The long term goals of this project are to achieve detailed understanding of the in vivo function of small, acid-soluble spore proteins (SASP) of Bacillus subtilis, and the regulation of the genes (ssp genes) which code for SASP as well as other co-regulated genes.
Specific aims are 1) with genes coding for major SASP determine the regions required for the initiation of their transcription in sporulation, by constructing upstream deletion mutations and analyzing their transcription in vivo and in vitro with oG RNA polymerase; 2) Test the specificity of oG RNA polymerase by constructing mutations in the -10 and -35 regions of ssp genes, and analyzing the transcription of these mutant genes in vivo and in vitro; 3) Determine those parts of major B subtilis SASP, ssp gene mRNAs or ssp genes which are involved in the feedback regulation of SASP synthesis, by analyzing the feedback regulation of ssp genes with various mutations, as well as different ssp-lacZ fusions. 4) Investigate the mechanism whereby oG synthesis is regulated by using an anti oG antibody to monitor oG synthesis during sporulation and by measuring the oG mRNA level and transcription start site(s) during sporulation of various B.subtilis strains; 5) Determine if the spore protease gene is part of an operon, and study the regulation of this gene (or operon) by measuring its in vivo transcription start site and expression in various asporogenous mutants, and its transcription in vitro with oG RNA polymerase; 6) Determine the supercoiling of various plasmids in cells and spores of various B.subtilis strains as well as the kinetics of change in these values during germination and sporulation. 7) In E.coli measure the supercoiling of plasmids carrying spp genes which can be expressed from an inducible promoter; 8) Determine the effect(s) of purified major and minor SASP on the supercoiling of DNA in vitro; 9) Determine the effects of various degrees of plasmid supercoiling on the expression of various forespore specific genes including ssp genes; 10) Continue studies on the killing of E.coli by induction of synthesis of high levels of oB type SASP; 11) Purify and characterize the minor SASP from B.subtilis spores, in particular those which are not antigenically related to major SASP, and attempt to correlate these proteins with genes and functions; 12) Test the possibility that the extreme conservation of the amino acid sequences of a/b type SASP is due to the interference of even a minor mutant a/b type SASP with the changes in DNA structure needed to give spore UV resistance; 13) Clone and sequence several a/b type ssp genes from Clostridial species.
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