H. influenzae is the most common cause of pyogenic meningitis in the United States. The peak incidence of H. influenzae meningitis is 6-9 months of age, and neurological sequelae are detected in approximately 50% of survivors. To expand our understanding of the molecular genetics of H. influenzae, we have developed a novel method for insertiona mutagenesis of the chromosome of H. influenzae. A mutagenizing elements, """"""""TSTE"""""""", has been constructed by ligation of the portion of the transposon Tn5 encoding kanamycin resistance with a synthetic oligonucleotide which includes the uptake sequence which enhances natural transformation of H. influenzae by several orders of magnitude. Chromosomal DNA fragment will be circularized by self-ligation. After digestion with a restriction enzyme which produces compatible sticky ends, the """"""""TSTE"""""""" element will be ligated, and the interrupted DNA fragments will be transformed back into the original strain. Kanamycin resistance transformants will have insertional mutations in the chromosome. The """"""""TSTE"""""""" insertional mutagenesis will be applied to genes encoding hydroxamate production. H. influenzae produces hydroxamates in high iron concentrations, and hydroxamate production by H. influenzae is highly conserved along clonal lines of virulent type b strains. To investigate this unique iron-related characteristic of H. influenzae a genetic analysis of defined mutants will be performed to identify the structural and regulatory genes responsible for hydroxamate production. These genes will be cloned, mapped with restriction enzymes, and regulatory regions will be sequenced to determine whether the """"""""iron box"""""""" operator found in other bacteria is present in H. influenzae. The hydroxamate have been purified, and the biological role of hydroxamate production will be analyzed by 1) determining the survival of isogenic strains in the presence of purified neutrophils, and 2) comparing the virulence of the isogenic strains; and 3) determining the ability of H. influenzae hydroxamates to interfere with the production of hydroxyl radical by iron sequestration. The molecular structure of the hydroxamates will be determined by mass spectroscopy and nuclear magnetic resonance.
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