Research objectives and content
The transport activities play an important role in the excretion of toxic compounds in Lactococcus lactis. Three kinds of transporters take place in this process. Two systems are involved in the excretion of cationic compounds (whose mechanisms have already been cloned and sequenced). One of them is very homologous to a multidrug transporter of human cells and play a crucial role in the resistance of carcinoma cells against anticarcinogenic agents. The third system excretes anionic compounds and also appears to excrete bile salts and to play an essential role in the colonization of Lactic Acid Bacteria (LAB) in the intestinal track. This system (BCECF) has functional homology to a multidrug transporter in liver cells.
The main objective of the project is the characterization of the BCECF transporter. Mutants have been isolated which lack the BCECF transporter or have the transporter over-expressed. By means of crossed
immonoelectrophoresis the BCECF transporter will be identified and purified. Aminoacid sequencing of the N-terminus will allow the design of nucleotide primers which can be used to identify the gene(s) encoding for this transporter. Subsequent studies will lead to the characterization of the gene(s) and the (over)expression of the transporter in Escherichia coli. In isolated inside-out membrane vesicles of these strains, the functional properties will be further characterized. Constructs will be made in which the transporter is put behind a His-tag to allow rapid purification over a high-affinity column. The experimental tools to conduct these studies are available at the host laboratory.
Training content (objective, benefit and expected impact)
The main training content is concerned with the molecular biology of the BCECF transporter in Lactococcus lactis. In this way, I will become familiarized with i) crossed immunoelectrophoresis techniques, ii) procedures of protein and DNA cloning and sequencing and gene expression in homo- and heterologous hosts, iii) methodology to characterize the functional properties of the cell membrane transporters of Gram positive bacteria.
Links with industry / industrial relevance (22)
BCECF transporter system is essential for the colonization of the intestinal track by Lactic Acid Bacteria. Furthermore, Prof. Konings is Scientific Advisor of three industrial companies and his group's work has led to several patents in the last few years. Finally, a close relationship also exists between Prof. Konings and the LABIP, the Industrial Platform associated to the European Commission LAB projects (BRIDGE T-Project and the BIOTECH G- Project).