We have struggled a lot with the best measures for plasmid cost and conjugative capacity. The challenges lie in 1) relevance, and 2) choosing an in vitro method. I often waffle (to my students’ disliking) about the best way to go about experiments measuring these parameters. If you look in the literature, there is some fantastic work that addresses in vitro measures of plasmid fitness cost (just google scholar “plasmid fitness cost”). In its most primitive form, you can measure the fitness cost of a plasmid by competing plasmid-free versus plasmid-containing bacteria in broth cultures over the course of several passages, measuring the proportions of each cell population on a daily basis (see here). Some great work by Eva Top’s group has come up with “sexier” and likely more accurate means to measure fitness cost of plasmids using multiple parameters and modeling approaches (see here). In our experience, any of these approaches result in a great deal of variation even when controlling for everything we can think of. I think this is because of the nature of the plasmids we are studying (IncA/C) which imposes a very small fitness cost on its hosts. Contrast this to previous work where fitness costs are much larger and easier to measure accurately. A second confounding component – I believe – is that rapid adaptation to IncA/C plasmids occurs when introduced into a host, but there are many factors about this we don’t understand (conditional dependency, cell density impact, temperature impact, role of host genetic background, plasmid-encoded transcriptional regulators, etc.).
We are finding that host background plays a huge role in the persistence and cost of carrying IncA/C plasmids. It has been shown that IncA/C plasmids will be lost over time in some in vitro approaches (see here), however we are finding that in wild type recipients these plasmids are rarely lost in vitro and impose virtually no fitness cost. My opinion is that doing these experiments in a strain like E. coli DH10B does not come even close to reality, and should be avoided.
As for conjugation, filter matings have become the method of choice for measuring conjugative ability of a plasmid. We have tried about every type of mating experiment we can think of, including time point filter matings, liquid conjugations, solid plate matings, etc. for IncA/C plasmids. Measuring conjugation with these plasmids seems to be more reliable and straightforward than measuring fitness cost, however we again see a variety of conjugative frequencies depending on donor and recipient. This is not at all unusual for a broad-host-range plasmid (see here) but it certainly drives me crazy when trying to determine the best approach for measuring conjugation – there are limitless possibilities. For these reasons, we have mostly stuck with E. coli K-12 (which I feel is at least a better choice than DH10B) as a donor/recipient for these experiments in an effort to first establish a baseline from which to pull what’s left of my hair out in future experiments.
I guess the point of this blog is to spew my frustrations with these types of experiments and remind myself the cautions of interpreting their results. Comments are welcome!