Cloning and characterization of the haemocin immunity gene of Haemophilus influenzae

Abstract

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 hmcI, 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.Most type b-encapsulated strains of Haemophilus influenzae, including strains demonstrated to be genetically diverse by multilocus enzyme electrophoresis (22), produce the bacteriocin haemocin (HMC), which is toxic in vitro to virtually all non-type b strains of H. influenzae, both encapsulated and nonencapsulated (16,37,38). Data suggest that HMC exerts its toxicity by inhibiting DNA synthesis in susceptible strains (34); however, little is known about the genetic determinants conferring HMC production or immunity in H. influenzae.Bacterial cells capable of bacteriocin production must be immune to the toxic effects of the bacteriocin protein. This immunity is usually mediated via the coproduction of a specific immunity protein. A number of bacteriocin immunity genes have been cloned and characterized. The majority of these are located within gene clusters that also encode bacteriocin structural, transporter, and accessory proteins (1,14,26). Although they generally encode relatively small peptides, little homology has been observed among characterized bacteriocin immunity genes. In this report, we describe the identification, characterization, and heterologous expression of hmcI, the gene responsible for conferring immunity against the H. influenzae bacteriocin HMC. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used are listed in Table 1. Escherichia coli strains were cultivated in LuriaBertani medium (LB) with appropriate antibiotics. Antibiotic concentrations (in micrograms per milliliter) used for plasmid selection in E. coli were as follows: carbenicillin, 40 to 50; chloramphenicol, 20 to 25; and ribostamycin, 40. H. influenzae strains were grown in brain heart infusion broth supplemented with 10 g of hemin and 10 g of ␤-NAD (Sigma Chemical Co., St. Lou...

Genetic analysis of a plasmid encoding haemocin production in Haemophilus paragallinarum

“…However, when the H. paragallinarum hmcI gene was cloned into the same vector (pGEM-T Easy, Promega) the resulting plasmid was unable to protect against haemocinmediated killing (data not shown), suggesting that the H. paragallinarum hmcI gene does not have a promoter immediately upstream. Differences in the s 70 210 region identified by Murley et al (1997) (TAAAAAT) and the same region of the H. paragallinarum sequence (TAAATTT) or the as-yet-unidentified 235 region of the gene may account for the lack of expression from the H. paragallinarum construct in E. coli.…”

“…The HmcI proteins also share homology with an uncharacterized protein from Mesorhizobium loti (31 % identity, 53 % similarity, NP 108470). Murley et al (1997) identified a s 70 210 region but could not identify a 235 region upstream of the H. influenzae hmcI transcription start site and showed that the HmcI protein could be expressed from this promoter in E. coli and provide protection from haemocin-mediated killing. However, when the H. paragallinarum hmcI gene was cloned into the same vector (pGEM-T Easy, Promega) the resulting plasmid was unable to protect against haemocinmediated killing (data not shown), suggesting that the H. paragallinarum hmcI gene does not have a promoter immediately upstream.…”

“…The haemocin is heat stable, but activity is destroyed by proteinase K. There are four Cys residues in the mature protein that may contribute to the retention of activity after heating. Mature HmcA Murley et al (1997). Both the H. influenzae and H. paragallinarum HmcI proteins have identical KHKRKAKK Lys-rich motifs.…”

“…The plasmid is 6286 bp in size and detailed analysis of the sequence revealed a haemocin operon (hmc), a putative integrase (int) and a putative replication (repB) protein (see Table 2). The haemocin operon is homologous to the H. influenzae haemocin operon (Murley et al, 1997(Murley et al, , 1998 and contains the hmcDCBA and I genes, responsible for haemocin production, processing, export and immunity. Approximately 1 kb of DNA sequence obtained has no homology with any DNA or translated protein in the GenBank database.…”

Cloning of the haemocin locus of Haemophilus influenzae type b and assessment of the role of haemocin in virulence

“…previously described the cloning and characterization of hmd, the H M C immunity gene (Murley et al, 1997); however, little is known about the genes responsible for genetically diverse bymultilocus enzyme electrophoresis (Musser et al, 1986), produce the bacteriocin haemocin (HMC), which is toxic in vitro to virtually all non-type b strains of H . influenzae, both encapsulated and nonencapsulated (LiPuma et al, 1990;Venezia & Robertson, 1975;Venezia et al, 1977).…”