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.

The controversy surrounding the function of DNA uptake

I'm holed up in Indio California, in a "RV Resort" for retirees, working on my grant proposal and checking out the local attractions (Washingtonia palms! The Salton Sea!).

Here's a few paragraphs from the proposal introduction, explaining the big question:

The consequences of DNA uptake are not in question. A cell that takes up DNA inevitably incurs the physiological costs of becoming competent and of transporting the DNA across its envelope. The cell also inevitably gets the incoming DNA’s nucleotides, reducing the demands on its biosynthetic or salvage pathways. Because DNA is abundant in natural environments, and nucleotides are very expensive to synthesize, the nucleotide benefit may be sufficient to compensate for the costs and thus to explain the evolution (origin and continuation) of competence. However if the incoming DNA recombines with the chromosome it may also change the cell’s genotype, which may increase or decrease the cell’s ability to survive and reproduce. The controversial question is whether natural selection on the machinery and regulation of DNA uptake has been influenced by these genetic consequences.

The conventional view is that bacteria take up DNA for recombination (i.e., that recombination has net benefits, and that these are sufficient to account for the evolution of competence). This derives partly from the now-discredited idea that sex in eukaryotes is easily explained by long-term benefits to the species, and partly from observation of ancient beneficial recombination events in bacterial genomes and recent ones in the laboratory. But there are also substantial costs to genetic recombination, because the homologous DNA in the environment comes from dead cells and is likely to carry excess deleterious mutations, and because recombination with heterologous DNA will usually the cell’s well-adapted genetic machinery. These genetic costs are easily overlooked because natural selection eliminates the cells incurring them.

Understanding the evolution of bacterial competence has major implications for our present far-from-satisfactory understanding of why sexual reproduction evolved in eukaryotes. The problematic hypothesis that meiotic sex evolved to create new combinations of genes is often supported by claims that bacterial ‘parasexual’ processes also evolved for this. Because conjugation and transduction are now known to be side effects of selection for more immediate benefits to cells or their genetic parasites, understanding competence is key. If the genetic consequences of competence have not shaped its mechanism or regulation, we will conclude that bacteria get all the genetic variation they need by accident, and thus that meiotic sex is a eukaryotic solution to a eukaryotic problem.

Direct experimental testing of proposed costs and benefits is not the best approach, because it is all too easy to create selection in bacterial cultures, and because laboratory conditions in no way replicate those of the natural environment. Rather, the best way to understand the evolution of competence is to understand its regulation and mechanism. Regulation is informative for all bacteria, as the genes that regulate competence evolved in the natural environment, and understanding the signals they respond to will give us a window on the consequences of competence that have been most beneficial. Because H. influenzae’s uptake specificity causes it to preferentially take up its own DNA, understanding the uptake mechanism responsible for this bias is a critical test of the importance of recombination.