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The P2 promoter from Bacillus subtilis sigma-A (sigma A) operon and the strong phi 29 phage G3b promoter were used to study their interactions with free sigma A and with RNA polymerase holoenzymes (E sigma A and E sigma 70). No binding of free sigma A to the tested promoters was observed, suggesting that the B subtilis free sigma A does not bind promoter by itself for the initiation of RNA transcription. Different footprints of B subtilis RNA polymerase holoenzyme (E sigma A) on the P2 and G3b promoters were detected. The footprint on the P2 promoter is mainly in the -10 downstream region of the bottom strand (noncoding strand) DNA and limited on the top strand (coding strand), whereas the footprints on both strands of the G3b promoter are very clear. These results suggest that the footprint regions of RNA polymerase on a promoter and the strength of its binding to the promoter depend on the properties of the specific promoter DNA sequence. It also suggests that the -10 and its downstream regions are more important than the -35 region for the formation of the E sigma A-P2 promoter open complex. Footprints of B subtilis E sigma A and E coli E sigma 70 on the same G3b promoter are very similar on the top strand but different on the bottom strand, with the footprint being about 17 bases wider (-4 to +13) in the case of E coli E sigma 70. Since this region contains most of the bases involved in promoter DNA melting, we suggest that E coli and B subtilis RNA polymerases have different efficiency in forming the open complex with heterologous promoter DNA during initiation of transcription.

The Bacillus subtilis degSU two-gene operon encodes a two-component regulatory system which positively controls expression of a variety of genes encoding degradative enzymes. Point superactivator mutations in either degS or degU increase the production of these enzymes. A specialized transducing SP beta phage which partially complements several of these mutations was isolated from a phage library of the B. subtilis chromosome. This phage was shown to contain the degU (wild-type) gene. This unexpected co-dominance is discussed.

B.subtilis phage M2 uses a protein, instead of RNA, as the primer of its DNA replication. Hence this protein encoded in the phage genome is called as the primer protein (PP). At the initiation of DNA replication, a hetero dimer complex with its own DNA polymerase and the PP supposed to interact with the terminal protein (TP), which is covalently bound to the template DNA (TP-DNA). PP contained an important adhesive amino acid sequence, Arg-Gly-Asp (RGD), near the carboxyl terminal. We have recently showed that the synthetic RGD peptide inhibited the transfection of phage M2. By site-directed mutagenesis, we introduced different amino acid into the RGD site of PP. These altered PP decreased obviously the priming activity in vitro.

The Bacillus subtilis phage phi 29 DNA polymerase, involved in protein-primed viral DNA replication, contains several amino acid consensus sequences common to other eukaryotic-type DNA polymerases. Using site-directed mutagenesis, we have studied the functional significance of a C-terminal conserved region, represented by the Lys-X-Tyr ("K-Y") motif. Single point mutants have been constructed and the corresponding proteins have been overproduced and characterized. Measurements of the activity of the mutant proteins indicated that the invariant Lys and Tyr residues play a critical role in DNA polymerization. Interestingly, substitution of the invariant Lys either by Arg or Thr, produced enzymes with an increased or a largely reduced, respectively, capability to use a protein as primer, an intrinsic property of TP-priming DNA polymerases. On the other hand, the viral protein p6, which stimulates initiation of phi 29 DNA replication by formation of a nucleoprotein complex at both DNA replication origins, increased (about 5-fold) the insertion fidelity of phi 29 DNA polymerase during the formation of the TP-dAMP initiation complex. We propose a model in which the special strategy to maintain the integrity of the phi 29 DNA ends, by means of a "sliding-back" mechanism, could also contribute to increase the fidelity of phi 29 DNA replication.

The thyPIG 3 gene from Bacillus subtilis bacteriophage IG 3 was cloned in the plasmid pHV 33. Two recombinant plasmids, pISL 61 and pISL 62 carrying that gene are effective in transforming to thymine prototrophy both Escherichia coli (by complementation) and B. subtilis (by complementation and recombination). The comparison of cloned fragment containing the thyPIG 3 gene and the thyP 3 gene from phage phi 3 T, by restriction analysis and DNA hybridization, suggests a strong homology between the two. The thyPIG 3 gene was mapped in this study in the central region of the IG 3 genome.

Using a new method to get stable steady state levels of competence in B. subtilis, we have obtained in our experimental conditions 0.1% competence as determined by the frequency of transfection with phage SPP1 DNA. The effect of growth rate (DT) and certain amino acids on the competence levels was also determined.

Partially purified extracts from sporulating Bacillussubtilis cultures transcribed different natural DNAs with different efficiencies. This template specificity results in an increased or a decreased synthetic activity with respect to extracts from vegetative cells, depending on the template used. With SPP1 DNA a decrease in activity occurs, whereas with T7 DNA an increased activity was observed, which is due to a higher efficiency of initiation. This is not an intrinsic property of RNA polymerase, but is due to some fraction(s) which can be separated from the enzyme. Together with invivo experiments on transcription and SPP1 phage production during sporulation, these results suggest a possible role of promoter recognition in sporulation.