On the consequences of premature retreat from small-lab basic research on Escherichia coli K-12

We were listening to a talk by Molly McQuilken, a PhD student in Amy Gladfelter’s lab. McQuiklen described her work, which involved a fairly cheap (ableit purpose-built) polarization microscope (from Rudolf Oldenburg and his group at Woods Hole MBL (Abrahamsson et al. 2015)) used to image pixels full of signal coming from fusions of yeast septin proteins to GFP. By which means, if the majority of the GFP moieties in the [rigid] septin-GFP fusions in a pixel are oriented in parallel, she can see that this is so and determine the orientation of the fusion proteins in the pixel. And thus, in each pixel, observe and start to understand the concerted actions of these molecules and their interactions with the rest of the cytoskeletal machinery, as (in S. cerevisiae) the gap connecting the mother and the bud forms an ever narrowing hourglass.

This work has been going well since at least 2011 (DeMay et al. 2011). Its current progress among other things illustrates is how marvelously empowered graduate students are these days. Lurking under the work (enabling that power) are extraordinary tools — the GFP, the recombinant methods that allow design and construction of the rigid Septin-GFP fusions, and the polarization microscope. But the main lesson that shined through the talk was the wonderfulness of this relatively small science, and the serene confidence that McQuilken inspired in her listeners that continued work: a) by this person b) in this lab c) working with these methods d) in cerevisiae— was going to reveal new knowledge about these highly-not-understood proteins.  Information that will be directly relevant to understanding, for example, how the otherwise difficult-to-gain-purchase-on mammalian orthologs of these proteins work (imagine that one needed to figure out how SEPT7 product, directs microtubules into filopodia when axons branch (Xie et al. 2007)).  That being an example of the kind of process that will certainly prove to be dysfunctional in some already known human neurodegenerative diseases. The kind of process that might one day be targeted by a drug therapy.

This rational experimentation in yeast to find out how septins work provides a textbook example of how the NIH, and within it, the NIGMS, keeps faith with the public.  By enabling young scientists to study basic biology, the work the NIH supports helps generate a stock of ideas and truths that will provide many of the hypotheses that power more applied research in mammalian organisms, and that work will enable development and validation of therapeutic interventions that reduce morbidity and mortality in humans.

Notably, this kind of work can be done and insights can be gained by motivated individual graduate students working in a well-understood, simple and highly tractable experimental organism.

Rodney mentioned something to the effect that it was a shame that work of this type– this marvelous, high-tech-but-accessible, tool-dependent, graduate-student-powered, 21st century small science, was no longer going on in E. coli.

And then imagined the counterfactual in which work on K-12 (and but let’s also imagine work on other coli strains, related species, and their phages) had been sustained at the levels of the 1960s-1970s-1980s.  And imagined that a much larger number of processes either exclusive to prokaryotes or that operate very differently in prokaryotes were under study by small labs armed with 21st century methods, in the same way yeast septation is now.  And then imagined that big genetics meetings featured talks on this kind of work by graduate students who were interacting with other graduate students working on other now poorly understood processes basic to the life of the same organism.   The assertion was (and is) that the antibiotic pipeline would not now be dry.

Restated, the semi-collective decision… in the mysterious ways those decisions get made…. by funding agencies and by scientists… this decision to scale down this sort of small lab work E. coli was premature.  It meant that questions that in prokaryotes of equivalent complexity to those being pursued by students in small labs in working on yeast are not being pursued in bacteria.  This retreat from E. coli was at least 30 years premature.   And humankind is now paying the price for that mistake.

I don’t expect very many readers to accept the above as a bare assertion, and there are some arguments against it that bear discussion, but to flesh out the argument and consider possible counterarguments I’ll first need to lay some groundwork.

One set of ideas I’ll need to introduce (and support) will be the shooting-fish-in-a-barrel criticisms of individual assertions advanced by soi disant systems biologists about how biological systems operate. There are a number of these these and it will take some time to lay them out.

Then, to illustrate the lost opportunity, to make the case that 25 years of application of current methods to E. coli would likely have generated ideas that would already have led to better antibiotics, so that human morbidity and mortality due to infectious disease might be significantly lower than it is now, I will need to introduce and support a second set of ideas.  These ideas go back to the 1990s, that time so many highly technological approaches to understanding biology seemed possible.  Starting from that time, I will need to give examples of real discoveries made and insights had, things that the techno-optimists and systems biologists did well and got right.

After that, I hope to be able to support a more sophisticated discussion of the different kinds of experimentation that leads to knowledge of function, and whether the current allocation of money and attention to the different methods is optimal for scientific knowledge or for human felicity.

And then to return to the counterfactual world in which a 21st level of understanding of dozens of processes specific to prokaryotes would be giving the industrial biotech and pharma structure a larger stock of ideas, and in which industry would be delivering new classes of antibiotics as one subset of the new small-molecule therapeutics that it knows how well to make.

It will likely take some months to introduce these topics.

References

Rothstein and Rothstein lab. https://systemsbiology.columbia.edu/faculty/rodney-rothstein, http://www.rothsteinlab.com

Mcquilken.   https://www.researchgate.net/profile/Molly_Mcquilken

Gladfelter lab.  http://www.dartmouth.edu/~gladfelterlab/

Abrahamsson, S., McQuilken, M., Mehta, S. B., Verma, A., Larsch, J., Ilic, R., Heintzmann, R., Bargmann, C. I., Gladfelter, A. S., and Oldenbourg R. (2015) MultiFocus Polarization Microscope (MF-PolScope) for 3D polarization imaging of up to 25 focal planes simultaneously.  Opt Express. 2015 Mar 23;23(6):7734-7754. PMID: 25837112

Oldenbourg lab.  http://www.mbl.edu/bell/current-faculty/oldenbourg-lab/

DeMay, B. S., Noda, N., Gladfelter, A. S., and Oldenbourg, R.  (2011)  Rapid and quantitative imaging of excitation polarized fluorescence reveals ordered septin dynamics in live yeast.  Biophys J. 2011 Aug 17;101(4):985-994. PMID: 21843491

Xie, Y., Vessey, J. P., Konecna, A., Dahm, R., Macchi, P., and Kiebler, M. A (2007). The GTP-binding protein Septin 7 is critical for dendrite branching and dendritic-spine morphology.  Curr Biol. 17(20):1746-1751. PMID: 17935997