Mutational analysis of the nucleoid-associated protein HBsu of Bacillus subtilis

The essential nucleoid-associated protein HBsu of Bacillus subtilis compris es 92 residues, 20% of which are basic amino acids. To investigate the role of the residues located within the DNA-binding arm, the arginine residues R58 and R61 were changed to leucine, while lysine residues K80 and K86 were replaced by alanine. All altered proteins exhibited a reduction in DNA bin ding capacity, ranging from 10% to 30% of HBsu wild type DNA-binding abilit y. To investigate the physiological effect of these mutations in B. subtili s, the indigenous hbs gene was replaced by the mutated genes. B. subtilis s train PK20, which carries the HBsu mutation R58L which exhibits the lowest DNA binding ability in vitro, showed the strongest retardation of growth co mpared to the wild type. Furthermore, PK20 cells displayed an increased rat e of cell lysis, diminished sporulation efficiency and a reduced level of n egatively supercoiled DNA. These observations suggest that the DNA binding ability of HBsu DNA is important for growth and differentiation and influen ces DNA topology.