Biofilm formation in Bacillus thuringiensis : Investigation of the roles of a putative cell surface adhesin and a chemotaxis-related protein responsive to cyclic-di-GMP

Field: Immunology and Microbiology (Life Sciences)

The Bacillus cereus group of bacteria is a subgroup in the Bacillus genus and consists of six different species. Research has evolved mainly around B. cereus, B. thuringiensis and B. anthracis, an opportunistic pathogen capable of food poisoning and infections in mammals, an insecticidal pathogen which can also be an opportunistic pathogen in mammals, and an opportunistic pathogen capable of causing cutaneous and or/systemic anthrax in mammals, respectively. These species are closely related based on analysis of chromosomal markers, but pathogenicity patterns vary mainly due to virulence factors carried on plasmids. Taxonomic differentiation has been, and continues to be, difficult, as many features used to classify the organisms are plasmid-borne.\nSeveral studies have shown that many strains in the B. cereus group are capable of producing biofilms, multicellular aggregates of cells that may contribute to virulence, antibiotic resistance and to reservoirs forming in food production facilities and hospitals. B. cereus, ubiquitously found as spores in soil, has been implicated in several cases of food spoilage. Research of biofilm formation in B. cereus is, therefore, important for economic and health- related purposes.\nOne research group reported in 2008 that two c-di-GMP responsive riboswitches, non- translated regions of mRNA located at the 5’-end that may govern further expression of a downstream gene, had been identified in Bacillus cereus strains ATCC 14579 and ATCC 10987. Sequence searches based on the sequences of these riboswitches showed that two similar riboswitches were present in B. thuringiensis 407, and two genes were located downstream of these riboswitches, whose proteins contained domains typical of cell surface adhesion (Bt407_1060) and chemotaxis (Bt407_0422) proteins, respectively. As c-di-GMP is an important regulator of biofilm formation in gram-negative bacteria, it was decided to proceed with functional studies of these two proteins. The aim of this thesis was to expand on initial research performed on this topic in the group.\nThe functional studies performed in this thesis confirmed that a gene deletion in Bt407_1060 (cell surface adhesion protein) resulted in reduced biofilm formation in a microtiter plate screening assay. Previous studies on adhesion proteins in gram-positive bacteria have shown \n\nan effect on biofilm formation when genes encoding the adhesion proteins were removed. No effect was observed on biofilm formation in a screening of the Bt407_0422 (methyl-accepting chemotaxis protein) gene deletion mutant compared to wildtype. Previous research in other bacteria has deemed chemotaxis as non-essential to biofilm formation, as opposed to the presence of flagella or motile behaviour, which seems of importance. However, in order to ratify these results, complementation and overexpression of the genes would be necessary. Future research should also focus on the role of c-di-GMP in the regulation of biofilm formation by B. cereus group bacteria to enhance our understanding of c-di-GMP regulated processes in gram-positive bacteria.