|Budget Amount *help
¥4,600,000 (Direct Cost : ¥4,600,000)
Fiscal Year 1995 : ¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1994 : ¥2,200,000 (Direct Cost : ¥2,200,000)
The production of Bacillus subtilis exoprotease are subject to regulation by a two-component regulatory system, DegS-DegU.In addition so-called accessory factors such as degR also function in this process. These factors work before DegS/U and seem to play a role in sensing environmental conditions. This study was undertaken to clarify through degR regulation the flow of signals that allow Bacillus subtilis cells to produce exoproteases. Following results were obtained.
1. Characterization of the proB gene that enhances the positive regulatory function of DegR.i) During the course of screening for a gene that enhances the production of the exoproteases in the presence of multicopy degR,we obtained the proB gene and mapped it at 1,373 kb on the physical map of B,subtilis. ii) ProB required DegS for its enhanceing effect, indicating that the ProB function is through the DegS/DegU signalling system. iii) Multicopy proB showed an inhibitory effect on degR expression.
2. Regulatory mechanism o
f degR expression. i) We determined the transcription start point by a primer extension analysis. Transcription of degR was found to be inhibited in the presence of multicopy proB.ii) When sigD,the structural gene for sigma^D, was disrupted, the expression of degR was aboished. iii) The inhibitory effect of ProB was dependent on DegS.iv) ProB inhibited the expression of hag that is also dependent on the sigma^D factor. v) Inhibition of degR expression by ProB was no more seen when the -10 sequence of the degR promoter was changed from a sigma^D type to a sigma^A type promoter. vi) ProB,however, did not affect the expression of sigD.
From these results, we conclude that ProB inhibits the function of sigma^D but not the synthesis of it, resulting in inhibition of sigma^D-dependent degR expression. ProB is an enzyme that functions in the proline biosynthesis, and therfore, the fact that it regulates degR expression suggests an involvement of a cell's nutritional condition in degR expression, leading to the production of the exoproteases. Less