Probable identification of a membrane-associated repressor of Bacillus subtilis DNA replication as the E2 subunit of the pyruvate dehydrogenase complex

Two Bacillus subtilis lysogenic libraries were probed by an antibody specif ic for a previously described membrane-associated inhibitor of B. subtilis DNA replication (J. Laffan and W. Firshein, Proc. Natl. Acad. Sci. USA 85:7 452-7356, 1988). Three clones that reacted strongly with the antibody conta ined an entire open reading frame. Sequencing identified one of the clones (R1-2) as containing the E2 subunit of the pyruvate dehydrogenase complex, dihydrolipoamide acetyltransferase. An AT-rich sequence in the origin regio n was identified initially as the site to which extracts from the R1-2 clon e were bound. This sequence was almost identical to one detected in Bacillu s thuringiensis that also bound the E2 subunit but which was involved in ac tivating the Cry1 protoxin gene of the organism, not in inhibiting DNA repl ication (T. Walter and A. Aronson, J. Biol. Chem., 274:7901-7906, 1999). Ho wever, the exact sequence was not as important in B. subtilis as the AT-ric h core region. Binding would occur as long as most of the AT character of t he core remained. Purified E2 protein obtained by use of PCR and an express ion vector reacted strongly with antibody prepared against the repressor pr otein and the protein in the R1-2 clone, but its specificity for the AT-ric h region was altered. The purified E2 protein was capable of inhibiting mem brane-associated DNA replication in vitro, but anti-E2 antibody was variabl e in its ability to rescue repression when added to the assay.