Alpha-amylase synthesis in Bacillus subtilis 168 is activated at the end of exponential growth, is apparently not inducible, and is repressed by more-readily-metabolized carbon sources, such as glucose. The mechanisms of Alpha amylase regulation are currently unknown. We find that the activation and the repression of Alpha-amylase synthesis are distinct, genetically separable events. We have determined that a cloned copy of the B. subtilis 168 Alpha amylase gene contains the flanking DNA necessary for the regulation of its expression. We have localized the regulatory DNA sequences to the 5' end of the cloned Alpha-amylase gene. We propose experiments to further characterize at the molecular level the activation and repression of Alpha-amylase synthesis, and to define the flanking DNA sequences involved in Alpha-amylase regulation. Specifically, we propose: 1. Quantitation of Alpha-amylase mRNA levels, 2. Determination of the start site(s) of transcription for the Alpha-amylase gene, 3. Characterization of in vivo-generated cis-regulatory mutations, 4. Characterization of in vitro-generated regulatory mutations. 5. Isolation & localization of mutations which act in trans. For the latter, we propose a novel technique for the isolation of mutations in genes encoding factors which act upon the flanking regulatory DNA to modulate Alpha-amylase expression. Elucidation of the mechanism of Alpha-amylase regulation may provide insights into the regulatory mechanism governing the expression of other early postexponential genes in B. subtilis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM034324-01
Application #
3285100
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1984-12-01
Project End
1987-11-30
Budget Start
1984-12-01
Budget End
1985-11-30
Support Year
1
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Earth Sciences/Resources
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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Kim, J H; Voskuil, M I; Chambliss, G H (1998) NADP, corepressor for the Bacillus catabolite control protein CcpA. Proc Natl Acad Sci U S A 95:9590-5
Voskuil, M I; Chambliss, G H (1998) The -16 region of Bacillus subtilis and other gram-positive bacterial promoters. Nucleic Acids Res 26:3584-90
Kim, J H; Chambliss, G H (1997) Contacts between Bacillus subtilis catabolite regulatory protein CcpA and amyO target site. Nucleic Acids Res 25:3490-6
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Voskuil, M I; Voepel, K; Chambliss, G H (1995) The -16 region, a vital sequence for the utilization of a promoter in Bacillus subtilis and Escherichia coli. Mol Microbiol 17:271-9
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Weickert, M J; Chambliss, G H (1989) Genetic analysis of the promoter region of the Bacillus subtilis alpha-amylase gene. J Bacteriol 171:3656-66

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