a gene in the signal transduction pathway that governs pro-σK processing in Bacillus subtilis.Genes Dev. 1991; 5: 456-466Crossref PubMed Google Scholar, 48Ricca E. Cutting S. Losick R. Characterization of bofA, a gene involved in intercompartmental regulation of pro-σK processing during sporulation in Bacillus subtilis.J. Bacteriol. 1992; 174: 3177-3184Crossref PubMed Google Scholar, 49Oke V. Losick R. Multilevel regulation of the sporulation transcription factor σK in Bacillus subtilis.J. Bacteriol. 1993; 175: 7341-7347Crossref PubMed Google Scholar, 50Rudner D.Z. Losick R. A sporulation membrane protein tethers the pro-σK processing enzyme to its inhibitor and dictates its subcellular localization.Genes Dev. 2002; 16: 1007-1018Crossref PubMed Scopus (90) Google Scholar). Kroos and Rudner continue to make outstanding contributions to our understanding of the signaling pathways linking the forespore to the mother cell in their own laboratories in Michigan State University and Harvard Medical School, respectively. Meanwhile, Amy Camp in my laboratory (51Camp A.H. Losick R. A novel pathway of intercellular signaling in Bacillus subtilis involves a protein with similarity to a component of type III secretion channels.Mol. Microbiol. 2008; 69: 402-417Crossref PubMed Scopus (0) Google Scholar, 52Camp A.H. Losick R. A feeding tube model for activation of a cell-specific transcription factor during sporulation in Bacillus subtilis.Genes Dev. 2009; 23: 1014-1024Crossref PubMed Scopus (88) Google Scholar) and Moran in his own laboratory at Emory University (53Meisner J. Wang X. Serrano M. Henriques A.O. Moran Jr., C.P. A channel connecting the mother cell and forespore during bacterial endospore formation.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 15100-15105Crossref PubMed Scopus (69) Google Scholar) determined that σG is linked to σE by a pathway involving a multiprotein channel. Adding more intricacy to the sporulation circuitry was the further discovery of sporulation-specific DNA-binding proteins (33Kroos L. Kunkel B. Losick R. Switch protein alters the specificity of RNA polymerase containing a compartment-specific sigma factor.Science. 1989; 243: 526-5289Crossref PubMed Google Scholar, 54Kunkel B. Kroos L. Poth H. Youngman P. Losick R. Temporal and spatial c