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The rise of Molecular Biology occurred in part via the spectacular convergence of few schools of thoughts located on each side of the Atlantic Ocean. This paper underlines the importance of the personal trajectory followed by Elie Wollman, as a shuttle between the Institut Pasteur and the Division of Biology of the California Institute of Technology in the elucidation of the phenomena of lysogeny.

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This text is a compendium of various lectures given to students working at the Institut Pasteur in Paris, in particular to the PPU program. Thanks to them and their mentors. The duet we formed with Francis-André Wollman on this last occasion was warm and inspiring. All the new data exposed here come from the “Pôle archives of our Institute”. I am greatly indebted in particular to D. Demellier,, S. Legout, C. Cecilio and M. Davty for their ceaseless attention to this project.. Charles Galperin was an inexhaustible source of references, recollections and inspirations.

1Elie Wollman’s contribution to the rise of molecular genetics is often underestimated. I stress here the cultural gap that existed in the immediate post-war period between the American and the pasteurian schools on the phenomenon of lysogeny as well as the cultural climate prevailing then in major laboratories. The evolution of the complex interactions which took place between André Lwoff and Max Delbruck owes a lot to Wollman. In turn, his initial attempts for demonstrating the insertion of the prophage into the chromosome of lysogenic bacteria paved the way for the success of the Jacob-Wollman ‘s tandem in the following years' Insertion of the local culture of the pasteurian tradition into the course of Molecular Biology took time. It owes a lot upon the generosity of the American scientific institutions in the immediate post-war period.

The ancestors

2The study of bacteriophages has been a favorite scientific topic at the Institut Pasteur. We have recently celebrated the one hundredth anniversary of the rediscovery of the bacteriophage by d’Herelle who christened its name. From the very start, the bacteriophage was a controversial subject. As magnificently recapitulated by Gunther Stent in his book “Molecular Biology of Bacterial Viruses”(1), Twort inoculated nutrient agar with small pox vaccine fluid. In fact, he was searching for nonpathogenic ancestors of animal viruses. He found nothing but contaminants. Upon further incubation, these micrococcal colonies underwent from time to time a glassy transformation. A normal microccocal colony, touched with a trace of this glassy material underwent the same transformation. This event was transmissible for an indefinite number of generations. The agent could be enormously diluted, it passed through the finest porcelain filters, but it lost its activity upon heating.

3Quite independently, d’Herelle came to similar findings. He was interested in the propagation of “diseases” in bacteria, entertaining the hope that they could provide a way to cure their hosts from bacterial infection. In Mexico, he noticed speckles in cultures of coccobacilli made in agar : the plated bacteria were locally killed. On his return to the Institut Pasteur, he made similar observations using filtrates of dysentery bacilli. The development of this capacity to generate speckles required three days. It persisted through 50 successive re-inoculations. The factor acted locally since it left circular areas where no bacterial growth was apparent. This “antidysentery microbe” did not grow by itself. For its transmission, its continuous presence in the contaminated strain was required. D’Herelle showed that an epidemic of bacillary dysentery could be stopped using filtrates of this invisible microbial antagonist. He concluded that the antidysentery microbe was an obligatory parasite, a “bacteriophage”. D’Herelle insisted on the killing power of the new agent. Once informed of Twort’s findings, he was not at all convinced that their findings could be the two facets of the same process.

4Gratia clarified the issue by performing rigorous cross-experiments ; he showed that the filterable agent isolated from glassy microccocal colonies- à la Twort- causes the transmissible lysis of liquid cultures of staphylococci. Reciprocally, isolation of a phage “à la d’Herelle” transformed the staphylococcal colonies into Twort’s glassy material. In 1922, he concluded “the two phenomena are identical, two different aspects of the same phenomenon, the transmissible lysis of bacteria“ (1))

5For forty years, the nature of this lytic principle has been the subject of vigorous debates. A specific field of contention concerned a discovery made in the twenties, notably by Bordet and his school : In certain strains of E. coli isolated from nature, each member of the strain could sporadically induce the lytic process and transfer this property to their progeny : they were lysogenic. In addition, Bordet et al showed that these strains could do so, even in the absence of any free phage. This last point was vigorously contested by d’Herelle, who maintained that the continuous presence of the phage in the contaminated strain was required. Many other controversies developed at this period, which have been masterly summarized in references (1) and (2). In short, around 1936, a clarification had progressively taken place. And three different hypotheses- - were proposed to explain the phenomenon :

6The exogenous hypothesis : a filtrating virus” parasites its bacterial host.

7Two endogenous hypotheses : the lytic power is a property of the infected bacteria and of its descent. It could be due to the fact either that the phage particle, within the bacteria acted as a kind of enzyme, or that the phage particle was redefined at each generation “within the hereditary substance of the bacteria”(2).

8Since 1925, at the Institut Pasteur, Elie’s parents, Eugene and Elisabeth, had entered the field. They were among the first to rule out d’Herelle’s contention and the parasitic hypothesis at least in the case of B megaterium. They used two strains of Bacillus megaterium, one lysogenic A, and the other, B, non-lysogenic. This experimental system allowed not only to follow the appearance of cells of the A type under various conditions but also to titrate the number of plaques they were able to form on strain B. In 1936, the Wollmans lysed a culture of strain A by lysozyme and look for the presence of infective phage in the lysate (3a). No preformed, active phage was ever detected, reinforcing previous observations made by Burnet and Mc Kie, who proposed that lysogenic bacteria, as they divide, perpetuate the ability to become infective via the constitution of a mysterious blueprint, in anticipation of being activated (4). Along the same lines, the Wollman’s couple observed that, upon phage infection, the lysogenic power disappears for a while before reappearing. This led them to formulate a striking proposal : In the life cycle of the phage, there occurs an alternation between infective and non-infective phases ; the non-infective phase is perpetuated in the lysogenic bacteria as part of the hereditary structures of the cell… Infective viruses are produced by or from the non infective phase (3b, 5).

9Why was it so difficult to come to a consensus in the late thirties ? Those people were first class experimentalists. They were also strongly interacting. But the molecular information at their disposal was extremely scarce : For example, crystallization of a true virus, TMV, was only reported in 1935 ; the fact that it consisted only of a nucleoprotein structure was still controversial. The clear-cut experiment by Avery et al showing that DNA was the transforming principle defined by Griffith in 1928 became available only in 1944. In the absence of such molecular data, experiments were in fact interpreted by analogy with observations made in related fields. For example, several tenants of the existence of a cryptic blueprint thought that its activation into a mature phage could occur at each division of the infected bacterium. Correlations were then searched between the number of phages released in a given experiment with the number of replication events occurring in the corresponding infected population. Last but not least, the various groups were handling different biological materials. They were not aware that the development of different viral types in infected bacteria followed different metabolic paths. The occurrence of the Second World War had a dramatic effect on the mutual understanding between the competing groups. It put an end to the specific developments occurring in European research units on lysogeny while, in American labs, rapid progresses were made on virulent phages, notably the T-even phages.

10We are looking to day at these quarrels having in mind a well-established classification that makes the distinction between virulent and temperate phages, the last class being the only one able to give the lysogenic response and the alternation between infective and non-infective phases. A molecular understanding of the events taking place in the development of virulent phages was simpler and was brilliantly undertaken in the forties. It led these advanced groups to interpret the older observations performed on temperate phages along the lines of the mechanisms they were elaborating for the development of virulent strains. No wonder that, at Caltech, the Vatican of the American school, the tentative explanations given for such strange behaviors were considered as problematic, as testified by the outrageous comment that Elie found in the margin of a paper by his parents : “nonsense !” (5, 3b).

Elie Wollman, a “go-between”

11For Elie Wollman, born in 1917, life did not follow a logical path. The war was a tragedy, in particular for the Wollmans’ and their son, as recalled by Francis-André in the companion paper (6). It had also a decisive influence on his career. Before the war he would have preferred not to work in the Institute, and certainly not with André Lwoff, who was practically a member of his family. However, he was seeking Lwoff’s advice. André advised him to work in his “service”. In fact, at the end of the war, Elie, as Monod or Jacob, was eager to catch up as quickly as possible with science. He accepted Lwoff’s proposal (7a).

12Today, we logically assume that Elie would have immediately started his PhD thesis on lysogeny. Lwoff was probably the Pasteurian who had the greatest understanding of this question at the time ; he was extremely close to Eugene Wollman. Lysogeny was at the heart of his preoccupations, but within a frame of thought that is difficult to apprehend to day. Following his master Chatton, André Lwoff was studying ciliates, fascinated by their behavior during the course of a reproductive cycle (8-9). In these species, a new kinetosome the spatial organizer for microtubules always proceeds from another kinetosome. How to account for this specific property, an example of what Chatton and Lwoff defined as a striking example of cellular morphogenesis ? They defined such phenomena as “units endowed with genetic continuity”, the theme of a remarkable colloquium Lwoff organized in 1948. André Lwoff considered at the time the occurrence of the perpetuation of lysogeny in certain strains as an example of such a concept. He was however deferring their experimental study in his laboratory. He said at the time : “Les cinétosomes me serviront de modèle et de guide pour l’attaque des bactéries lysogènes”(8).

13In reality, E. Wollman found his research subject thanks to Jacques Monod who entered the same service“ one week after him in 1945 (7a). Monod had already identified and quantitatively studied the biphasic growth process observed in bacterial cultures when the growth medium was changed, a specific sugar replacing another one. Why not to follow viral infection in the same manner ? Why not to record the perturbations occurring in the metabolism and its regulation during a viral infection ?

14The sequential steps of a viral infection leading irremediably to lysis had been already partially characterized, in particular in the rising American school. So Elie Wollman started his work on virulent strains, too. He knew nothing about bacterial genetics at the very start. He learnt to isolate his own set of virulent phages and to play the usual game of selecting mutants of the recipient bacteria resistant to the viral infection, and searched for viral suppressors. His main achievement was probably to demonstrate with Monod that a strong interference existed between the development of the phage and the metabolism of the infected bacteria, He detected mutants affected in amino acid metabolism which did not irreversibly adsorb his phages. These last studies came out to be quite similar to the ones developed at the same period by Gunther Stent, in Max Delbrück’s laboratory at Caltech. Elie Wollman successfully asked for a Rockefeller grant in 1947 and was allowed to continue his studies with Gunther Stent at Caltech, the mecca for the research on this type of bacteriophages ; he stayed in Pasadena for two years between 1948 and 1950.

15He discovered there an amazing cultural mix, something that did not exist in France at the same period. People from very different fields were working together on a program orchestrated by Delbrück. Jean Weigle was a pure physicist as Delbrück himself. Wollman was closely working with Stent, a physico-chemist by training. It was a great and vivid atmosphere (5).

16Delbrück was extremely impressive : A bright physicist, as many of his post-war friends, he was eager to direct his efforts towards new frontiers. Following Bohr, enthusiastic after reading the very influential little book by Schrödinger, ”what is life ?”, he thought that the real question was now to understand the hidden principles which govern Biology.

  • 1 In describing this "dull eventuality", Delbruck delivered in 1942 the most prescient description of (...)

17It is fascinating to read his famous assay “A physicist looks at biology”, written at the time of Elie’s arrival at Caltech (10) : Biology had to turn to a rigorous reductionist approach, along the methodology, which have been efficient in modern physics. They had to focus on the simplest objects amenable for their studies. Furthermore, Delbrück entertained the paradoxical hope that as results were accumulating, a serious tension will appear between facts and theories and the reduction will fail. For him, it would be rather dull if the biology of to-morrow were going to reduce simply into a complex type of biochemistry1*. He was expecting that a strong principle, perhaps of similar heuristic value as the ones governing quantum physics will then emerge in Biology. This utopia was as challenging for him as was the postulate of the existence of units of genetic continuity for Chatton and Lwoff.

18Delbrück was not don Quichotte. For him the greatest challenge was reproduction. He decided to select a biological object, having the simplest possible structure, the best-defined mode of development, onto which physical reasoning and physical experiments will have the greatest impact. He chosed the T even virulent phages.

19When Elie Wollman arrived, the project was already full-fledged. Elie observed that Delbrück had introduced a very special way of thinking :

Try to have systems as simple as possible
Try to examine each simple aspect you could dissect
Try to fit it into a coherent picture
This is what we call now a model system .(after (7b))

20Success came quite quickly ; the publications of the collective now known as the phage group illustrate the efficiency of the approach, greatly helped in this post-war period by the fast developments of techniques adapted to this purpose, and by the efficiency of communication of new results in a friendly community.

21What was going on at Pasteur, meanwhile ? In the midst of Elie’s absence, Lwoff decided that time was now ripe for him to tackle lysogeny (9). As specified in the review he published with Antoinette Gutmann in 1951, he wanted to solve three questions in that order :

  1. Can the faculty of producing phages really be perpetuated without intervention of external viruses ?

  2. How do lysogenic bacteria liberate the phages which they produce ?

  3. If this property appertains to a minority of the recipient bacteria, then what factors induce this production of bacteriophages in a population of potentially lysogenic bacteria ? (11).

22Lwoff was convinced that a definitive answer to the two first questions required a revolutionary approach, freed from a priori analogies, the observation of single cells and of their descent. Using a micromanipulator designed in the laboratory, he was able with Antoinette Gutmann to follow the persistence of the lysogenic power in the daughter cells isolated into micro drops for at least 19 divisions. As divisions of B. megaterium “ diplo-bacilli ” took place, one of the sister cells was maintained in the observation micro-chamber, while the other one was used for maniplations similar to the ones proposed earlier on by the Wollman’s parents. In doing so, Lwoff and Gutmann showed the absence of any free phage in the supernatant of the bacterial descent, ruled out the possibility that secreted phages could remain adsorbed on the bacterial cell-wall, and demonstrated the absence of any internal phage particle during the whole process. Finally they noticed the rare lysis of some single colonies of the lineage. Lysis appeared to be a rare stochastic event, unrelated to the reproduction cycle of the infected host (11).

23The temporal relationship between bacterial lysis and phage release could be followed using the same technical approach. For Max Delbrück, “true” lysogeny corresponded to the case where the phage was liberated in the absence of lysis (12). This bizarre proposal was completely ruled out. Lwoff observed that lysis always preceded phage release, stochastic events leading to a very variable amount of free phages. This led to a new definition of lysogenic bacteria “ in which the capacity to produce bacteriophages is perpetuated without the intervention of exogenous bacteriophages. (11). Therefore, each bacterium of a lysogenic strain harbors and maintains a non-infective structure, the prophage, which endows the cell with the ability to give rise to an infective phage in a small fraction of the population. (1).

24Was it possible to induce this stochastic process on an entire culture of B. megaterium ? The reader is referred to the delicious account given by Lwoff on this saga in the tribute to Delbrück, “The prophage and I” (12). After many failures with Lou Siminovitch and Niels Kjeldgaard, Lwoff discovered that UV induction was successful. It was now clear that in the lysogenic state, induction could trigger an otherwise infrequent event, the access to the vegetative phase, itself preceding lysis, the last two events proceeding very likely as in the case of virulent phages .

  • 2 This was not without reservation though. See letter from A. Lwoff to E. Wollman, July 1950 (14).

25Elie was kept informed of all these advances when he was working at Pasadena. The archives of the Institut Pasteur keeps three letters from A. Lwoff to Elie, dated respectively December 49, June and July 1950. (In the last letter Lwoff mentions the success “d’un procédé d’une simplicité enfantine pour induire à coup sûr la lyse bacteriophagique intrinsèque de 100 % des bactéries d’une population lysogène”. He maliciously adds “naturellement, vous devez vous demander comment” (13). It is clear that Elie played at this time a crucial role as a mediator. He was asked to write the section on lysogeny for the proceedings of the conference on the recent advances on comparative studies on Viruses, held by Delbrück’s laboratory at Caltech in March 1950. The corresponding “synopsis” shows that maintenance of lysogeny through the action of the prophage, as defined by Lwoff was now fully accepted at Caltech (14). Delbrück even asked André Lwoff to perform with Elie’s help some demonstrations on the topic in his laboratory2 (13).

26In summary, solid experimental strategies on both sides of the Atlantic led to a synthesis, admirably summarized by Lwoff in 1953 ((15) and Figure X). This synthesis put an end to the apparent conflict between the findings of the American groups on virulent phages and the studies conducted for so long at the Institut Pasteur on temperate bacteriophages.

27Elie Wollman came back to Paris in 1950. At this time, Lwoff’s synthesis was still in progress and the molecular events associated with the induction process were totally unknown. More than ever Elie was convinced that bacterial genetics were going to provide the key for this type of investigation (7b). Before coming to his own achievements, I have to describe the specific atmosphere prevailing in Lwoff’s service, the famous attic.

The attic dwellers :

28André Lwoff was head of the laboratory situated in the attic. As reminded by Melvin Cohn in his superb contribution (16), the “Service de physiologie cellulaire”, “was located ins a dimly lit space with a low ceiling and small windows that cradled a laboratory so primitive that it might well have been put together with furniture and equipment gathered at a garage sale. Besides the support personnel, the scientific staff consisted of essentially two groups. One was headed by Lwoff with Lou Siminovitch and Elie Wollman, joined somewhat later by François Jacob, Gunther Stent, Ernest Borek and Niels Kjeldgaard. The other was headed by Jacques Monod with Annamaria Torriani, Germaine Cohen-Bazire, Martin Pollock and myself, to be joined, somewhat later, by David Hogness, Seymour Benzer, Michael Doudoroff, and Roger Stanier. It is clear that it was a great international mix, where American scientists dominated.

29Mel Cohn praised Lou Siminovitch and gave us a hint of the great dependence of the service on the American help. The debt that the Lwoff laboratory owes to Lou Siminovitch can never be overestimated… I recall his patience in teaching the technicians and members of the scientific staff how to use the newly inherited Beckman spectrophotometer, and of course when the instrument broke down usually due to careless use, the plea to him to repair it. It was Lou’s upbeat disposition that was endearing, and added to that a keen critical mind made him the hub of the laboratory. …France was recovering from war and everything from food to fuel was in short supply and rationed. Monod managed to obtain Rockefeller Funds to build a cold room and buy a high speed, reasonable volume centrifuge that was to be housed in that space. in early 1950 we baptized the cold room, a small walk in refrigerator of a type then coming into vogue in Paris butcher shops. No sooner christened, when André using his “droits de seigneur” made claim on the space under the table to store his favorite Sancerre wine, a small but delicious sacrifice. However, that wasn’t enough as I needed to be able to carry out experiments with isotopes and for that no funds for a Geiger counter were available. So in a joint venture with Lou Siminovitch we visited many physics labs around Paris, begging for abandoned equipment that we could use to build one. Thanks to Lou’s expertise and a few miracles, we put together the instrument that permitted the introduction of isotope research into the attic lab ...We tend to concentrate on the scientific staff... However, here I would like to digress a moment to talk about Sarah Rapkine. She began as a technician with Lou Siminovitch but over time took charge of the total administration of the lab. She was a close friend of Bethsabée de Rothschild who helped the lab philanthropically. Sarah was a mother figure to everyone who came to work in the lab, finding living quarters for them as well as guiding them through all kinds of personal problems.

30Mel then takes Anna-Maria Torriani’s example, a singularly remarkable being who along with her husband, Luigi Gorini, had spent over 10 years in the Italian underground fighting Mussolini’s brand of fascism. He mentioned how quickly novices deprived of a significant scientific background became a first-class scientist. Mel’s marvelous assay helps us to appreciate how these foreign scientists, generally emerging from the war, blended with the French spirit in a warm atmosphere.

31How an intellectual dialogue developed behind ? All collected testimonies insist on the strong influence exerted on the group by the three founders of the French school of genetics, Lhéritier, Teissié, and above all, Boris Ephrussi. Their studies were performed on different systems, but in each case they had to postulate that cytoplasmic elements exerted a control on gene expression. Elie insists on the fact that this school was very much freed from academic duties, and worked on what seemed attracting for them, independently of any teaching commitment (7b). The great physiologist, J.T. Bonner, spent a sabbatical year in Paris in 1953. He wrote : An informal seminar, including Ephrussi and a number of others, met periodically in the late afternoon to discuss modern problems in developmental biology. It included Jacques Monod and André Lwoff. I realize that already these first-rate people were plotting the molecular revolution... at that time their vision into the future was extraordinary prescient. He concludes by saying : ...The group at the Pasteur, plus Ephrussi plus Fauré (his French mentor) seemed so different from many of the other scientists that I encountered in France that year (17).

32In summary, both at the very local level, the attic, and in this restricted Parisian circle, a school of thought - as defined by Lwoff later on- was taking ground : a school of thought is clearly articulated, very demanding at the bench, growing as a cooperative process involving local as well as foreign friendly interactions. Elie Wollman’s main achievements, accomplished in their greater part through an intimate cooperation with François Jacob, stand as a magnificent testimony of its success.

The great achievements

33After he had completed his writings on the work he performed at Caltech, Elie started to investigate the nature of the prophage in Lwoff’s laboratory. The first step was to determine whether or not the genetic unit specifying the prophage was linked or not with a gene of the harboring bacterium. For this purpose, he decided to rely on a recently emerging technique, genetic recombination. Genetic crosses implying markers specifying the use of metabolites were barely starting. This implied to work with E. coli where a curious phenomenon had been observed by Joshua Lederberg and his school, and studied since 1948 : Mixtures of specific E. coli strains gave rise to progenies where emerged from time to time colonies carrying a stable cocktail of the genetic markers of their parents. Furthermore Esther Lederberg, Joshua’s wife, had recently discovered that the strain used for these assays was lysogenic for a phage, called lambda, and that the phage could be cured after UV irradiation. The strains could adopt two allelic forms at the prophage location, + or - .

34Elie therefore settled the corresponding techniques at Pasteur, with the hope to see how the genetic marker characteristic of the presence of lambda segregated or not with the gal marker in the progeny. To day, the experiment seems simple to carry on and its interpretation straightforward. It was not so in 1952. With the Lederberg strains an efficiency of recombination of the order of 10-6 was to be expected at best. Segregation studies were heroic. The chemical nature of the genetic material was simply suggested to be DNA. The simple alternative we would consider to-day -the prophage could either be present or absent on the unique chromosome of E coli- was far from being accepted. Added complexities emerging from Lederberg’s laboratory were explained at the time by a distinct hypothesis : the prophage, when present, was carried out by an ensemble of “cytoplasmic particles” whose expression could be controlled by a master gene present on the main chromosome of the recipient cells (8).

35From the segregation pattern of the recombinants, Elie did show that the presence of the prophage was strongly linked to specific genes of the host. The account of his findings at the meeting organized by A and M Lwoff at Royaumont was well received (Jacob notes in the Statue Within : Elie managed to convince his people that even bacteria had the right to copulate (19)). The year later, 1953, Elie published a paper which retrospectively appears as a model of lucidity and intellectual honesty (18) : He had demonstrated the existence of a genetic linkage for the prophage, a fact which undoubtedly indicated that the prophage was present on the chromosome of the host, if the simpler hypothesis given above was correct. Elie did not hide another puzzling observation : recombination was successful only when the lysogenic character was carried by certain types of strains : William Hayes studying recombination in London had just shown that what we call to-day the transfer of the genetic information was polar : To classify his strains, he had to postulate two “sexual” types a “male” and a “female F-”, distinguished by the fact that the mixture of two “female” strains never gave rise to recombination.

36François Jacob arrived in 1950 in Lwoff’s service. As pointed out by Melvin Cohn, it was with Elie that François began to reveal his genius. He loved working on the genetics of the lysogenic phages using techniques that were surprising and rewarding (16). Their cooperation blossomed between 1953 and 1957. Through Jacob’s or Wollman’s accounts, we are reading an adventure novel, so intimately are geared the local experimental findings, the findings at large on the same subject, and the quick impact of nascent molecular advances. Constant luck for the prepared minds ! The findings of the period look as the sequence of Bonaparte victories in the Italian campaign, the victories being led this time by the stubborn cooperation of the two fellows.

37 As Wollman, Jacob was fascinated by the key question of the time : Elie’s experiments worked only when the prophage was transmitted to the recombinants by the female partner. They decided to work together on this enigma. In 1953, new possibilities were opening : Elie had brought to the lab new strains from Hayes. These variant strains of the male class, the so-called Hfr strains, had acquired the property to be extremely efficient providers of recombinants. An elaborate genetic map was established around the gal locus. Crosses implying the lysogenic character showed that the lambda marker was inserted at a consistent position in this local map. Furthermore, the general picture was also changing : Watson had announced in Royaumont a decisive discovery by Hershey and Chase : at the start of a virulent infection only the nucleic acid of the male was found in the recipient. In April 1953, came the twin notes by Watson and Crick on DNA. These reports probably suggested to the duo that the male phage was acting as a syringe during phage infection, The model implying that recombination arose because of an indiscriminate mixing of two types of “cytoplasmic particles” was loosing ground in front of a scheme implying polarity.

38Why not therefore to incorporate into the mirror genetic crossings performed routinely in the laboratory :

39an assay which monitored the possible development of mature phages after the mating ? Crossings were performed as usual in the afternoon. François Jacob reports in his book his amazement as he inspected the plates the next morning : In the first type of crossings, when recombination failed, phages were developing in the recipient bacteria ! Conjugation did proceed (the phage was infectious) but recombination aborted because the prophage was induced. Nothing similar took place in the second case. Lysis was prevented whenever the prophage was carried by the recipient strain. The cytoplasm of a female lysogenic strain contained some substance coded by the prophage that inhibited a possible infection and allowed the recombination process to take place. As concluded by both Elie and François, a fundamental question in the study of lysogeny, (the prophage expresses in the cytoplasm of its host a substance acting as a repressor) was solved by experiments based on conjugation. Conversely, the nature of the conjugation process was elucidated through the introduction of a lysogenic marker in the genetic crosses. Sexuality provided again the suitable image to label the mechanism implied by the experiment, erotic induction !

40The next step in the campaign was to get a better understanding of the copulation process itself. Wollman and Jacob knew the beginning of the story : a simple mixing was followed by an adsorption process. They knew also the final result : conjugation led to a segregation of the lysogenic character at a specific place on the genetic map. But what about the time course of the event, the recombination process itself ?

41Jacob noted in the Statue Within (19) : And Elie was visited by a strange idea. One could imagine the following scenario for conjugation : male and female pair off ; after a certain period, transfer of chromosome from male to female ; genetic recombination will then occur within this intermediate. Why not to try to interrupt the process ? If this is performed too early, no transfer would have taken place. If the two partners were separated after the transfer, then the recombinants ought to form. Hence Elie’s suggestion to mix males and females ; then some moment later, to separate the partners abruptly by placing the “couples” in a Waring blender. A sort of coitus interruptus. This type of device had been bought by Lise, Jacob’s wife. It had been stored in the lab ; it was immediately used in a recombination assay implying two markers. The next morning, inspection of the culture plates. Wonder of wonders. No recombinant at the start of the experiment, then the number of recombinants increased over the time allowed for the coitus (19). A surprise though : the two characters had a different kinetics of appearance, since one arrived 15 minutes before the other. Was it due to the fact that the two genes were present on two different chromosomes ? After great discussions, the experiment was repeated with the incorporation of more markers, a total of five, sparse over the whole genetic map : They arrived one after the other, in the very order in which they were known to occur on the genetic map. The blender has not only separated the couples. It has also cut the chromosome of the male as it was entering the female bacterium ! The process was slow enough to yield estimates for the different times of entrance, as well as for the different times required for their integration. Genetic markers, present on the chromosome, were behaving as a sequence of signposts as the injection proceeded.

42The novel was not over : The experiment was repeated, this time with different Hfr strains. Deception : The order was not the same ; sometimes it looks even chaotic. It took time to solve the puzzle. The chromosome could be read in one direction or in reverse. And the factor responsible for fertility was inserted in different locations in each original Hfr strain (20-21). The whole sequence was however still injected and read with a different efficiency. This finding implied that the E. coli chromosome was circular, a revolutionary finding soon confirmed by Cairns by electron microscopy.

43The story continued, marked by the findings of many foreign postdocs coming to Lwoff’s attic : In particular, the location of the sites of insertion of other prophages on the genetic map, Congruence of physical, chemical, and genetic assays for the accurate location of more than thirty bacterial genes all over the map. But the main advances were already there. In six years, the picture coming from “erotic induction” and from the coitus interruptus had definitively changed our understanding of the conjugation process. In 1948, the two strains were viewed as establishing an undifferentiated cohabitation during which they exchanged their genetic material after mixing. This view was now replaced by an asymmetric representation. A sequence of mechanistic steps commanded genetic exchange on DNA, now the accepted template of genetic information.

44In this late period, Wollman’s greatest achievement was certainly the writing of a major book, La sexualité des bactéries, in cooperation with François Jacob (20), following Elie’s PhD thesis. The monograph was enlarged in its English version,” Sexuality and the genetics of bacteria”, a classic for all the biologists of my generation (21). After another stay in the States, Elie decided to spend all his energy on the restoration of the conduct of Science at the Institut Pasteur, and at the promotion of Molecular Biology in France, as it has been explained earlier on (6).

45We have mainly focused on the achievements made in Paris. However, a close intertwining occurred between many results produced and accepted over the world. International cooperation boosted technical improvements and deeply affected the way the problems were formulated. An amazing acceleration of progresses thus took place when Lwoff, Wollman then Jacob tackled the subject.


46The study of lysogeny has been pivotal for the early development of Molecular Biology (See for example (22). Since the very beginning of the XXth century, the Institut Pasteur has been at the heart of the discovery of lysogeny, has been the theater of the initial controversies, and has been the place where the major discoveries on the subject have been made. As beautifully explained in “Sexuality and the genetics of bacteria”, the critical characterization of three different techniques to promote DNA exchange between bacteria-transformation, conjugation and transduction- has founded the development of molecular genetics in bacteria, and the transactions operating on their genes (20-21). As it is often the case three unexpected discoveries occurred concomitantly, the first evidence for the existence of a negative control exerted by a cytoplasmic protein on the expression of developmental viral genes, a new way for mapping the host genome of E. coli, the discovery of its circularity.

47These findings had a very long-range impact. Discoveries made in eubacteria led to the search for equivalent mechanisms in eukaryotes. Elie used to stress that the definition of the status of the prophage paved the way for the search and the discovery of viral oncogenes. He mentioned also in one of his interviews that lysogenic bacteria provided a link between two concepts, infection and heredity (7b).

“These two concepts seemed (at the time) completely opposed. You had the heredity, which was something which was vertical, two parents, and so on… and you have the infection, which was something from the outside, but it had no relation to heredity… If there had not been infectious heredity, the progress could not have been what it has been. The progress came more from infectious heredity than from vertical heredity.”

48The most striking illustration of these considerations is the importance gained by the role played by lateral gene transfer in evolution. Molecular biologists, for so long inspired by vertical heredity, slowly realized that genetic transmission is horizontal too !

49But this success has been hard to reach ! True, the general climate of the post-war period favored intense intellectual exchanges. And there were strong analogies between the ways the conduct of Science was envisaged on both sides of the Atlantic. Rigorous methodologies had taken precedence over inspiring but inadequate grand perspectives. But the war had wiped off cultural exchanges for a while. The Lwoff and Guttmann’s single cell experiments, beautiful as they are, announced in fact modern cellular physiology, while, on the other side, the phage group was already fully engaged in Molecular Biology. I have tried to show that it was Monod and Wollman who really implanted molecular genetics into the attic. Elie should be credited indeed for having imported genetic recombination at a period when success in this venture was not at all guaranteed. The generosity of the American institutions had been crucial and persisted at later times when coalescence between two traditions, the one on virulent phages, the other on lysogeny was accomplished. In a deep sense, the famous Lwoff’s diagram, given in figure X, materializes the coalescence of two scientific traditions as Molecular Biology took its flight.


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(1) Stent Gunther S., Molecular Biology of Bacterial Viruses, Freeman, San Francisco and London, 1963.

(2) Gayon Jean and Burian Richard M, Eugène et Elisabeth Wollman : la question de la lysogénie, in L’invention de la Régulation Génétique, Loison L. and Morange M. édit., Éditions rue d’Ulm, Paris, 2017, p. 31-65.

(3a) Wollman Eugène and Wollman Elisabeth, Régénération des bactériophages chez le B. megatherium lysogène, C.R.Soc. Biol., 1936, 122, p. 190-192.

(3b) Wollman Eugène and Wollman Elisabeth, Les phases des bactériophages, C.R.Soc. Biol., 1937,124, p. 931-934.

(4) Burnet F. M. and Mc Kie M., Observations on a permanently lysogenic strain of B. enteridis gaertner , Austral. J. Exp.Biol. Med. 1929, 6, p. 277-284.

(5) Wollman Elie, Bacterial conjugation in Phage and the origin of Molecular Biology, Cairns J. Stent G.S., Watson J. edit., Cold Spring Harbor Laboratory Press, New-York, 1966, p. 88-99.

(6) Wollman Francis-André, « Elie Wollman, un homme de conviction. Une défense de la recherche incarnée par des femmes, des hommes et leurs institutions », Histoire de la recherche contemporaine, tome VII, 2018-2.

(7) Archives Institut Pasteur, Elie Wollman papers, 7a) WOL. A1 02 Textes autobiographiques : Après des études secondaires... 7b) WOL A1 04 Interviews : Transcription de l’entretien avec R. Buryan, 1984. 7c) WOL.A1 02 Textes autobiographiques : Ayant l’un et l’autre...

(8) Galperin Charles, Le bactériophage, la lysogénie et son déterminisme, History and Philosophy of the life sciences, 1987, 9, p. 175-224.

(9) Loison Laurent (edited by), André Lwoff, une autobiographie, Hermann, Paris, 2017.

(10) Delbrück Max, A physicist looks at Biology in Phage and the origin of Molecular Biology , Cairns J., Stent G.S. and Watson J. edit., Cold Spring Harbor Laboratory Press, New-York, 1966, p. 9-22.

(11) Lwoff André and Gutmann Antoinette, Investigations on a lysogenic Bacillus megaterium, Annales de l’Institut Pasteur, 1950, 78, p. 711-739 (English translation).

(12) Delbrück Max, Bacterial viruses or bacteriophages, Biol. Rev. 1946, 21, p. 30-40.

(13) Benzer Seymour et al. A syllabus on procedures, facts and interpretations in phage, in Viruses 1950, Max Delbrück edit., Division of biology of the California Institute of Technology Bookstore, Pasadena, 1950, p. 100-147.

(14) Archives Institut Pasteur, Elie Wollman papers, WOL.B2 Dossiers de correspondance, Three letters from A. Lwoff to E. Wollman, December 1949, June and July 1950.

(15) Lwoff André, Lysogeny, Bacteriol. Rev. 1953, 17, p. 269-337.

(16) Cohn Melvin, The Institut Pasteur attic dwellers : their origins, their paths to discovery

Res. Micr. ,2014,165, p. 318-324.

(17) Bonner John T., Lives of a microbiologist, Adventures in a Century of Extraordinary Science, Harvard University Press, Cambridge, 2002.

(18) Wollman Elie, Sur le déterminisme génétique de la lysogénie, Ann. Inst. Pasteur, 1953,84, p. 281-293.

(19) Jacob François, The statue within, An autobiography (passim), Urwin-Hyman, London, 1988.

(20) Wollman Elie and Jacob François, La sexualité des bactéries, Masson, Paris, 1959.

(21) Jacob François and Wollman Elie, Sexuality and the Genetics of Bacteria, Academic Press Inc., London, 1961.

(22) Morange Michel, A history of Molecular Biology, Harvard University Press, Cambridge, 1998.

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1 In describing this "dull eventuality", Delbruck delivered in 1942 the most prescient description of what this type of biology would be, and indeed has been !

2 This was not without reservation though. See letter from A. Lwoff to E. Wollman, July 1950 (14).

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Henri Buc, « Elie Wollman and the question of lysogeny », Histoire de la recherche contemporaine, Tome VII N°2 | 2018, 212-224.

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Henri Buc, « Elie Wollman and the question of lysogeny », Histoire de la recherche contemporaine [En ligne], Tome VII N°2 | 2018, mis en ligne le 15 avril 2019, consulté le 22 août 2019. URL : ; DOI : 10.4000/hrc.2510

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Henri Buc

Worked as an enzymologist at the Institut Pasteur. He developed methodologies to characterize the behavior of allosteric enzymes, and to critically examine the adequacy of the MWC model to account for their kinetic properties. He then studied nucleic acid polymerases, their behavior as molecular motors. More recently, he turned into an historian of sciences, concentrating in particular on the critical use of scientific archives to enlighten the interactions of scientists as they cooperate on a common project. He is presently Directeur de recherches honoraire at the Institut Pasteur

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