Haemophilus influenzae is able to use hemoglobin as a sole source of heme, and heme-repressible hemoglobin binding to the cell surface has been demonstrated. Using an affinity purification methodology, a hemoglobinbinding protein of approximately 120 kDa was isolated from H. influenzae type b strain HI689 grown in heme-restricted but not in heme-replete conditions. The isolated protein was subjected to N-terminal amino acid sequencing, and the derived amino acid sequence was used to design corresponding oligonucleotides. The oligonucleotides were used to probe a Southern blot of EcoRI-digested HI689 genomic DNA. A hybridizing band of approximately 4.2 kb was successfully cloned into pUC19. Using a 1.9-kb internal BglII fragment of the 4.2-kb clone as a probe, hybridization was seen in both typeable and nontypeable H. influenzae but not in other bacterial species tested. Following partial nucleotide sequencing of the 4.2-kb insert, a putative open reading frame was subcloned into an expression vector. The host Escherichia coli strain in which the cloned fragment was expressed bound biotinylated human hemoglobin, whereas binding of hemoglobin was not detected in E. coli with the vector alone. In conclusion, we hypothesize that the DNA fragment encoding an approximately 120-kDa heme-repressible hemoglobin-binding protein mediates one step in the acquisition of hemoglobin by H. influenzae in vivo.

The complete repeating unit of the human ribosomal RNA gene has been reconstructed by the cloning of approximately 27 kilobases (kb) of non-transcribed spacer. The structure of this tandemly repeated gene can now be studied in its entirety. We report the analysis of spacer DNA by molecular cloning and its organization in the genome by Southern transfer analysis. These studies reveal both length and sequence variation of the spacer. Sequence variations are distributed throughout the spacer while the length variations exist near the 5' end of the transcript and just beyond the 3' end. The human spacer shares extensive homology with primates but little with other mammals. Within the primates the degree of homology reflects the rapid evolutionary changes characteristic of the primate group.

Inoculation of the replication-defective retrovirus DEF27 (BM5d), packaged as an amphotropic virus pseudotype, into C57BV6J mice leads to development of murine AIDS. Disease development showed a long incubation period (20 to 24 weeks), was associated with amplification of the BM5d provirus in splenocytes and lymph nodes, and was independent of the presence of exogenous or endogenous replication-competent helper viruses. However, both the onset of disease and amplification of the defective provirus were significantly enhanced by coinfection with the replication-competent B-cell-tropic ecotropic helper virus BM5e. The part of the BM5d viral genome that was essential for the pathogenicity was determined by making precisely engineered alterations in the reading frame of the gag and pol genes of BM5d proviral DNA and examining the ability of the altered amphotropic BM5d pseudotypes to induce the disease in C57BV6J mice. The results show that expression of the MA (p15) and p12 regions of the gag gene is sufficient for pathogenicity of the BM5d retrovirus.

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...

Binding of biotinylated human hemoglobin to Haemophilus influenzae was detected when organisms were grown in heme-deplete, but not heme-replete, conditions. Hemoglobin binding was completely inhibited by a 100-fold excess of unlabelled human hemoglobin or human hemoglobin complexed with human haptoglobin. Binding was only partially inhibited by rat hemoglobin, bovine hemoglobin, human globin, and bovine globin, and not at all by heme, human serum albumin, bovine serum albumin, human transferrin, or myoglobin. Hemoglobin binding was saturable at 16-20 ng of hemoglobin per 10(9) cfu. Binding of human hemoglobin was detected in serotypes a-f and serologically non-typable strains of H. influenzae, as well as Haemophilus haemolyticus but not Haemophilus parainfluenzae, Haemophilus aphrophilus, Haemophilus parahaemolyticus, or Escherichia coli.