Field of Science

Bacillus? Acinetobacter? Thermus?!?

I'm leaning towards doing the experiment that tests whether competent bacteria benefit nutritionally from the DNA they take up. There are two main issues to resolve. First is the design of the experiment, second is which organism to use.

I was originally thinking of simply measuring cell growth in medium with and without added DNA. This will work only if the effect of DNA is large. For it to be large, the cells will need to take up quite a lot of DNA while they are growing. If they only take up DNA when they run out of other nutrients, the effect is likely to be quite small. I would need to measure differences in growth or survival over long periods in stationary phase, rather like what Steve Finkel has done with E. coli.

An alternative to growth experiments is measuring competition. This would involve mixing cells that can take up DNA with cells that can't (probably cells with a knockout in a DNA uptake gene), and growing them in medium with and without DNA. This growth could be done over multiple dilution/growth cycles, potentially amplifying any advantage of DNA uptake. Using a non-competent mutant provides a nice internal control, but it's important that the mutation not reduce growth under conditions where DNA is not available. A minor effect might be acceptable, but I don't want to have to use statistical wizardry to show the effect of DNA.

What bacteria should this be done with? I wouldn't use H. influenzae - their nutritional requirements are too complex and the uptake specificity is a big complication. My first thought was Bacillus subtilis. I've worked with them before, and they are easy to make competent and can be grown on very simple defined media. So I spent much of yesterday reading papers about the regulation of competence in B. subtilis.

What a regulatory nightmare! I don't know whether B. subtilis gene regulation is intrinsically complex or whether the people who work on it just delight in digging up more complexities, but nobody seems to be trying to make functional sense of it. They do propose some absurd evolutionary just-so stories, such as that B. subtilis has prophage in its genome so cells can lyse under stressed conditions, allowing other cells to take up their genes.

Competence in B. subtilis is a bit of a phenotypic nightmare too - when food runs out most of the culture forms spores but about 10% of the cells instead become competent. While competent they're unable to replicate their DNA (I think) or septate their cells, so they grow into long filaments that divide into many short cells once the competence fit has passed (I saw the movie so I know this is true). Nobody who works on B. subtilis competence seems concerned by this....

I had already known about most of the B. subtilis complications (though not the DNA replication/septation arrest), but being reminded of them made me start thinking about alternatives. The first I thought of was Acinetobacter. These guys are easy to grow and nutritionally simple, like B. subtilis. Their competence development appears to be a lot simpler - there's no sporulation involved. Cells in lab culture express all the competence genes when they get into stationary phase, but they can't actually take up DNA until they're diluted into fresh medium. Then they gradually lose competence during exponential growth. This would make them quite suitable for a multi-cycle competition experiment.

But while reading about Acinetobacter I was reminded about competence in Thermus thermophilus. In lab culture these cells take up DNA all the time (less in stationary phase), giving transformation frequencies of about 1% for most genes. This might give a growth advantage large enough to be detected in a single-step growth experiment. Mutants carrying knockouts of DNA-uptake genes are readily available and well characterized. I think T. thermophilus's nutritional requirements are quite simple, but growing it is likely to be complicated because it's a hyperthermophile - lab cultures are usually grown at 60-70°C! We do have a spare shaking waterbath that could be set this high, but for competence assays and cell counts we'd want to grow cells on solid medium, and I don't even know if one can use agar at this temperature.

I'm going to email the T. thermophilus expert (Beate Averhoff, in Frankfurt) asking if she can send me a pdf of a recent chapter she wrote for a methods book (our library doesn't have it), and whether she thinks it's easy to work with.

No comments:

Post a Comment

Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="">FoS</a> = FoS