LACK OF EXPRESSION OF THE GLOBAL REGULATOR OXYR IN HAEMOPHILUS-INFLUENZAE HAS A PROFOUND EFFECT ON GROWTH PHENOTYPE

A pBR322-based library of chromosomal DNA from the nontypeable Haemoph ilus influenzae TN106 was screened for the expression of transferrin-b inding activity in Escherichia coli, A recombinant clone expressing tr ansferrin-binding activity contained a 3.7-kb fragment of nontypeable H. influenzae DNA, Nucleotide sequence analysis of this insert reveale d the presence of two complete open reading frames encoding proteins o f approximately 26 and 34 kDa, Mini-Tn10kan transposon mutagenesis at different sites within the open reading frame encoding the 34-kDa prot ein resulted in the abolition of transferrin-binding activity in the r ecombinant E. coli clone. The deduced amino acid sequence of the 34-kD a protein had 70% identity with the OxyR protein of E. coli, this latt er macromolecule is a member of the LysR family of transcriptional act ivators. When a mutated H. influenzae oxyR gene was introduced into th e chromosome of the wild-type H. influenzae strain by allelic exchange , the resulting oxyR mutant still exhibited wild-type levels of transf errin-binding activity but was unable to grow on media containing the heme precursor protoporphyrin IX (PPIX) in place of heme. This mutant also exhibited reduced growth around disks impregnated with heme sourc es, Supplementation of the PPIX-based growth media with catalase or so dium pyruvate resulted in normal growth of the H. influenzae oxyR muta nt. Provision of the wild-type H. influenzae oxyR gene in trans also p ermitted the growth of this mutant on a PPIX-based medium, Exogenously supplied catalase restored the growth of this mutant with heme source s to nearly wild-type levels. These results indicate that expression o f a wild-type OxyR protein by H. influenzae is essential to allow this organism to protect itself against oxidative stresses in vitro.