The Research Associate's experiments, part 2

The second big component of the Research Associate's recent work is on regulation of (and existence of) competence in E. coli.  (The rest of her work is described in the previous post.)  We've shown that E. coli has homologs of all but one of the genes known to be needed for competence in H. influenzae, and these genes are induced when the competence-specific regulator Sxy is artificially induced.  BUT, we haven't been able to detect transformation, and we don't know of any conditions that naturally induce Sxy.  Because Sxy in E. coli appears to work very similarly to Sxy in H. influenzae, we're also hoping that some questions will be more easily answered in E. coli, which is a more tractable system.

Questions:  I.  About the regulation of Sxy expression in E. coli

Q. 1:  How is transcription of the E. coli sxy gene induced? 

We don't know.  Nobody knows.  See previous posts.

Q. 2:  Is the E. coli sxy gene post-transcriptionally regulated?

First, we needed to find the 5' end of the sxy transcript?  The RA has now mapped this (after some strenuous battling with experimental artefacts).  It's well upstream of the start codon (~100 nt?), as is the case in the Pasteurellaceae and in Vibrio cholerae.  So there's lots of potential for post-transcriptional regulation, as has been shown in H. influenzae and V. cholerae.

Prediction of secondary structure of the mRNA sequence using the program mfold shows that it can fold into complex secondary structure, but we'd need experimental evidence to show that this structure is biologically significant (mfold will fold just about anything).

Questions:  II.  About what E. coli Sxy protein does for E. coli

The results of the experiments described below are quite consistently negative, so the RA's strategy is to cover all the possibilities so we can at least write a solid negative-result paper.

Q. 3:  How does Sxy stimulate transcription of genes with CRP-S promoters?

CRP-S sites are variant CRP sites where transcritional activation requires both CRP and Sxy.  We have published genetic evidence that stimulation of transcription at CRP-S sites depends on direct interaction between the CRP and Sxy proteins - assays using cross-complementation between H. influenzae and E. coli induce transcription much more strongly if the CRP and Sxy proteins both come from the same species.  We also have unpublished evidence.  In vitro, far-Western blots show that CRP and Sxy physically interact, and pull-down experiments show that pulling down Sxy brings down CRP.

But Sxy doesn't bind to CRP-S sites in bandshift experiments (in vitro) whether or not CRP is present, and adding Sxy doesn't improve CRP's poor ability to bind to them.

A plan?  Construct a fusion of the candidate regulatory parts of the sxy gene (= promoter, 5' untranslated region and 5' part of the coding sequence) to a reporter gene.  Then mutagenize the sxy part and screen for changes in expression.  The mutagenesis could be done randomly with degenerate PCR oligos (???) or be site-directed.  Changes in expression would give clues about regulation, and any high-expression mutations could be tested for induction of transformability (see below).

Here's another idea.  How about we put the E. coli sxy gene, with all its candidate regulatory sequences, into a H. influenzae sxy knockout, mutagenize the regulatory region by transformation with a degenerate sxy PCR product of some sort, and then select for hypercompetence mutants as we did to get the original H. influenzae sxy mutants?  Then we can sequence the sxy genes to find mutations that increase expression (or activity).  I suppose the E. coli genes could be on a plasmid (or plasmids) rather than in the chromosome.  I can't remember whether we've shown that E. coli sxy complements a H. influenzae sxy knockout - it should, based on the E.coli experiments.

We should also just first do a time course etc. to see how replacing the Sxy gene changes induction.  For the time course we'd probably want to put the E. coli CRP gene in too, so we'd get higher induction of the CRP-S genes and thus more sensitive transformation assays.

Questions:  II.  About what the E. coli CRP-S regulon does

Q. 4.  Will expression of E. coli Sxy ever induce transformation in strain K-12?

The RA has developed an assay for chromosomal transformation in E. coli, using chromosomal DNA of a strain carrying a KanR insertion in the crp gene, or a TetR insertion in the purE gene.  Sxy is artificially induced, from either a high-copy plasmid or a low-copy plasmid.  BUT, the big problem is that we have no positive control - we don't have any conditions or genotypes where giving cells this DNA does produce transformants.  Transformation requires both that the cells take up DNA and that the DNA recombines into the chromosome.  In H. influenzae transformation is a very sensitive assay for DNA uptake because the recombination is quite efficient.  But in E. coli we have no idea whether transformation would be a usual outcome of DNA uptake.

One solution is to artificially enhance the efficiency of recombination by turning on the phage-derived recombineering genes she's been using to create mutations.  In principle this means we only need to assay for uptake.  So she's done this, inducing the recombineering genes by heat-shock (they're in the chromosome under the lambda CI857 ts promoter).  The target for recombination is the H. influenzae comM gene on a plasmid, and the recombining DNA is a PCR product (i.e. dsDNA) of a comM::SpcR fragment.  When the SpcR fragment is introduced into the cells by electroporation, she gets thousands of transformants.  The cells also contain an IPTG-inducible sxy gene, and when this is induced along with the recombineering genes, she gets a few SpcR colonies without electroporation.

Are these natural transformants?  She wants to rule out the possibility that the temperature shock used to induce the recombineering genes somehow allows some SpcR DNA to get into the cell.  She's going to do this by using a different recombineering strain, one where the phage genes are on a plasmid and induced by adding the sugar arabinose rather than by heat shock.  Another advantage of using this strain is that, if she gets what appear to be transformants, she will be able to test the effect of knocked-out competence genes by simply moving the recombineering plasmid to one of our Keio strains carrying chromosomal competence-gene knockouts.

However we still need to worry about the difference between single-stranded and double-stranded DNA coming into the cell - I think the recombineering proteins handle these very differently.  One control would be to denature the PCR product by heating it before giving it to the cells.

She's also tried inducing sxy in the  K-12 strain that Steve Finkel used to show that (1) E. coli cells can take up DNA and use it as a nutrient, and (2) this uptake needs homologs of H. influenzae competence genes.  In principle we know that this strain can take up DNA, but our visiting student last year was unable to replicate this result.  In an case, this strain did not transform with her marked chromosomal DNA.

Q. 5.  Does expressing Sxy promote recombination?

She also tested transformation by electroporation without inducing the recombineering proteins.  Whether or not Sxy was induced, she saw no transformants.  This tells us that Sxy does not activate recombination pathways.


Q. 6.  Does providing the H. influenzae pilF2 gene enable E. coli K-12 to transform when Sxy is induced?

The only H. influenzae gene needed for competence that's not in E. coli (K-12 and other sequenced strains) is pilF2.  It's in all competent Pasteurellaceae and in Vibrio cholerae.  (I'm sure I already wrote this somewhere but I can't locate it now.)  She's provided it on the sxy expression plasmid, both under its own promoter and under the IPTG-inducible promoter, but these cells didn't transform either.

Q. 7.  Will expression of E. coli Sxy ever induce transformation in non-K-12 strains?

She's tested many strains of the ECOR collection (a reference set of diverse E. coli strains isolated from many different locales and hosts) for transformation during growth and stationary phase in the rich medium LB, using her chromosomal DNA (probably crp::KanR).  No transformants were seen.  She also tested the cultures for expression of the Sxy-induced pilin protein; none was produced.  With the help of a summer volunteer undergrad she's now going to put an inducible sxy plasmid in these strains and retest them.

Q. 7.  Does expression of E. coli sxy and the CRP-S regulon provide E. coli with benefits other than competence?

The RA hasn't been working on this specifically, but we need to think about it if her results continue to be negative.  If it's true that E. coli cannot take up DNA by the natural competence mechanism, why does it have intact versions of all the necessary genes except pilF2, under a Sxy-controlled CRP-S regulon?  If these genes were just leftovers from a competent ancestor, we would expect to see some loss-of-function mutations in them, if not in K-12 then in some of the other sequenced strains.  (Has anyone checked the alleles in the other sequenced strains?)

How carefully have we checked the phenotype of the E. coli sxy knockout?  Might it be defective at anything?  In the RA's E. coli sxy paper (2009) we compared long-term survival of wildtype and sxy-knockout cells in LB.  The mutant reached the same initial cell density as the wildtype, and it survived long-term culture just as well.  But it did not compete equally with wildtype when the two strains were cultured together, a phenotype that Steve Finkel's work suggested was due to inability to sue DNA as a nutrient.