Monitoring the Dissemination of the Broad-Host-Range Plasmid pB10 in Sediment Microcosms by Quantitative PCR

Sebastien Bonot and Christophe Merlin
Applied and Environmental Microbiology Vol. 76 No. 1 2010

There have been many discoveries about the process of horizontal gene transfer and our understanding of this subject has increased greatly in recent years. However, the work that can be done on this process is hindered by limitations inherent in laboratory settings. The results of current experiments are difficult to apply to real-world situations with diverse microbial communities. For example, it is believed that we are only able to cultivate approximately 1% of environmental bacteria under laboratory conditions. Also, genetic information transferred under laboratory conditions may show narrow host expression. Quantifications using culture-based techniques may lead to an underrepresentation of the extent of gene transfer that has occurred, especially in complex environments. Recent work involving green fluorescent protein allows us to see a more realistic representation of the gene transfer that would occur in these complex environments. However, the authors suggest that these studies are still not completely representative of natural environments because the elements under study still require genetic manipulation. PCR and other molecular techniques can get around these problems, but are not typically used due to the fact that so many DNA markers are shared among different genomes. In this paper the authors show that with carefully designed, specific primers they were able to use quantitative PCR (qPCR) to monitor the dissemination of the broad-host-range plasmid pB10 in sediment microcosms. E. coli DH5α was chosen to be the donor strain because it had poor chances of survival since the sediment microcosms under study were not its natural habitat. Also, the high numbers of genetic alterations that this lab strain has undergone were likely to further reduce the donor’s chances of survival.

To follow the fate of plasmid pB10 with qPCR the researchers needed to develop primers specific to the plasmid, as well as to the donor bacteria, E. coli DH5α. The researchers were unable to develop traditional primers specific to pB10 due to the fact that the plasmid has many similarities to other genetic elements. The researchers came up with a very interesting solution to this problem. They used the fact that bacterial genomes have a unique structure based on a combination of DNA blocks instead of just specific DNA sequences. The researchers developed unique primers for pB10 that prime on both sides of a junction of these building blocks. They used the same technique to design primers for DH5α. qPCR tests were performed on total environmental DNA to determine whether or not these primers were truly specific. Amplification was only achieved when DNA was present from pB10 or DH5α.

Tests were run to determine the ability of qPCR to quantify DNA from pB10 and DH5α when in complex environments. Samples were inoculated with known amounts of DH5α/pB10, followed by immediate total DNA extraction and qPCR quantification. 20% of the expected pB10 DNA was recovered, and 0.25% of DH5α DNA was recovered. The researchers postulate that this discrepancy occurred because plasmid DNA is much easier to recover than chromosomal DNA. They also determined that these results mean they must analyze qPCR results based on appearance/disappearance of DNA instead of absolute quantities.

The researchers then performed an experiment to monitor the fate of pB10 in sediment microcosms. The microcosms used were blended river and sediment samples. These were inoculated with pB10/DH5α. Microcosms were maintained for 5 days and total community DNA was sampled at intervals. The researchers found that pB10 concentrations remained stable, while DH5α DNA was lost completely after 48 hours. This suggests that pB10 invaded the microcosm relatively early, before DH5α was lost. Controls that were inoculated with naked pB10 DNA showed a rapid loss of plasmid DNA that disappeared completely after 48 hours. This shows that the plasmid could not have been in the environment extracellularly during the experiment.

This experiment is noteworthy because it monitors the dissemination of a broad-host-range plasmid in a complex environment using molecular tools. I found the use of DNA block primers over conventional sequence primers quite interesting. Perhaps this will lead to new methods of studying horizontal gene transfer in complex environments. The use of qPCR to do this would allow researchers to study horizontal gene transfer in these environments with minimal disturbances to the microbial communities. The researchers plan to expand on their current work by including factors such as spatial structure in future analyses.

Ryan Simmons
University of Idaho