Molecular Characterization of KatY (Antigen 5), a Thermoregulated Chromosomally Encoded Catalase-Peroxidase of Yersinia pestis

Abstract

ABSTRACT The first temperature-dependent proteins (expressed at 37°C, but not 26°C) to be identified in Yersinia pestis were antigens 3 (fraction 1), 4 (pH 6 antigen), and 5 (hereafter termed KatY). Antigens 3 and 4 are now established virulence factors, whereas little is known about KatY, except that it is encoded chromosomally, produced in abundance, possesses modest catalase activity, and is shared by Yersinia pseudotuberculosis , but not Yersinia enterocolitica . We report here an improved chromatographic method (DEAE-cellulose, calcium hydroxylapatite, and Sephadex G-150) that yields enzymatically active KatY (2,423 U/mg of protein). Corresponding mouse monoclonal antibody 1B70.1 detected plasminogen activator-mediated hydrolysis of KatY, and a polyclonal rabbit antiserum raised against outer membranes of Y. pestis was enriched for anti-KatY. A sequenced ∼16-kb Y. pestis DNA insert of a positive pLG338 clone indicated that katY encodes an 81.4-kDa protein (pI 6.98) containing a leader sequence of 2.6 kDa; the deduced molecular mass and pI of processed KatY were 78.8 kDa and 6.43, respectively. A minor truncated variant (predicted molecular mass of 53.6 kDa) was also expressed. KatY is similar (39 to 59% identity) to vegetative bacterial catalase-peroxidases (KatG in Escherichia coli ) and is closely related to plasmid-encoded KatP of enterohemorrhagic E. coli O157:H7 (75% identity). katY encoded a putative Ca 2+ -binding site, and its promoter contained three homologues to the consensus recognition sequence of the pCD-encoded transcriptional activator LcrF. rbsA was located upstream of katY , and cybB , cybC , dmsABC , and araD were mapped downstream. These genes are not linked to katG or katP in E. coli .