HI0659 progress and plans

I'm making progress in figuring out how the knockout of gene HI0659 prevents cells from becoming competent.  I don't know the answer yet, but I've ruled out some alternatives.  We know that the mutation blocks both DNA uptake and transformation, so the defect is not at the translocation or recombination steps - the mutant cells must either fail to induce their competence genes correctly or be blocked at some point in DNA uptake (assembly of the uptake machinery or uptake itself).

The first experiments were to see if treatments or mutations that normally induce competence would override the competence defect of the knockout strain (strain RR3112, HI0659::spc, but for convenience here I'll just call it HI0659-).  Competence induction requires that the CRP protein bind to its cofactor cyclic AMP (cAMP) and then induce transcription of competence genes, which is normally synthesized under competence-inducing conditions.  Adding cAMP restores competence to cells unable to synthesize it, so I tested whether the HI0659- competence defect was corrected by adding cAMP.  It's not.

I could also test whether the HI0659 mutation interferes with the ability of CRP, by assaying the strain's ability to ferment CRP-regulated sugars.  But I don't need to do that because one of the other experiments I've done (described below) shows that CRP regulation works normally in HI0659 mutants.

I also tested whether competence is restored to the HI0659- strain by mutations that cause expression of competence genes under conditions that normally repress this (hypercompetence mutations).  We have two sets of these mutations, in the sxy gene and in the murE gene.  I made double mutants by transforming these strains with DNA of strain RR3112, selecting for its SPcR cassette, and tested their competence.  They were not competent at all, even after normal MIV induction, so the defect isn't that the competence genes just require stronger-than-normal induction.

The next test asked whether HI0659- cells fail to induce competence genes.  This is a bit odd to think about since HI0659 is itself a competence gene whose transcription is induced by Sxy and CRP+cAMP, but maybe once some HI0659 gene product is made it increases or stabilizes transcription or translation of the other genes, or protects transcripts from degradation.  As I explained here, we have 'reporter' strains that let us detect transcription of the comA and rec2 competence genes because these gene's promoters have been fused to a lacZ gene, whose beta-galactosidase product is easy to detect with a colorimetric assay.

I introduced the HI0659- mutation into four fusion strains, two carrying a comA::lacZ fusion and two carrying a rec2::lacZ fusion, and assayed their production of beta-galactosidase and their competence.  Here are the results:

All the HI0659 mutants have the same beta-galactosidase levels as their HI0659+ parents (yellow bars and tubes).  Only the comA parent was included in this assay (the leftmost column), but you can see the induced and uninduced activities of both parent strains in the previous post.  Importantly, all the HI0659- strains were completely non-transformable (blue bars), confirming that they had replaced their HI0659+ allele with the HI-659- allele.

This result tells us that the HI0659 mutation does not act by interfering with normal transcription or mRNA stability of comA or rec2.  It's possible that it specifically affects the expression of another of the competence genes, but this is unlikely.

We've been preparing to do 'RNA-Seq' analysis of the HI0659 mutant - this analysis uses Illumina of other 'next-gen' sequencing of reverse-transcribed mRNAs to measure the amounts of transcripts present in the cell.  We have been hoping that it would reveal changes in transcription caused by the mutation, but the lacZ fusion results make that unlikely.

The RNA-Seq analysis is expensive and quite a lot of work - should we still do it?  The controls we'd need to do would give us lots of solid information about the regulation of competence, complementing the microarray analysis we did ten years ago.

I have one more analysis to do, suggested by the bioinformatics analysis I did a couple of weeks ago, described here.  The bioinformatics suggested that the HI0660/HI0659 gene pair might be a toxin-antitoxin system (or derived from one), with HI-0660 being the 'toxin' and HI0659 the 'antitoxin'.  If so, then HI0569's job is likely to be preventing HI0660 from doing something that prevents competence.  This is consistent with the normal phenotypes of the HI0660::spc and HI0660 unmarked mutants.  They both take up DNA and transform normally, even though the HI0660::spc insertion might be expected to interfere with expression of the downstream HI0659 gene and thus reduce competence.  If HI0659's job is just to stop the HI0660 product from doing something that prevents competence, then  the competence defect of the HI0659 mutant should be corrected by adding a HI0660 knockout.

This is simple in principle (just transform the HI0660 cells with HI0659::spc DNA), but complicated by how small the two genes are and how close they are to each other.  We have the E. coli plasmids carrying the mutations, and my plan is to instead construct a new plasmid that's deleted for both genes, with one spcR cassette inserted, and transform wildtype cells with this DNA to get the desired double mutant.  How easy this construction is will depend on whether the HI0660/0659 genes and the spcR cassette have convenient restriction sites, so I'm going to spend this afternoon looking for them.