Test systems to study transcriptional regulation and promoter activity in Bacillus megaterium.

Plasmid-located (multi-copy) and chromosomally located (single-copy) promoter test systems were developed for Bacillus megaterium by making use of the homologous beta-galactosidase-encoding bgaM gene. The multi-copy system facilitates rapid promoter analyses and promoter trapping, whereas the single-copy system, integrated into the chromosome, allows investigation of tightly regulated promoters. As a prerequisite for both the multi- and the single-copy systems, a beta-galactosidase-deficient B. megaterium strain was generated by deletion mutagenesis. Both test systems were verified using the promoter of the xylose operon (P( xylA )) from B. megaterium along with its repressor (XylR). As expected, expression levels in the two systems differed significantly, although expression of the bgaM reporter gene was induced by xylose in both cases, thereby proving the functionality of both the multi- and the single-copy system.

Molecular characterization of the operon comprising the spoIV gene of Bacillus megaterium DSM319 and generation of a deletion mutant.

According to sequence analysis, the spoIV-locus of Bacillus megaterium DSM 319 is 1,185 bp long; it is the second gene of a sporulation operon, which altogether contains three open reading frames. The ORF preceding spoIV encodes a putative polypeptide with 94 amino acids; the 3rd ORF of the operon has 972 bp corresponding to 324 amino acids. The operon is flanked on both sides by palindromic sequences, probably representing Rho-independent terminators. A primer extension analysis revealed that mRNA synthesis starts immediately downstream of a promoter, which is similar to the consensus sequence of Bacillus subtilis sigma(E) dependent promoters. Both the -35 and the -10 region are within the terminator region of the preceding operon. Gene knockout experiments and reporter gene assays with a newly developed system based on the heterologous Paenibacillus macerans glucanase gene (bgl) confirmed sigma(E)-dependent transcription. Two open reading frames of a further upstream operon were also identified. Northern analysis revealed that transcription of these ORFs comes about in late sporulation phases. The genetic organization of the spoIV comprising operon and adjacent loci clearly resembles that of the B. subtilis yqfa-phoH gene cluster. Thus our findings are of general significance for endospore-forming bacteria.