Cloning and characterization of the haemocin immunity gene of Haemophilus influenzae.
The bacteriocin haemocin is produced by most type b strains of Haemophilus influenzae, including strains of diverse genetic lineage, and is toxic to virtually all nontypeable H. influenzae strains. An H. influenzae transformant bearing a plasmid with a 1.5-kbp chromosomal fragment 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 immunity gene to the 3' open reading frame (ORF) within this chromosomal fragment. Subcloning of this ORF demonstrated that it was sufficient to confer haemocin immunity on wild-type haemocin-susceptible H. influenzae strains as well as haemocin-susceptible strains of Escherichia coli. This ORF, designated hmcl, encodes a 105-amino-acid protein with an estimated molecular mass of 12.6 kDa. Primer extension analysis revealed a putative transcriptional start site 34 bp upstream of the start codon, and the presence of a promoter immediately upstream of hmcI was confirmed by cloning the gene into a promoterless chloramphenicol acetyltransferase vector. To characterize the hmcI gene product, a His-HmcI fusion protein was constructed.
Murley
Y M
YM
Department of Microbiology/Immunology, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19129, USA.
Edlind
T D
TD
Pozsgay
J M
JM
LiPuma
J J
JJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001430:Bacteriocins / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006193:Haemophilus influenzae / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D017421:Sequence Analysis; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D001430:Bacteriocins; C104637:HmcI protein, Haemophilus influenzae; D011993:Recombinant Fusion Proteins; C030520:haemocin