Functional dissection of the large adhesion protein

There are numerous microorganisms which have the ability to switch between being in planktonic stateor form biofilms, these biofilms are complex communities of microorganisms attached to surfaces orassociated with interfaces. This concept has a lot of relevance in ecology due to the fact that thesemicrobial communities are often composed of multiple species that interact with each other and theirenvironment. The longest gene in the Gram-negative bacterium Pseudomonas putida genome encodesLapA, a >9000 amino-acid surface adhesin essential to surface adhesion and biofilm formation. LapA isa complex protein, containing numerous functional domains and a large array of repeated sequences.However, the exact function of any of these elements in LapA is unknown.The strategy of our project is the construction of different lapA variants containing internal deletions ofthe putative functional domains, and study the role of each of these in LapA-dependent phenotypes .Each version will be inserted in the chromosome of a Δ lapA mutant using a Tn 7 -based delivery system.An Initial synthetic construct containing the complete N-terminal and C-terminal domains and a 3xHAtag for immunodetection, but lacking all repeated sequences is already available, and constructsbearing progressively shorter N-terminal domains are underway. Addition of different numbers ofrepeats will be tested afterwards.Phenotypic assays will include swimming and adhesion assays using different surfaces, biofilmformation curves and microscopic assessment of biofilm morphology under different conditions. Weexpect that this approach will provide useful insight into the functions of the different domains of LapAand the dynamics of biofilm development in P. putida