Role of Motility and its Regulation in Escherichia coli Biofilm formation

Biofilms are multicellular communities of bacterial cells that usually grow on surfaces. Bacteria in biofilms are non-motile and are surrounded by a complex meshwork containing proteins, polysaccharides and DNA, the so-called biofilm matrix. The matrix provides stability and rigidity to the biofilm and is important for sticking cells to the surface and towards each other. The transition from the motile planktonic growth state to the sessile biofilm state requires therefore tight regulation of motility and matrix synthesis. In this work, I have investigated the roles and regulation of motility during biofilm formation of Escherichia coli. I could show that flagella play roles at different stages of biofilm formation, providing swimming motility at biofilm initiation and structural integrity in mature biofilms. Swimming motility is required for initial cell attachment to the surface and helps to shape mature biofilm structures. My results show that during biofilm initiation, smooth swimming at the surface leads to hydrodynamic entrapment, thereby promoting cell attachment. Additionally, swimming speed seems to influence surface attachment with enhanced swimming speed increasing attachment. Swimming speed is regulated by the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), which is a key factor in the transition from motility to sessility. C-di-GMP on the one hand inhibits motility via the motor binding protein YcgR and on the other hand promotes the synthesis of biofilm matrix factors. With the requirement of swimming for cell attachment, I could show that c-di-GMP plays a dual role during the course of biofilm formation, inhibiting biofilm initiation through motility inhibition and promoting biofilm maturation through upregulation of adhesion factors. Cells with permanently decreased c-di-GMP levels show increased cell attachment, however, have defects in biofilm maturation and architecture. In contrast, cells with permanently increased c-di-GMP levels show decreased cell attachment, but are able to form elaborate three-dimensional structures in mature biofilms. The effect on attachment is mediated by the motor binding protein YcgR, which regulates swimming speed. Based on the above described results, I could characterize a new regulator of c-di-GMP signaling in Escherichia coli. My results suggest that the bacterial dynamin YjdA, together with the small protein YjcZ, regulates c-di-GMP production by the diguanylate cyclase YegE. This is represented on the phenotypic level by changes in swimming motility, cell attachment and biofilm maturation in the yjdA deletion. It thereby phenotypically copies the deletion of the cyclase YegE. Additionally, the c-di-GMP dependent interaction of YcgR with the flagellar motor is decreased similarly in both yjdA and yegE deletion strains. Last but not least, bacterial two-hybrid experiments show an interaction between YjdA and YegE. My results further suggest the aid of the bacterial flotillins HflK/C in the regulation of YegE by the dynamin YjdA. Altogether, the results in this thesis provide an overview of the roles of flagella and motility during biofilm formation of Escherichia coli, which includes a requirement of swimming during biofilm initiation, thereby tight regulation of c-di-GMP during the course of biofilm formation, and a structural role of flagella at the later biofilm stage