Functional characterization of the HasA(PF) hemophore and its truncated and chimeric variants: determination of a region involved in binding to the hemophore receptor.
Hemophores are secreted by several gram-negative bacteria (Serratia marcescens, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Yersinia pestis) and form a family of homologous proteins. Unlike the S. marcescens hemophore (HasA(SM)), the P. fluorescens hemophore HasA(PF) has an additional region of 12 residues located immediately upstream from the C-terminal secretion signal. We show that HasA(PF) undergoes a C-terminal cleavage which removes the last 21 residues when secreted from P. fluorescens and that only the processed form is able to deliver heme to the S. marcescens outer membrane hemophore-specific receptor, HasR(SM). Functional analysis of variants including those with an internal deletion of the extra C-terminal domain show that the secretion signal does not inhibit the biological activity, whereas the 12-amino-acid region located upstream does. This extra domain may inhibit the interaction of the hemophore with HasR(SM). To localize the hemophore regions involved in binding to HasR, chimeric HasA(PF)-HasA(SM) proteins were tested for biological activity. We show that residues 153 to 180 of HasA(PF) are necessary for its interaction with the receptor.
Unité des Membranes Bactériennes, Institut Pasteur (CNRS URA2172), 75724 Paris Cedex 15, France.
S
S
Létoffé
K
K
Omori
C
C
Wandersman
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins; D002846:Chromatography, Affinity; D006090:Gram-Negative Bacteria / Q000235:genetics* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012706:Serratia marcescens / Q000235:genetics / Q000378:metabolism; D015010:Yersinia pestis / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; C093956:HasA protein, Serratia marcescens; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins