Cloning, molecular characterization, and application of rice epiphytic Bacillus pumilus promoter fragments

To establish a constitutive, high-efficiency expression system for Bacillus pumilus (B.P), we cloned random chromosomal DNA into promoter probe shuttl e vector ECE7 and selected for strong promoter activity by chloramphenicol resistance of transformed B. pumilus cells. The nucleotide sequences of nin e chromosomal fragments were determined. These DNA fragments range from 300 to 2200 bp in size. The transcription strength of these promoters was esti mated by determination of CAT enzyme production in both E. coli and B. pumi lus. Transcription start (TS) sites of the cat mRNA were located by primer extension by using total RNA. Preliminary analysis showed that three of the promoter sequences contain - 35 and - 10 regions like E. coli RNA polymera se sigma (70) and B. subtilis sigma (43) consensus sequences. One is simila r to B. subtilis a, the other two have no conserved sequences like any of t he typical consensus sequences of the known sigma factors so far. To estima te the feasibility of the utilization of these promoters, one promoter frag ment was subcloned and used to drive the expression of green fluorescent pr otein (GFP) in B. pumilus cells. This is the first report of B. pumilus pro moters randomly cloning from total DNA and molecular analysis of their cons ensus sequences.