Microbial Genomics: Standing on the Shoulders of Giants

Professor Stanley Falkow, part III

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?’.

This is the third installment from ‘Standing on the Shoulders of Giants’ featured giant, Professor Stanley Falkow.

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

Read The Early History of Plasmids: Part II – The R-Factors

In the last installment of Professor Stanley Falkow’s feature he gives a real sense of the discussions that took place at a point in time when we were setting definitions that are now in common parlance. When you consider the impact of these defining structures on our understanding of evolution, their key role in molecular biology and the task we face in understanding and combating antimicrobial resistance, they represent some of the most important discoveries in science. Here, Professor Falkow presents the final in a series of fascinating insights into our molecular biology history that picks up the debate around the definition of episomes or plasmids from the previous installments.

The death knell for the term episome, long live the plasmid!

The meeting held at Ciba House has been analyzed in considerable detail by Mathias Grote. I attended this meeting 'Bacterial plasmids and episomes', which took place in 1968 (Wolstenholme & O'Connor 1969) and perhaps a few personal observations may be useful here. To begin with the organizers, Bill Hayes, Salvador Luria and Martin Pollock decided that the meeting would have a decidedly genetic view of the subject. I presented the only paper dealing directly with the molecular nature of the extrachosomal elements. The opening remarks by Bill Hayes set the theme. He argued that the term ‘episome’ should be retired (with honor) because chromosome insertion is a necessary criterion for the definition. Instead he suggested the adoption of Lederberg’s term ‘plasmid’ as it defined, simply, an extrachromosomal element. This meeting had no way to enforce this recommendation but in the end, episome was rapidly abandoned and the term plasmid was finally embraced, and so it has been ever since!

Yet, reading this Symposium volume again for the first time in many years I was taken by the general uncertainty of all of the presentations and the discussion that followed. The discussion on the nature of R-factors revolved around Tsutomu Watanabe’s view that R-factors were single units of transmission that had their origin in the acquisition of resistance genes from a bacterial chromsome and E.S. Anderson’s view that R factors were composite genetic elements. Some R-factors were like those described by Watanabe but yet there was another kind of R-factor, equally common – one that consisted of a transfer factor and non-self transmible element carrying one or more resistance genes. The differences were explained to some extent by the fact that Watanabe studied R-factors from E. coli and Shigella and Anderson mostly studied plasmids from Salmonella. Yet, Anderson’s view was confirmed shortly after this meeting was held.

In 1968 we stood on the cusp of the molecular characterization of plasmids. The application of the lysis method of Godson and Sinsheimer was used by Don Clewell and Don Helinski to develop the ‘cleared lysate’ and the demonstration of covalently closed circular molecules of plasmids in E. coli and, further, that these covalently closed molecules could be converted to an open circular form. This lysis method permitted the proof by Roy Clowes, Stanley Cohen and my own laboratory that there were indeed non-conjugative plasmids, as postulated by Anderson, and that these were relatively small in size, about 5X106 daltons (using the measurement standard of the time), and that there were conjugative plasmids, those capable of self transfer, that were at least 4 times as large and most often in the 60X106 – 100x106 dalton range. Of course, we now appreciate plasmids that are linear and quite a bit larger. I suppose that the definition distinguishing a plasmid from a bacterial chromosome is still that the plasmid is an accessory genetic element that is not essential for ordinary growth in the natural habitat of the microbe in question.

The Ciba Sympoisium in 1968 also highlighted for the first time the potential promise both at the basic level and in medicine of R-plasmids in the staphylococci and the molecular basis for restriction and modification that were assoiated with the inheritance of some R-plasmids. I think there was an admirable admixture of the old wise gurus of genetics, as personified by Hayes, Luria and Wollman together with medical microbiologists, veterinarians and the young geneticists. In my judgment the importance of this meeting is not that the term ‘plasmid’ was embraced in the place of ‘episome’ but rather it’s a report of the state of the thinking of the members of plasmid field just at the dawn of the juxtaposition of genetics, and the molecular biology of the first genetic elements that were so experimentally amenable.

Plasmids are important for bacterial pathogenicity

Once again, I take a personal aside in my narrative. I had worked as a medical bacteriologist for about six years before I became interested in bacterial genetics and molecular biology. Molecular biology was so new that the first definition of molecular biology used by Jacob and Monod was not far from correct; “Molecular Biology is what is published in the Journal of Molecular Biology”.

A number of my initial gambits designed to investigate bacterial pathogenicity had ended in failure and frustration. I knew that there must be pathogenicity genes that were special but I couldn’t do a single experiment to prove it. I was told it was just wishful thinking. That was the driving force for me to enter the field of entrachromosomal elements in 1961 and I was especially keen to work in this area after the discovery of R-factors, However, at the Ciba Symposium in 1968 I encountered something I hadn’t known existed before, and which had a profound impact on my ideas about bacterial pathogenicty. Naomi Datta asked me to join her and H. Williams (Willie) Smith at a pub for a drink after the second day’s meeting sessions ended. Willie, a veterinarian, was interested in the work I presented on the molecular nature of R-plasmids and asked if it could be extended to other plasmids. I was unaware of his work and asked Willie what plasmids he had in mind. In the minutes that followed, I was treated to an unaffected explanation of one of the most exciting things I had ever heard in medical microbiology. In his soft Welsh accent, he told me that E. coli were the cause of an infectious diarrhea in pigs and other farm animals. He described marvelous experiments that established that the epidemic E. coli strains carried a transferable plasmid that encoded one or more enterotoxins. He further astounded me by saying a second plasmid was necessary for the bacteria to adhere to the pig’s intestinal cells. He offered to send me any or all of his strains and said simply; “Just promise me you’ll let me know what you find”. I was at the time drinking my first pint of English bitter, and this gracious offer caused me to try to swallow the stuff and say “Yes” simultaneously with somewhat embarrassing consequences for my shirt and innocent bystanders. Willie was a great experimental scientist and we enjoyed a long relationship exchanging a number of letters about our research and slanderous gossip. It was because of him that we established E. coli as the causative agent for human ‘travelller’s diarrhea’ we also establshed that the conjugative plasmids encoding Enterotoxin (Ent), Hemolysisn (Hly) and animal specific Adherence factors (K88 amd K99) could act to mobilize non-conjugative resistance plasmids. Thus, almost any type of transmissible plasmid could act as an R-factor as long as it could mobilize the non-conjugative entrachomosomal elements encoding antibiotic resistance genes.

The non-conjugative plasmids – the major building blocks for molecular cloning

A formal scheme of classification based on incompatibility was developed in the early 1970s, mainly by Naomi Datta and Bob Hedges; incompatibility was defined by inability of two plasmids to be propagated stably in the same cell line. Plasmids incompatible with each other were assigned to the same incompatibility group. Conjugative plasmids were classified according to their incompatibility, a property directly related to replication. My laboratory worked closely with Datta and Hedges to define the molecular basis for incompatibility. We showed that the plasmids of any particular compatibility class were found to be relatively homogeneous with respect to their general molecular size, G + C content, and common polynucleotide sequences. Plasmids of different compatibility classes showed less than I5% sequence similarity. What was surprising to me was that there was very little polynucleotide similarity between members of the different incomptibility groups – a finding that was repeatedly confirmed. There appeared to be many different ‘species’ of plasmids in nature. Yet, it was puzzling that these plasmids encoded for similar antibiotic resistance traits and often used identical enzymatic mechanisms to inactivate antimicrobial activity. 

The discovery of non-conjugative plasmids and the idea of plasmid mobilization had a profound impact on those of us who were interested in the ecology of plasmids. At a more basic level it was clear that the mobilization event did not entail any detectable stable covalent attachment of the transferable element and the non-transfereable element. The non-conjugative replicons as they were called were found in recipient cells as separate entities. Moreover, in DNA-DNA duplex studies there was no evidence that most of the discrete nonconjugative plasmids were related to one another. The availablity of the small replicons encoding antibiotic resistance made it possible to focus on autonomous chromosome replication at a level of detail heretofore impossible.

I was particularly intrigued by the number of extrachromosomal elements that could be detected in clinical isolates of all species of medically important bacteria when one simply looked at the number of distinct types of circular molecules one could detect in a cleared lysate subjected to dye-bouyant gradient centrifugation. Plasmids were a general kind of genetic currency greater in number and variety than had been dreamed of, and more often than not there was not a precise phenotype associated with the possession of plasmids molecules. They were seen as ‘cryptic’. But then, not that long after, there were plasmids associated with crown galls mediated by Agrobacterium and nitrogen fixation in Rhizobium - and that was just the beginning.

The major breakthroughs in understanding plasmids came within a few years of the Ciba Symposium (1968). The adoption of the ‘cleared lysate’ method for the physical fractionation of circular plasmid DNA from the larger mass of linear chromsosomal DNA permitted the isolation of all kinds of extrachromosomal elements from their naturally occurring host bacteria. As this was applied widely it became evident that non-conjugative plasmids were far more ubiquitous than imagined. Moreover, in the absence of antibiotic resistance or other selectable markers it was difficult to detect trasmissible genetic elements. The use by Anderson of mobilizable non-conjugative R-plasmids as an indicator of the presence of conjugative plasmids led to their discovery in many different species of bacteria and the realization that there was a pool of transmissible self-replicating genes widely distributed in the microbial world.

To my mind, the last piece of the puzzle necessary to bring about recombinant DNA technology came about by the discovery by Mandell and Higa, refined by Stanley Cohen, Annie Chang and Christine Miller, that plasmid DNA, including circular molecules, could be used to transform bacterial species. We had, at last, all the means of DNA manipulation readily available to analyze these marvelous small, self-replicating entities. We could study the basic principles of translation and gene expression in small molecules capable of autonomous replication. DNA transformation likewise gave us the capacity to genetically modify these self-replicating entities and do detailed genetic and molecular characterization of these small chromosomes.

The relatively few years between the discovery of the F-plasmid and bacterial conjugation to the isolation and characterization of their molecular and biological properties and the understanding of the central role they play in microorganisms and other living things, spans only a scant 30 years. But those years and these studies parallelled the birthing of molecular biology. The stage was set for the advances which were to take place in restriction enzyme chemistry and their role in biology which led to the discovery of recombinant DNA technology. The stage was set as well for the expansion of the plasmid field into their role in the biology of virtually the entire microbial world, and not just to antibiotic resistance and pathogenicity. It was the beginning of plasmid biology as applied to more life forms than just bacteria.

This is the end of Stan’s story, and in his words: "the dawn of the next genetic revolution: molecular cloning". I hope like us you have enjoyed hearing in his unmistakable voice the story of plasmids and episomes. I would like to end this installment by recognising Professor Stanely Falkow as one of the people we think about as ‘Standing on the Shoulders of Giants’, and by thanking him for his generosity in time and ideas to so many and for so many wonderful quotes.

“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.”

Professor Stanley Falkow, Professor Emeritus Stanford University

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.

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