| A critical early stage in bacterial sporulation is an asymmetric division that divides the organism into two distinct cell types, the prespore and the mother cell. These two cell types have radically different developmental pathways. The smaller prespore eventually becomes the dormant spore while the larger mother cell lyses. The concerted action of a series of a factors whose activities are compartmentalized determines these radically different fates. sigma F is the first expressed of the sporulation-specific sigma factors, and the activation of the other sigma factors depends on it. Few genes have been identified that are under the control of RNA polymerase containing sigma F (E-sigmaF). Thus, at the start of this study the promoter sequence recognized by E-sigmaF was very poorly defined.;The strongest known a sigmaF promoter, P spoIIQ, was used as base for analysis in order to elucidate a consensus for sigmaF promoters. A vector, pEIA98, was designed in such away that oligonucleotides containing either randomized --10 or randomized --35 regions could be fused to a promoterless lacZ gene. A clone bank for the random --10 region (R10) was made in pEIA98 and a portion was used to transform B. subtilis. Transformant colonies were screened for beta-galactosidase activity with the chromogenic substrate X-gal. Approximately 55,000 transformant colonies were obtained of which approximately 800 were blue, indicating beta-galactosidase activity. 500 of the 800 clones were subjected to additional screenings for sigma F activity. 328 clones passed the screens and 35 clones were selected at random for further characterization.;A random clone bank was also made for the --35 region (R35) and a portion of the R35 library was transformed into B. subtilis. 64 transformant colonies were blue on plates containing X-gal out of a total 3,300. All 64 clones were subjected to additional screenings for sigma F activity. 44 clones passed the screens and 18 clones were selected at random for further characterization. Data from the sequences of R10 and R35 clones and from site-directed mutagenesis studies have helped establish a consensus for sigmaF promoters. The data revealed that there is not a strong sigmaF consensus sequence for the --10 region while there is a strong consensus sequence for the --35 region. |
