Bacterial bioreporters and pollutant effects
Whole cell bacterial bioreporters are bacteria specifically engineered to react to the presence of chemical signals with the production of an easily quantifiable marker protein. In most cases, an existing regulatory system in the bacterial cell is exploited to drive expression of a specific reporter gene, such as bacterial luciferase, green fluorescent protein, beta-galactosidase or others. This is achieved by fusing the DNA for a promoterless reporter gene to an extra copy of the selected regulatable promoter and introducing this construction into the bacterial cell. Regulatory systems that have been applied include those for heavy metal resistancies (to obtain heavy metal responsive sensors), for organic compound degradation (to obtain organic compound sensors), and for cellular stress responses (to obtain general toxicity sensors).
Most whole-cell bacterial bioreporters are applied by incubating the cells in aqueous solution with the target compound(s) and analyzing the activity of the reporter protein (i.e., the reporter signal) after a previously calibrated induction period. Concentrations of the target chemical in unknown samples are inferred by comparing the reporter signal to that with a series of standard concentrations and incubated under exactly the same conditions. The reporter signal is usually only proportional to the target chemical within a specific concentration range. At higher target concentrations, the sensor output becomes saturated or even diminishes because of toxic effects to the sensor cells.
In principle it is also possible to have bacterial bioreporters measuring volatile compounds in the gas phase. Bioreporters expressing the green fluorescent protein can be used to measure the bioavailability of target chemicals to single cells. Current projects in our group focus on phenanthrene bioavailability, on development of new protocols for working with arsenic biosensors, on creating multisensor platforms and on mutagenizing regulatory proteins to obtain more and other effector recognition specificities.
Within the framework of the European project BACSIN, we are investigating the usefulness of applying specific bacterial strains to remediate toxic compounds. Here we study the bacteria Sphingomonas wittichii (which degrades dibenzofuran), Arthrobacter chlorophenolicus and Alcanivorax borkumensis. With the help of transcriptomic and genetic studies we are trying to unravel which stress pathways become activated when such bacteria are re-implanted in a contaminated environment, and how this affects their capability to degrade the target chemicals.
In another project we are trying to understand the effects that pollutants and low concentrations may have on aquatic microbial communities. We have studied the effects of fungicides and biocides on bacterial communities in Lake Geneva, and are now developing more sensitive detection techniques for pollutant stress, inhibition of bacterial reproduction and changes in microbial physiology. We also study how we can detect growth of bacteria at very low pollutant concentrations using flow cytometry and ultra high throughput microcultivation plates.
One central question in many of the application studies is the detection of the inoculated strain and the possible changes in the original microbial community. For this, we are applying various PCR-based techniques, among which terminal restriction fragment length polymorphisms (T-RFLP). Microbial diversity techniques are also used in a project which focuses on identifying and characterizing soil bacteria which may interact with hyphae of arbuscular mycorrhizal fungae. This project was carried out in collaboration with Ian Sanders of the Department of Ecology and Evolution.
BIOMONAR: Development of new aquatic sensors ...>>
Arsenic reporter cells embedded in agarose microbeads within a microfluidics cage. Picture: Nina Buffi, EPFL.
Subsection of a 20 µm well ultrahigh throughput cultivation disk.