Análisis del patrón espaciotemporal de flotilinas bacterianas

The structure of cellular membranes is not homogeneous, in fact it is compartmentalised. When it comes to bacterial membranes, the existence of certain zones with differential lipid composition called Functional Membrane Microdomains (FMMs) has been described. The most characteristic type of protein found in these FMMs is flotillin, a transmembrane protein that is capable of oligomerizing, interacting with other cellular proteins to favour complex formation and is key in maintaining the architecture of FMMs. Flotillins are present in almost all bacteria including Bacillus subtilis, where their coding sequences are found in operons alongside two other genes. The aim of this report is to study the spatio-temporal production patterns of the two flotillins found in Bacillus subtilis, FloA and FloT. FloA has been described to be constitutive and organize into small-sized and numerous foci whereas FloT is mostly found during stationary phase grouped into bigger and fewer foci. To achieve the stated goal, new B. subtilis strains have been developed by integrating both flotillin genes, labelled with gfp, alongside their native promoters in different combinations. For the development of these strains two genetic backgrounds have been considered, B. subtilis 168 and B. subtilis 168 double deletion mutant for both the operon of floA and floT. To the determine the production pattern of both flotillin-GFP fusion proteins, Western blot analysis at different growth stages were performed, as well as fluorescence microscopy imaging of cultures at stationary phase. From this work it can be concluded that the timing of flotillin production is dependent on the native promoter and that flotillin stabilization is highly dependent on the production of the proteins coded by the other two genes found in the operon, especially for FloT. Finally, the microscope used for fluorescence imaging did not generate images with a high enough resolution to distinguish foci pattern formation in most of the strains.