Molecular characterization of the operon comprising the spoIV gene of Bacillus megaterium DSM1319 and generation of a deletion mutant

According to sequence analysis, the spoIV-locus of Bacillus megaterium DSM3 19 is 1,185 bp long; it is the second gene of a sporulation operon, which a ltogether contains three open reading frames, The ORF preceding spoIV encod es a putative polypeptide with 94 amino acids; the 3rd ORF of the operon ha s 972 bp corresponding to 324 amino acids. The operon is flanked on both si des by palindromic sequences, probably representing Rho-independent termina tors. A primer extension analysis revealed that mRNA synthesis starts immed iately downstream of a promoter, which is similar to the consensus sequence of Bacillus subtilis sigma(E) dependent promoters. Both the -35 and the -1 0 region are within the terminator region of the preceding operon, Gene kno ckout experiments and reporter gene assays with a newly developed system ba sed on the heterologous Paenibacillus macerans glucanase gene (bgl) confirm ed sigma(E)-dependent transcription. Two open reading frames of a further u pstream operon were also identified. Northern analysis revealed that transc ription of these ORFs comes about in late sporulation phases. The genetic o rganization of the spoIV comprising operon and adjacent loci clearly resemb les that of the B. subtilis yqfa-phoH gene cluster. Thus our findings are o f general significance for endospore-forming bacteria.