Adaptation or selection? Old issues and new stakes in the postwar debates over bacterial drug resistance

Stud. Hist. Phil. Biol. & Biomed. Sci. 38 (2007) 159–190

Studies in History and Philosophy of Biological and Biomedical Sciences www.elsevier.com/locate/shpsc

Adaptation or selection? Old issues and new stakes in the postwar debates over bacterial drug resistance Angela N. H. Creager Department of History and Program in History of Science, Princeton University, 129 Dickinson Hall, Princeton, NJ 08544-1017, USA Received 17 March 2005; received in revised form 16 June 2006

Abstract The 1940s and 1950s were marked by intense debates over the origin of drug resistance in microbes. Bacteriologists had traditionally invoked the notions of ‘training’ and ‘adaptation’ to account for the ability of microbes to acquire new traits. As the field of bacterial genetics emerged, however, its participants rejected ‘Lamarckian’ views of microbial heredity, and offered statistical evidence that drug resistance resulted from the selection of random resistant mutants. Antibiotic resistance became a key issue among those disputing physiological (usually termed ‘adaptationist’) vs. genetic (mutation and selection) explanations of variation in bacteria. Postwar developments connected with the Lysenko affair gave this debate a new political valence. Proponents of the neo-Darwinian synthesis weighed in with support for the genetic theory. However, certain features of drug resistance seemed inexplicable by mutation and selection, particularly the phenomenon of ‘multiple resistance’—the emergence of resistance in a single strain against several unrelated antibiotics. In the late 1950s, Tsutomu Watanabe and his collaborators solved this puzzle by determining that resistance could be conferred by cytoplasmic resistance factors rather than chromosomal mutation. These R factors could carry resistance to many antibiotics and seemed able to promote their own dissemination in bacterial populations. In the end, the vindication of the genetic view of drug resistance was accompanied by a recasting of the ‘gene’ to include extrachromosomal hereditary units carried on viruses and plasmids.  2006 Elsevier Ltd. All rights reserved. Keywords: Antibiotic resistance; Plasmid; Bacterial genetics; Bacteriology; Neo-Darwinian synthesis; Evolution

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1. Introduction The development of antimicrobial drugs, particularly antibiotics, has long been touted as one of the great medical success stories of the twentieth century. In 1935, publication of the effectiveness of Protonsil against streptococcal infection ushered in the rapid development of sulfa drugs, overtaken within a decade by the promise of penicillin and streptomycin.1 Yet troubling observations that infectious agents could become resistant to such drugs surfaced early on in bacterial chemotherapy.2 As William Summers has remarked, ‘No sooner were new antibiotics announced than reports of drug resistance appeared: sulfonamide resistance in 1939, penicillin resistance in 1941, and streptomycin resistance in 1946’ (Summers, 2002, p. xix).3 Thus, even as observers at the end of World War II hailed the end of infectious diseases, drug resistance was already a recognized problem—and its origin, like the mechanism of antibiotic action, remained unknown. In the early postwar period, the antibiotic resistance problem became a testing ground for different views on bacterial variation. Many scientists resorted to explanations of ‘adaptation’ and ‘training’ to account for microbial drug resistance. Throughout the 1930s, bacteriologists had shown that microbes could adapt to their nutritional environment by synthesizing new enzymes. These acquired characteristics could persist for several generations, and some researchers thought they could become hereditary. Yet these changes were regarded as induced, not mutational, and there was little reason to differentiate between individual cell and culture in conceptualizing the adapted bacteria. In the 1940s, a new generation of bacterial geneticists began challenging the adaptationist explanation for bacterial variation in general and drug resistance in particular, arguing that the trait appeared by random mutation as a strictly heritable trait in microbes, and that exposure of a population of bacteria to antibiotics simply selected for this pre-existing variant. At stake in this debate over drug resistance was the nature of bacteria as organisms. Bacteria had not previously been regarded as ‘genetic’ organisms—they did not possess chromosomes, nor could they exhibit Mendelian patterns of inheritance, since they lacked the morphological apparatus associated with the genetics of sexual reproduction. As Julian Huxley described bacteria in 1942, They have no genes in the sense of accurately quantized portions of hereditary substance; and therefore they have no need for the accurate division of the genetic

1

Protonsil was one of large number of aniline dyes tested for chemotherapeutic activity against bacteria. Within months of the publication of the Protonsil results, its constituent sulfonamide (sulfanilamide, the noncolored moiety of the red dye molecule) was shown to be just as effective. The first paper on the novel antibiotic properties of streptomycin was Schatz, Bugie, & Waksman (1944); early papers on penicillin are cited below. For a mid-century perspective on the development of bacterial chemotherapy, see Chain (1954). On the history of sulfa drugs, see Lesch (1993, 1997); on penicillin, see Steffee (1992), Bud (1998), and Gaudillie`re (2002), Ch. 1; on streptomycin, see Wainwright (1991). Moberg (1999) gives a general account of the early studies of drug resistance. Nearly all of the antimicrobial drugs discussed in this paper are antibiotics in the sense Selman Waksman defined the term in 1942—that is, they are chemical compounds produced by microorganisms that inhibit the growth of or destroy other microorganisms (Waksman, 1956). 2 This observation predated antibiotics: in a 1907 publication, Paul Ehrlich noted that certain protozoa were resistant to his recently developed anti-trypanosomiasis agent (Ehrlich, 1907). 3 For examples of these original publications, see MacLeod & Daddi (1939); Maclean, Rogers, & Fleming (1939); Abraham et al. (1941); Rammelkamp & Maxon (1942). See also Dubos (1942); Moberg (1999); Steffee (1992).

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system which is accomplished by mitosis. The entire organism appears to function both as soma and germplasm and evolution must be a matter of alteration in the reaction system as a whole. That occasional ‘mutations’ occur we know, but there is no ground for supposing that mutations are similar in nature to those of higher organisms, nor, since they are usually reversible according to conditions, that they play the same part in evolution.4 Microbiologists did not need evidence of the same sort of hereditary determinants found in higher organisms to classify and characterize bacterial species.5 Yet bacteriologists’ interests in ‘physiologic plasticity’ had long been a source of frustration to some geneticists; in 1916, L. J. Cole and W. H. Wright contended that bacteriologists, by believing that conditions could induce changes in bacterial cultures and that these could become fixed, refused to abandon Lamarckianism.6 Bacterial geneticists of the 1940s echoed Cole and Wright’s characterization, but in a changed context. The Lysenko affair—which resulted by 1949 in the Soviet suppression of Mendelian genetics as a manifestation of capitalist ideology—politicized debates everywhere over heredity and made charges of Lamarckianism especially polemical. At the same time, the neo-Darwinian synthesis provided a new theoretical framework for viewing drug resistance as the selection of random resistant mutants in a population of sensitive bacteria. Growing support for the genetic explanation of resistance fits well with what Stephen Jay Gould has called the ‘hardening of the modern synthesis’, as more pluralistic views of evolutionary change gave way to a strict emphasis on natural selection working on genetic variation as the mechanism for evolution at all levels (Gould, 1983). Indeed, microbial drug resistance became an oft-cited exemplar of the principles of Darwinian genetic selection in the decades after World War II, marginalizing alternative explanations of resistance. The very language of the neo-Darwinian synthesis conflicted with a long tradition of bacteriological explanation: the evolutionary meaning of adaptation, now with strongly genetic overtones, threatened to displace the physiological meaning of adaptation, as bacteriologists had long used the term in discussing acquired traits.7 Yet, ironically, the thorough-going geneticization of bacteria by the 1960s ended up having a subversive effect on genetic orthodoxy, with its ‘nucleocentrism’ (Sapp, 1994). Although bacterial geneticists called upon their experiments on the origin of antibiotic resistance to demonstrate that mutation and selection could account for any bacterial 4 Huxley (1942), pp. 131–132. It should be noted that other microbes such as yeast and fungi provided useful exceptions to the predominance of asexual reproduction in unicellular organisms. 5 Arkwright (1930), p. 356, argued that the terms chromosome and mutant are inappropriate for bacteria, even though ‘true hereditary variation occurs in bacteria, and that in this process the hereditary apparatus is so changed that the new characters reappear continuously in the offspring’. 6 ‘In fact, it is among bacteriologists and paleontologists, of all biologists, that a belief in the inheritance of acquired characteristics has its strongest hold. In the case of the bacteria, the belief is based on the not uncommon observation that when a particular condition produces a change in a culture of organisms, and cultivation is continued a sufficient time under the condition, the variation may become so thoroughly fixed that there is no return to normal, even tho [sic] cultivation under the original conditions is resumed. In other words, it is believed that the change has been impressed on the organism by the conditions of the environment, and such changes are called impressed variations’ (Cole & Wright, 1916, p. 211, also quoted by Brock, 1990, p. 49). Cole and Wright use the term ‘physiologic plasticity’ on p. 210. 7 This is not to suggest that scientists confused the two meanings of adaptation. Joshua Lederberg drew a careful distinction; Lederberg (1951a), pp. 93–96.

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variation, researchers eventually determined that most genes for antibiotic resistance were not carried on bacterial chromosomes. The understanding of gene had to be recast in order to include the extrachromosomal pieces of nucleic acid that carry resistance genes and are laterally transmitted between bacteria, even those of different species. This revised notion of the gene became operationalized in the use of engineered drug-resistant plasmids for cloning genes in the 1980s. 2. The problem of bacterial variation Behind the skepticism about bacteria as true genetic organisms was an older controversy regarding the biological stability of bacterial cultures. At one level, the germ theory established—or, more accurately, posited—the biological constancy of bacteria as organisms. Robert Koch’s ‘postulates’ hinged on the assumption that a single bacterial species caused a single infectious disease. Koch made use of the taxonomic system for bacteria published in 1872 by botanist Ferdinand Cohn, who took morphological characteristics of bacteria as stable markers (Mazumdar, 1995, Ch. 2).8 Medical bacteriologists tended to use these monomorphic ideas of bacterial stability pragmatically, along with laboratory techniques of isolation, to culture and classify pathogenic bacteria (Amsterdamska, 1987). The everpresent threat of contamination made observations of bacterial variability seem dubious.9 By the 1920s, observations of morphological and physiological variation in bacteria began to be taken more seriously, particularly after Joseph Arkwright’s 1921 finding that variations in the agglutination behavior of bacteria correlated with changes in both virulence and the appearance of colonies—termed ‘smooth’ versus ‘rough’ (Arkwright, 1921).10 Bacteriologists at several institutions characterized these variant forms in bacteria such as Pneumoccocus and Pneumobacillus, and investigated the conditions under which growth of the ‘smooth’ type could yield rough or intermediate colonies.11 Observations of the spontaneous lysis of cultures through ‘bacteriophagy’ provided another example of bacterial instability, whether one subscribed to Fe´lix d’Herelle’s theory that the phenomenon was due to bacterial viruses or Jules Bordet’s view that lysis was caused by an endogenous factor (Hadley, 1927, p. 7). D’Herelle noted that certain bacterial cultures acquired resistance to bacteriophage; such a change correlated with loss of agglutinability 8 Gradmann (2001) offers an astute account of the debates between ‘monomorphists’ and ‘pleomorphists’ in the German context in the latter part of the nineteenth century, and points out that there was a paucity of empirical evidence supporting the ‘speciecist’ position at this time. 9 This is the received view; see, for example, Brock (1990), Ch. 3. However, Mendelsohn (2002) challenges the conventional wisdom that bacterial variation was so neglected. 10 As Amsterdamska points out (1987), agglutination was an important aspect of laboratory techniques for diagnosis, as used in testing for typhoid. 11 Hadley (1927) and Arkwright (1930) give overviews. The line of research on culture forms is now remembered most often for Frederick Griffith’s experiments in 1928 on the heritable ‘transformation’ of rough, avirulent type I pneumococci to smooth, virulent type II pneumococci. In 1944, Oswald T. Avery, Colin M. Macleod, and Maclyn McCarty published their experiments showing that this factor inducing this heritable transformation was DNA. What appears in retrospect as a muted reception to this important paper has inspired many historical explanations, including its publication in a medical journal and its framing in bacteriological rather than genetic terms. Others, however, have pointed to citation patterns showing that the 1944 paper was not neglected by bacterial geneticists and phage researchers, even if it was not fully appreciated. For examples of the many perspectives on this issue, see Hotchkiss (1966), Wyatt (1972), and Portugal & Cohen (1977), Ch. 7. An excellent historical account of Avery’s research is given by Amsterdamska (1993).

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in many different strains (D’Herelle, 1926, p. 218). Some bacteriologists, such as Frank MacFarlane Burnet, expressed interest in whether these variations were similar in origin to mutations in higher organisms, and whether such adaptations represented heritable changes.12 However, the majority of bacteriologists were unconcerned with whether bacterial variation was hereditary, an issue which seemed to have little relevance to their pragmatic, often clinical, research programs (Amsterdamska, 1987).13 Over the course of the 1930s, a different line of research, growing out of nutritional studies of bacteria, accounted for bacterial variation in terms of ‘chemical adaptation’. In 1900, Fre´de´ric Dienert had shown that yeast grown in glucose could not immediately ferment galactose, but if cells were transferred to a galactose-containing medium that also had a nitrogen source, they could acquire the ability to grow on galactose (Dienert, 1900). This acclimatization took place also in media containing disaccharides such as melibiose and lactose, which contain galactose, but growth in the presence of other sugars, such as maltose and sucrose, did not confer the ability to ferment galactose. Conversely, cells that could grow on galactose, if transferred back to glucose-containing media, lost their ability to ferment galactose. Bacteria seemed able to respond to the presence of certain foodstuffs by generating de novo enzymes that could metabolize them (Summers, 2003; Barnett, 2004). Subsequently, Henning Karstro¨m’s work on the specificity of different bacteria in fermenting carbohydrates led him to suggest that, in many cases, the previous medium of a bacterial culture determined what the cells could ferment. His 1937 publication ‘Enzymatische Adaptation bei Mikroorganismen’ became a touchstone for the emerging literature on microbial growth and nutritional requirements (Karstro¨m, 1937). Karstro¨m drew a distinction between those enzymes that are synthesized only in the presence of their specific substrates— adaptive enzymes—and enzymes that are always present in the cell, or constitutive enzymes. The widespread attention to microbial adaptation reflected the growing importance of biochemistry, with its focus on enzymes, to bacteriology. (By contrast, the earlier interest in morphological and serological variation had relied on older bacteriological techniques and microscopy.) Exemplary of the biochemical approach was the work of Marjory Stephenson at F. Gowland Hopkins’s Cambridge laboratory. By studying lactose fermentation in brewer’s yeast (Saccharomyces cerevisiae) and in the intestinal microbe Escherichia coli (E. coli), Stephenson and her colleagues cast new light on the relationship of enzyme adaptation to cell growth (Kohler, 1985a, p. 178). Stephenson’s coworker John Yudkin presented the scientific problem in terms of two competing explanations, offering a dichotomy that would become pervasive in the literature on bacterial variation: The influence of the substrate on the production of organisms possessing the hydrogenylases [enzymes] may be pictured as either (1) a natural selection, or (2) a chemical adaptation. In the former case, one must suppose that there exists in all cultures a small but definite number of cells possessing the enzyme . . . Enzymes, then, arising by some such mutation become, according to this hypothesis of natural selection, 12 Neeraja Sankaran has analyzed Frank MacFarlane Burnet’s marginalia and private annotations to demonstrate that when reading d’Herelle’s 1926 book (Le bacte´riophage et son comportement, translated as The bacteriophage and its behavior), Burnet considered whether the emergence of secondary cultures resistant to bacteriophage might be attributed to pre-existing genetic variation. She dates his reading to late 1927 or early 1928 (Sankaran, 2006). 13 Bacteriologists in the 1920s also considered the possible role of bacterial life cycles as a source of morphological variation; on this issue see Amsterdamska (1991).

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of biological value to the organism. Those members possessing them are therefore at an advantage and tend to multiply at the expense of the others. A strain is thus formed in which the majority of the members possess the enzyme in question. : : : More plausible is the second suggestion, that of chemical adaptation. On this view, an adaptive enzyme arises as a response to its chemical environment. It would then partake of the nature of an acquired character in higher organisms. With the removal of the stimulus, the character is lost by the descendants of the organism. They still, of course, retain the power to develop that character in conditions similar to those in which the parent organism developed it. (Yudkin, 1932, p. 1869; emphases added) The group at Cambridge published several experiments demonstrating that adaptation in their systems arose from the synthesis of completely new enzymes in the bacteria after the cells encountered the substrate. The new enzymes appeared even in the absence of any cell growth, so that they could not be due to the selection and growth of mutant cells that harbored the enzyme (Yudkin, 1932; Stephenson & Stickland, 1933; Stephenson & Yudkin, 1936; Stephenson, 1937). Even when specific enzymes were not identified or invoked, adaptation was woven into a broader framework for explaining how bacteria survived environmental change. The great increase in nutritional studies of bacteria provided the context for much of this research.14 Many bacteriologists reported being able to change the nutritional requirements of bacterial strains: the scientist could ‘train’ an auxotrophic strain (one that required a particular amino acid or nutrient) to grow without the substance by subjecting the culture to a decreasing concentration of it.15 In reality, the meaning of ‘training’ and its relationship to adaptation remained murky; many bacteriologists used these terms interchangeably, but Yudkin argued that ‘training’ represented the selection of an inheritable change (mutation), not an environment-induced adaptation (Yudkin, 1932, 1938). This framework of adaptation carried over to observations of bacterial drug resistance. As Rene´ Dubos observed in 1942, ‘it may be profitable to keep in mind that susceptible bacterial species often give rise by ‘‘training’’ to variants endowed with great resistance to these [antibacterial] agents’ (Dubos, 1942, p. 672). When bacterial cultures that were initially inhibited by antibiotics began growing again, the phenomenon was reminiscent of the auxotropic strains that overcame their nutritional requirements. Medical bacteriologists who cultured strains from penicillin-treated patients found a substantial fraction (as high as one third) to be resistant to the usual drug dose. Bacteriologists rapidly demonstrated that resistance could be generated in laboratory strains as well by culturing bacteria with antibiotics. From 1945 onward there was a flurry of literature on the growth and characteristics of antibiotic-resistant strains isolated from both patients and laboratory stocks.16

14

On the rapid growth of research on bacterial nutrition in the 1930s, see Kohler (1985b), pp. 62 ff. Francis Ryan cites twelve ‘instances of the adaptations of strains of bacteria and yeasts to dispense with complete growth-factor requirements’ (Ryan, 1946, p. 224). 16 See Bailey and Cavallito (1948) for a review of this literature; the earliest publication of this type that they cite is North and Christie (1945). However, sulfonamide-resistant gonococci (responsible for gonorrhea) had been identified as early as 1938 (though not apparently published for five years). Military uses of sulfonamides to treat common streptococcal infections during World War II also led to observations of drug resistance: ‘Mass prophylaxis with sulfonamides reduced the frequency of streptococcal infections from 6.7 cases per 1,000 persons in 1943 to 5.1 in 1944, yet in the following year the rate rose to 7.4 cases per 1,000 persons because of the spread of resistant strains’ (Dowling, 1977, p. 112). 15

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In the 1940s, British chemist Cyril Hinshelwood took up the investigation of bacterial ‘training’. As he asserted, ‘The problem of the bacterial cell throws down a challenge to the physical chemist’ (Hinshelwood, 1944, p. 150). Hinshelwood made his name in the 1920s through his study of chemical reactions, especially his use of kinetics to account for chain reactions. He was especially interested in the analogy between the autocatalytic behavior of chain reactions and apparently self-perpetuating patterns associated with bacterial growth and metabolism. Because bacterial cells in a constant medium produce enzymes and cell substances at a constant rate, he argued, ‘they must all obey the law of autosynthesis’ (Hinshelwood, 1946, p. 22). According to his view, the reaction of enzyme with substrate produced not only product but more enzyme as well.17 He asserted that ‘bacteria possess nothing in the way of a definitely differentiated structure’, so that their responsiveness to environmental conditions could be understood in strictly physico-chemical terms (ibid., p. 20). Hinshelwood drew on his chemical interpretation to explain how bacterial cells could adapt and multiply in the presence of antibiotics, even though the drugs initially inhibited growth (ibid., p. 1). By exposing initially susceptible cultures of bacteria to higher and higher concentrations of antibiotic, he induced antibiotic resistance in a stepwise fashion. In his view, the bacteria were being ‘trained’ to survive (Hinshelwood, 1944). Hinshelwood identified this change in response to antibiotics as an example of ‘adaptation’, which might be attributed either to kinetic changes in the cellular enzymes induced by the drug, or to natural selection of pre-existing resistant strains. He concluded from his studies that ‘while selection may be, and indeed must be, superimposed upon other adaptive mechanisms, there is little profit, with bacterial cells at least, in regarding it as the primary factor governing the adjustment of the economy to resist drugs or to utilize new sources of material [e.g. sugars]’ (Hinshelwood, 1946, pp. 97–98). Hinshelwood’s 1946 book The chemical kinetics of the bacterial cell presented a comprehensive physico-chemical theory of bacterial variation, including sections on nutritional adaptation, resistance to heat, and effects of radiation, but his chief example was drug resistance.

3. The fluctuation test In the early 1940s, research by Max Delbru¨ck and Salvador Luria offered a very different understanding of the source of bacterial variation.18 These two e´migre´ researchers met in 1940 and began a fruitful collaboration on bacteriophage research, joining forces during the summers of 1941 at Cold Spring Harbor and 1942 at Delbru¨ck’s laboratory at Vanderbilt (Selya, 2002).19 Delbru¨ck, a former physicist, and Luria, a physician who had previously worked in Enrico Fermi’s laboratory, borrowed from radiation physics a statistical approach to their experiments. Just as the effects of ionizing radiation had to be understood statistically, one could envision the bacterial cell as a ‘black box’, in which 17

Hinshelwood’s conception here relied on conflating the kinetics of enzyme action with the logarithmic multiplication of bacterial cells in a growth medium. 18 Alfred Hershey also collaborated with Delbru¨ck and Luria; the three of them are generally considered the founders of the ‘phage group’. For the canonical account, see Cairns, Stent, & Watson (1966). 19 For more on Delbru¨ck, see Kay (1985) and Fischer & Lipson (1988). On the choice of phage, see Summers (1993).

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the ‘output’ from phage infection could be statistically analyzed, to distinguish unexpected events from low frequency but usual effects (Kay, 1985). Luria and Delbru¨ck began with d’Herelle’s observation that after lysis, the tube of an infected bacterial culture could again become turbid with cells, due to the growth of a bacterial variant resistant to the bacteriophage. In their view (and following Yudkin’s earlier interpretation), two hypotheses could explain the appearance of these variants: either (1) these were spontaneous mutations conferring resistance to bacteria, that were subsequently selected for in the presence of phage, or (2) exposure to the phage was inducing a specific adaptation to a resistant state.20 They noted that ‘d’Herelle and many other investigators believed that the virus by direct action induced the resistant variants’, whereas Burnet and a few others ‘believed that the resistant bacterial variants are produced by mutation in the culture prior to the addition of virus’ (Luria & Delbru¨ck, 1943, p. 491). They devised an experiment to discern which explanation was correct. Luria realized (by way of analogy to a slot machine, according to his autobiography) that a fluctuation in the number of resistant bacteria (isolated either as colonies or resistant cultures) would signal that the change was mutational, not an induced adaptation.21 (See Fig. 1.) For true hereditary changes, a mutation early after inoculation would occur infrequently but result in a large number of resistant cultures—a ‘jackpot’. By contrast, acquired immunity was expected to occur at a more regular rate in bacteria that survived infection. Thus one could discriminate between the two mechanisms through the degree of fluctuation in the frequency with which resistant variants appeared—‘large fluctuations are a necessary consequence of the mutation hypothesis’ (ibid., p. 494). As it turned out, their experiment showed ‘jackpot’ effects: fluctuations in the number of variants resistant to bacteriophage among the otherwise identical cultures were significantly greater than would be expected from the acquired immunity hypothesis. This observation became a landmark for bacterial genetics, as one of the first clear demonstrations that inheritance in bacteria was not Lamarckian.22 Not that all microbial geneticists viewed their interpretation as irrefutable. In response to a draft of the paper, Tracy Sonneborn pointed out to Luria that what they viewed as spontaneous mutations could be acquired resistance that persisted for several generations (Selya, 2002, p. 82).23 Luria and Delbru¨ck’s paper was published in 1943, when researchers throughout the US, even at Cold Spring Harbor, were seeking ways to contribute to the scientific mobilization. Milislav Demerec, Director of the Department of Genetics at Cold Spring Harbor, saw in microbial genetics an opportunity ‘to take up problems brought about by

20

Luria and Delbru¨ck do not cite Yudkin, but their analysis bears a striking resemblance to his interpretation in the 1932 paper quoted above. 21 See Luria (1984), pp. 74–79, for his account of the experiment and see Brock (1990), pp. 58–63, and Selya (2002), pp. 78–84 for insightful analysis. 22 As William Summers (1991), p. 172, states, ‘In the views of most molecular biologists, it was the work of Salvador Luria and Max Delbru¨ck on bacteriophage-resistant variants of Escherichia coli that established the validity of bacteria as organisms for genetic studies. Thus, it was not until the mid-1940s that bacterial genetics gained the respectability and scientific legitimacy long accorded to fruit flies and corn’. On later challenges to the Luria–Delbru¨ck paper by John Cairns and colleagues, see Keller (1992). 23 As Selya (2002), p. 83, points out, Tracy Sonneborn saw several drafts of the Luria–Delbru¨ck paper, and the authors introduced some changes to address his criticisms. Selya also makes clear that the experimental design and its execution was Luria’s; Delbru¨ck assisted in analyzing the data.

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Fig. 1. ‘Diagram explaining the theory of the fluctuation test. The early stages in the development of several small cultures are shown. Resistant bacteria are dark. Left: Anticipated results if resistant bacteria are induced by contact with phage. In this case, the proportion of resistant cells in the population should be more or less the same in each culture. Right: Anticipated results if resistance arises randomly by mutation. Families of resistant siblings should develop, and depending on when the mutation occurred, the sizes of these resistant clones should vary widely’. Diagram and caption from Brock (1990), p. 59.

the war’ (Demerec, 1943, p. 123).24 One of the leading projects involving microbiologists (as well as chemists) was development of penicillin. Pharmaceutical companies were initially skeptical that the yield of penicillin from the mold Penicillium chrysogenum could be increased enough to make microbial fermentation more promising than chemical synthesis as a means of production (Neushul, 1993; Neushul, 1998). Demerec, in collaboration with biophysicist Alexander Hollaender (then at the National Institutes of Health), proposed to the US Office of Scientific Research and Development (OSRD) to address this problem through genetic methods—namely, employing X-ray and ultraviolet radiation to produce higher-yielding mutants of Penicillium strains. (Hollaender and Demerec were 24 In 1944 Demerec quoted from his initial proposal to Vannevar Bush that since 1941 he and Dr. Hollaender had been considering ‘the possibilities of utilizing modern genetic methods for improving the yield of various microorganisms used in the production of chemicals important in the war’. Hollaender had already been involved in ‘developing by irradiation a strain of Aspergillus terreus which has a high efficiency in the production of itaconic acid’ (letter from Milislav Demerec to W. M. Gilbert, 24 March 1944, Milislav Demerec Papers, American Philosophical Society, Series I., Box 18, Folder 5). By the same token, Demerec acknowledged that the war effort did not produce as great a demand for geneticists as for other specialists (Demerec, 1943, p. 123).

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already investigating radiation-induced mutants in Neurospora crassa.)25 The OSRD’s Committee on Medical Research rejected their proposal in 1942 on account of ‘the speculative character of the whole penicillin enterprise, and particularly the mutant aspect of it’.26 But in the spring of 1944, as pressure mounted to identify higher-yielding strains of Penicillium, Demerec’s project received the go-ahead. Irradiation of Penicillium chrysogenum with X-rays by Demerec’s group produced a mutant, X-1612, capable of yielding 500 units of penicillin per milliliter of culture. This strain was rapidly put into industrial production.27 During the same year that Demerec began the war-related Penicillium project, he also extended Luria and Delbru¨ck’s fluctuation approach to study the emergence of antibiotic resistance—using penicillin from E. R. Squibb.28 Having established that he could produce new strains of Staphylococcus aureus more resistant to penicillin than the original standard stock, Demerec investigated the origin of these resistant bacteria. (Demerec was already using Staphylococcus aureus in the bioassay for the penicillin-yield project.) Implicitly, Demerec’s use of Luria and Delbru¨ck’s experimental design drew an analogy between resistance to antibacterial infectious agents (bacteriophage) and resistance to antibacterial chemical substances (antibiotics). Demerec followed precedent (not only that of Luria and Delbru¨ck, but also of Yudkin) in presenting two possible explanations: resistance as an acquired characteristic, or resistance as an inherited mutation (Demerec, 1945a, p. 19). To discern which of these mechanisms was operating in the resistant strains he produced, Demerec set up a fluctuation test. If the penicillin induced resistance, one would expect it to occur at the same frequency in colonies from almost any culture. If, on the other hand, resistance occurred as a consequence of random genetic mutations, then one would expect only bacteria from a single culture—genetic clones—to yield similar numbers of resistant bacteria. Samples taken from separate cultures, according to the genetic interpretation, should show a wide distribution of numbers of resistant colonies, depending on whether a mutation occurred early or late in the growth of the culture.29 Demerec’s experiment showed a wide variation in the number of resistant bacteria in samples from independent cultures, which confirmed a genetic interpretation of the origin of resistance. (See Fig. 2.) ‘Thus penicillin acts as a selective agent which suppresses nonresistant bacteria’ (ibid., p. 23).

25

Lily Kay (1989) has shown how the biochemical genetics of Neurospora crassa also received a crucial boost from the war effort. 26 Letter from A. N. Richards to Milislav Demerec, 21 December 1942, Milislav Demerec Papers, American Philosophical Society, Series I., Box 18, Penicillin Project, Folder 5. See also the letter recounting this episode from Milislav Demerec to Franklin C. McLean, 9 October 1945, Folder 3. 27 In 1948 a patent was issued on Demerec’s method for producing high-yielding strains. See Milislav Demerec Papers, American Philosophical Society, Series I., Box 18, Penicillin Project, Folders 1–3. 28 There was only limited civilian distribution of penicillin in 1944, and Demerec’s publication, submitted in December of that year, does not make clear whether his contract with the government helped him access the compound. The supply increased in 1945. See Richards (1964). On the contemporary beginning of the antibiotic resistance project, see Demerec (1944), pp. 109–110. 29 ‘If resistance originates by mutation, then, the variation in number of resistant bacteria between samples taken from separate cultures should be much greater than between samples taken from the same culture’ (Demerec, 1945a, p. 19).

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Fig. 2. ‘Number of bacteria resistant to concentration of 0.064 Oxford units of penicillin per cc. of agar medium in samples taken from a series of independent cultures and similar samples taken from a single culture which assay 2.3 · 108 bacteria per cc’. Caption and table from Demerec (1945a), p. 20.

Demerec also showed that penicillin resistance was transmitted as a permanent inherited trait, passed through twenty transfers of culture in non-penicillin-containing broth. At the same time, his studies addressed one of the weaknesses of the genetic interpretation, namely the fact that penicillin resistance developed in a gradual, step-wise fashion.30 His evidence suggested that resistance was a complex characteristic, involving a number of genetic changes or mutations. In this view, selection could account for the ‘training’ effect with penicillin—one could elicit an ‘increase in resistance proceeding exponentially with the number of mutant genes involved’ (Demerec, 1945b, p. 138). After the war, use of the fluctuation test became a boon for bacterial geneticists. Luria extended the fluctuation approach to show that mutations accounted for sulfonamide resistance in Staphyloccus aureus (Oakberg & Luria, 1947). Demerec showed that streptomycin resistance was similarly genetic in origin (Demerec, 1948). Moreover, the fluctuation test proved applicable for traits beyond resistance to antibacterial agents such as phage and antibiotics. Evelyn Witkin showed that resistance to radiation in E. coli was mutational in origin, and Francis Ryan demonstrated that mutations could restore independence from nutritional requirements (on the amino acid histidine for E. coli and the 30 By the early 1950s, bacteriologists spoke of two patterns for the bacterial acquisition of antibiotic resistance: the slow, step-wise pattern characteristic of penicillin, and resistance that developed after few exposures (or even just a single exposure), characteristic of the response to streptomycin. See English and Gelwicks (1951).

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pyrimidine uracil for Clostridium septicum).31 Thus armed with the fluctuation test, bacterial geneticists challenged bacteriologists on their own turf: nutritional training. 4. Drug resistance and debates over bacteria as genetic organisms Bacterial geneticists were staking a greater claim than simply the mutational origin of antibiotic resistance. As Luria stated in 1946, If a case could be made : : : for similarity of the processes of mutation in bacteria and in higher organisms—that is, for the existence of discrete, gene-like hereditary units in bacteria—then these organisms might prove to be invaluable material not only for the study of physiological genetics, but also for an attack on the problems of gene structure and mutability. (Luria 1946, p. 130) Making this case was not trivial. Not only were genetic techniques unfamiliar in bacteriology but also classical genetics was developed to study diploid and sexually reproducing creatures, whereas bacteria reproduced asexually—by simple binary fission. As George Beadle stated in 1945, The genetic definition of a gene implies sexual reproduction. It is only through segregation and recombination of genes during meiosis and fusion of gametes that the gene exhibits its unitary property. In bacteria, for example, in which cell reproduction is vegetative, there are presumably units functionally homologous with the genes of higher organisms, but there is no means by which these can be identified by the techniques of classical genetics. (Beadle, 1945, p. 18) Beadle’s own work on the bread mold Neurospora crassa showed how one could extend the techniques of classical genetics to microbes, albeit to one with a sexual life cycle. Using nutritional mutants and taking advantage of the fact that bread mold has a diploid phase before sporulating, Beadle and Edward L. Tatum showed that particular nutritional requirements arose as a consequence of genetic mutations (Kay, 1989). Tatum was able to isolate similar growth-factor-requiring mutants in E. coli, and even created double mutants, but as crossing was not possible, these strains were merely evidence for the heritability of growth factor requirements in bacteria (Brock, 1990, p. 79). Beyond the identification of microbial mutants, the similarity of hereditary units in bacteria to those in ‘higher’ organisms rested on indirect evidence, such as the similar mutational effects of radiation on Drosophila, bacteria, and even viruses (Gowen, 1941). Then in 1946, using double nutritional mutants of strain K-12 acquired from Tatum, Joshua Lederberg was able to demonstrate mating in E. coli (Lederberg & Tatum, 1946, 1947; Tatum & Lederberg, 1947).32 This breakthrough opened up bacteria to the techniques of genetic crossing. The mechanism of genetic recombination in E. coli was unclear—there was no evidence for a diploid state, even though linkage relations could be established. The 31

Witkin (1946); Ryan (1946); Ryan, Schneider, & Ballantine (1946). On the wide-ranging applicability of the fluctuation test, see Luria (1947). Francis Ryan and Joshua Lederberg (1946) introduced the term ‘prototroph’ for revertents that lost their dependency on a nutritional requirement. 32 Brock offers a discussion of the innovative features of their prototrophic recovery technique, which opened the way—even more than Luria and Delbru¨ck’s fluctuation test—to a thorough-going research stream in bacterial genetics (Brock, 1990, p. 82).

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important point was that, in principle, genes in E. coli could be mapped. Within a year, Lederberg had produced the first genetic map of a bacterium (Lederberg, 1947; Creager, 2004). Lederberg and Tatum’s first linkage map included resistance to bacteriophage as a marker (in addition to several growth factor requirements), and Lederberg soon extended the repertoire of traits to include antibiotic resistance. Mapping a gene for antibiotic resistance in E. coli strongly supported the contention that the trait was under genetic control. Despite these achievements, the community of mainstream bacteriologists kept an agnostic stance towards the genetic explanation of antibiotic resistance. As John Hays Bailey and Chester J. Cavallito wrote in a review of antibiotics for the Annual Review of Microbiology in 1948,33 There are two theories as to the origin of resistant organisms. One theory attempts to explain their occurrence as an effect of the antibiotic on the bacterium, resulting in a metabolism so altered that the organism can grow in a normally inhibitory concentration of the antibiotic; the other, proposed by Demerec and Luria, may be termed the genetic theory . . . In the presence of the antibiotic the majority of the population is inhibited and only those few resistant forms or mutants can develop. (Bailey & Cavallito, 1948, p. 169) The authors offer no appraisal of these competing interpretations. Similarly, Windsor Cutting, in his review of antibiotics the next year, gave an equivocal (if muddled) account of the two theories: ‘Resistance may result from the growing out of a few naturally resistant organisms in a culture until they form the mass of the organisms, or may be induced in otherwise susceptible organisms through mutations which give rise to strains better adapted to resist penicillin’ (Cutting, 1949, p. 139). The following year (1950), C. Phillip Miller and Marjorie Bohnhoff opened their review of ‘The development of bacterial resistance to chemotherapeutic agents’ with the two general theories, induced adaptation or spontaneous mutation and selection (Miller & Bohnhoff, 1950, p. 201). They acknowledged that the latter alternative had ‘gained wide acceptance’, yet the following year, James G. Horsfall and Albert E. Dimond reviewed the work on antibiotics without entering into the ‘problem of resistance’, for, ‘it does not appear necessary to our arguments to enter the battleground of the mutation versus the adaptation theory’ (Horsfall & Dimond, 1951, p. 207).34 This dichotomy between adaptationist and mutationist explanations is pervasive in the literature, offered as often by agnostic reviewers as by advocates of one interpretation or the other. Yet the alternatives were not as clear-cut as such overviews tended to suggest. Bacterial geneticists sought to clarify that they did not argue with adaptation, but rather wanted to distinguish true non-hereditary adaptation from the selection of genetic variants.35 But to further complicate matters, adaptation was increasingly interpreted in terms of heredity—not via mutation and selection, but cytoplasmic heredity. 33 The first issue of the Annual Review of Microbiology was published in 1947, and every volume for the first ten years had at least one review on antibiotics. Coverage of the topic remained strong into the 1960s. 34 Horsfall and Dimond continue, ‘The statistical method developed by Luria & Delbru¨ck which has provided much of the evidence for the mutation theory has been the subject of criticism, since variables other than mutation are included in the population. On the other hand, those who are critical of the adaptation theory point to the existence of strains resistant to concentrations of antibiotics greater than that in which they had been trained . . . [F]or the present, the subject must still be considered to be under discussion’ (Horsfall & Dimond, 1951, p. 207). 35 See, for example, Lederberg & Lederberg (1952) and Cavalli-Sforza & Lederberg (1956).

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Several lines of experimental evidence beyond adaptation pointed to the existence of hereditary cytoplasmic entities in flies, microbes, and plants (Sapp, 1987). In 1941, Sewall Wright had referred to ‘cytoplasmic proteins which possess the essential genic property’ as plasmagenes, in contrast with nuclear genes (Wright, 1941, p. 501).36 C. D. Darlington subsequently published a paper on plasmagenes in Nature, pointing to the similarities between cellular plasmagenes and viruses, which also behaved as self-perpetuating genetic entities (in an infected cell). He argued that there were in fact two distinct systems of non-Mendelian nuclear inheritance: plastids in plants (which could be seen in a microscope) and molecular agents of cytoplasmic inheritance that included plasmagenes (Darlington, 1944).37 Sol Spiegelman further tailored the notion of plasmagenes to propose a theory of enzyme formation that would specifically account for adaptation. He argued that a cytoplasmic gene was formed from the chromosomal gene, and was subsequently replicated without assistance from the nucleus.38 Instead, the presence of the enzyme substrate stabilized and so maintained the plasmagene in the cytoplasm over time (Spiegelman & Kamen, 1946). Accordingly, enzyme adaptation might be hereditary, yet not genetic in the sense that classical geneticists meant—traceable to the permanent genetic material, nuclear chromosomes. Rather, the perpetuation of the cytoplasmic heritable factor would rely on environmental cues, such as sugar or other substrates critical to growth. The legitimacy of such cytoplasmic genes was controversial in the late 1940s, especially in the US, where classical genetics dominated discussions of heredity.39 (In Europe, and especially Germany, chromosome theory did not eradicate strong interest in cytoplasmic heredity, especially from botanists and embryologists (Harwood, 1993).) What did it mean to have a gene that was not part of the nuclear genome and not subject to the laws of Mendelian inheritance? Muller, like several other prominent geneticists, attacked the peculiarity of plasmagenes, arguing that the best-documented cytoplasmic agents were infectious and, hence, exogenous to the cell (Sapp, 1987, pp. 117–18). Many of these cytoplasmic agents were microbial, from Tracy Sonneborn’s kappa factor in Paramecium to the DNA transforming factor in Pneumoccocus. This created a potential quandary for bacterial geneticists. Just as they sought to establish that the genes of microbes could be analyzed using the tools of classical genetics, observations of cytoplasmic inheritance in microbes reinforced older suspicions that Mendelian–Morganian genetics—even chromosome theory—did not reach to bacteria. These debates, moreover, became politically charged as Soviets seeking to suppress genetics invoked observations of cytoplasmic heredity as evidence for the limitations of

36 This notion (plasmatischen Gene) was already being used widely in Germany, as introduced by Winkler (1924). Jonathan Harwood offers an excellent account of the German research into cytoplasmic investigation, including Winkler’s contributions (Harwood, 1984). 37 Darlington classified the ‘molecular’ system, including plasmagenes, with ‘maternal’ inheritance; he termed the plastid system ‘corpuscular’, since the bodies could be seen. Much of the evidence for cytoplasmic determinants of heredity derived from embryology. For an extended analysis of the analogies between plasmagenes and viruses, see Creager (2002), Ch. 6. 38 Carl Lindegren offered a similar model (Lindegren, 1945; Brock, 1990, §10.2). I use the term nucleus for bacteria, as many bacteriologists did in the 1940s, acknowledging that the nature of such an entity was quite controversial. However, Rene´ Dubos in his 1945 book, The bacterial cell, argued that the bacterial nucleus consisted of a single linear unit of genes, like a chromosome in higher organisms. 39 T. H. Morgan had declared (famously) in 1926, ‘In a word, the cytoplasm may be ignored genetically’ (Morgan, 1926, p. 491).

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the Mendelian framework. Trofim Lysenko, who for a time successfully led the Soviet attack on modern genetics, argued that ‘Mendelism–Weismannism–Morganism’ manifested the assumptions of bourgeois capitalism in its attribution of traits to idealistic genes irrespective of environmental conditions. Lysenko presented ‘Michurinist’ biology as an alternative, defending the inheritance of acquired characteristics as demonstrated by vernalization experiments.40 As a result of his rise to power, several of the most prominent Russian geneticists were imprisoned, including Nikolai Vavilov and Georgii Karpechenko, both of whom died; many others lost their positions.41 Classical genetics, where it still existed, went underground in the USSR (Adams, 1980; Krementsov, 1997). While many Western scientists had been sympathetic to the socialist organization of science in the Soviet Union, the death of Vavilov in 1943 (just after he had been elected fellow of the Royal Society of London) led to a scientific campaign against Lysenkoism. Bacterial geneticists Luria and Demerec joined with other prominent geneticists in the US, including Theodosius Dobzhansky, Curt Stern, L. C. Dunn, and H. J. Muller to try to support the cause of Russian geneticists by publishing critical reviews of the recently published translation of Lysenko’s book.42 Despite the campaign in Western periodicals against Lysenkoism, the Cold War gave the upper hand to the Michurinists in the USSR (Krementsov, 1996). Lysenko’s public denunciation of Mendelian genetics at the Lenin Academy of Agricultural Sciences in 1948 marked the official abolition of genetics in the Soviet Union.43 Proponents of Lysenkoism regularly cited work on cytoplasmic and infective heredity as evidence of the inadequacy of Mendelian genetics. The same year he was president of both the Genetics Society of America and the American Society of Naturalists, Tracy Sonneborn found his research on Paramecium invoked in support of Lysenko’s views.44 Other biologists were cited for their openness to non-Mendelian inheritance: J. B. S. Haldane and Darlington had both argued in favor of the existence of Lamarckian patterns of inheritance (although Darlington did not believe these were the principal mechanisms of evolutionary change). Moreover, the transformation of Pneumococcus strains, as observed by Frederick Griffith in the 1920s, was commonly viewed as evidence of Lamarckian inheritance. The isolation of the transforming factor by Oswald Avery, Colin MacLeod, and Maclyn

40 ‘Michurinist’ biology was named after the Russian horticulturalist Ivan Michurin. The key experiments on vernalization were nearly all agricultural in nature, many being conducted on cereals. 41 Vavilov died shortly after his release from prison of malnutrition. See Soyfer (1994). 42 An English translation of Lysenko’s book Heredity and its variability (translated by Dobzhansky) was published in 1943. On the involvement of geneticists in the West, see Krementsov (1996), pp. 242–244. For many left-leaning geneticists (especially in England and Europe) the Lysenko affair was what led them to abandon the Communist Party. The notable holdout was J. D. Bernal, who retained his Party membership and an openness to Lysenko’s point of view. 43 On Stalin’s editing of Lysenko’s speech, see Rossianov (1993). On the international repercussions of the 1948 speech, see Krementsov (1996). Roll-Hansen (2005) offers a different treatment of the events. 44 As Sonneborn wrote to Dobzhansky late in 1948, ‘You will be interested to see a translation of an article by I. I. Prezent . . . This article confirms my suspicion that the Communist devils are quoting from my scriptures as if I were a supporter of Lysenko. Darlington writes me that the English Communists quote me in support of Lysenko; and Newsweek did the same in this country. So you see I have a very personal interest in trying to set matters straight’ (Sapp, 1987, p. 175). Sonneborn used his positions to lead a counteroffensive against Lysenkoism, in coordination with Muller (president of the Human Genetics Society), Dobzhansky, and Ralph Cleland (president of the Governing board of the American Institute of Biological Sciences). Sonneborn’s presidential address to the American Society of Naturalists took up the challenge of Lysenkoism (Sonneborn, 1950; Sapp, 1987).

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McCarty reinforced this impression by showing that the acquired trait was attributable to a transmissible molecule, DNA (Griffith, 1928; Avery, MacLeod, & McCarty, 1944). The organizational efforts of geneticists in the US to counter Lysenkoism culminated in the 1951 publication of Genetics in the twentieth century by the Genetics Society of America (Dunn, 1951; Sapp, 1987, p. 180). The contributions, which were first presented at a conference honoring the fiftieth anniversary of the rediscovery of Mendel’s work, celebrated both Mendelism’s practical achievements (e.g. hybrid corn) and its recent intellectual triumphs, most notably contributions from Dobzhansky and Huxley addressing the neo-Darwinian synthesis. As Joshua Lederberg has noted, the preoccupation with the ‘primacy of the nucleus for the genetic determination of cell traits’ meant that cytoplasmic contributions to heredity tended to be marginalized.45 Bacterial genetics was sparsely represented.46 At the disciplinary level, bacterial geneticists felt they were fighting an uphill battle— well into the 1950s—to get bacteriologists to accept a genetic approach to microbes. They had good reason to feel outnumbered. By Robert Kohler’s estimation, there were less than a dozen bacteriology laboratories in the world until the 1940s that systematically investigated bacterial physiology (Kohler, 1985a, p. 163).47 By contrast, laboratories of medical bacteriology numbered in the hundreds (Spath, 1999, Ch. 1).48 Evelyn Witkin has described the concerted efforts of bacterial geneticists to get a hearing in the Society of American Bacteriologists in terms of a ‘fifth column’.49 They were not above polemical tactics. In a 1947 review, Luria stated that ‘bacteriology [was] one of the last strongholds of Lamarckianism’ (Luria, 1947, p. 1).50 This was a damning charge in the context of Lysenkoism. Haldane, in his ambivalent criticism of Lysenkoism, stated that ‘A number of people in this country agree substantially with Lysenko’s views on heredity, notably

45

Lederberg, unpublished manuscript, 1952, as quoted in Lederberg (1998), p. 1. Lederberg contributed ‘Genetic studies in bacteria’ and Sonneborn contributed ‘The role of the genes in cytoplasmic inheritance’. Beadle discussed his work on chemical genetics in Neurospora and Richard Goldschmidt made brief mention of bacteriology (among a laundry list of other fields that genetics had impacted). See Dunn (1951). 47 Kohler’s count of ten laboratories of bacterial physiology includes the Pasteur Institute in Paris, the Rockefeller Institute in New York, agricultural schools at the universities of Iowa, Wisconsin, and Helsinki, laboratories in the biology departments at Stanford University, University of Delft, and the California Institute of Technology, the biochemistry department at Cambridge University, and the bacteriology departments of Middlesex Hospital in London and the London School of Hygiene. This is probably an underestimate. Joshua Lederberg has said that bacteriologists at Cornell and Illinois, uncounted by this tally, should be acknowledged (personal communication, 31 July 2002). Along similar lines, Thomas Brock argues that Berkeley, Cornell, and Rutgers should be included in such a list, and not Caltech (personal communication, 11 November 2002). Even with these corrections, the overall dominance of the medical orientation remains undisputed. 48 As is often the case, the boundaries between ‘basic’ and ‘medical’ bacteriology were not as distinct as this terminology suggests. Nonetheless, other historians such as Keith Vernon have agreed that there was not a consolidated discipline of microbiology before World War II, and theoretical research remained secondary during the first half of the twentieth century, when microbes were studied in the diverse contexts of public health, breweries, dairies, sewage treatment, and agriculture (Vernon, 1990). 49 Evelyn Witkin, conversation with author, July 18, 2002. The organization was renamed the American Society for Microbiology in 1961. 50 Similarly, Lederberg declared in 1949, ‘The exposition of the problem of directed adaptive variation has been fully discussed. Spontaneous mutation and natural selection are adequate to account for most adaptive changes in bacterial populations. From a general biological standpoint, exceptions to this rule would be very instructive, but except for induced lysogenicity, claims of lamarckian responses in bacteria have not been sufficiently fortified by experiment’ (Lederberg, 1949, p. 1). 46

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Professor Hinshelwood of Oxford’ (Haldane, 1948).51 Thus the politics of Lysenkoism intersected with the debate over bacterial resistance to antibiotics (Hinshelwood’s key example), and this occurred in the popular press as well. A science journalist for the New York Times reported (albeit incorrectly) that Lysenko’s views on heredity were confirmed by National Institutes of Health researcher Harry Eagle in his studies of adaptation and antibiotic resistance (Plumb, 1954).52 Charges of Lamarckianism notwithstanding, most bacteriologists did not embrace bacterial genetics.53 In addition to the established tendency to explain new bacterial traits in terms of ‘training’ and ‘adaptation’, few bacteriologists were familiar with the kind of statistical arguments that underlay genetic interpretation, especially in experiments such as the fluctuation test.54 K. R. Eriksen questioned whether applying the fluctuation test to the origin of antibiotic resistance was even justified (Eriksen, 1953; Eagle & Saz, 1955, p. 201). Furthermore, new research on the mechanism of drug resistance, particularly concerning bacterial production of penicillinase, cast doubt on whether the laboratory-generated resistant mutants were similar to those that arose as a consequence of therapeutic use. Waclaw Szybalski showed that only 1 in 10,000 penicillin-resistant mutants obtained by selection in the laboratory was a penicillinase-producer (Szybalski, 1953). By contrast, ‘more than 99 per cent of the highly penicillin-resistant staphylococci isolated from patients produce penicillinase’ (Eagle & Saz, 1955, pp. 201–202). Even so, some bacteriologists acknowledged the utility of genetic explanations. In 1948, the prominent British bacteriologist Paul Fildes published a paper showing that the phenomenon of ‘training’ he had previously observed could be accounted for by mutation followed by selection (Fildes & Whitaker, 1948). Harry Eagle wrote approvingly of the genetic explanation for antibiotic resistance, yet he did not rule out bacterial adaptation to low concentrations of antibiotic (Eagle, 1954, 1955). Moreover, his acceptance of the validity of the genetic explanation did not mean that he assimilated genetic methods in his research on the mode of action of antibiotics. Instead, he relied on the same sorts of growth studies that bacteriologists had employed productively for decades. In fact, Eagle turned in the mid-1950s to investigate the growth requirements of animal cells in culture, resulting in his best-known scientific legacy; the chemically defined minimal essential growth medium he developed still bears his name.

51

I have not been able to ascertain whether this charge was simply based on Hinshelwood’s views on bacterial heredity and drug resistance, or on his political views. I have not seen evidence that Hinshelwood involved himself in the Lysenko controversy. 52 According to Lederberg, the story in the New York Times relied on a gross misunderstanding of Eagle’s research. See Lederberg to the American Civil Liberties Union, 8 January 1955, available at http://www.profiles. nlm.nih.gov/BB/A/D/C/T/. 53 An indication of how gradually bacterial genetics was assimilated into bacteriology can be seen by perusing a standard textbook, Topley and Wilson’s principles of bacteriology and immunology, (4th ed.) (Wilson & Miles, 1955). In the arena of research on antibiotic resistance, Antibiotics & Chemotherapy published numerous bacteriological studies from 1951 to 1962, but only a handful employed genetic methods. 54 As Luria stated in trying to account for Hinshelwood’s influence over bacteriologists, ‘I have often noticed . . . that biologists are readily intimidated by a bit of mathematics laid before them by chemists or physicists. It was one of the blessings of my too short stay among physicists to be immunized against mathematical humbug’ (Luria, 1984, p. 74; also quoted in Brock, 1990, p. 57). I have found one paper by bacteriologists that did use the fluctuation test in studying the pattern of resistance of E. coli to polymyxin B, neomycin, and streptomycin: Waisbren, Carr, and Struxness (1951).

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Others accepted the validity of selectionist mechanisms while taking a different view of the process than geneticists. For example, Fredy Roland and C. A. Stuart argued that streptomycin resistance in Salmonella typhi arose as a result of a ‘directed mutation’ (Roland & Stuart, 1951). Along similar lines, Mark Lepper’s review of antibiotic resistance for the Annals of Internal Medicine depicted the emergence of resistant strains as attributable to three, not two, possible mechanisms: adaptation, in which organisms ‘find a way to protect themselves’, mutation resulting from exposure to the antibiotic, or overgrowth of pre-existing mutants (Lepper, 1955, p. 300). (He placed production of an ‘inactivating enzyme’, i.e. penicillinase, in a completely separate category.) If bacterial geneticists viewed most bacteriologists as uncomprehending of—or even resistant to—their approach, they found a warmer reception in the mainstream genetics community. Advances in Genetics, a serial published first in 1947, gave attentive coverage to microbial genetics. (Then again, the editor in chief was Demerec.) Even more significantly, Dobzhansky included a section on ‘Mutation and selection in microorganisms’ in his 1951 edition of Genetics and the origin of species. He called attention to the experimental suitability of microorganisms for ‘studies of mutation and selection’, which had been long underappreciated by researchers. Occurrence of changes in bacterial strains has been known for about half a century, but their interpretation had a Lamarckian flavor, as implied by the words ‘dissociation,’ ‘adaptation,’ ‘training,’ etc., used in this connection. It took the brilliant analysis by Luria and Delbru¨ck (1943) and by Demerec and Fano (1945) to open this field for genetic study. (Dobzhansky, 1951, p. 87) Dobzhansky went on to discuss the work by Demerec and Luria (among others) on the mutational origin of microbial antibiotic resistance: Demerec (1945[a]) and Luria (1946) showed that penicillin-resistant strains of Staphylococcus aureus arise by mutations which survive in the presence of enough penicillin in the medium to kill most individuals of the parental strain. Resistance to very high doses of the drug can be built by summation of several mutational steps. This accounts for the gradual adaptation of bacterial strains to unusual environments, which was known in bacteriology for a rather long time but was misinterpreted in a Lamarckian fashion (ibid., pp. 89–90). Dobzhansky’s was a key text for the neo-Darwinian synthesis, and it made antibiotic resistance a crucial—and observable—example of natural selection at work. In this sense, bacterial genetics had obvious utility to geneticists, and to the project of unifying Darwinian evolution and Mendelian genetics.55 For their part, bacterial geneticists continued to accumulate more elegant experimental evidence that selection of mutants could account for drug resistance. Joshua and Esther Lederberg’s method of replica plating provided a new experimental approach for challenging the adaptationist explanation. The Lederbergs made an impression of a Petri dish containing confluent bacterial growth on a piece of velveteen, then transferred the colonies (using the same fabric impression) to subsequent plates. (See Fig. 3.) If transferred colonies grew on selective media (e.g. media containing an antibiotic), one could obtain direct 55

On the project of the neo-Darwinian synthesis, see Smocovitis (1996).

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Fig. 3. A diagram showing how replica plating works for selecting resistant (in this case, phage-resistant) bacterial mutants. From Lederberg & Lederberg (1952), p. 403; reprinted by permission of the American Society for Microbiology.

evidence (in contrast to Delbru¨ck and Luria’s statistical evidence) that occasional colonies not previously exposed to antibiotic could grow in its presence. The adapted cells were ‘not randomly distributed in space’ on the Petri dish, yet another indication that the occurrence of mutations was random and preceded exposure to the selective environment (Lederberg & Lederberg, 1952, p. 404).56Adaptation itself, especially enzyme adaptation, was being reinterpreted to accord with the precepts and methods of bacterial geneticists, most notably by Jacques Monod.57 In the mid-1950s, attention in the press to the problems of drug resistance gave the old debates a new visibility (Rosenkrantz, 1995). Large hospitals had become incubators of antibiotic-resistant strains. By 1955, the fraction of staphylococcus strains isolated from hospital patients found to be penicillin-resistant approached three-quarters (Finland, 56 As Lederberg (1989), p. 397, has noted since, replica plating ultimately spawned a ‘major industry’ of blots, such as Northern and Southern, for the molecular identification of particular variants. 57 See, for example, Monod (1956). The ‘geneticization’ of enzyme adaptation was not an easy or obvious process; see Gaudillie`re (1992). On the early abandonment of the term adaptation itself, see Cohn, Monod, Pollock, Spiegelman, and Stanier (1953).

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1955).58 Newspaper stories warned the public about a false confidence that antibiotics would vanquish infectious disease. The New York Times alerted readers in 1955 that the ‘War on bacteria [was] seen as backfiring’; two years later another by-line in the paper cautioned, ‘Resistance to antibiotics is cited by surgeons for world-wide epidemic’.59 This aura of heightened concern amidst growing therapeutic use of antibiotics provided the backdrop to a Ciba Foundation Symposium on Drug Resistance in Micro-Organisms, held in 1957. The program was focused specifically on the ‘mechanisms of development of drug resistance’, which the organizers regarded as a neglected aspect of bacterial chemotherapy (Wolstenholme & O’Connor, 1957, p. v). Battle lines were clearly drawn from the outset of the meeting, which opened with Hinshelwood (now Sir Cyril) and his longtime collaborator A. C. R. Dean on the defensive. ‘[I]t may be well to begin by mentioning views which have at one time or another been attributed to us, but which we have never held and of which no expression could be quoted from any of our publications’ (Dean & Hinshelwood, 1957, p. 4). They protested that they had never doubted that DNA carries ‘essential characters of a cell’ (ibid.), and considered ‘arguments invoking the name of Lamarck to be largely meaningless or irrelevant’ (ibid., p. 7). Where they did part company with the bacterial geneticists, who were overwhelmingly represented at the Symposium, was in questioning whether drug resistance should be viewed as a manifestation of ‘stable heredity’ (ibid., p. 6), and whether classical genetics could be said to apply to asexual bacteria. Discussion was vigorous, with Lederberg, Luca Cavalli-Sforza, Demerec, William Hayes, and Guido Pontecorvo arguing for the genetic point of view. Besides the Russian contributors, Yudkin and his collaborators provided one of the only papers supporting ‘Lamarckian induction’ (in their words), although they favored a pluralistic view in which mutation and selection accounted for some cases.60 The underlying disagreement had less to do with antibiotics than with whether all hereditary bacterial variation was ultimately attributable to genes. As Bernard Davis summarized the issue: ‘The real rub comes in [Hinshelwood’s] claim that such reversible adaptive resistance, if carried through enough generations will gradually develop (by some process other than random mutation plus selection) into a stable, inheritable resistance. Most biologists would be sceptical about the existence of such gradual, non-mutational stabilization of an adaptation’.61 However, some participants wanted to allow for heritable changes in response to the environment. B. Gyo¨rffy responded to Davis by asserting that ‘the terms ‘‘heritable’’ and ‘‘genetic’’ . . . are not synonymous’, insofar as changes might arise via ‘environmental influence’ and be inherited for several generations without involving ‘real genetic change’.62 With such basic issues (still) at stake, even Cavalli-Sforza 58

Harry Marks has drawn my attention to a pandemic of staphylococcal infections, characterized by high rates of antibiotic-resistant strains, in the two decades after World War II (personal communication). 59 Plumb (1955, 1957). The second headline mentioned was the subtitle to ‘Hospitals found in germ danger’. 60 Thornley, Sinai, and Yudkin (1957). A few years earlier, Yudkin (1953) had proposed a new theory of acquired resistance, which he distinguished from both Hinshelwood’s induction view and the bacterial geneticists’ interpretation of mutation and selection. However, his ‘acquired drug resistance by clonal variation and selection’ included assumptions that would be entirely untenable to bacterial geneticists. For instance, he supposed that a dividing cell would lead to two daughter cells with different degrees of resistance. He argued its plausibility given that the enzyme systems likely responsible for drug resistance need not be equally divided between daughter cells, but he left entirely vague the relationship of such enzyme systems to bacterial genes. 61 Bernard Davis, contribution to discussion following paper of L. L. Cavalli-Sforza (1957), p. 44. 62 B. Gyo¨rffy, contribution to discussion following paper of L. L. Cavalli-Sforza (1957), p. 44.

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and Lederberg’s impressive indirect selection experiment with test tube cultures could not be expected to bring closure to the debate.63 The reviewer of the published symposium volume for Antibiotics & Chemotherapy recommended the volume to those interested in ‘drug resistance, bacterial genetics, bacterial physiology, and biochemistry’ but concluded that ‘the question of drug resistance has not been answered unequivocally’ (Reedy, 1959, p. 247).

5. Plasmids from explanation to technology Antibiotic resistance was not only a topic of scientific debate and public concern, but also a resource for manipulating bacteria in the laboratory. Because one could select positively for drug resistance (resistant variants being the only bacteria to multiply in culture media containing an antibiotic), antibiotics were used widely in laboratories of microbial genetics as a means of selection. In fact, even in sensitive bacteria, penicillin could be used to selectively promote the growth of metabolic mutants (Davis, 1948; Lederberg & Zinder, 1948). Drug resistance also served as a key genetic marker in the study of ‘sexuality’ in bacteria. In 1952, Hayes crossed streptomycin-resistant strains of E. coli with streptomycin-sensitive strains, and found that mating (recombination) depended on an extrachromosomal factor, soon termed the ‘F’ factor (for fertility) (Hayes, 1952). This entity, which behaved as an infectious particle, was carried by some strains (termed F+ or ‘male’) and absent in others (F or ‘female’).64 The F factor had the ability to replicate independently of the chromosome, but when it was transferred between cells, it could carry bacterial genes with it. Thus, in an ironic twist, the very capacity of E. coli to exhibit mating, the trademark of Mendelian organisms, relied on an entity that displayed cytoplasmic inheritance. Indeed, Joshua Lederberg had been cautioning that those struggling against Lamarckianism in microbiology might be misled into discounting nonnuclear genetic units, which were indispensable to bacterial genetics.65 Admittedly, the word ‘plasmagene’ had lost its appeal.66 However, under the new unifying framework 63

Cavalli-Sforza’s contribution to the Ciba Symposium (1957) recapitulated and updated the collaboration with Lederberg (Cavalli-Sforza & Lederberg, 1956). Lederberg has referred to their experiment as a lean ‘Euclidean’ demonstration, using only test tubes and pipettes, the bacteriological equivalents of a straight-edge and compass, rather than the ‘new’ technologies like replica-plating with velveteen swatches (personal communication, 31 August 2002; see also Lederberg, 1989). 64 As Brock (1990), p. 91, puts it, ‘In addition to coding for maleness, the F factor also codes for its own ability to replicate independently of the chromosome and for its transfer to other cells. F cells that receive the F factor as a result of mating thus become F+ and are able themselves to transfer F. the F factor therefore behaves as an infectious particle. This infectiousness of F was the property that first led to its discovery’. See Bivins (2000) for analysis of the sexualized language. 65 Lederberg urged that the various genetic units outside the nucleus be considered along a spectrum of ‘infective heredity’, with ‘deleterious parasitic viruses at one extreme, and integrated cytoplasmic genes like plastids, at the other. Within this interval, we find a host of transition forms; kappa, lysogenic bacteriophages, genoids, tumorviroids, male-sterility factors, Ephrussi’s yeast granules, etc. . . . The objection has been voiced that this viewpoint is an attempt to relegate plasmagenes to pathology. I rather think that it may broaden our genetic point of view if we consider the likenesses as well a the dissimilarities between pathogenic viruses and plasmagenes’ (Lederberg, 1951b, p. 286). 66 This was especially due to Spiegelman’s theory that plasmagenes were responsible for the formation of adaptive enzymes, against which Jacques Monod fought strenuously (and, ultimately, successfully). See Gaudillie`re (1992).

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of ‘infective heredity’ developed by Lederberg, there remained a need for some way to refer to non-nuclear genetic units. Drawing on the similarities between Pneumococcus transformation, bacterial transduction, and viral infection, he proposed in 1952 that plasmid serve ‘as a generic term for any extra-chromosomal hereditary determinant’ (Lederberg, 1952, p. 413; Sapp, 1994, pp. 157–164). As it turned out, Lederberg’s emphasis on agents of extrachromosomal heredity proved especially relevant to the ongoing research on microbial drug resistance in the late 1950s.67 In postwar Japan, dysentery remained a serious infectious disease despite good sanitary conditions, and the role of drug resistance was carefully investigated (Watanabe, 1963). From dysentery patients that had been treated with chloramphenicol, researchers isolated strains of Shigella (the pathogen responsible for dysentery) that were resistant to several other drugs, including sulphonamides. This observation presented a paradox for the genetic explanation. As Thomas Brock puts it, ‘Since chloramphenicol should not be a selective agent for all antibiotic resistance genes, this observation could hardly be attributed to mutation’ (Brock, 1990, p. 107). Tomoichiro Akiba, Toshio Fukushima, and their colleagues in Tokyo investigated the origin of concurrent resistance to streptomycin, chloramphenicol, tetracycline, and sulfonamide in Shigella. They demonstrated that multiple drug resistance in intestinal E. coli could be transferred to Shigella. Their analysis drew on both in vitro studies involving mixed cultivation of strains and clinical cases of resistance. They found that transfer relied, like bacterial mating, on cell-to-cell contact, but it was not associated with the F factor (Akiba, Koyama, Ishiki, Kimura, & Fukushima, 1960).68 Multiple drug resistance became another example of infective heredity, this time associated with an extrachromosomal entity called R factor (Watanabe, 1963). Because the factor appeared to be able to integrate into the genome or exist autonomously within a bacterial cell, it fit into Franc¸ois Jacob and Elie Wollman’s category of episomes.69 However, as evidence for ‘reversible attachment to the chromosome’ was not general for drug resistant genes, Lederberg’s broader term ‘plasmid’ became favored in discussing transferable drug resistant genes.70 Most microbial drug resistance was subsequently shown to be attributable to transferable genetic elements such as R factors, rather than to chromosomal mutations, as bacterial geneticists had originally thought.71 Plasmids themselves went from being objects of study to experimental tools by the 1970s. In the ongoing research into antibiotic resistance factors, investigators found they could distinguish the genes that enabled replication and transfer of the plasmid from those that actually conferred drug resistance on the host microbe. Working within this area in 1968, Stanley Cohen at Stanford University set out to isolate and characterize the components of multiple resistance plasmid R1.72 Initially, his group’s efforts focused on improv67

On the larger significance of Lederberg’s contribution, see Sapp (1994), Ch. 10; (2003), Ch. 19. Two other groups, those of K. Ochiai and of S. Mitsuhashi (the latter at Gunma University in Maebashi), also made important contributions to understanding the transmissibility of drug resistance among Enterobacteriaceae. See Mitsuhashi (1993). 69 Jacob and Wollman’s definition of an episome was a genetic structure that is added to the genome and that, once inside the cell, may exist either independent of the chromosome or integrated into it. The category arose from the perceptions of similarity between F factor and lysogenic phages. See Brock (1990), pp. 185–186. 70 The phrase is from R. P. Novick (1963), as quoted by Lederberg (1998), p. 4. 71 R factors can confer a variety of other protective traits to their bacterial hosts; see Smith (1967). 72 My narrative draws on Cohen (1993). 68

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ing the technical procedures for manipulating and studying plasmids. Leslie Hsu, a medical student in his laboratory, found that treatment of bacteria with calcium chloride led bacteria to readily take up isolated plasmid DNA. Fortunately, plasmid genes conferring antibiotic resistance allowed for easy selection of those bacteria that had been so ‘transformed’.73 At this same time, Herbert Boyer was studying a plasmid from an antibiotic resistant strain of bacteria recovered from a patient at his medical school, the University of California, San Francisco. This plasmid carried a gene for a restriction enzyme (an enzyme that could digest the nucleic acid of other microbial species) called EcoRI.74 Many other biochemists were actively investigating restriction enzymes, and two different groups at Stanford showed that EcoRI produced cohesive ends when it cut double-stranded DNA (Sgaramella et al., 1972; Mertz & Davis, 1972). Boyer and Cohen began collaborating, and soon transformed these objects of research into tools for recombining DNA from different sources. In 1974 they used a drug-resistant plasmid as a vector for the first gene cloned from a eukaryotic organism, the frog Xenopus laevis, and expressed it in E. coli (Morrow et al., 1974).75 By the 1980s, after safety concerns about such genetic constructs had abated—and once the techniques became so standardized as to be published in a ‘cookbook’ (Maniatis, Fritsch, & Sambrook, 1982)—drug-resistant plasmids began to proliferate in biological and biomedical laboratories, not as objects of study but as tools for genetic engineering.76 The most widely used cloning vector, a plasmid dubbed pBR322, was constructed to contain both ampicillin- and tetracycline-resistance genes and many conveniently placed restriction sites (Bolivar et al., 1977). (See Fig. 4.) Once foreign genetic material was inserted so as to disrupt the tetracycline-resistant gene, colonies that were resistant to ampicillin but sensitive to tetracycline could be recovered for further screening in order to identify colonies containing plasmids that carried the gene of interest. This became the most popular strategy for cloning genes in the 1980s. Thus research on drug resistance in bacteria proved instrumental (quite literally) to the emergence of genetic engineering, a trajectory with its own far-reaching consequences for biology and medicine.77

73 Cohen, Chang, & Hsu (1972). Their method drew on observations about calcium chloride treatment and cell permeability to DNA made at University of Hawaii by Mandel & Higa (1970). 74 See Boyer (1971). Matthew Meselson and Robert Yuan had isolated the first restriction enzyme, and Werner Arber, Hamilton Smith, and Daniel Nathans shared the 1978 Nobel Prize for their work on restriction enzymes (including using them to make the first physical map of a viral genome). For a discussion and citations, see Morange (1998), pp. 186–187. 75 Researchers in several laboratories were working to construct hybrid DNA molecules. Cohen and Boyer’s team made their first inter-generic hybrid construct in 1973. Cohen, Chang, Boyer, & Helling (1973). Workers in Paul Berg’s laboratory produced hybrid DNA molecules combining genetic material from different species, for example, the tumor virus SV40 with an altered form of phage lambda. Jackson, Symons, & Berg (1972). Disagreements over credit, which I do not attempt to adjudicate, concern not only the scientific competition between laboratories but also the Cohen–Boyer patent application, which did not include all of their own coauthors. For a historical assessment of the Cohen–Boyer collaboration, see Hughes (2001). 76 On the safety concerns about recombinant DNA, see Watson & Tooze (1981) and Wright (1994). 77 For an insightful reflection, see Rheinberger (1995).

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Fig. 4. The circular restriction map of pBR322. The molecular weight is given in the middle of the circle (2.6 · 106 daltons), and the diagram shows the points at which each restriction enzyme cleaves the circular plasmid. The ampicillin resistance gene is indicated by Apr, and the tetracycline resistance gene by Tcr. Reprinted from F. Bolivar et al. (1977), p. 103; used with permission of Elsevier Science.

6. Conclusions In the end, ‘adaptation versus mutation’ turned out to be a false dichotomy. In 1960, Jacob and Monod’s ‘operon’ model enabled a fruitful separation of questions of gene mutation from the cell’s response to the environment, or gene regulation.78 They demonstrated that E. coli adjusted the amount it synthesized of b-galactosidase (a well studied adaptive enzyme) through the use of a genetic ‘switch’, which activated or repressed gene 78 This statement is a little oversimplified—by gene mutation, I am referring to the mutation of a structural gene (like that encoding an enzyme). Mutations of regulatory genes (which encode repressors and other regulatory proteins) could affect regulation. This distinction between structural and regulatory genes was a crucial contribution of Jacob and Monod. The earliest publication of the operon model is Jacob, Perrin, Sanchez, & Monod (1960); the classic papers are Jacob and Monod (1961a,b).

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transcription depending on the availability of the enzyme’s substrate. Such control mechanisms allow bacteria to respond to their environment (e.g. the availability of particular sugars as carbon sources) while synthesizing as few enzymes as possible to survive. Thus the French researchers preserved a place for the importance of the environment (even if not Lamarckian heredity) in their articulation of molecular genetics. Adaptation as gene regulation need not contradict the reality of altered genes via mutation. At another level, the purview of the gene had to be expanded to account for traits such as drug resistance. Ingenious microbes showed biologists that selection could operate beyond the realm of nuclear genes. Extrachromosomal determinants of heredity, especially plasmids, were largely responsible for conferring drug resistance on microbes. The revised notion of the gene as a hereditary entity that could reside outside the chromosome(s) drew strength from research on the genes of viruses, an experimental material of choice for the first generation of molecular biologists.79 In the end, the consensus that emerged around the genetic origin of drug resistance came with an admission that these acquired traits were inherited without the direct involvement of nuclear chromosomes. But this was probably meager consolation to Hinshelwood. Thus as a scientific controversy, the debate over the origins of drug resistance was resolved by the mid-1960s, and early misconceptions could be seen on both sides. The emergence of molecular biology in the meantime, with Delbru¨ck and Luria as favored contenders for scientific paternity, have made the very existence of the debate over the origin of antibiotic resistance dim in the historiography.80 In comparison with the bacterial geneticists involved with the debate, those who questioned the mutation and selection explanation of antibiotic drug resistance (who, in the end, were not wrong to doubt that genes on the bacterial chromosome accounted for all observed resistance) have received scant historical attention. In part this is due to the fact that the postwar debates over drug resistance derived much of their fire from broader scientific controversies, particularly over Lysenkoism and cytoplasmic heredity, which have been studied in their own right. Nonetheless, the history of research on antibiotic resistance, as I have tried to argue, had significant consequences for biology as well as medicine. Two points deserve reiteration. First, the debates over bacterial drug resistance show an unexpected (if incomplete) overlap of interests between evolutionary biologists and biomedical researchers. The genetic explanation for antibiotic resistance received a boost from the neo-Darwinian synthesis, and in turn was invoked as an unequivocal example of selection in action. But by the same token, as Jan Sapp has argued, the fact that cytoplasmic inheritance (like symbiosis) was left out of the neo-Darwinian synthesis hindered scientists, at least those in the West, from fully appreciating the importance in microbial evolution of lateral gene transfer via plasmids (Sapp, 2005). The role of multiple drug resistant plasmids in accounting 79

See Creager (2002), Ch. 6. The touchstone for giving Luria and Delbru¨ck pride of place in the development of molecular biology is Cairns, Stent, & Watson (1966). There is a large and contentious historiography on the ‘origins’ of molecular biology, which I will not attempt to cite or summarize here. See Creager (2002) for discussion and references. One issue has concerned dating—did molecular biology begin in the 1930s with the Rockefeller Foundation’s investments, in the 1940s with the phage group, in the 1950s with Watson and Crick’s double helical model of DNA and the first institutional uses of the term molecular biology (for the lab in which they collaborated), or in the 1960s, with the launching of a journal and graduate programs? Soraya De Chadarevian (2002) makes a good case for considering the 1950s as the time when ‘molecular biology’ connoted a new field to historical participants (irrespective of that field’s antecedents). 80

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for most clinical incidence of antibiotic resistance changed the thinking of microbiologists about how natural selection on bacteria operates, but had longer-term implications as well. The importance of lateral gene transfer in the evolution of microbes (since genes are transferred not only within but also between species) eventually brought into question sequence-based assumptions about establishing phylogenetic relationships. Second, the trajectories of research associated with bacterial drug resistance had profound technological consequences, as resistance genes became routine tools for genetic selection and, subsequently, genetic engineering. Hans-Jo¨rg Rheinberger has illuminated the way in which epistemic objects, once brought into view and under control, can become epistemic tools for searching out other biological entities (Rheinberger, 1997). This dynamic was clearly at work in the discovery of plasmids as agents of antibiotic resistance and their subsequent development as technologies for shuttling genes between species. Antibiotic resistance genes and plasmids became crucial instruments in the age of molecular cloning. In this respect, the study of drug resistance had an enormous material impact on late-twentieth century biology in ways that could hardly have been imagined two decades earlier by participants in the debate over the origin of resistance. Acknowledgements Research on this project was supported by the author’s NSF CAREER grant, ‘Life science in the atomic age’, SBE 98-75012. Joshua Lederberg offered many excellent suggestions on earlier versions of this paper, and Evelyn Witkin provided valuable guidance to the literature and first-hand recollections. Attendants of presentations of this paper at Johns Hopkins’ Department of Science, Medicine, and Technology (2002), Princeton’s History of Science Program Seminar (2004), and UCLA’s Center for Society and Genetics (2006) raised key issues that spurred new research and writing. I also thank Olga Amsterdamska, Thomas Brock, John Ceccatti, Michael Gordin, Christoph Gradmann, Nikolai Krementsov, Ole Molvig, Joseph November, Jan Sapp, Rena Selya, Leo Slater, Betty Smocovitis, Alistair Sponsel, William Summers, Dan Todes, Norton Wise, and two anonymous referees for their suggestions and criticisms of the paper. In the final editing stage, Doogab Yi corrected and improved the manuscript. Finally, while I alone bear responsibility for the paper, including any errors or misinterpretations it contains, I hold Harry Marks responsible for getting me so interested in the topic that I couldn’t let it go. References Abraham, E. P., Chain, E., Fletcher, C. M., Gardner, A. D., Heatley, N. G., Jennings, M. A., & Florey, H. W. (1941). Further observations on penicillin. Lancet, 2, 177–189. Adams, M. (1980). Science, ideology, and structure: The Kol’tsov Institute, 1900–1970. In L. L. Lubrano, & S. G. Solomon (Eds.), The social context of Soviet science (pp. 173–204). Boulder, CO: Westview Press. Akiba, T., Koyama, K., Ishiki, Y., Kimura, S., & Fukushima, T. (1960). On the mechanism of the development of multiple-drug-resistant clones of Shigella. Japanese Journal of Microbiology, 4, 219–227. Amsterdamska, O. (1987). Medical and biological constraints: Early research on variation in bacteriology. Social Studies of Science, 17, 657–687. Amsterdamska, O. (1991). Stabilizing instability: The controversy over cyclogenic theories of bacterial variation during the interwar period. Journal of the History of Biology, 24, 191–222. Amsterdamska, O. (1993). From pneumonia to DNA: The research career of Oswald T. Avery. Historical Studies in the Physical and Biological Sciences, 24, 1–40.

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