Field of Science

Does H. influenzae need DNA uptake genes to form lab biofilms?

This morning I had another Skype conversation with the (most recent) former post-doc.  We mostly talked about the toxin/antitoxin work.  One question that came up was whether the antitoxin knockout strain was unable to form simple biofilms as well as to take up DNA.

The kind of biofilm I mean is a simple film of cells that might stick to the surface of the glass or plastic container the cells are being cultured in.  Formation of such films depends on the species (do its cells have a sticky surface), on the genotype (how much of the sticky substances are being produced), on the container properties (glass? polystyrene? polypropylene) and on the culture conditions (cells may stick more easily if the culture is not being shaken).

Here's a diagram of the basic assay; the the amount of crystal violet depends on how many cells were stuck to the tube surface.

Many components of the cell surface can contribute to its stickiness, but we're interested in the effects of type 4 pilin (T4P) structures on the cell surface, because these are used both for adherence to surfaces and for DNA uptake. If our wildtype H. influenzae cells consistently form biofilms, and if this depends on the expression of the normal DNA uptake machinery, then we can test whether the DNA-uptake defect of our antitoxin knockout mutant is accompanied by a defect in forming biofilms.

Why do we care about this?  We know that this mutant has near-normal expression of the genes needed for DNA uptake, so why can't it take up DNA?  If the controls show that biofilm formation requires the uptake machinery, and the mutant does not form normal biofilms, we'll conclude that the toxin interferes with assembly of the basic T4P machinery.  If the mutant does form biofilms, we'll conclude that the toxin specifically blocks the DNA-uptake activity of the T4P machinery that has been assembled and is able to stick to surfaces, perhaps by blocking the retraction step that pulls the DNA in.

The experiments are quite straightforward.  Versions of this assay have been done on various H. influenzae clinical isolates, but not to examine the roles of the type 4 pilus machinery.  We'd use one of our competence-negative regulatory mutants, probably a sxy knockout.  The lab down the hall does similar assays with Campylobacter - I'll ask their advice before proceeding.

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