Paper Review: Robust growth of Escherichia coli

I’m going through a 2010 paper by Wang et al studying the cell growth of E.Coli as individual cells age. The paper is pretty remarkable, the team managed to track the growth of cells for hundreds of generations, but I have a lot of criticisms/questions. Let’s dive in!

The team used a specially built “mother machine”. It’s a high throughput liquid culture variation of a standard soft lithographic technique, kinda the reverse of the “baby maker” used in other biological studies. It locks cells in place and diffuses fresh medium over them, allowing the study of the mother while washing all the progeny away. The mother cell is referred to as the “old pole mother”.

They studied two strains, both marked with yellow fluorescent protein. Growth was measured by live microscopy.

The results:

1) The long term growth rate of individual cells is very constant. From the begining of the experiment to the end, the cell has the same average growth rate

2) The short term growth rate is extremely variable. The rate of a cells previous growth has zero predictive power over  rate of it’s next growth, ie the mother cell “forgets” it’s previous rate of growth. Short term growth is purely random, the rates fall along a Gaussian distribution.

3) After around 50 generations of one of the strains the mothers began filamentation – the spikes in the graph below. Successive filamantions occurred more rapidly, following a power law.

 

Filamentation is an indication of the SOS response. The authors tested a mutant phenotype lacking SOS response and found it decayed rapidly and randomly. They therefor concluded that the slower death rate in their WT cells could not be due to stochastic age-independent forces because they did not observe the same exponential decay. They speculate that the eventual death of WT cells after hundred of generations is due to the accumulation of an unknown lethal factor or the physically aging cell wall of the old pole mother.

Now my criticism, maybe I’m just confused so I’d be very grateful if anyone could clear this up for me. The weight of these comments are about aspects of aging I wish were addressed in the paper and not any logical or methodological fault. Overall the paper is pretty rocking.

Growth of E. coli is symmetrical. Assuming everything was equal, if the daughter cells were saved instead of discarded and had the same growth rate of the mothers you could not distinguish replicative age, every cell in the culture would have undergone the exact same amount of divisions. So after the 200 or so divisions that it takes for the mothers to die, every single E. Coli cell should die. But this can’t be true, since E.coli is still around. Somehow the replicative clock is being reset.

By physically trapping the cells in a distal position the cells must have been affected in some way. The filamentous growth effect, how the intervals get shorter and shorter, particularly stands out to me as evidence that age-dependent deleterious effects are going on. The accumulation of some type of asymmetrical damage is occurring. But what? What’s going on in these cells? The cell wall is locked in position, but I have absolutely no idea what this means to the inheritance of the contents of the cell. Are the old template DNA strands more likely to end up in the “mother” than by chance, older proteins, oxidatively damaged structures?  This is what I wish the authors tested. The paper misses it’s potential as it could have been much more informative.

There’s a bromide based thymide analogue that would make tracking DNA relatively simple, it would just involve making mothers with it and washing with regular thymide, tracking inheritance by testing the antibiotic florescence on the daughter runoff.

On a somewhat but not completely unrelated note the Wikipedia page of the day is the “immortal DNA strand hypothesis”. The observation is that in some stem cells the stem cells always retain the old pole of DNA, the template strand instead of the freshly made strand. The idea is that this system reduces the accumulation of mutations in the constantly replicating stem cells,  passing on any replicative errors to terminal cells, as a way of preventing serious genetic defects like cancer from arising

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A buddy of mine alerted me that Mcdicks is having free coffee week. I’ve been there three time today. I love mcdonalds coffee, it’s dark and bitter and they burn the beans super hot. Much better than the rubbish they have in the cafe below our lab

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