Sunday, February 07, 2021

The quiet revolution of Oswald Avery


Avery in his lab, 1948. Source: Rockefeller Archive

Imagine a world where we build skyscrapers more than 300 meters high, where we know of nuclear fission, where the first digital computers have been designed, where the first color television has been produced, but a world where we do not even know what stuff genes are made of! [1] This is the world of the early 1940s, before Oswald Avery achieved his ‘quiet revolution’ – to borrow an expression from the biologist and author Matthew Cobb  on the subject.

I knew of Avery and his experiments – as do all microbiologists – from the textbooks and microbiology class: working on pneumococci, Avery showed in 1944 that the active compound in bacterial transformation was DNA. In that he was providing an explanation to the illustrious findings of Griffith in the late 1920s, who demonstrated that, in infected mice, avirulent mutants of Streptococcus pneumoniae could be effectively transformed into the deadly kind via an unknown process involving contact with heat-killed virulent cells (again, textbook knowledge).

Alas, textbooks usually fail to convey much excitement about such groundbreaking episodes of human inquiry. To be fair, this is of course not the textbooks’ mission. This said, I can’t remember that my microbiology class fared much better in that task – often the amount of material to be covered in class does not allow for much dwelling into the historical context.

So I was thrilled to (re)discover the story of Avery in Cobb’s excellent book, Life's Greatest Secret: The Race to Crack the Genetic Code (2015), which explores in depth the work that led to the resolution of the DNA structure and later to the deciphering of the genetic code. Cobb spends a whole chapter on Avery’s work and life, and brings to light many fascinating aspects.

The career of Oswald T. Avery (1877-1955) centered in the study of pneumonia bacteria and the immunological response they elicit. We have to remember that, before antibiotics became widespread, pneumonia was among the deadliest infectious diseases in the world [sadly we should also stress that millions of people still die of pneumonia every year, although this number is decreasing]. Avery had joined the Rockefeller Institute Hospital in New York in 1913 in order to pursue this research, with the goal of developing therapies against pneumococcus. He stayed at Rockefeller thirty years, until his retirement in 1943.

Avery was interested in why certain pneumococci are more virulent than others. In the 1920s, he found that virulent strains typically embed themselves within a large polysaccharidic capsule that protects them from the human body’s defenses. In 1928, in London, Fred Griffith found that pneumonia bacteria devoid of capsule could be transformed into virulent ones producing capsules, simply by putting them into contact with dead capsule-producing bacteria. But what was the process? Griffith thought incorrectly that the avirulent bacteria were using the capsules of the dead virulent bacteria as a template to make new ones.

From the early 1930s, Avery worked to isolate what they called ‘the transforming principle’ from cultures of pneumococci, and to identify its chemical nature, with the help of the Canadian scientist Colin MacLeod. They managed this feat by using a messy, dangerous procedure with a commercial kitchen cream separator that leaked and produced tons of aerosols – I kid you not. They also found that calcium chloride precipitated nucleic acids into a white, gooey substance (if you have done DNA extraction you know how that looks like). The white precipitate, which contained nucleic acids and polysaccharides, was very potent in transforming bacteria, as demonstrated by MacLeod, who had to leave the lab in 1941.


Avery lab around 1920. Source: Rockefeller Archive

At that time another young researcher joined the Avery lab. His name was Maclyn McCarthy, and he made a decisive contribution by showing that the polysaccharides could be enzymatically digested in the precipitate without it losing its transforming ability. It became increasingly clear that most of the transforming precipitate was made of DNA. What is more, some enzymes active on DNA were able to block transformation.

This should have been the nail in the coffin of that story, however, resistance to grant DNA that extraordinary role was strong: DNA shows no variation whatsoever, how could it be the transforming principle? Proteins are varied and very low amounts show activity – surely they are the stuff responsible for transformation. And you can never be sure that your precipitate is totally protein-free!

So while all the evidence pointed at DNA as the agent of transformation, many were still convinced that it could not be – a powerful expectation bias that complicated the acceptance of Avery’s work. He and his team continued to work tirelessly to strengthen the case for DNA. Avery noted, in a letter to his brother:

‘We have isolated highly purified substance of which as little as 0.02 of a microgram is active in inducing transformation… this represents a dilution of 1 part in a hundred million – potent stuff that – and highly specific. This does not leave much room for impurities – but the evidence is not good enough yet.’

In 1943, Avery proposed the connection between DNA and genes. He had not solved the problem of protein impurities – after all, you cannot prove a negative – but at that time the attitude towards DNA was changing, weakening the opposition to its role. Avery started to write down all their results, a pile of evidence from different types of experiments. He wrote in that paper:

‘The inducing substance has been likened to a gene, and the capsular antigen which is produced in response to it has been regarded as a gene product.’

The paper, authored by Avery, McLoed and McCarthy, was published in February 1944 in the Journal of Experimental Medicine. Its initial reception by the scientific community was good and, in the next couple of years, Avery and McCarthy continued to publish, adding more evidence to their case. Avery received the Gold Medal of the New York Academy of Medicine. Erwin Chargaff, inspired by Avery, started to study DNA composition. In 1945, André Boivin at the Institut Pasteur demonstrated transformation in E. coli, supporting Avery’s findings on the importance of DNA.

After 30 years of hard work, the quiet revolution was very much underway. Avery received many awards for his work on pneumococcus, albeit never the Nobel Prize... 

Reference: Cobb, Matthew. (2015) Life's Greatest Secret: The Race to Crack the Genetic Code.   Basic Books, 464 pages.

 [1] In 1933, Thomas Hunt Morgan noted, during his Nobel Prize acceptance lecture: ‘There is no consensus of opinion amongst geneticists as to what the genes are – whether they are real or purely fictitious.’

Sunday, March 10, 2019

A view of microbial life in soil

Image source

All soils teem with microscopic life that is invisible to the naked eye, and for that reason often overlooked. Yet, microorganisms are an essential component of all soils, and through their metabolic activity (mineral weathering, production and recycling of organic matter, and more) soil microbes actually play a major role in soil formation, development, and fertility.

The images presented here show diverse microorganisms inhabiting a pasture soil. I sampled this soil on the Uetliberg, in Zürich, Switzerland, in 2018. Samples were chemically fixed and observed with scanning electron microscopy. Post-acquisition pseudocoloring was applied to the images - in other words, colors were added to the original black and white images to facilitate visualization of the microbes and soil particles. (The pseudocolors do not represent the real colors of the samples.)

All images are courtesy of the Soil and Terrestrial Environmental Physics group of Dani Or at ETH Zurich. SEM image acquisition and coloring by Anne Greet Bittermann, ETH Zurich - ScopeM.

We do not know the precise identity of these microbes (we would need DNA sequencing for that), but given their shape and size some level of characterization is possible. Most of them are bacteria, which are the most abundant cellular organisms in all soils.

Sunday, July 22, 2018

Self-organization of bacterial populations

Fluorescent bacteria in glass microchannels

In many environments, hundreds, sometimes thousands of different microbial species coexist as mixtures of cells of various sizes and shapes. For example, each human being teems with their very own and unique microbial mixture (trillions of cells), as the human microbiome project unveiled a few years ago. Microbial diversity in soil is equally possibly even moreastounding, with a gram of rich soil capable to host thousands to millions of distinct bacterial and archaeal species, as well as hundreds of fungal and protistan species. A recent study in the magazine Nature highlights the results of the earth microbiome project, which aims at revealing the extent of  microbial diversity on our planet.

Yet, despite this wealth of diversity, microbial communities are not simply soups of species – there is order hiding behind this curtain of complexity. Actually, the more we look, the more we find patterns of microbial organization in the natural world. Some patterns are obvious and have been known for a long time, such as the distinct layers of microorganisms visible with the naked eye in sections of microbial mats. The vast majority of patterns, however, reveal themselves only at the scale of individual microbes, that is, at the microscopic scale. See, for example, the beautiful arrangements revealed in  the lab of Jessica Mark Welch at the University of Chicago. 

Microbial populations are thus often spatially organized at small scale, and in a very defined and refined way. But how does it work? Obviously, microbes don't organize following some kind of blueprint that is imposed on the community!... But what then? Part of the answer seems to reside in so-called
self-organization processes. With such processes, patterns emerge from the individual behavior of cells that can only sense the conditions in their local environment and react accordingly. This in appearance simple process at the individual level can lead to seemingly complex patterns of organization at larger scale. Think of bird murmuration, or of how ants can form bridges with their own bodies! I also discussed spatial patterns of bacterial organization triggered by metabolic cooperation in a previous post.

In a recent paper whose lead author is my colleague Benedict Borer, we examined some of the basic processes that can lead to bacterial spatial self-organization. (In that study we were specifically interested in pore networks that mimic the spatial structure of soil aggregates, but the processes that matter here are valid in other kinds of environments as well.) The idea is as follows. In a given habitat, bacterial populations with distinct metabolic capabilities and food preferences would spontaneously arrange in space in order to optimize their use of the available resources. We thought there must be two necessary conditions for that. First, that the bacterial cells have some level of motility (which could be flagellar motility, such as swimming, or simply movement provide by growth and cell division). Second, that gradients of carbon and nutrients are present in the habitat (i.e. there must be some spatial heterogeneity for the microbes to respond to).  

We chose to work with two bacterial species that differ on their ability to respire: one is a strict aerobe (Pseudomonas putida), which only respires using oxygen as final electron acceptor, while the other (Pseudomonas veronii) is a facultative anaerobe that can respire with oxygen but also with nitrate as final electron acceptor. Both species are motile thanks to flagella that they can use to swim within liquid films. To facilitate observation, the two species were each tagged with a distinct fluorescent protein (green or red). The two bacterial populations were inoculated into micrometric pore networks with various structures,  and which allowed us to create gradients of oxygen (coming from the periphery of the network) and of carbon source (citrate, coming from the center of the network). Although initially well-mixed in the center of the network, after a week of incubation the two bacterial species segregated in the network to form two distinct and coexisting populations (see figure below). One grew preferentially where the oxygen was more abundant (the obligate aerobe), while the other could occupy the anoxic niche at the center of the network, which also contained more carbon.
The two bacterial populations, initially well-mixed, grow and segregate in the pore network as function of their respiration metabolism. From Borer et al., 2018

Interestingly, this spatial organization did not occur when carbon source and oxygen were provided together (i.e., there was no counter-gradients of oxygen and carbon). Another intriguing result was that the coexistence of the two species, as seen in the figure, could not been achieved in well-mixed liquid environments (vials or flasks), because one of the species would always win over and dominate the community (see below). This illustrates how the habitat spatial structure can help limit competition and maintain species coexistence.

Experimental and modeling results show competitive exclusion in homogeneous cultures (a) and coexistence in structured pore networks (b). The percentage corresponds to the connectivity in the network (100% is highest connectivity).
Admittedly, our system is artificial and our community only composed of two species... Yet,  the basic mechanisms that we observed most probably also apply to more complex environments and could participate to the final structure of the community. Bacteria have a remarkable faculty of optimizing their distribution and activity in complex habitats, which we could demonstrate with our simple mathematical and experimental models.

Sunday, September 17, 2017

The Vital Question, by Nick Lane - Part 2

Mitochondrion. Source: US NIH
" So what about sex, or the nucleus, or phagocytosis? [...] If each of these traits arose by natural selection which they undoubtedly did and all of the adaptive steps offered some small advantage which they undoubtedly did then we should see multiple origins of eukaryotic traits in bacteria. But we don't. This is little short of an evolutionary 'scandal'. "
Nick Lane The Vital Question (2015)

In my previous post I commented on the first part of Nick Lane's book, which deals with the proton-motive force and the origin of life. This second post focuses on the second half of the book, which explores the origin of complex life (i.e. eukaryotic cells).

If the first half of the book was equally part history of the field and part new hypotheses, the second half leans clearly more towards hypothetical ideas albeit including many facts, and backed with rigorous thinking. Lane's big idea is the following. Most traits that differentiate eukaryotes from prokaryotes (cell and genome sizes, nucleus, introns, sexual reproduction) ultimately follow from a single and singular event: the endosymbiosis event that created mitochondria. For Lane, the organelles that power respiration in all eukaryotic cells are the one special ingredient that permitted the development of cellular complexity.

Sunday, July 09, 2017

The Vital Question, by Nick Lane

“In the end, respiration and burning are equivalent; the slight delay in the middle is what we know as life.”
This quote, from Nick Lane’s book The Vital Question (2015), is both poetic and true, which is the mark of great popular science writing. What Lane’s book attempts to do (and in my opinion succeeds in doing) is to radically change our perspective on life by showing us the crucial role played by energy. 

Lane is a biochemist at University College London and already the author of three books. I think there’s something to be said about popular science written by scientists, as opposed to science journalists, in the sense that they can sometimes achieve much more than educating. For example, they can fundamentally change our understanding of some topics (that certainly happened to me on some occasions). Actually, reading The Vital Question reminded me of reading Richard Dawkins’ The Selfish Gene many years ago, and Lane’s book did for me with biochemistry what Dawkins did with genetics and evolutionary theory: it opened a window into a fascinating new landscape. 

Friday, March 17, 2017

Cooperation shapes the spatial patterns of bacterial organization

Cooperative bacterial strains colonizing a surface
Bacteria colonize surfaces in all environments. That could be the surfaces of soil aggregates, of rocks in a stream bed, of plant leaves, of animal skin, or that could be the surface of your showerhead... On such surfaces microbes establish complex communities ('biofilms') that can contain many different interacting species. These various species are usually not randomly distributed in the biofilm, but rather organized depending on their environmental preferences (for example some like well-aerated areas, others not so much...) and on the type of interactions that they have with each other. This can result in complex patterns of organization that manifest at the microscopic scale and up to the millimeter scale. Such patterns are not trivial, as they can sustain microbial activity and functions that would not be possible in a well-mixed environment, which has importance for biotechnology applications as well.

In a new study published this month, we examined the role of cooperation in shaping spatial patterns of bacterial organization on wet surfaces. The paper is available online and is entitled 'Cooperation in carbon source degradation shapes spatial self-organization of microbial consortia on hydrated surfaces'. Our idea was that a feeding dependency between two partners would directly control their distribution in space, hence imposing a specific pattern. We used a simple model system made of two bacterial strains that could grow using the chemical compound toluene (a hydrocarbon), but only when they were working together as a 'team' (a bacterial consortium in the jargon).

Tuesday, September 06, 2016

Symplasmata: a curious case of multicellularity in bacteria

Cells in a symplasmatum and surrounded by a capsule,
seen with transmission electron microscopy.

'Curiouser and curiouser’, famously said Lewis Carrol’s Alice, as she was experiencing some very peculiar events in Wonderland. I have sometimes felt like Alice when I was studying the curious behavior of the bacterium Pantoea agglomerans [1], during my time in the Lab Leveau at UC Davis.

At first sight, Pantoea agglomerans looks quite ordinary. It grows as rods a few micrometers long, it can swim with flagella and it feeds on all sorts of sugars. It belongs to the family Enterobacteriaceae, and thus it is a distant cousin of E. coli. You can find P. agglomerans in all sorts of environments, but it is particularly good at colonizing the surface of plants, and in certain cases it competes with pathogens and thus keeps its plant host healthy (that is, it can serve as a biocontrol agent). Because it is a very good leaf colonizer, we have used it in many studies of bacterial life in the ‘phyllosphere’ (the aerial surfaces of plants), such as the one described in this previous post

Now here’s what special, and actually seemingly unique, about Pantoea bacteria. Under certain conditions, instead of dividing and spreading as individual cells, the bacteria stay close together and form an aggregate containing up to hundreds of tightly packed cells. Aggregation is not uncommon in bacteria but, in the case of Pantoea, cells are constrained by a fibrillar layer, and surrounded by a thick capsule made of polysaccharides, which indicates some level of cooperation and resources sharing (see image on top of the post). The resulting sausage-shaped structures are called symplasmata [2]. Interestingly, the species name 'agglomerans' (forming into a ball), which was coined by the great Dutch microbiologist and botanist Martinus Beijerinck in a paper dating from 1888, probably refers to the species' ability to form symplasmata [3]. Although symplasmata have been known for a very long time, their importance and function in the environment is still a mystery. We have observed symplasmata on bean leaf surfaces, and others have described them attached to the roots of rice plants (Achouak et al., 1994). What is their ecological role? Does it benefit the plant as well? We do not know yet.