Bacillus pumilus mutants were generated by targeted deletion of a set of genes eventually facilitating genetic handling and assuring biological containment. The well-defined and stable mutants do not form functional endospores due to the deletion of yqfD, an essential sporulation gene; they are affected in DNA repair, as Delta uvrBA rendered them UV hypersensitive and, thus, biologically contained; they are deficient for the uracil phosphoribosyl-transferase (Delta upp), allowing for 5-fluorouracil-based counterselection facilitating rapid allelic exchanges; and they are readily transformable due to the deletion of the restrictase encoding locus (Delta hsdR) of a type I restriction modification system. Vegetative growth as well as extracellular enzyme production and secretion are in no case affected. The combination of such gene deletions allows for development of B. pumilus strains suited for industrial use and further improvements.