Field of Science

Relative activities of comM from strains Rd and NP

A month or so ago I described four experiments I wanted to do.  I've now done the last of them, testing whether the comM gene of strain NP could be partly responsible for that strain's 100-fold lower transformability.

...and the answer appears to be...  NO  (with one qualification).

The experiment was to compare the abilities of plasmid-borne Rd and NP comM genes to restore full competence to Rd cells whose chromosomal comM gene had been deleted.  The RA made all the strains for me - all I had to do was measure their transformation frequencies after fully inducing competence by transfer to starvation medium.  The first four bars in the graph below show the results:



Both comM alleles restore normal competence to the Rd knockout when cloned in the forward orientation but not when cloned in the reverse orientation.  I think each insert has its own CRP-S promoter, but the plasmid also carries the E. coli lacZ promoter which, I suspect, interferes with expression of inserts whose promoters face in the reverse orientation.

The motivation behind this experiment was a recombinant strain identified by an undergraduate who was working with the postdoc.  This strain transforms only 10% as well as Rd; it has been sequenced and we know it contains a single 40 kb segment of NP sequence.  The only known competence gene in this 40 kb segment is comM, so they hypothesized that a partially defective NP comM might be responsible for the recombinant's 10-fold reduced competence and partly responsible for NP's 100-fold lower competence.  This new result suggests that this hypothesis is wrong.

The qualification is that strong expression from a plasmid could mask lower expression or catalytic activity of NP's comM gene.  The gold standard experiment would be to replace the Rd chromosomal allele with the NP version and vice versa.  For various technical reasons we haven't been able to do this yet.

A rotation student replaced the recombinants NP comM allele with the knocked-out Rd version - this reduced its transformation frequency by another 10-fold, about the same as the TF of a simple RD ∆comM strain.

I had tried to transform a ∆comM strain of NP with the Rd comM allele, but the ∆comM mutant the RA made was, unexpectedly, completely non-transformable.  She has now given me another NP ∆comM isolate to test.  That's the last column in the chart; the red star indicates no transformant colonies at all, so this isolate too is completely non-transformable.  This result is consistent with the similarity of the Rd and NP comM plasmid results; they both suggest that the NP comM gene is fully functional, and that some other difference(s) must be responsible for its lower transformability.

So why does the recombinant have lower transformability?  Before designing any more experiments I need to be better able to think about the relative chromosomal locations of the various selectable markers and competence genes we're interested in.  To this end I'm having our work-study student take a break from glassware-washing and media preparation to do what she calls an 'arts and crafts' project - making us a poster showing the locations of all these genes drawn on a circular chromosome.  I  just need to give her a list of the genes/ locations to include on her poster.

No comments:

Post a Comment

Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="http://www.fieldofscience.com/">FoS</a> = FoS

Post a Comment