Field of Science

Which E. coli is best?

I haven't yet tested whether different growth/non-growth conditions alter the expression of the ppdA fusion (though I did get the sxy manuscript resubmitted today), but a paper I came across reminded me of another issue I need to consider.

The paper is an opinion piece titled "Laboratory strains of Escherichia coli: model citizens or deceitful delinquents growing old disgracefully?"; it just came out in the journal Molecular Microbiology (Mol. Micb. 64:881-885). The authors argue that the standard K-12-derived strains of E. coli that microbiologists and molecular biologists typically use are not at all representative of the strains out in the natural environment. For one thing, lab strains have lost about 20% of their genomes. These are (by definition) non-essential genes, but their loss no doubt affects cellular metabolism, so that the metabolic interactions we see in K-12 strains may be quite different than those in natural strains. Furthermore, the culture conditions we use (rich broth, lots of oxygen, no competitors) are unlikely to ever occur in nature. Their long maintenance under these and other unnatural conditions means that the lab strains will have evolved by accumulating cryptic mutations that are beneficial under lab culture conditions but that may have very different and perhaps harmful effects in the natural environment.

I already knew this. It has important implications for my search for conditions that induce expression of competence genes in E. coli. Right now I'm working with standard lab strains, but it's all too possible that one of their ancestors lost the ability to express the genes encoding homologs of H. influenzae's competence genes, or to assemble the proteins into functional DNA uptake machinery.

So I should perform my tests on a less lab-adapted, more 'natural' strain as well as on the K-12 strain the ppdA::lacZ fusion is in. But this raises two problems. First, which strain should I use? I do have a fairly-ancestral K-12 strain, but really I should use a 'wild' strain recently isolated from the environment. The NCBI Microbial Genomes page lists 8 completely sequenced E. coli genomes; their sizes range from 4.6 million bp (K-12) to 5.6 million bp (O157:H7). And the various wild strains have genomes that are quite different from each other - does this mean I would need to test many strains before giving up? I'm also not meticulous enough to be trusted with a strain that's seriously pathogenic to humans, so the two O157:H7 strains are out, as is the uropathogenic strain*.

Second, I'll need to transfer the necessary genes into this strain; first a lacZ mutation making it Lac-, then the ppdA::lacZ fusion so I can assay induction by Sxy. This might mean that I need to get my P1 transductions working after all (I had been thinking I could let them slide now that I've found that the ppdA::lacZ fusion can be used as an indicator of CRP-S induction by Sxy). I don't need P1 to move in the ppdA fusion because it's on a plasmid, but transduction would certainly be the easiest way to move a lac- mutation in. If I'm lucky, maybe some of the wild strains are naturally Lac-. (Probably not; most screens for wild E. coli start by treating anything that's Lac- as not E. coli.)

* NCBI also lists 6 sequenced 'Shigella' genomes - we now know that the bacterial strains assigned to the genus Shigella are really variants of E. coli. But I have absolutely no intention of working with these very pathogenic bacteria.

17 comments:

  1. I can weigh in here, as I just spent 6 years doing a PhD in a pathogenic E. coli lab (O157:H7 and uropathogenic). I think your main challenge will be getting P1 to work in your organism of choice. Although CFT073 was my favorite model uropathogen, we never were able to get P1 transduction working in that strain, always relying on recombineering, Tn7 and Tn5 to deliver and alter genes.


    There are numerous reports of P1 transduction working in UTI89, a uropathogen Scott Hultgren works with at Washington University, and I believe they have lac- strains. Cloning by mail is always easier.

    If your ppd::lacZ fusion is marked in some way, you can also you lambda-Red mediated recombination (recombineering) to pseudo-transdcuce the construct. Basically, if the genomic insertion point for the ppd::lacZ is very similar between the E.coli strains you have in mind, you PCR up the the region (+-200 bases or so), then electroporate into Lambda-Red competent E.coli. Lambda Red recombination has been published to work in at least EDL933, UTI89, CFT073, MG1655, W3110, and RS218

    I am not sure what biosafety precautions are required for H.in, but I would suggest that, to start, O157:H7 and the Shigella strains aren't worth the risk. Even in our lab, we avoided breaking these strains out if there was any way we could manage otherwise. Their infectious dose (<10 organisms) just make things a bit tense around the lab. If you do end up going the O157:H7 way, I am sure Brett Finlay has some strains you can use

    Uropathogenic E. coli, on the other hand are pretty low risk, just light BL2 precautions: lab coat, gloves, and autoclaving everything. CFT073, though, is particularly complement resistant, and if it gets in the human bloodstream via puncture, sepsis can quickly arise.

    ReplyDelete
  2. Wow, open science proves its worth!

    Thanks for the caution about P1; I thought that might be a problem. We have the recombineering tools and I've just needed an incentive to get them working for us, in E. coli and hopefully in H. influenzae too.

    ReplyDelete
  3. In my Mol Biol & Evol paper we published data and a tree of E. coli sequenced genomes that illustrates how similar they are. Personally, I would be inclined to look at at least one of two of the ECOR strains; these represent the natural diversity of E. coli and I vaguely recall that many were isolated from the faeces of healthy zoo animals, rather than from sick humans... (and most of those from sick humans were, I think, uropathogenic). I worked with all of them for four years without any ill-effect.

    ReplyDelete
  4. This comment has been removed by a blog administrator.

    ReplyDelete
  5. Hawaii warriors quarterback 2007 heisman award winner and top nfl draft pick plays with hawaii warriors. You can buy sports tickets for the hawaii warriors football games.

    Colt Brennan
    Colt Brennan bio
    Quarterback Colt Brennan
    2008 nfl draft Colt Brennan
    Colt Brennan hawaii heisman
    Hawaii Quarterback Colt Brennan
    Colt Brennan Bio
    sports tickets
    bondage sex toys
    buy sex toys buy sex toys
    tickets
    Hiroki Kuroda

    ReplyDelete
  6. [url=http://www.ld-harvest.com/3-2-1.htm]模块电源[/url]
    [url=http://www.ld-harvest.com]高压变频[/url]
    [url=http://www.ld-harvest.com/3-1-1.htm]高压变频器[/url]

    模块电源
    高压变频
    高压变频器

    ReplyDelete
  7. This comment has been removed by a blog administrator.

    ReplyDelete

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