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Learning about prophages from François Jacob
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Research in Microbiology xx (2014) 1e3 www.elsevier.com/locate/resmic
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Learning about prophages from François Jacob
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Armin Dale Kaiser Department of Developmental Biology, Stanford School of Medicine, Stanford, CA, USA Received 30 April 2014; accepted 6 May 2014
Keywords: François Jacob; Lambda repressor; cI, cII, and cIII; Escherichia coli chromosome; Fþ (donor) and F (recipient) cells; Prophage
Late in August 1954, François Jacob picked up me, my wife Mary, our 3-month old daughter, and all our luggage from the Paris boat-train station. Jacob and his wife, Lise who had lived in America during WWII, drove us in his black Citroen down the brightly lighted Champs Elysees in the normally heavy evening traffic. To show us the beauty of Paris at night, he drove us slowly in the right curb lane until another car stopped directly in front of us to talk to two ladies on the sidewalk. Forced to stop behind the other car, Jacob pulled angrily away from the curb. In so doing he clipped the other car's bumper. After proceeding a short distance, the other car again forced us to stop at the curb. One of the two men from the other car got out, stood just outside Jacob's door, and motioned for him to get out. Jacob did, and an argument ensued with the other man grasping Jacob by the lapels of his jacket. After about 2 min, they abruptly ceased their argument and Jacob and his attacker were agreeing to have a drink together later in the week. The other man had seen the tricolored threads in Jacob's jacket lapel button-hole, which signified that they had fought under Charles de Gaulle in Normandy where Jacob, as a medical officer in the 2nd Armored Division had taken a large dose of flak in his right side. Only part of the shrapnel could be removed. The part that could not prevented Jacob from standing long enough to practice the surgery for which he had trained. Instead, Jacob had decided to try his hand at microbiological research with Andre Lwoff at the Institute Pasteur. My family had come to Paris in order that I could study with Jacob as his first of many post-doctoral fellows.
E-mail addresses: [email protected], [email protected].
Jacob's thesis, presented when he was 34 years old, is a masterful analysis of prophage as defined by Lwoff [1] in Pseudomonas pyocyanea and in Escherichia coli. In addition to experiments on prophage, Jacob presents in his thesis a phage by phage cross that demonstrates recombination between a genetic marker for large plaques and a marker for clear plaques, now known to control virulence. My research at CalTech had consisted of a series of two and three-factor crosses involving alleles for small plaques, later known as red1; mi, for minute plaques, later known as R, or endolysin; cI, cII, and cIII, later known to specify and to regulate lambda repressor synthesis. At the same time, Jacob and his Pasteur colleague Ellie Wollman had isolated their own set of plaque size and virulence mutants in lambda and had mapped them [2]. Our postdoctoral research plan was to test whether the lambda crosses carried out in Paris and in Pasadena were mapping the same chromosome. And indeed they were. From E. coli cultures, acquired from several Paris hospitals, Jacob and Wollman had isolated a valuable new set of tools: a set of temperate bacteriophages that were serologically related to lambda and whose prophages had different specificities of lysogenic immunity [3]. Although the lambda repressor had not yet been isolated at this time, the different specificities of immunity meant that the series had different repressor proteins, as well as inserting into different locations on the E. coli chromosome. Then by crossing phage 434, 82, or 21 with lambda marked with cI, cII, or cIII, it was possible to show that lambda differed from 434 only at its cI locus, implying that this locus encoded the repressor and the left and right operator sites [4]. Other crosses showed that cII and cIII controlled the amount of repressor that was made, but not its specificity, which was the function of cI. Jacob and Wollman collected these heteroimmune phages because they were trying
http://dx.doi.org/10.1016/j.resmic.2014.05.010 0923-2508/© 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur. Please cite this article in press as: Kaiser AD, Learning about prophages from François Jacob, Research in Microbiology (2014), http://dx.doi.org/10.1016/ j.resmic.2014.05.010
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A.D. Kaiser / Research in Microbiology xx (2014) 1e3
to clarify the structural relation between the prophage, and the bacterial chromosome. These experiments led Jacob and Wollman to discover that the donor bacterial chromosome was transferred linearly to the recipient with what came to be known as the “spaghetti experiment” [3]. Picture someone sucking a single strand of spaghetti through pursed lips. To prove that prophage lambda was nothing more than a continuous segment of E. coli chromosomal DNA just beyond the galactose metabolism locus, Jacob and Wollman were making crosses that involved lambda lysogens (Jacob and Wollman 1961). The protocol involved separating Fþ (donor) and F (recipient) cells that were engaged in conjugal pairing away from each other at different times after mixing them, using the shearing action of a Waring blender borrowed from Jacob's kitchen at home. Their Fþ and F strains differed by several genetic markers: ability to make threonine, ability to make leucine, ability to metabolize galactose, and the presence of distinctive lambda prophages. Threonine, leucine and gal were transferred in perfect linear order. But, unexpectedly when prophage lambda was transferred to the recipient, the recipient always lysed, giving rise to a burst of infective lambda phage particles. When bursts of lambda phage were included in their scoring, prophage lambda segregated like a biochemical marker slightly to the right of the gal (galactose) locus. In other words, when the prophage was transferred from Fþ to F, the prophage was unexpectedly de-repressed e a result full of new information about repressors that Jacob extracted later. Members of Lwoff's Service de Physiologie Microbienne in 1954 included Jacob, Wollman, Jacques Monod (who became the Chef-de-Service of Biochemistry), Pierre Schaffer,
Georges Cohen, their students, research assistants and their postdoctoral fellows. A few of those scientists are pictured in Fig. 1 attached. Lwoff's own laboratory, evident in the picture, opened onto a common 100 ft long hallway with glassware storage cabinets along its sides. It was customary for Jacob, Wollman, and Monod to discuss their experimental results at tea dispensed at 10 am from a tiny corner cabinet, outside Sarah Rapkine's secretarial office at one end of the long hallway. A dense knot of morning tea drinkers that included Jacob, Wollman, and Monod circulated next to the tea to take lumps of sugar. Jacob used three lumps for his large, conical tea vessel. It was, as I recall, Monod who suggested the spaghetti image for Wollman's experiments at tea one day. After such lighthearted, public discussions over tea, Jacob and Wollman planned the afternoon's experiment with their research assistants in private. Those teatime discussions taught me how important the free, playful exchange of ideas can be for scientific progress. Wollman and Jacob were an effective and productive scientific team. Jacob, the more impatient of the two, would write the first draft of a joint paper. Then Wollman, a more cautious experimentalist, would go over the draft to check the facts, the sufficiency of the data as well as the clarity of presentation. Jacob had trained to be a surgeon and was expert in anatomy, histology, and physiology; yet from his youth he was a scholar who loved words and treasured his French heritage of rationality. Writing seemed to come to him naturally; he enjoyed explaining his results to others. His biological science was an attempt to understand how genes and the environment interact to produce a particular phenotype. While Jacob fought as a soldier in the French Army, as
Fig. 1. Some members of the Service de Physiologie Microbienne in 1955. From left to right: Dorothy Lane, Tom Anderson, Jacqueline George, Andre Lwoff, Rene Cohen, Elie Wollman, François Jacob, Dale Kaiser. Please cite this article in press as: Kaiser AD, Learning about prophages from François Jacob, Research in Microbiology (2014), http://dx.doi.org/10.1016/ j.resmic.2014.05.010
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A.D. Kaiser / Research in Microbiology xx (2014) 1e3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
his grandfather had done, Jacques Monod was a spellbinding leading strategist for the Resistance, who fought the Germans in France. Philosophically, Monod was a Cartesian thinker: if an idea can be expressed clearly, it is likely be part of the truth. Jacob, the unusually talented experimentalist and Monod, the theoretician, became a powerful scientific team because they understood each other very well and thought along the same lines. The collaboration between Jacob and Monod can be viewed as part of the grand effort to explain what a gene is and how a gene carries out its unique function. Genetic crosses, like those performed by Wollman and Jacob, showed that the prophage or any bacterial gene is a segment of DNA with a unique position on the chromosome and a particular nucleotide sequence. So defined, Jacob and Monod pointed out in 1959 [5], using Cartesian logic that there must be two kinds of genes: genes that determine the structure of some particular protein, and genes that specify how the structural genes are regulated. Regulatory genes include the operators, where a repressor binds DNA; the promoters, where RNA polymerase binds DNA to initiate messenger RNA synthesis; and the enhancers. Enhancers had not been discovered when Jacob and Monod were theorizing, but enhancers control where a sigma factor binds DNA on the chromosome. Jacob's genetic arguments may have reached their widest scope when he and Monod compared in great detail the induction of phage lambda from E. coli with the
3
induction of the enzyme, b-galactosidase in E. coli. From that comparison, they realized that, although inducers had to be added in both systems that “inductions” of bacteriophage and of enzymes actually consisted in the loss of repression. In both systems specific repressors were blocking messenger RNA synthesis, and both lambda and b-galactosidase repressors were proteins, not RNA as some had suggested. When Ptashne isolated lambda repressor and Gilbert isolated the b-galactosidase repressor, and both were shown to bind specific operators, the messenger theory was confirmed beyond reasonable doubt. References [1] Lwoff A. Lysogeny. Bacteriol Rev 1953;17:269e337. [2] Wollman EL, Jacob F. Genetic study of a temperate bacteriophage in Escherichia coli II. The mechanism of genetic recombination. Ann Pasteur Inst 1954;87:674e91. [3] Jacob F, Wollman E. Sexuality and the genetics of bacteria. In: Ch VII zygotic induction and its genetic effects. New York: Academic Press; 1961. pp. 93e100. Ch XIV the phenotypic expression of the genetic material. pp. 255e67. [4] Kaiser AD, Jacob F. Recombination between related temperate phages and genetic determination of immunity specificity and prophage localization. Virology 1957;4:509e21. [5] Jacob F, Monod J. Genes de structure et genes de regulation dans la bioysnthese des proteines. Comptes rendus Acad Des Sci Genetique Biochim 1959;249:1282e4.
Please cite this article in press as: Kaiser AD, Learning about prophages from François Jacob, Research in Microbiology (2014), http://dx.doi.org/10.1016/ j.resmic.2014.05.010
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RESMIC3278_proof ■ 28 May 2014 ■ 1/3
MODEL
Research in Microbiology xx (2014) 1e3 www.elsevier.com/locate/resmic
Q1
Learning about prophages from François Jacob
Q2
Armin Dale Kaiser Department of Developmental Biology, Stanford School of Medicine, Stanford, CA, USA Received 30 April 2014; accepted 6 May 2014
Keywords: François Jacob; Lambda repressor; cI, cII, and cIII; Escherichia coli chromosome; Fþ (donor) and F (recipient) cells; Prophage
Late in August 1954, François Jacob picked up me, my wife Mary, our 3-month old daughter, and all our luggage from the Paris boat-train station. Jacob and his wife, Lise who had lived in America during WWII, drove us in his black Citroen down the brightly lighted Champs Elysees in the normally heavy evening traffic. To show us the beauty of Paris at night, he drove us slowly in the right curb lane until another car stopped directly in front of us to talk to two ladies on the sidewalk. Forced to stop behind the other car, Jacob pulled angrily away from the curb. In so doing he clipped the other car's bumper. After proceeding a short distance, the other car again forced us to stop at the curb. One of the two men from the other car got out, stood just outside Jacob's door, and motioned for him to get out. Jacob did, and an argument ensued with the other man grasping Jacob by the lapels of his jacket. After about 2 min, they abruptly ceased their argument and Jacob and his attacker were agreeing to have a drink together later in the week. The other man had seen the tricolored threads in Jacob's jacket lapel button-hole, which signified that they had fought under Charles de Gaulle in Normandy where Jacob, as a medical officer in the 2nd Armored Division had taken a large dose of flak in his right side. Only part of the shrapnel could be removed. The part that could not prevented Jacob from standing long enough to practice the surgery for which he had trained. Instead, Jacob had decided to try his hand at microbiological research with Andre Lwoff at the Institute Pasteur. My family had come to Paris in order that I could study with Jacob as his first of many post-doctoral fellows.
E-mail addresses: [email protected], [email protected].
Jacob's thesis, presented when he was 34 years old, is a masterful analysis of prophage as defined by Lwoff [1] in Pseudomonas pyocyanea and in Escherichia coli. In addition to experiments on prophage, Jacob presents in his thesis a phage by phage cross that demonstrates recombination between a genetic marker for large plaques and a marker for clear plaques, now known to control virulence. My research at CalTech had consisted of a series of two and three-factor crosses involving alleles for small plaques, later known as red1; mi, for minute plaques, later known as R, or endolysin; cI, cII, and cIII, later known to specify and to regulate lambda repressor synthesis. At the same time, Jacob and his Pasteur colleague Ellie Wollman had isolated their own set of plaque size and virulence mutants in lambda and had mapped them [2]. Our postdoctoral research plan was to test whether the lambda crosses carried out in Paris and in Pasadena were mapping the same chromosome. And indeed they were. From E. coli cultures, acquired from several Paris hospitals, Jacob and Wollman had isolated a valuable new set of tools: a set of temperate bacteriophages that were serologically related to lambda and whose prophages had different specificities of lysogenic immunity [3]. Although the lambda repressor had not yet been isolated at this time, the different specificities of immunity meant that the series had different repressor proteins, as well as inserting into different locations on the E. coli chromosome. Then by crossing phage 434, 82, or 21 with lambda marked with cI, cII, or cIII, it was possible to show that lambda differed from 434 only at its cI locus, implying that this locus encoded the repressor and the left and right operator sites [4]. Other crosses showed that cII and cIII controlled the amount of repressor that was made, but not its specificity, which was the function of cI. Jacob and Wollman collected these heteroimmune phages because they were trying
http://dx.doi.org/10.1016/j.resmic.2014.05.010 0923-2508/© 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur. Please cite this article in press as: Kaiser AD, Learning about prophages from François Jacob, Research in Microbiology (2014), http://dx.doi.org/10.1016/ j.resmic.2014.05.010
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A.D. Kaiser / Research in Microbiology xx (2014) 1e3
to clarify the structural relation between the prophage, and the bacterial chromosome. These experiments led Jacob and Wollman to discover that the donor bacterial chromosome was transferred linearly to the recipient with what came to be known as the “spaghetti experiment” [3]. Picture someone sucking a single strand of spaghetti through pursed lips. To prove that prophage lambda was nothing more than a continuous segment of E. coli chromosomal DNA just beyond the galactose metabolism locus, Jacob and Wollman were making crosses that involved lambda lysogens (Jacob and Wollman 1961). The protocol involved separating Fþ (donor) and F (recipient) cells that were engaged in conjugal pairing away from each other at different times after mixing them, using the shearing action of a Waring blender borrowed from Jacob's kitchen at home. Their Fþ and F strains differed by several genetic markers: ability to make threonine, ability to make leucine, ability to metabolize galactose, and the presence of distinctive lambda prophages. Threonine, leucine and gal were transferred in perfect linear order. But, unexpectedly when prophage lambda was transferred to the recipient, the recipient always lysed, giving rise to a burst of infective lambda phage particles. When bursts of lambda phage were included in their scoring, prophage lambda segregated like a biochemical marker slightly to the right of the gal (galactose) locus. In other words, when the prophage was transferred from Fþ to F, the prophage was unexpectedly de-repressed e a result full of new information about repressors that Jacob extracted later. Members of Lwoff's Service de Physiologie Microbienne in 1954 included Jacob, Wollman, Jacques Monod (who became the Chef-de-Service of Biochemistry), Pierre Schaffer,
Georges Cohen, their students, research assistants and their postdoctoral fellows. A few of those scientists are pictured in Fig. 1 attached. Lwoff's own laboratory, evident in the picture, opened onto a common 100 ft long hallway with glassware storage cabinets along its sides. It was customary for Jacob, Wollman, and Monod to discuss their experimental results at tea dispensed at 10 am from a tiny corner cabinet, outside Sarah Rapkine's secretarial office at one end of the long hallway. A dense knot of morning tea drinkers that included Jacob, Wollman, and Monod circulated next to the tea to take lumps of sugar. Jacob used three lumps for his large, conical tea vessel. It was, as I recall, Monod who suggested the spaghetti image for Wollman's experiments at tea one day. After such lighthearted, public discussions over tea, Jacob and Wollman planned the afternoon's experiment with their research assistants in private. Those teatime discussions taught me how important the free, playful exchange of ideas can be for scientific progress. Wollman and Jacob were an effective and productive scientific team. Jacob, the more impatient of the two, would write the first draft of a joint paper. Then Wollman, a more cautious experimentalist, would go over the draft to check the facts, the sufficiency of the data as well as the clarity of presentation. Jacob had trained to be a surgeon and was expert in anatomy, histology, and physiology; yet from his youth he was a scholar who loved words and treasured his French heritage of rationality. Writing seemed to come to him naturally; he enjoyed explaining his results to others. His biological science was an attempt to understand how genes and the environment interact to produce a particular phenotype. While Jacob fought as a soldier in the French Army, as
Fig. 1. Some members of the Service de Physiologie Microbienne in 1955. From left to right: Dorothy Lane, Tom Anderson, Jacqueline George, Andre Lwoff, Rene Cohen, Elie Wollman, François Jacob, Dale Kaiser. Please cite this article in press as: Kaiser AD, Learning about prophages from François Jacob, Research in Microbiology (2014), http://dx.doi.org/10.1016/ j.resmic.2014.05.010
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A.D. Kaiser / Research in Microbiology xx (2014) 1e3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
his grandfather had done, Jacques Monod was a spellbinding leading strategist for the Resistance, who fought the Germans in France. Philosophically, Monod was a Cartesian thinker: if an idea can be expressed clearly, it is likely be part of the truth. Jacob, the unusually talented experimentalist and Monod, the theoretician, became a powerful scientific team because they understood each other very well and thought along the same lines. The collaboration between Jacob and Monod can be viewed as part of the grand effort to explain what a gene is and how a gene carries out its unique function. Genetic crosses, like those performed by Wollman and Jacob, showed that the prophage or any bacterial gene is a segment of DNA with a unique position on the chromosome and a particular nucleotide sequence. So defined, Jacob and Monod pointed out in 1959 [5], using Cartesian logic that there must be two kinds of genes: genes that determine the structure of some particular protein, and genes that specify how the structural genes are regulated. Regulatory genes include the operators, where a repressor binds DNA; the promoters, where RNA polymerase binds DNA to initiate messenger RNA synthesis; and the enhancers. Enhancers had not been discovered when Jacob and Monod were theorizing, but enhancers control where a sigma factor binds DNA on the chromosome. Jacob's genetic arguments may have reached their widest scope when he and Monod compared in great detail the induction of phage lambda from E. coli with the
3
induction of the enzyme, b-galactosidase in E. coli. From that comparison, they realized that, although inducers had to be added in both systems that “inductions” of bacteriophage and of enzymes actually consisted in the loss of repression. In both systems specific repressors were blocking messenger RNA synthesis, and both lambda and b-galactosidase repressors were proteins, not RNA as some had suggested. When Ptashne isolated lambda repressor and Gilbert isolated the b-galactosidase repressor, and both were shown to bind specific operators, the messenger theory was confirmed beyond reasonable doubt. References [1] Lwoff A. Lysogeny. Bacteriol Rev 1953;17:269e337. [2] Wollman EL, Jacob F. Genetic study of a temperate bacteriophage in Escherichia coli II. The mechanism of genetic recombination. Ann Pasteur Inst 1954;87:674e91. [3] Jacob F, Wollman E. Sexuality and the genetics of bacteria. In: Ch VII zygotic induction and its genetic effects. New York: Academic Press; 1961. pp. 93e100. Ch XIV the phenotypic expression of the genetic material. pp. 255e67. [4] Kaiser AD, Jacob F. Recombination between related temperate phages and genetic determination of immunity specificity and prophage localization. Virology 1957;4:509e21. [5] Jacob F, Monod J. Genes de structure et genes de regulation dans la bioysnthese des proteines. Comptes rendus Acad Des Sci Genetique Biochim 1959;249:1282e4.
Please cite this article in press as: Kaiser AD, Learning about prophages from François Jacob, Research in Microbiology (2014), http://dx.doi.org/10.1016/ j.resmic.2014.05.010
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