Microbial Genomics: Standing on the Shoulders of Giants

Professor Stanley Falkow, part I

Described as the ‘father of molecular microbial pathogenesis’ and 2004 Marjory Stephenson Prize winner. Professor Falkow discusses his own research, which by his own account, is divided into three sections: firstly, antibiotic resistance/plasmid biology; secondly, plasmids that contributed directly with bacterial pathogenicity, and finally, his work away from virulence plasmids to investigate 'what is a pathogen?’.

Microbial Genomics aims to capture the biological insights that are possible from applying the plethora of new ‘omics’ technologies and approaches. However, in focusing on the ‘new’ it is important to highlight the origins of microbial genomics too, and celebrate those that laid down the foundations for, and continue to have a deep influence on, the work we currently do.

To help us introduce MGen’s feature ‘Standing on the Shoulders of Giants’ we asked one of our Giants and the Society’s 2004 Marjorie Stephenson Prize  winner, Professor Stanley Falkow, Stanford University, to give his perspective on an area of his career highlighting his own ‘Giants’. By his own account, Professor Falkow’s professional career was divided into three sections: firstly, antibiotic resistance/plasmid biology; secondly, plasmids that contributed directly with bacterial pathogenicity, where Professor Falkow recognises H. Williams Smith as one of the most inspirational people he ever met; and finally, he transitioned his work away from virulence plasmids to investigate 'what is a pathogen?'.

What follows here is the first instalment of a series of short excerpts of a larger article detailing The Early History of Plasmids by Professor Stanley Falkow. For the final installment we will present the article together as one with references. For now, hear the voice of Professor Emeritus Stanley Falkow, Stan to many.

The Early History of Plasmids: Part I – Plasmids and Episomes

The origin of the term plasmid is easy enough.  In 1952, Joshua Lederberg wrote in a review article, “I propose plasmid as a generic term for any extrachromosomal hereditary determinant. The plasmid itself may be genetically simple or complex.” The definition was straightforward and it was intended to dissipate the controversy as to whether factors like kappa in Paramecium, sigma in Drosophila, the milk factor for mammary cancer and other vertically transmitted elements in mice were ‘viruses’ or ‘genes’.  In part, this definition was influenced by Esther and Joshua Ledernberg’s discoveries of phage λ and the fertility factor F.  Yet, fertility in bacteria and its relation to prophages, together with the startling discovery by Zinder and Lederberg of phage mediated transduction, was far from clear. Lederberg firmly believed that the product of bacterial conjugation was a zygote, in which the two parental bacterial cells contributed equally to produce a haploid recombinant cell.  On a personal note, about the same time in 1953, when I was a student majoring in Bacteriology at the University of Maine, I came upon a monograph entitled Papers in Microbial Genetics - bacteria and bacterial viruses selected by Joshua Lederberg.  This was one of the first collections of papers of its kind in the field of microbial genetics prepared for a course presented at the University of Wisconsin in the spring of 1951. I found it difficult reading. I remember distinctly coming face to face with a figure depicting a schematic representation of the possible linkage relationships resulting from a bacterial mating.  Finally, with a great sigh, I put the book down and decided it was just far too confusing for me to understand! Suffice it to say, the term plasmid was not enthusiastically embraced, nor used by the early microbial geneticists.

The same year Lederberg suggested using the term ‘plasmid’ was the same that William Hayes had reported in his  experiments (termed the Pallanza Bombshell), which showed that the transfer of genetic material in E. coli is not reciprocal. One bacterial cell acts as donor, and the other bacterial cell acts as the recipient.  The first Hfr strains were isolated by Hayes and Cavalli-Sforza. Bill Hayes remarked the next year at a Cold Spring Harbor Symposium that temperate phages and F factors might have a similar function in disseminating bacterial genes among bacteria, although he also noted that “perhaps Hilair Belloc's poem 'The Microbe' can express, better than I can say, my feelings on this matter:”

All these have never yet been seen –
But Scientists, who ought to know,
Assure us that they must be so...
Oh! Let us never, never doubt
What nobody is sure about!'

F and the temperate phages did share a number of unique properties. They are dispensible. Microbes do not require them as essential factors for their survival (at least in the laboratory). They are, or were, transmissible factors that could be shared by different microorganisms. They could be both extrachomosomal autonomous genetic elements but they could, as well, enter (recombine) with the chromosome but then also return to the autonomous state sometimes carrying along adjacent bacterial genes. In their different states of existence, integrated or autonomous, they seemed to have different replicative as well as regulatory properties. In 1958, based on these and other similarities, Jacob and Wollman called these elements episomes. The term episome was quickly adopted by the bacterial genetics community.  However, it is noteworthy that in 1961, Jacob and Wollman still hedged their bet ‘as to the nature and size of episomic elements, the available information is still rather meager. It may be suspected that in all cases they contain nucleic acids.’ Thus, lysogenic viruses and F, particularly in the autonomous state proved nebulous.

Again, on a personal note to me, one of the most important insights into the whole question of F came from a relatively short paper published by Ed Adelberg and Jacob in 1959, in which they show that when F is integrated into the chromosome, it can sometimes ‘pop back out’, and, that sometimes when it does it brings along a souvenir in the form of one, or sometimes quite a few, bacterial genes which were located next to F and are now replicated as part of F as well as being present in the bacterial chromosome. The strains are diploid for these genes since bacteria ordinarily carry one single set of genes: they are haploid. When these complex sex factors are mated with recipient bacteria, the frequency of transmission of the F-factor and the carried pieces of DNA approach 100%. They termed this F-duction or sexduction. The discovery was important in many ways. It not only crystallized the idea that phage as well as the sex factor had many similar properties in common, but, the availability of these bits of chromosome attached to F was also an important keystone in understanding bacterial regulation as well as bacterial gene transfer. Monod worked at the Pasteur Institute just down the hall from Jacob, Wollman and Lwoff, along with a growing number of Americans who were flocking to Paris to share in these exciting times.  Monod worked on another major scientific mystery of the time: the induction of enzyme synthesis, especially β-galactosidase. In Escherichia coli there is no trace of this enzyme when grown in any medium devoid of lactose. Add lactose and instantly the enzyme is produced (induced like a temperate phage is induced by UV) and begins its job of breaking down this sugar into components that the microbe can use for energy. Jacob and Monod had begun to see similarities in their work, on lysogenic bacteria, and on enzyme induction.  Sexduction helped bring these concepts together because it was possible to construct strains that had one set of mutations on an episome and another different combination of genes on bacterial chromosomes.

The study of these strains led to a better understanding of gene regulation. In short, genes worked using an ‘on-off switch’ that worked on groups of genes which had a common function.  The principle worked for understanding many of the properties of enzymes and viruses and the F factor.  Indeed, it went a long way in understanding a number of biological questions: how does the information in the DNA molecule direct the synthesis of the protein to do the cell’s work? What code is used? How does a bacterial cell, or any cell, suddenly go from being devoid of any trace of an enzyme or bacterial virus to within a very short time producing an enzyme that breaks down a needed sugar or in a burst of suicidal activity produce an enormous number of bacterial viruses released in a final cellular agonal gasp?

It was 1960! We had these interlacing concepts beginning to emerge from microbial genetics and we had a series of observations from which a pattern of surprising biological similarties seemed to coalesce. Yet, in 1960, we still had no messenger RNA nor a genetic code. The formal proof of the circularity of the bacterial chromosome and its accessory genetic elements was not universally appreciated. We were not even sure about the molecular nature of episomes nee’ plasmids.

1961 was an extraordinary year for biology! The idea of the central dogma, the concept of messenger (m)RNA, repressors, operons and the universality of the genetic code crystalized in this year. The concepts today are, rather matter-of-factly, taught as part of elementary science education in schools throughout the world. I lack the words to describe the mixture of excitement and anxiety (“What would be left to discover!”) I felt as a 27 year old young scientist, to be living at this time. All of these wonderful ideas and experiments happened within a relatively short period of time.  I had a newly minted Ph.D. from Brown and had just started working at the Walter Reed Army Institute of Research with L.S. (Lou) Baron on the genetic transfer of E. coli genes to Salmonella typhosa (typhi in current parlence). I could not create a coherent picture of the strange behavior of the E. coli genes in Salmonella and their instability or, what any of this had to do with pathogenicity.  Fortunately, I had also just started to work on a naturally occurring transmissible ‘lactose sexduction factor’ isolated from the typhoid bacillus (which I unfortunately called F0lac). There were not many major differences with F-lac except, of course, that F0lac came from nature. However, my years of training as a medical bacteriologist was reflected in my genetic experiments in that I attempted to transfer the mobile lac element to microorganisms like Serrratia marcescens which had a strikingly different G+C content than the donor Salmonella F0-lac strain – and was successful. Subsequently, Julius Marmur and I worked together to visualize plasmids/episomes distinct from chromosomal DNA by CsCl density gradient centrifugation – it was a form of biological fractionation to separate the extrachromosomal DNA from the chormosomal DNA.  We concluded: “genetic exchange can occur among organisms which differ in DNA base composition. The results presented in this study are consistent with the genetic evidence that episomal elements are deoxyribonucleic acid and that the transferred material is not integrated into the genome of recipient bacteria”. I noted in my paper describing this work that I extended the genetic observation just a bit further by transferring a newly discovered episome that conferred antibiotic resistance among enteric bacteria sent to me by Susumu Mitsuhashi in Japan. I easily transferred this episome into Serratia and could visualize the extra band of eposome DNA.  Little did I know that this off-hand experiment with this transmissible sex factor carrying antibiotic resistance would, in a few years time, go on to dominate a major portion of my experimental life for the next two decades.

Professor Falkow receiving the honorary degree of Doctor of Science.

This is the end of the first part of the story I was asked to tell. These early discoveries are sometimes forgotten in the excitement generated by the new findings that exploded from the application of recombinant DNA methods. The notion of transposons and their importance, both from an evolutionary standpoint and from an experimental standpoint, came after gene cloning. We discarded physical fractionation of plasmid DNA for agarose gel electrophoresis a few years later and this, in turn, revolutionized several aspects of epidemiology. We had moved from sequencing at most a few hundred nucleotide pairs a day in 1978 to today’s entire chromosomes in a morning. 

The good old days are now! We should not dwell on the past to be sure but we should not overlook the preface to it all because it still serves to teach us. It is the basis of current wisdom.

Acknowledgement: Professor Falkow composed these thoughts when preparing for Cold Spring Harbor Symposium Plasmids, History and Biology (held 21–24 September 2014) where he gave an oral presentation that can be found here. When told about the aspirations, aims and scope of Microbial Genomics he provided this article to help launch 'Standing on the Shoulders of Giants'. The journal would like to thank Professor Falkow for his contribution.

Read Part II of Professor Falkow’s feature article which will focus on the discovery and characterisation of R-factors.

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