Looking for a diploma topic in (micro)biology?
If you're a (micro)biology student interested in environmental aspects but with a good deal of molecular biology, we might be able to offer you a few exciting topics. If you are interested, please come and discuss with us. Contact: Jan Roelof van der Meer.
Evolution of Catabolic Pathways
ICEclc is a conjugative DNA element that is normally integrated in the chromosome of its host bacterium Pseudomonas knackmussii. Transfer is only initiated in a few percent of cells during stationary phase, which we have called 'transfer competent' cells. The formation of transfer competence is thought to be the consequence of a 'bistable' decision (i.e., cells in a population follow two different processes: active transfer or no transfer at all). Cells that follow the transfer process express several promoters on ICEclc simultaneously, whereas those promoters are silent in other cells. Interestingly, these promoters are also silent in other Pseudomonas species that do not have ICEclc. The main goal of the project is to better understand how bistability is generated and, in particular, to find possible sequence features in bistable promoters that may determine bistability.
1) Visualization of a conjugative machinery involved in horizontal gene transfer
Horizontal gene transfer is one of the most fascinating evolutionary processes among prokaryotes, by which genes can be exchanged between species. Rapid adaptation of bacteria, to for example, antibiotic resistance or toxic compound metabolism, are thought to be a major consequence of horizontal gene transfer and selection. Despite the fact that a lot is known on the molecular aspects and the outcomes of horizontal gene transfer, there is very little understanding of the dynamic process itself and how it affects cells which act as the DNA donors. Here we propose to develop a system which may help to visualize DNA donating cells by making a hybrid fluorescent-protein-fusion to a component of the conjugative machinery called VirB4..
The aim of the project is to construct a hybrid fluorescent-protein-VirB4 fusion in Pseudomonas and follow location of the machinery in individual DNA transferring cells.
2) Molecular characterization of the DNA binding properties of a bacterial transcription regulator
Bacterial transcription factors often exert their action by binding to specific regions on the DNA up- or downstream of promoters. DNA binding is therefore a clearcut property to define the role of suspected transcription regulators and their target gene(s).
This project is part of a larger study which focuses on understanding the behavior of a mobile DNA element named ICEclc in Pseudomonas. ICEclc is a curious mobile DNA, which is integrated into the genome of its host, but which can excise in a small proportion of cells during stationary phase. Excised ICEclc can transfer to new recipient cells, where it again integrates. One of the key questions at this point in the larger project is the mode of regulation that decides to "activate" ICEclc excision in some cells but not in (most) others. Previously, we have identified a cluster of three regulatory genes named mfsR-marR-tciR, whose expression is controlled by MfsR itself. Genetic studies further showed that it is likely the TciR protein, which then activates one or more promoters in the ICE that leads to excision. TciR is member of the class of so-called LysR transcription regulators. In order to demonstrate its regulatory role more directly, we would like to study the binding properties of TciR to the DNA of its (suspected) target promoter(s).
The aim of the project is to purify the TciR protein and to study its binding to a set of suspected target promoters from ICEclc.
3) Escaping the bad host? What triggers transfer of a bacterial mobile DNA
Horizontal gene transfer through mobile DNA elements plays a crucial role in shaping genomes and is a key player in bacterial evolution and adaptation. Intriguingly, experimental evidence suggests that mobile DNA elements "react" to the state of the host cell and that cellular damage may trigger subsequent gene transfer.
Our group has been studying a mobile DNA in Pseudomonas named ICEclc, which is normally integrated and silent in the host chromosome, but becomes active in 3-5% of individual cells in stationary phase. Activation starts a cascade of gene expression leading to ICEclc excision and finally conjugative transfer. Interestingly, cells which have been grown on a toxic chlorinated aromatic compound have higher activation rates. We have recently demonstrated that cells in which ICEclc becomes activated show more Reactive Oxygen Species (ROS) than other cells in which ICEclc remains silent. In addition, cells with activated ICEclc are those with on average higher levels of the RpoS sigma factor.
Several hypotheses may explain the ICEclc activation process. In the “bad host hypothesis”, cells which somehow or by chance accumulate ROS are more prone to elicit the ICEclc activation and transfer cascade. ROS may directly trigger ICEclc activation or indirectly may lead to higher levels of the starvation factor RpoS in cells, which then have a higher chance to become transfer proficient. In the alternative hypothesis, all cells are the same and ICEclc activates totally random, independent of any pre-discernable biochemical markers.
The aim of the project is to test the bad host hypothesis and to demonstrate or refute a link between specific biochemical markers appearing in individual cells, and activation of the the ICE. We will first pursue the idea of ROS damage leading directly or indirectly via RpoS to cells activating ICEclc.