Inactivation of Blasticidin S by Bacillus cereus. VI. Structure and Comparison of the bsr Gene from a Blasticidin S-Resistant Bacillus cereus

Abstract

Two types of recombinant plasmids containing 600 bp Nde I fragments that coded the bsr gene in opposite directions were obtaind. Nucleotide sequencing shows that the bsr encodes a 140 amino acid protein with a putative molecular weight of 15560, the same as that of purified blasticidin S (BS)-deaminase (BSR), on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (15500). Upstream of the open reading frame, a Shine-Dalgarno (SD) sequence, frequent inverted repeats, and the σ^A and σ^B promoter sequences are observed. The tarnscriptional start point was determined to be the A located 7 bases downstream from the putative σ^A promoter (⁹¹TTGATC and ¹¹³TAAAAT) by the primer estension method and site directed mutagenesis at the -10 or -35 promoter region.A comparison of the amino acid sequence of BSR with that of BS-deaminase from Aspergillus terreus (BSD) showed 27.2% homology. Low degrees of homology were also observed with cytidine deaminase and deoxy cytidine monophosphate (CMP) deaminase. Four conserved amino acid motifs were observed, VGAx₆ G, C(orH)AEx₆A, SPCGxCR, and Gx₈ELIP (x_n indicates a nonspecific residue and its position). It is possible that the three Cys residues and the Glu in the conserved motifs comprise the active center. Site-directed mutagenesis of the Cys residues supports this possibility.