Way back, before we could measures changes in gene expression caused by transfer to MIV, we know that it induced two independently measurable phenotypic changes. The first was that cells became able to take up DNA and recombine some of it into the chromosome. The other was that phage recombination was increased about 100-fold.
What is ‘phage recombination’? When cells are co-infected with two different mutant strains of the same phage, their DNAs can recombine in the cell while they are replicating, giving rise to some phage genomes that have neither mutation. For H. influenzae, we used three different temperature-sensitive mutants of the phage HP1, which could form plaques at 32°C but not at 40°C. Co-infecting non-competent cells with any two of these gave a very low frequency of recombinant phage, measured as plaques on lawns of cells grown at 40°C. But co-infecting competent cells gave about 100-fold more recombinant plaques.
This increased phage recombination was interpreted (by me and others) as evidence that transfer to MIV induced both DNA-uptake machinery and recombination machinery. I was looking for a way to distinguish between mutations that just knocked out a component of the uptake machinery and those that knocked out the regulatory machinery cells used to decide to become competent. So I used phage recombination to categorize mutants as regulatory or mechanistic - hypothesizing that mutations that eliminated both uptake and phage recombination were probably regulatory, as they affected two independent processes, whereas mutations that knocked out uptake but still induced phage recombination probably affected just a component of the uptake machinery.
Now we know which genes are induced by transfer to MIV. None of them qualify as recombination machinery. But this wasn't bothering bother me for two reasons. First, I’d decided that cells take up DNA as a source of nutrients, so I didn’t expect recombination to be induced. Second, I’d forgotten all about the induction of phage recombination and my former interpretation of it.
But today I was reading my old (before we did microarrays) notes about all the competence genes we knew of, and one note mentioned a mutant that had very little phage recombination but that I now know is part of the CRP-S regulon. This gene is comM; it specifies a protein that somehow protects incoming DNA from being degraded by a nuclease or nucleases present in the cytoplasm. It does DNA uptake but its transformation frequency is very low because the DNA is degraded before it can recombine.
EUREKA! This makes sense. The reason phage DNAs can recombine better in normal competent cells than in non-competent cells is that competence induces comM, and the ComM protein prevents the nuclease from degrading the phage DNA recombination intermediates. Recombination intermediates have unusual DNA structures (exposed single strands, ends of strands, and four-way connections called Holliday junctions) which are vulnerable to general and specialized nucleases.
Another gene, dprA, does something similar. Mutations in dprA also allow incoming DNA to be rapidly degraded, and DNA brought in by the DNA uptake machinery doesn’t survive long enough to recombine with the chromosome. In my old notes I think I have data for its phage recombination - I bet it’s low. (Later - yes, it is.)
I have been hypothesizing (without any direct evidence) that both comM and dprA have evolved to be competence-induced because their jobs are to protect stalled replication forks from nucleases. Stalled replication forks have a lot in common with recombination intermediates; they often get into tangles that are structurally indistinguishable from the Holliday intermediates produced by recombination. I wonder if I can use phage recombination in some way to help sort this out?
(Note to self: The above sounds great, but don't forget the complication that the rec2 mutant also has low phage recombination, which is unexpected because we've been pretty sure it acts in DNA transport across the inner membrane.)
Too Easy, but What the Hell
6 hours ago in Angry by Choice