FIG. 1.
(A) Construction of pP43NMK, a B. subtilis expression vector for the production and secretion of MPH. See the text for a detailed description of the construction of pP43NMK. P43 and SP represent the P43 promoter and the nprB signal peptide-encoding sequence from B. subtilis, respectively. ori, amp, rep, and km represent the sequences coding for the ColE1 replication origin, ampicillin resistance marker, replicase, and kanamycin resistance marker, respectively. The arrows show the transcription directions for these genes. (B) Nucleotide sequences of the P43 promoter fragment, nprB signal peptide-encoding sequence, and part of the mpd gene. The deduced amino acid sequence of the neutral protease B signal peptide is underlined. The “−10” and “−35” regions of the promoters (recognized by σA and σB factors), the transcription start site (“+1”), and the ribosome binding site (RBS) are indicated. The vertical arrow indicates the predicted cleavage site for the signal peptidase I.