| dc.description.abstract | Biofilms are communities of microorganisms establishing on surfaces like minerals, air-liquid interfaces or living material like plants or humans. Bacteria living within biofilms are protected from environmental factors like mechanical stress, antimicrobial compounds and constituents of the human immune system and may thus cause serious problems in for example medicine and in the food industry. Bis-(3’-5’)-cyclic dimeric guanosine monophosphate, also known as cyclic di-GMP (c-di-GMP), is a second messenger shown to play a central role in controlling the transition from a motile, planktonic to a sedentary biofilm-associated lifestyle in various bacteria, mostly characterized in Gram-negative species. Increased levels of c-di-GMP have been found to stimulate formation of biofilms and reduce motility. c-di-GMP is synthesized by diguanylate cyclase (DGC) enzymes containing GGDEF domains, and degraded by phosphodiesterase (PDE) enzymes containing EAL domains. Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are members of the B. cereus group of Gram-positive, rod-shaped spore forming bacteria and have shown to be of great economical and medical importance. Their chromosomes exhibit a close similarity and genes encoding species-specific virulence factors are mostly plasmid-borne. While B. anthracis is the cause of anthrax, an acute and possible lethal disease in mammals, B. cereus is an important cause of food poisoning in humans, and B. thuringiensis a commercially utilized insect pathogen. B. thuringiensis 407 (Bt407) is a model strain used for studying the genetics of B. thuringiensis. The genome of Bt407 carries ten genes predicted to encode proteins with GGDEF and/or EAL domains. In this thesis, three of these proteins were functionally characterized: Cdg135 (containing a GGDEF and an EAL domain, both with conserved active sites required for enzymatic activity), Cdg141 (containing a GGDEF domain predicted to lack DGC activity) and Cdg113 (containing a predicted enzymatically inactive EAL domain). Results from the current thesis indicate that Cdg135 is probably involved in controlling biofilm formation and motility in Bt407, possibly by exhibiting DGC activity and catalyzing the synthesis of c-di-GMP. Cdg135 was shown to positively affect the transcription of a gene encoding a predicted cell wall bound adhesion protein. This gene, an ortholog to the B. cereus ATCC 14579 gene with locus tag BC_1060, referred to as bspA (Bacillus surface protein A) in the current thesis, is located downstream of a c-di-GMP-sensitive “on-riboswitch”, and the corresponding BspA gene product was shown to stimulate biofilm formation in Bt407. The effects of BspA are, however, not essential for the ability of Bt407 to form biofilms, as Cdg135 over-expression could compensate for bspA loss. Cdg141 did not seem to influence biofilm formation in Bt407, but excessive amounts of Cdg141 in the cell probably results in toxic effects, as the over-expression strain was deficient in growth. Results from biofilm assays indicated that Cdg113 has positive effects on biofilm formation in Bt407. As Cdg113 is predicted to lack DGC activity, it could function as an effector molecule, regulating biofilm formation upon binding c-di-GMP. | eng |