Cloning and characterization of the gene encoding Pasteurella haemolytica FnrP, a regulator of the Escherichia coli silent hemolysin sheA

Abstract

A Pasteurella haemolytica A1 gene was identified from a recombinant library clone that expressed hemolysis in host Escherichia coli cells. The gene, designated fnrP, had sequence identity to E. coli fnr, a global transcriptional regulator of genes required for conversion to anaerobic growth. FnrP complemented anaerobic deficiencies of a fnr-null mutant strain of E. coli and increased expression of the Fnr-dependent, anaerobic terminal reductase gene, frdA. FnrP was purified, identified by immunoblotting, and shown to be nonhemolytic. When FnrP was expressed in E. coli DeltasheA, a null mutant of the cryptic hemolysin SheA, the transformants were nonhemolytic, indicating that FnrP activates this silent hemolysin.

FIG. 1

Identification of FnrP in the fractions eluted during affinity purification and their correlation with hemolysis. Top, immunoblot of the eluted column fractions collected during partial purification of FnrP. Proteins were separated by electrophoresis and blotted with anti-FnrP polyclonal antibody. Bottom, hemolytic activity of the eluted column fractions on 2.5% ovine blood agar. Column A, fraction eluted at pH 4.0; column B, fraction eluted at pH 6.0.