CLONING AND CHARACTERIZATION OF THE HAEMOCIN IMMUNITY GENE OF HAEMOPHILUS-INFLUENZAE

The bacteriocin haemocin is produced by most type b strains of Haemoph ilus influenzae, including strains of diverse genetic lineage, and is toxic to virtually all nontypeable H. influenzae strains, An H. influe nzae transformant bearing a plasmid with a 1.5-kbp chromosomal fragmen t capable of conferring haemocin immunity on a haemocin-susceptible H. influenzae mutant was selected by using partially purified haemocin. Deletional and site-directed mutagenesis localized the haemocin immuni ty gene to the 3' open reading frame (ORF) within this chromosomal fra gment. Subcloning of this ORF demonstrated that it was sufficient to c onfer haemocin immunity on wild-type haemocin-susceptible H. influenza e strains as well as haemocin-susceptible strains of Escherichia coli. This ORF, designated hmcI, encodes a 105-amino-acid protein with an e stimated molecular mass of 12.6 kDa. Primer extension analysis reveale d a putative transcriptional start site 34 bp upstream of the start co don, and the presence of a promoter immediately upstream of hmcI was c onfirmed by cloning the gene into a promoterless chloramphenicol acety ltransferases vector. To characterize the hmcI gene product, a His-Hmc I fusion protein was constructed.