Friday, July 31, 2009

Plasmid Conjugation in an Activated Sludge Microbial Community

By Ruoting Pei and Claudia K. Gunsch


Vol 26, No 4, 2009, p. 825

This is jet another paper describing plasmid transfer and their role in so called bioaugmentation in natural kind of environments.

Bioaugmentation involves the addition of exogenous micro-organisms that have the ability of degrading the compound of interest. Using this process, new biodegradation pathways can be added, which inherently improve the metabolic conversion of the contaminants. The main problem of this method is that the laboratory strains used to introduce new genetic traits usually cannot grow in natural environmental conditions. This means that bioaugmentation can fail due to the poor establishment and=or survival of new strains under field environmental conditions. The second process used for enhancing natural biodegradation capabilities is biostimulation which consists of adding nutrients (e.g., carbon, nitrogen, electron acceptor, etc.) to promote the growth of indigenous microorganisms. This method is preferred over bioaugmentation because no additional bacterial strains are introduced but it requires the presence of indigenous microorganisms, which are capable of breaking down the contaminant of interest. To overcome the limitations associated with biostimulation and bioaugmentation, it might be possible to combine these two strategies into a technique called genetic bioaugmentation. This technique would consist of adding bacteria with the needed genes and inducing their horizontal gene transfer (HGT) to indigenous bacterial species utilizing natural prokaryotic adaptation mechanisms.

In this paper authors described pWWO plasmid transfer from Pseudomonas putida strain to bacterial population present in activated sludge. The pWWO plasmid is well known for its toluene degradation capability. The plasmid DNA was tagged with transposone containing gfp gene driven by lac promoter. In the donor strain which overproduce LacI repressor gfp is not expressed. In other bacterial hosts gfp is expressed and gives bright green fluorescence which can be measured. Using flow cytometry authors showed that plasmid was actually transferred into bacterial community of activated sludge with number of bacteria expressing GFP protein up to 6%. They showed that the highest number of transconjugants is reached at the third day of experiment and it depends on the donor to recipient ratio with the best results at the 1:20 ratio. Authors tried to connect the plasmid transfer with the actual capability of activated sludge to toluene degradation. This experiment did not really work as the level of toluene degradation remained unchanged during the time of the experiment.

The paper stays in a main stream of microbial ecology and engineering facing the problem of global pollutions and waists utilization. It is very important to use new methods for detecting plasmid transfer in natural environment. Authors were facing the same problem as in previously published papers that there are some limitations in using fluorescent proteins as a plasmid infection marker. The true plasmid bearing cells are not known until gfp gene is expressed and protein is properly matured to give strong fluorescence, which can be detected by flow cytometry. Authors cannot also link the presence of TOL phenotype (toluene degradation) with the number of plasmid bearing cells as the sludge microbial community shows already high toluene degradation activity.

Perhaps, it is not a very “big” paper, but it is a small step toward understanding how the plasmid transfer occurs in natural environment and how can we use this to save our polluted world.

Jaroslaw Krol, PhD


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