Molecular characterization of KatY (antigen 5), a thermoregulated chromosomally encoded catalase-peroxidase of Yersinia pestis

Abstract

The first temperature-dependent proteins (expressed at 37 degrees C, but not 26 degrees 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 approximately 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 Ca2+-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.

FIG. 1

Elution profiles of pigmented material (A₄₀₅) containing KatY from a cell extract of Y. pestis KIM10 (substrain D28) during stepwise chromatography on columns containing DEAE-cellulose (A [the insert illustrates details of the area of KatY elution shown in the box]), calcium hydroxylapatite (B), and Sephadex G-150 (C). A₂₈₀ (○), A₄₀₅ (●), and A₆₁₅ for Blue dextran (▴) were used to determine the void volume for the column containing Sephadex G-150. See Materials and Methods for details.

FIG. 2

Immunoblots prepared with rabbit polyclonal antiserum raised against whole cells of Yersinia pestis KIM10 substrain D27 (A), rabbit polyclonal antiserum raised against purified outer membranes of Y. pestis KIM10 substrain D28 (B), or mouse MAb 1B70.1 raised against KatY. Lanes: 1, crude cell extract of Y. pestis KIM10 (substrain D28); 2, eluate obtained upon chromatography on DEAE-cellulose; 3, eluate obtained upon chromatography on calcium hydroxylapatite; 4, eluate obtained upon chromatography on Sephadex G-150. The positions of the α, β, γ, and δ forms of KatY are shown in the right margin.

FIG. 3

Immunoblot prepared with mouse MAb 1B70.1 raised against KatY versus whole cells of Y. pestis KIM10 substrain D28 carrying pPCP (lane 1), Y. pestis KIM10 substrain D47 cured of pPCP (lane 2), Y. pestis EV76 carrying pPCP (lane 3), Y. pestis EV76 cured of pPCP (lane 4), Y. pseudotuberculosis PB1 transformed with pPCP (lane 5), Y. pseudotuberculosis PB1 lacking pPCP (lane 6), E. coli HB101 transformed with pLG338 containing katY (lane 7), and E. coli HB101 alone (lane 8). The positions of the α, β, γ, and δ forms of KatY are shown in the right margin.

FIG. 4

Sequence of a 3,460-bp fragment from the Y. pestis KIM10 (substrain D46) genome that contains the ORF (amino acids 82 to 2292) encoding catalase-peroxidase katY and the ORFs (amino acids 2372 to 2752 and 2903 to 3430) that are homologous with the cybC and cybB genes encoding cytochromes b₅₆₂ (61) and b₅₆₁ (44) of E. coli, respectively. A potential ribosome-binding site (RBS) and putative −10 and −35 promoter areas of katY are underlined. An inverted repeat sequence 12 bp downstream of the termination codon and potential binding sites 1, 2, and 3 for the LcrF-like transcriptional regulator upstream of katY are shown by dashed arrows under and over the nucleotide sequence, respectively. N-terminal amino acid sequences of the native (amino acids 24 to 31) and truncated (amino acids 250 to 254) KatY that were determined by protein sequencing are designated by italic letters. Peroxidase motifs (amino acids 96 to 109 and 255 to 268) are in boldface and are underlined.

FIG. 5

Physical map of the Yersinia pestis KIM10 (substrain D46) katY region. katY is represented by the hatched box, the rest of the chromosomal region is represented by a gray bar, and selected restriction sites present in the segment are indicated. The homologue genes identified in the region sequenced upstream and downstream of KatY are represented by gray bars below the chromosome. Arrows indicate the direction of transcription. Base pair coordinates are depicted on the top part of the figure. DMSO, dimethyl sulfoxide.