Haemocin Production by Encapsulated and Nonencapsulated Haemophilus influenzae

LiPuma, John J.; Sharetzsky, Christine; Edlind, Thomas D.; Stull, Terrence L.

doi:10.1093/infdis/165-supplement_1-s118

Cited by 6 publications

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“…Because of the presence of the double-glycine leader sequence in ORF4, we sought to examine the role of this ORF in HMC production. This was done by creating an ORF4-deficient mutant using TSTE (LiPuma et al, 1992). Southern blot analysis of EcoRI-digested chromosomal DNA from Ela and the ORF4 insertion mutant, designated H206, confirmed the insertion of the 2·2 kbp cassette into the chromosome of Ela ().…”

Section: Resultsmentioning

confidence: 99%

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

et al. 1998

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The bacteriocin haemocin (HMC) is produced by most type b strains of Haemophilus influenzae, including strains determined to be genetically diverse, and is toxic to virtually all non-type b strains of H. influenzae, both encapsulated and non-encapsulated. Examination of the deduced amino acid sequences of several genes upstream of the previously identified HMC immunity gene (hmcl) revealed several features common to class II bacteriocins of certain Gram-positive bacteria. Mutagenesis of the open reading frame immediately upstream of hmcl resulted in a loss of the HMC production phenotype. When an HMC-producing strain of H. influenzae and the HMC-deficient isogenic mutant were compared for invasion in the infant-rat model, the HMC-producing strain was found to invade significantly earlier; however, a significantly higher number of rats infected with the isogenic mutant became bacteraemic as compared with those infected with the HMCproducing parent.

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Section: Resultsmentioning

confidence: 99%

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

et al. 1998

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“…Equal volumes of this bacterial suspension and ppHMC were incubated at 37ЊC for 90 min and then cultured on sBHI agar containing carbenicillin. Surviving transformants were screened for HMC immunity and production (17).…”

Section: Methodsmentioning

confidence: 99%

Cloning and characterization of the haemocin immunity gene of Haemophilus influenzae

et al. 1997

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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...

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Haemophilus

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2010

Topley &Amp; Wilson's Microbiology and Microbial Infections

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Haemocin Production by Encapsulated and Nonencapsulated Haemophilus influenzae

Cited by 6 publications

References 0 publications

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

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

Cloning and characterization of the haemocin immunity gene of Haemophilus influenzae

Haemophilus

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