Yesterday I talked to my colleague down the hall about a possible joint project. She reminded me that one of her grad students has done quite a bit of work on DNA in Campylobacter jejuni biofilms; the student has a mutant that makes faster/thicker biofilms, and whose biofilms have much more DNA than is usual. The biofilms also fall apart in the presence of DNase I. She gave me some papers to read, one by the student and two others about DNA in Pseudomonas biofilms.
Despite quite a few publications about DNA in biofilms, none consider the role of competence. So I still like the idea of testing whether biofilm formation by C. jejuni and by H. influenzae is enhanced by the ability to bind (and take up?) DNA.
I also did some reading about Campylobacter competence. There aren't many papers, but there's some nice work characterizing genes needed for DNA uptake, and characterizing how competence varies under different culture conditions. We'd need to send for a suitable mutant, one that was unable to take up DNA but had no growth defect. Ideally we'd want to know that the cells couldn't bind DNA, or couldn't turn on the competence genes, but I'll have to do more reading to see if such a mutant is known.
All the H. influenzae biofilm work appears to have been done with 'nontypable' strains (clinical strains lacking a capsule, which are important causes of ear infections), rather than with the Rd strain we normally use. I was hoping that these experiments might have used Rd as a control, but apparently not. I'd much prefer to do these experiments with Rd, but we'll have to check whether it readily forms biofilms in culture. If not, we might be able to use one of the nontypable strains that we've found to be readily transformable, but we'd need to introduce a hypercompetence mutation into it.
The minimal experiment would use glass culture tubes, some of which had been precoated with DNA. Some DNA sticks to plain glass, although I can't find out how much. A lot more sticks in the presence of saturating sodium iodide but that's not an option for living cells. So we could try just filling tubes with moderately concentrated solutions of H. influenzae or C. jejuni DNA, maybe 10 µg/ml, leaving them for a few hours, and then letting the DNA dry on overnight. (There's evidence that C. jejuni, like H. influenzae, prefers to take up its own DNA, although there's no obvious uptake sequence in its genome.)
We'd probably be wise to rinse the tubes with culture medium in the morning, to remove DNA that wasn't stuck to the glass surface. Then we'd add dilute cultures of hypercompetent (sxy*) or sxy- H. influenzae, or wildtype or noncompetent C. jejuni, and let the cultures incubate overnight at 37°C. We would also include cultures with added DNase I as another negative control. After overnight culture (or shorter), we'd wash out the non-attached cells, stain the biofilm with crystal violet, and measure the staining.
Another experiment would measure whether being competent helps cells attach to an existing biofilm, presumably by attaching to the DNA. For this experiment we'd grow biofilms in the ordinary way, using whatever strain makes good biofilms. Then we'd add antibiotic resistant H. influenzae or C. jejuni cells (competent and non-competent), and give them time to attach. Then we'd wash away all the unattached cells, break up the biofilm by adding DNase (or ???) and plate on antibiotic plates to count the newly-attached cells. To distinguish binding to DNA from binding to other biofilm components, we could swamp the culture with free DNA - that should abolish DNA-specific attachment. (This control could also be done for the minimal experiment.)
Welcome to the 4th Reich part 1.
16 hours ago in Angry by Choice