Tuesday, September 16, 2008

Impact of conjugal transfer on the stability of IncP-1 plasmid pKJK5

Bahl MI, Hansen HL & Sørensen SJ (2007. FEMS Microbiol Lett 266:250-6.

“With great power comes great responsibility”. As strange as it may seem, Churchill’s famous quote can be applicable to the bacterial as well as the human world. Many bacteria contain plasmids that confer to them the “power” to do many impressive things: resist antibiotics or heavy metals, break down toxins in the environment, become virulent, and some even give bacteria the power to conjugate (mate) with other bacteria. All this power comes at a cost, however. Having a plasmid that gifts a bacterium with novel traits also means that the bacterium has to invest in maintaining the plasmid. A variable amount of a bacterium’s resources will have to be diverted to keeping the plasmid and its various functions up and running. Thus, plasmids often affect bacterial growth rates, causing the plasmid bearing bacteria to grow and reproduce more slowly than their plasmid free neighbors. Therefore, it is commonly thought that a plasmid is only truly beneficial if there’s an immediate selective advantage to having it (for instance, having a plasmid that keeps you alive in the presence of antibiotics is great when being actively doused in antibiotics, but the costs of the plasmid might not be worth it when the flow of antibiotics stops). In times where such selective pressures are removed the percentage of plasmid harboring cells will often decrease due to factors such as the slower growth rate and the occasional loss of plasmids in one of the daughter cells during segregation (Bergstrom et. al., 2000).

All this said, the authors of a recent paper suggest that plasmid loss in the absence of selective pressures may not be such a sure thing after all. They propose that when bacteria have a plasmid and frequent access to each other (as is the case in bacterial biofilms or microcolonies) conjugation can more than compensate for plasmid loss in a population. By constructing fluorescing bacteria the authors of this paper were able to see and quantify plasmid stability in bacterial populations that could and could not conjugate. They therefore were able to study the impact of conjugal transfer on the stability of an IncP-1 plasmid in bacterial populations, as the name of their article implies.

The authors carried out this study using Escherichia coli MC4100 and Kluyveria sp. MB101. A gene cassette coding for kanamycin (Km) and streptomycin (Sm) resistance, as well as a green fluorescing protein (GFP) was inserted into the chromosome of each of the above-mentioned bacteria. In liquid broth the constitutively expressed gfp can be detected by flow cytometry using an argon ion laser, whereas an epi-flouresence microscope was used for visual detection of fluorescing colonies on solid media. The production of GFP is regulated by a lac operon, and so is repressed in the presence of a functional lacI gene (which is not present in either of the constructed strains of bacteria). In order to test the importance of conjugation on plasmid stability, the authors inserted an entranceposon containing a lacIq1 gene into plasmid pKJK5. The entire genome of pKJK5 had been previously sequenced, and so the authors were able to use PCR to screen for neutral insertions (e.g. pMIB4) and insertions that disrupted conjugation (e.g. pMIB8) (Haase et al., 1997). The authors introduced these lacI-containing plasmids into E. coli MC4100 and Kluyveria sp. MB101. Therefore, any bacterium containing pKJK5 or one of its derivatives would not fluoresce, due to the lacI suppression of GFP production. Thus, this method allowed the authors to quantify the percentage of plasmid harboring and plasmid free cells.

Using this system, the authors were also able to compare the stability of plasmid pKJK5 in the presence and absence of conjugation. In a culture initially containing 100% pMIB4, three days (and many generations) later more than 99.99% of the cells still contained the lacI plasmid even without selection for it. On the other hand, in bacteria that couldn’t conjugate (those containing pMIB8) only around 99.43% or 99.13% of the E. coli and Kluyvera sp., respectively, still were plasmid harboring in bacterial mats. As with similar experiments involving stability of conjugation-deficient bacteria conducted by Sia et al (1995), this suggests that conjugation plays a significant role in sustaining an IncP-1 plasmid in bacterial mats. But that’s not all. Not only can conjugation promote plasmid persistence in a population, but according to the authors it can also account for the infectious spread of plasmids throughout a mat population within three days, even when starting from only an initial 25% of the population containing the plasmid. Again, this is only true if conjugation is possible. With the pMIB8 plasmid the total plasmid-containing population actually decreased, likely due to segregational loss.

Whereas conjugation may compensate for segregational loss in high-density bacterial mats, the same cannot be said of lower density, well mixed liquid broth cultures. It appears that the percentage of plasmid containing cells decreased in populations harboring either pMIB4 or pMIB8, although the decline was less dramatic in those populations that could conjugate. So what does it matter if plasmid stability in bacterial mats differs from that in liquid media is different? Well, for one, other than the thermos of chicken soup that’s been rolling around in the back of your car for a week, bacterial populations in nature may not be accurately modeled by the perfectly mixed broth cultures common to most labs. This means that in general, we may be underestimating the role of conjugation in plasmid stability due to unrepresentative experimental systems.

There is a vast range of applications of studies in horizontal gene transfer in general. In some cases we may want to limit plasmid stability in populations such as in the fight against antibiotic resistant strains. In other cases, as with bioremediation, we may want to encourage plasmid stability so that plasmids that we introduce into bacteria allow the bacteria to do our clean-up work for us. In either case, we need a solid understanding of how, when, and under what conditions plasmids are more or less stable. That’s not to say that conjugation is the only important factor in stability. As mentioned above, and in the author’s paper, segregational loss, relative growth rates, and transfer frequency all contribute to overall plasmid stability. This article doesn’t discount the importance of these other factors, but rather emphasizes the need to respect conjugation as a major player that can, given the right conditions, act parasitically in its spread through a population, even when it doesn’t benefit the host bacterium. So maybe the bacteria aren’t as “responsible” for the process as we originally thought. Maybe the plasmids themselves are the ones with the real power after all.

This study also opens up a question for the philosophers of science out there (although it’s a question much too broad for one blog, so an answer won’t be attempted here). That question is one raised by Richard Dawkins, and pertains to the idea of the selfish gene. If plasmids behave parasitically, does that support the selfish gene idea? Could the results of this article be applied to an argument that the population isn’t always the level that we should think about when considering evolution, if it’s the plasmids and not their host bacteria that run the show? Maybe, maybe not, but it’s a fun debate either way, and something to think about.


Bahl MI, Sørensen SJ &Hansen HL (2004) Impact of conjugal transfer on the stability of IncP-1plasmid pKJK5 in bacterial populations. FEMS Microbiol Lett 232:45-49.

Bergstrom CT, Lipsitch M & Levin BR (2000) Natural selection, infectious transfer and the existence conditions for bacterial plasmids. Genetics 155: 1505–1519.

Haase J & Lanka E (1997) A specific protease encoded by the conjugative DNA transfer systems of IncP and Ti plasmids is essential for pilus synthesis. J Bacteriol 179.

Sia EA, Roberts RC, Easter C, Helinski DR & Figurski DH (1995) Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2. J Bacteriol 177: 2789–2797. 5728–5735.

Julie Hughes, Ph.D. student
Department of Biological Sciences, University of Idaho

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