Thinking outside the rod (or coccus)

With kind permission by the organizers, we reproduce here as a lightly edited transcript Roberto's talk (via live video) at the meeting on 'Major ideas in quantitative microbial physiology: Past, Present and Future' held in June this year in København, Denmark. STC readers will find that what recently began here in STC as his Transient Thoughts on Transduction has now evolved into a wide-ranging Thinking About the Physiology of Natural Bacterial Populations, the title of his talk.
 

by Roberto
 

Roberto on video from Bogotá, Colombia.

I would have really loved to attend this meeting in person. I had great expectations of return­ing to Copenhagen. We were there in 2019 [together with Mechas Zambrano] and this was going to be a dream come true. It's very sad not to be there. But I want to thank the organizers for inviting me and allowing me to present some of my thoughts, though now at long distance.
 
 
 
 
 

 

Stream in Isle of Skye, Scotland. Image Ro­­berto Kolter

The title of my talk is: "Thinking About the Physiology of Na­tural Bacterial Populations." I place the emphasis on 'think­ing' since these days that is mostly what I do. I'd like to share with you one small thought, which I find fascinating since it relates to natural microbial populations, such as those that you find in settings like this beautiful stream and its surroundings. Keep this image in mind as I'll come back to it at the end (Figure 2).
 
 
 
 
 
 
 

Thinking outside the rod (or coccus)  I want to share with you today a thought that will require you to think outside the rod. Not only outside the rod, also outside the coccus (Figure 3).
 

Let me explain to you what I mean.
 
 
 
 
 

Let's start with how we normally consider the physiology of the bacterial cell. A basic tenet in our experiments is the concept that a bacterial cell will divide, and daughter cells will inherit identical copies of the genome. The genome is assumed to remain the same and only very rarely will there be genetic variation. When all cells are dividing at the same rate we say the culture is undergoing balanced growth.
 
 
 
 
 

In addition to balanced growth, we can also consider what happens when cells are starved, when the morphology changes from a rod to a coccus. If you provide these cells a surface to form a biofilm, you see development of physiolo­gical heterogeneity (Figure 5). But the genetic content of each cell remains the same. So far so good. This is pretty much the way we consider bacterial physiology to be able to analyze cells in a quantitative way, make models, etc..
 
 
 
 
 

But what do I mean by thinking outside the rod? There is a lot of work done on how the out­side changes what goes in­side the cell. But there is one aspect always present in envi­ronmen­tal settings that is usually removed in physiological studies of bacteria. I'm talking about bac­teriophages. One aspect of bacteriophages that I want to address here is the concept of abortive transduction (Figure 6). It's a process that fascinates me and is hopefully familiar to many of you. But let me tell you about the history of its discovery. A year or so after Zinder and Lederberg dis­covered transduction in Salmonella in 1952 ─ almost exactly 70 years ago – Bruce Stoker, who was working on motility of Salmonella, went to Lederberg's lab because he was interested in doing genetics on Salmonella motility.
 

Trails produced by Salmonel­la pa­ra­ty­phi-B strain SW 966, treated with lysate of S. typhimurium strain TM 2; the treat­ed organisms were plated at the centre of a 5 cm Petri dish of semisolid medium, which was incubated at 37°C for 42 h. magnification ×1. Source

Abortive transduction  Stocker, who had non-motile mu­tants and could do some genetics using transduction, made a remarkable observation. I share with you this image (Fig­ure 7), which I think this is one of the most remarkable pic­tures of early bacterial genetics. I will ex­plain it because it is key to understanding some of my thinking today. They in­fected a non-motile strain with phages that had grown on a motile strain and spotted the mixture on moti­lity agar. I love this image because it almost looks like a modern art depict­ion of a dancer. At the center is the growth of the non-mot­ile strain. But the legs and arms, they called trails. And I want you to think back to 1952 when they were doing these experiments and how clever they were to figure out what was happening. This is before DNA was known for certain to be the genetic material. They saw that the trails were a se­ries of non-motile microcolonies, along lines stretching away from the site of inoculation, and concluded that these were the results of abortive trans­duction.
 
 
 

The fate of P22-derived trans­ducing DNA. Once transducing DNA has en­tered the cell it can undergo degrada­tion and/or recombination with homo­logous recipient sequences (complete transduction, shown on the left). More frequently the DNA is abortively trans­duced (shown on the right). Source

What do they mean by that? They meant that a cell had ob­tained genetic information through transduction that made it motile, but that this transducing DNA was not replicating. Every time a cell swam away and divided, only one progeny cell contained the motility gene and could continue swim­ming away. The non-motile one remained, leading to micro­colony formation. There could be 50 or more microcolonies in a trail, meaning that for 50 or more generations these cells contained DNA that was not replicating but was not destroyed. How the DNA was kept was a mystery. Fast for­ward almost fifty years. In 1997, Benson and Roth published a paper explaining the mechanism of what was happening. They discovered that in the process of normal, complete transduction, a linear piece of chromosomal DNA has phage proteins associated to one end and the RecBCD complex at the other end that mediates recombina­tion. But at a much higher frequency there was no recombination because this DNA was circularized (Figure 8). This circle of DNA remained stable in one cell for many generations, the process of ab­ortive transduction dramatically affected the physiology of some cells, they could now swim!
 
 
 
 

Physiological implications  Now I want to go to the physio­logical implications of abortive transduction and how this might be working in the environment. The biofilm I showed before is all the same color, a single species with the same genome (Figure 9). But I'm suggesting that such a popula­tion will also have some cells that transiently contain not-replicating, autonom­ous DNA circles sitting in the cytoplasm that will only be inherited by one daughter cell, not both. You have a lot of cells with different pieces of DNA from other cells. When dealing with one strain, all cells can test the physiological effects of the same DNA, no big deal. But when you consider na­tural populations containing cells from different strains or species, you have a remarkable pheno­menon. The physiology of individual cells is not going to be dictated by the genome of just that cell, but also by the visiting, transient, non-replicating pieces that might arrive into those cells through abortive transduction, which happens at a much greater frequency than stable trans­duction and recombination.
 

Given the numerous phage protection mechanisms such as CRISPR or retrons, it is very likely that the most frequent phage-mediated events are those where phages serve as vehicles to bring in a piece of DNA that come from the chro­mosome of another cell. I find this fascinating because it tells you that the population is exploring alternative physio­logies. Individual cells test physiologies as a result of not only their genome, but also visiting DNA. We are, of course, still thinking of cells that are dividing every now and then.
 
 
 

This has tremendous evolutionary implications when we think of a cell that is almost never dividing or dividing ex­tremely slowly and testing DNA from adjacent cells through horizontal gene transfer. We typical view a cell's physiology as follows. Cells divide, and the progeny are genetically the same (Figure 11). By changing the environment, we can de­termine the effects on cell physiology. And from there de­velop quantitative models, etc.
 
 
 
 

Evolutionary implications  I am suggesting that we need to approach these studies under conditions of extremely long generation times. We need to think about what might be hap­pening in natural populations, such as in soils or wa­ter, and develop quantitative approaches that measure ac­curately the rates of horizontal gene transfer under such conditions. It may be that when division times are in the range of months or even years, horizontal gene transfer frequencies will be extremely high per division. Extremely slow dividing cells could modify their genomes through horizontal gene transfer many times before they divide. That is something that we don't normally think about. Adjacent progeny cells could thus come to have a different genetic content. The reality is that environmental populations are not strictly clonal. They often have dif­ferent pieces of DNA. We need to take quantitative approaches of microbial physiology and evo­lution to settings in the environment.
 

I am certainly not the only one thinking along these lines. A couple of papers have been particularly inspiring because they emphasize the need to address this question. Their abstracts can serve as an invocation for you to go and read them and think about what this means.
 

A paper from Eric van Nimwegen's lab entitled "Whole gen­ome phylogenies reflect the distri­butions of recombination rates for many bacterial species" points us in the direction of where we should go:

Our analyses indicate that prokaryotic genome evo­lution is driven by recombination that occurs at a very wide distribution of different rates between different lineages, and there is now a strong need for identifying what sets these rates, and the development of new mathe­matical tools and models that can accurately describe this kind of genome evolution.

That is, standard thinking about recombination rates doesn't explain evolution of genomes out there; we need more quantitative approaches to address these inconsistencies.
 

Another paper with the fascinating title "Using Cartesian Doubt to Build a Sequencing-Based View of Microbiology" is a thought-provoking paper based on metagenomic ap­proach­es. They make a shocking statement that I think is worth thinking about.

The technological leap of DNA sequencing generated a tension between mo­dern meta­gen­omics and historical microbiology. We are forcibly har­monizing the output of a mo­dern tool with centuries of experimental knowledge de­rived from culture-based micro­biology. As a thought experiment, we borrow the notion of Cartesian doubt from philo­sopher Rene Des­cartes, who used doubt to build a philo­sophical framework from his incorrigible statement that "I think therefore I am." We aim to cast away preconceived notions and conceptualize microorganisms through the lens of metagenomic sequenc­ing alone. Specifically, we pro­pose funding and building ana­lysis and engineering me­thods that neither search for nor rely on the assumption of independent genomes bound by lipid barriers containing discrete functional roles and taxonomies.

There you have it. The metagenomic data is being forced into genomes in cells, while in fact the genetic content might reflect a lot of transient visitors as I was suggesting, for example, through abortive transduction.
 

But I want to leave you with some parting philosophical thoughts and keep these thoughts – and all the work that we do – in perspective. I want to take you back to the big picture, the beautiful stream scene I showed at the start.
 

Droplets on a leaf that even­tu­ally end up in the ocean. Images Roberto Kolter

Keeping these thoughts (and our work) in perspec­tive  It's useful to remember how at every little leaf, at every small grain of soil and in every drop of water, there's an ex­citing mi­crobial world. Each one of us has been fascinated by going into a given system. Consider each one of those systems as a single drop. We are fascinated by the universe within that little drop. But we must remember what these drops are. I want to encourage everybody to continue stu­dying their little drop because in fact, there is so much beau­ty inherent in the system that each one of us studies. But it is not a bad idea to always keep in mind the big pic­ture. And it's a real­ly big pic­ture. It is not just that every drop will eventually join that enormously more complex rushing stream (Figures 2 and 15). Realize that the stream, every drop of water in that stream, will eventually find its way to the infinitely larger ocean of all of Earth's microbial physiology and ecology. Thanks.