Isolation und Charakterisierung der sporulationsspezifischen Protease LonB von Bacillus subtilis

The use of proteases for the regulation of physiological and regulatory processes in Microorganisms is a widely distributed phenomenon. Most of all proteolytic breakdown is dependent on the energetic status of the cell. The genome of Bacillus subtilis codes for two members of the family of ATP-dependent Serine proteases.The synthesis of the first one,which is LonA, increases in response to heat stress, ethanol, osmotic and oxidative stress and after treatment with puromycin. Furthermore a role in the negative regulation of SigA-activity under non sporulation inducing conditions is suspected. The gen of the second protease, lonB, is located immediately upstream of lonA. This study shows that transcription of lonB is controlled by the first forespore specific sigma factor, SigF, and that it?s expression is restricted to that compartment. DNA-array analysis of cells after artificial induction of sigF expression revealed a significant increase of the amount of lonB transcript and Northern analysis showed a monocystronic transcript. Flourescence of strains carrying a lonb-GFP fusion was exclusively detected in the forespore and depended on the presence of the transcription factor SigF. Transcription was initiated at a specific start point that was identified by primer extension analysis and that is located downstream of sequences similar to known sigF dependent sequences. Both LonB and LonA were proven to be present in crude cell extracts using specific polyclonal antibodies. LonB appeared 90 min after onset of sporulation, whereas LonA could be found allready during vegetative growth. In vitro both peptidase and ATP-dependent protease activity could be demonstrated for both enzymes, and an associated ATPase activity is very likely. Inactivation of lonA and lonB by insertion showed no effect on the phenotype of vegetative and sporulating cells, and transcription of genes controlled by the sporulation sigma factors SigF, SigG and SigK was not affected in a lonB-mutant. Comparative 2D-gel analysis of wildtype and lon-mutants lead to the assumption that LonB may influence the ammount of the mothercell specific serin protein kinase PrkA, but as LonB and PrkA are restricted to different compartments the effect may be indirect.