Topics for MSc students

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.

Applied microbial ecology

1) Microbial degradation of sub-MIC (minimum inhibitory concentration) of antibiotics

Antibiotics have been naturally produced by bacteria and fungi for millions of years. However, during the last 70 years or so, humans have produced and used large amounts of antimicrobial drugs for both clinical, veterinary and agricultural purposes. Many of those antibiotics end up in the environment at low concentrations (high ng/L to low µg/L), where they are suspected to lead to adverse effects on wild-life and to selective conditions for appearance of antibiotic resistance. However, antibiotics might also be used a carbon and energy source by other bacteria, which can inactivate them. Up to this date, microbial degradation of antibiotic compounds at low environmentally relevant concentration is unknown. In particular, the question of whether the permanent presence of antibiotics in the environment is the consequence of poor degradation or of being too toxic remains unresolved.

The aim of the project is to study degradation of a select set of antibiotics under low concentrations and to isolate bacteria which can use such antibiotics as sole carbon and energy sources.

2) Understanding bacterial degradation of oxybenzone

Oxybenzone is a substance frequently added to suncreams that ends up in low concentrations in aquatic systems. The environmental fate of oxybenzone is poorly understood. Previously, by enrichment techniques, our laboratory has isolated a number of bacteria from Lake Geneva which seem to use oxybenzone as sole carbon and energy substrate for growth. Since the metabolic pathway for oxybenzone degradation is unknown we would like to find out which genes may be implicated in oxybenzone metabolism. For this purpose, we will use transposon mutagenesis and screening.

The aim of the project is to investigate the nature of the oxybenzone metabolic pathway by isolating and characterizing mutants which are unable to use the compound as growth substrate.

3) Characterizing the soil interactome

Bacteria in nature are mostly found in complex communities of multiple species rather than as single individual species. How such communities form and are maintained as a function of environmental parameters and species-species interactions is still poorly understood. Soils are a typical example of a complex environment where thousands of prokaryotic (and other) species live together. Soils have crucial ecosystem functions but, unfortunately, many soils are contaminated through human activities and lose essential functional aspects. One of the ideas to restore functionalities to deteriorated soils is to inoculate with one or more specific microorganisms, which can, for example, help to remove contamination, after which the soil community can re-equilibrate itself. Simple as it seems, however, inoculation of individual species mostly does not lead to their establishment in the soil and them expressing their intended functional characteristics. One of the reasons for absence of successful establishment may be unfavorable interactions with members of the existing community. This project will focus on a new methodology to measure multiple interactions of newly inoculated bacterial species and resident soil bacteria, in order to judge the possible success of their establishment and functional activity. As inoculant we will focus on a bacterium named Pseudomonas veronii which was isolated from an oil-contaminated site in Czech Republic, which is inoculated with the purpose of increasing biodegradation rates of frequently spilled solvents.

The aim of the project is to assess success of establishment of a bacterial inoculant in soil from the types of growth interactions with resident bacteria.

Development of whole-cell biosensors


1) Engineering bacterial chemoreceptors for toluene

Motile bacteria use chemotaxis to sense their environment and swim in the direction of certain chemical compounds (attractants) or away from others (repellants). Chemotaxis starts with the interaction of an attractant or repellant molecule (the ligand) with membrane-located receptor proteins, named methyl-accepting chemotaxis proteins (MCP). Ligand-binding induces a bias in the flagellar motor to favor straight swimming, moving the cell toward higher attractant concentrations (or away from repellants).

Our lab is interested to exploit bacterial chemotactic behavior to engineer biosensors that can rapidly detect and report the presence of specific chemicals. We would like to exploit the natural variability of MCPs to bind chemical ligands and place these in a standardized sensor-"chassis", a bacterial cell for which we can measure chemotactic swimming or intercept the chemotaxis signaling pathway. Such as chassis could be Escherichia coli or Pseudomonas putida.

Escherichia coli possesses five MCPs (Tar, Tsr, Trg, Tap and Aer) that bind mostly amino acids, peptides and sugars. Soil bacteria, such as Pseudomonas putida, carry other MCPs, many of which with unknown ligand-binding properties. Interestingly, P. putida DOT-T1E, a toluene-degrading bacterium possesses a chemoreceptor called McpT that enables chemotaxis towards toluene and other aromatic compounds (Lacal et al., 2011). Heterologous expression of MCPs can damage the cell but we recently managed to stably clone the mcpT gene from P. putida MT53 (a relative to DOT-T1E) under control of the trg promoter in E. coli. This is the starting basis for the project.

The aim of the project is to engineer chemoreceptors for toluene and to study its usefulness as rapid toluene biosensor.




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