A Midcentury Watershed: the Transition from Microbial Biochemistry to Molecular Biology

A small number of microbiological discoveries in the 1940s were responsible for the dramatic transition from microbial biochemistry to molecular biology and consequently for the movement of microbiology from the periphery to the center of biology.

It took some time for zoologists and botanists, representing the major biological disciplines at that time, to realize that this transition had occurred. Thus, when two eminent microbiologists, A. J. Kluyver from Delft in the Netherlands and C. B. van Niel from Pacific Grove in California, were invited to give the prestigious John M. Prather lectures at the Department of Biology of Harvard University in April of 1954 they were warned not to expect the members of their audience to have any knowledge of microbiology. The aim of the lectures, which were published in 1956, is evident from their title, The Microbe’s Contribution to Biology (6).

The first three lectures dealt with earlier biochemical studies of energy generation in a wide array of microorganisms. The manner in which many of these microorganisms obtain energy differs greatly from the mechanisms of energy production in animals and plants; nevertheless, as the lectures pointed out, the studies of energy production provided strong evidence for the unity of biochemistry, in that, in all living cells, chemical energy was generated by the transfer of electrons and protons to a variety of receptors.

The last two lectures described the more recent studies, carried out in the 1940s, which showed that mutations in bacteria are spontaneous, that bacteria can exchange genetic material, that this material is DNA, and that the bacterial cell can form enzymes in specific response to the presence of compounds in the growth medium. These were the studies which provided evidence for the unity of genetics and established microbiology as a discipline at the center of biology.

The earliest of the papers describing these discoveries was published by Luria and Delbrück in 1943 (9). It was well known at that time that, upon exposure to an agent capable of killing them, some bacteria survive and generate descendants resistant to the agent. Luria and Delbrück provided clear evidence that the mutations to resistance are spontaneous and not induced by the presence of the bactericidal agent in the environment.

The next important paper was by Avery et al. in 1944 (1). It was known at the time that the pathogenic organism Streptococcus pneumoniae changes it colonial morphology upon continued cultivation in laboratory media from “smooth” to “rough” due to a loss of ability to produce a polysaccharide capsule and becomes nonpathogenic. Avery and his collaborators were able to transform rough bacteria to the smooth form by the addition of DNA isolated from smooth bacteria and thus identified DNA as the genetic material of the cell.

Another important advance was the publication in 1946 of a paper by Lederberg and Tatum (7) describing the transfer of genetic material from one strain of Escherichia coli to another by conjugation and the publication of a paper in 1952 by Zinder and Lederberg (15) describing the transfer of genetic material from one bacterial strain to another by means of a temperate phage (transduction).

Of less fundamental importance, but of great practical value, was the demonstration independently by Davis (3) and by Lederberg and Zinder (8) in 1948 that auxotrophic bacterial mutants could be selected by the use of penicillin.

Finally, of great importance was the discovery by Monod et al., published in 1951 (11), that the substance capable of inducing the enzyme β-galactosidase in E. coli did not need to have any affinity for the enzyme, thus disproving the hypothesis that the process of induction depends on an interaction of the inducer with the enzyme.

It took some time to appreciate the significance of these findings for the field of microbiology. This is illustrated by the fact that only one of these discoveries, pneumococcal transformation, is discussed in two text books of microbial physiology published, respectively, in 1948 and 1949, the second edition of The Chemical Activities of Bacteria by Gale (4) and the third edition of Bacterial Metabolism by Stephenson (13). Gale comments cautiously that “it is tempting to think that this (the addition of a minute amount of nucleic acid) is equivalent to adding a gene to the genetic makeup of the organism.” Otherwise, there is no discussion of genetics or mutation found in this textbook. Stephenson also describes the transformation experiment; she states emphatically that “the importance of the observation is that it carries irrefutable proof that nucleic acid controls enzyme production.” Nevertheless, these comments are found in a chapter on enzyme variation and adaptation where no clear distinction is made between properties acquired by mutation or transiently in response to changes in the environment.

However, 5 years later the importance of the discoveries made between 1943 and 1951 was fully recognized. In the textbook An Introduction to Bacterial Physiology by Oginsky and Umbreit published in 1954 (12) there are separate chapters dealing with genetics and adaptation. Although the major portion of the book is devoted to the description of the chemical activities of bacteria, the transition from microbial biochemistry to molecular biology is clearly documented.

I am very much aware of this transition since my graduate study from 1945 to 1948 and the early period of my independent research career from 1949 to 1952 coincided with this transition (10). The research for my thesis in the Department of Biochemistry, College of Physicians and Surgeons, Columbia University, under the direction of Erwin Chargaff, was directed toward the elucidation of the steric requirements of polyhydroxy derivatives of cyclohexane for oxidation by Acetobacter suboxydans, a typical problem of microbial biochemistry, having its origin in a study by the French microbiologist Bertrand in 1898 (2). When I started my independent research as a member of the Department of Bacteriology and Immunology of Harvard Medical School, I extended my study of the metabolism of cyclic polyhydroxy compounds to the degradation of myo-inositol by Klebsiella aerogenes. In contrast to the limited ability of A. suboxydans to convert myo-inositol to a monoketone, K. aerogenes was able to metabolize myo-inositol in the presence of oxygen to CO₂ and H₂O and in its absence to a mixture of CO₂, ethanol, and acetic acid and it could use this compound as the sole source of carbon and energy. Although my initial interest was to work out the pathway of inositol degradation, a standard project of microbial biochemistry, I quickly became intrigued by the fact that the enzymes of inositol metabolism were inducible and that it was not known at that time whether induction caused the conversion of an inactive precursor to the active enzyme or the synthesis de novo of a protein not present in the uninduced cell. The fact that auxotrophic mutants could be isolated by penicillin selection allowed us to investigate the effect of depriving the cells of specific amino acids, purines, and pyrimidines on their ability to form the enzymes required for the degradation of myo-inositol (14). The fact that amino acid deprivation greatly reduced the formation of these enzymes in response to myo-inositol strongly suggested, as was proved conclusively subsequently for the induction of β-galactosidase by Hogness et al. (5), that induction resulted in the synthesis de novo of the enzymes.

The mutants we had isolated had interesting properties, for example a guanine auxotroph excreted xanthosine and a histidine auxotroph required much less histidine when glucose, rather than myo-inositol, was the major source of carbon. These observations led us to investigate the mechanism and regulation of the interconversion of purine nucleotides, the role of purine nucleotides in the biosynthesis of histidine, and the global mechanisms responsible for the control of enzyme formation in response to the availability of carbon and nitrogen sources (10). In other words, I had made the transition from the study of microbial biochemistry to that of molecular biology.

In the 1950s the importance of the new field of molecular biology was recognized. Kluyver and van Niel had emphasized the unity of biochemistry and genetics and demonstrated the importance of the study of microorganisms for biology. Because of their rapid growth and the ease with which mutants could be selected, it was apparent that microorganisms were ideally suited for the study of molecular biology. This development altered the course of my own career when, in 1955, the administration of the Massachusetts Institute of Technology decided to make the Department of Biology a center for molecular biological research and teaching. Salvador Luria was appointed Professor of Microbiology in 1958, and I joined him in 1960 after a sabbatical leave in the laboratories of Monod and Jacob at the Pasteur Institute in Paris. Our task was to instruct undergraduate and graduate students in the rapidly expanding fields of bacterial genetics and physiology as disciplines at the center of molecular and cellular biology. I am still happily engaged in this endeavor.