Functional Characterization of the HasA PF Hemophore and Its Truncated and Chimeric Variants: Determination of a Region Involved in Binding to the Hemophore Receptor

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Abstract
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.