Identification and characterization of a trans-activator involved in the regulation of encapsulation by Bacillus...

Production of the plasmid-pXO2-encoded capsule by Bacillus anthracis is required for full virulence of the organism. The induction of capsule synthesis in vitro requires growth in the presence of bicarbonate and CO2; however, little else is known about the regulation of capsule synthesis and the role it plays in the expression of virulence. Recently, transposon Tn917 mutagenesis of B. anthracis plasmid pXO2 identified genes involved in capsule production and genes associated with virulence in inbred mice. One mutant, UUP5, had an 8.2-kb deletion located outside of the capsule structural gene region (cap). UUP5 was reduced significantly in capsule production and in virulence as compared to the wild-type (wt) parental strain. Using a HindIII-generated pXO2 library, we examined fragments contained in the deleted region and showed that electroporation of the mutant with a cloned 2.3-kb HindIII fragment restored capsule production to wt levels. Sequence analysis of the 2.3-kb fragment revealed a 1449-bp open reading frame (ORF) encoding a 483-amino-acid (57 kDa) protein, in good agreement with the 55-kDa protein detected by in vitro transcription/translation. Construction of a frameshift mutant that replaced the 55-kDa protein with a truncated 34-kDa moiety abrogated the complementing activity of the fragment in UUP5. mRNAs specific for cap and for the 1449-bp ORF were detected in mutant UUP5 transformed with the unaltered fragment and grown in the presence of bicarbonate, but not in air. No cap-specific mRNA, and very low levels of ORF-specific mRNA, were detected in UUP5 containing the frameshift mutation.(ABSTRACT TRUNCATED AT 250 WORDS)