Inactivation of Blasticidin S by Bacillus cereus(6)Structure and Comparison of the bsr Gene from a Blasticidin S-Resistant Bacillus cereus

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

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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 σA and σB promoter sequences are observed. The tarnscriptional start point was determined to be the A located 7 bases downstream from the putative σA 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 (xn 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.

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