A gene cluster involved in the utilization of both free heme and heme:hemopexin by Haemophilus influenzae type b

The utilization of heme bound to the serum glycoprotein hemopexin by Haemophilus influenzae type b (Hib) strain DL42 requires the presence of the 100-kDa heme:hemopexin-binding protein encoded by the hxuA gene (M. S. Hanson, S. E. Pelzel, J. Latimer, U. Muller-Eberhard, and E. J. Hansen, Proc. Natl. Acad. Sci. USA 89:1973-1977, 1992). Nucleotide sequence analysis of a 5-kb region immediately upstream from the hxuA gene revealed the presence of two genes, designated hxuC and hxuB, which encoded outer membrane proteins. The 78-kDa HxuC protein had similarity to TonB-dependent outer membrane proteins of other organisms, whereas the 60-kDa HxuB molecule most closely resembled the ShlB protein of Serratia marcescens. A set of three isogenic Hib mutants with cat cartridges inserted individually into their hxuA, hxuB, and hxuC genes was constructed. None of these mutants could utilize heme:hemopexin. The hxuC mutant was also unable to utilize low levels of free heme, whereas both the hxuA and hxuB mutants could utilize free heme. When the wild-type hxuC gene was present in trans, the hxuC mutant regained its ability to utilize low levels of free heme but still could not utilize heme:hemopexin. The hxuA mutant could utilize heme:hemopexin when a functional hxuA gene from a nontypeable H. influenzae strain was present in trans. Complementation analysis using this cloned nontypeable H. influenzae hxuA gene also indicated that the HxuB protein likely functions in the release of soluble HxuA from the Hib cell. These studies indicate that at least two and possible three gene products are required for utilization of heme bound to hemopexin by Hib strain DL42.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  S. E. Thomas,et al.  The 100 kDa haem:haemopexin‐binding protein of Haemophilus Influenzae: structure and localization , 1994, Molecular microbiology.

[3]  E. Hansen,et al.  A functional tonB gene is required for both utilization of heme and virulence expression by Haemophilus influenzae type b , 1994, Infection and immunity.

[4]  P. Williams,et al.  Identification and characterization of an iron-regulated hemopexin receptor in Haemophilus influenzae type b , 1994, Infection and immunity.

[5]  J. Musser,et al.  Expression of the Haemophilus influenzae transferrin receptor is repressible by hemin but not elemental iron alone , 1993, Infection and immunity.

[6]  V. Braun,et al.  Amino acid replacements in the Serratia marcescens haemolysin ShlA define sites involved in activation and secretion , 1993, Molecular microbiology.

[7]  S. Calderwood,et al.  Role of iron in regulation of virulence genes , 1993, Clinical Microbiology Reviews.

[8]  S. Payne,et al.  Cloning and characterization of the Vibrio cholerae genes encoding the utilization of iron from haemin and haemoglobin , 1993, Molecular microbiology.

[9]  T. Lucier,et al.  Storage reservoirs of hemin and inorganic iron in Yersinia pestis , 1993, Infection and immunity.

[10]  V. Braun,et al.  Serratia marcescens forms a new type of cytolysin. , 1992, FEMS microbiology letters.

[11]  K. Hantke,et al.  Hemin uptake system of Yersinia enterocolitica: similarities with other TonB‐dependent systems in gram‐negative bacteria. , 1992, The EMBO journal.

[12]  W. Thomas,et al.  The synthesis and function of the Escherichia coli hemolysin and related RTX exotoxins. , 1992, FEMS microbiology immunology.

[13]  S. E. Thomas,et al.  Use of electroporation to construct isogenic mutants of Haemophilus ducreyi , 1992, Journal of bacteriology.

[14]  C. Slaughter,et al.  The hbpA gene of Haemophilus influenzae type b encodes a heme-binding lipoprotein conserved among heme-dependent Haemophilus species , 1992, Infection and immunity.

[15]  M. Hanson,et al.  Identification of a genetic locus of Haemophilus influenzae type b necessary for the binding and utilization of heme bound to human hemopexin. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[16]  B. C. Lee.  Isolation of an outer membrane hemin-binding protein of Haemophilus influenzae type b , 1992, Infection and immunity.

[17]  P. Daskaleros,et al.  Iron uptake in Plesiomonas shigelloides: cloning of the genes for the heme-iron uptake system , 1991, Infection and immunity.

[18]  M. Hanson,et al.  Molecular cloning of a gene involved in lipooligosaccharide biosynthesis and virulence expression by Haemophilus influenzae type B , 1991, Molecular microbiology.

[19]  C. Higgins,et al.  TonB; a model for signal transduction between membranes. , 1991, Biochemical Society transactions.

[20]  J. Tommassen,et al.  Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein. , 1991, Journal of molecular biology.

[21]  R. Welch.  Pore‐forming cytolysins of Gram‐negative bacteria , 1991, Molecular microbiology.

[22]  M. Hanson,et al.  Molecular cloning, partial purification, and characterization of a haemin‐binding lipoprotein from Haemophilus influenzae type b , 1991, Molecular microbiology.

[23]  K. Postle.  TonB and the Gram‐negative dilemma , 1990, Molecular microbiology.

[24]  E. Hansen,et al.  Characterization of a mutant of Haemophilus influenzae type b lacking the P2 major outer membrane protein , 1990, Infection and immunity.

[25]  E. Hansen,et al.  Effect of mutations in lipooligosaccharide biosynthesis genes on virulence of Haemophilus influenzae type b , 1990, Infection and immunity.

[26]  J. Konisky,et al.  Genetic suppression demonstrates interaction of TonB protein with outer membrane transport proteins in Escherichia coli , 1990, Journal of bacteriology.

[27]  R. Welch,et al.  Nucleotide sequencing of the Proteus mirabilis calcium-independent hemolysin genes (hpmA and hpmB) reveals sequence similarity with the Serratia marcescens hemolysin genes (shlA and shlB) , 1990, Journal of bacteriology.

[28]  V. Braun,et al.  Subcellular location and unique secretion of the hemolysin of Serratia marcescens. , 1989, The Journal of biological chemistry.

[29]  J. Tomb,et al.  Transposon mutagenesis, characterization, and cloning of transformation genes of Haemophilus influenzae Rd , 1989, Journal of bacteriology.

[30]  C. Slaughter,et al.  Primary structure of the porin protein of Haemophilus influenzae type b determined by nucleotide sequence analysis , 1989, Infection and immunity.

[31]  J. Konisky,et al.  Evolutionary relationship between the TonB-dependent outer membrane transport proteins: nucleotide and amino acid sequences of the Escherichia coli colicin I receptor gene , 1989, Journal of bacteriology.

[32]  E. Hansen,et al.  Cloning of the gene encoding the major outer membrane protein of Haemophilus influenzae type b , 1988, Infection and immunity.

[33]  V. Braun,et al.  Molecular characterization of the hemolysin determinant of Serratia marcescens , 1988, Journal of bacteriology.

[34]  P. Sparling,et al.  Effects of serum carrier proteins on the growth of pathogenic neisseriae with heme-bound iron , 1987, Infection and immunity.

[35]  P. Matsudaira,et al.  Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. , 1987, The Journal of biological chemistry.

[36]  S. Wold,et al.  Signal peptide amino acid sequences in Escherichia coli contain information related to final protein localization. A multivariate data analysis. , 1987, The EMBO journal.

[37]  T. Stull.  Protein sources of heme for Haemophilus influenzae , 1987, Infection and immunity.

[38]  R. Kadner,et al.  Nucleotide sequence of the gene for the ferrienterochelin receptor FepA in Escherichia coli. Homology among outer membrane receptors that interact with TonB. , 1986, The Journal of biological chemistry.

[39]  R. Kadner,et al.  Nucleotide sequence of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli , 1985, Journal of bacteriology.

[40]  T. A. Loftus,et al.  A minor high-molecular-weight outer membrane protein of Haemophilus influenzae type b is a protective antigen , 1985, Infection and immunity.

[41]  E. Hansen,et al.  Immunogenic proteins in cell-free culture supernatants of Haemophilus influenzae type b , 1984, Infection and immunity.

[42]  J. Devereux,et al.  A comprehensive set of sequence analysis programs for the VAX , 1984, Nucleic Acids Res..

[43]  T. Close,et al.  M13 bacteriophage and pUC plasmids containing DNA inserts but still capable of β-galactosidase α-complementation , 1983 .

[44]  E. Hansen,et al.  Monoclonal antibodies directed against a cell surface-exposed outer membrane protein of Haemophilus influenzae type b , 1982, Infection and immunity.

[45]  J. Shine,et al.  Determinant of cistron specificity in bacterial ribosomes , 1975, Nature.

[46]  D. Smith,et al.  Haemin and nicotinamide adenine dinucleotide requirements of Haemophilus influenzae and Haemophilus parainfluenzae. , 1974, Journal of medical microbiology.

[47]  M. Vogt,et al.  Defined Nongrowth Media for Stage II Development of Competence in Haemophilus influenzae , 1970, Journal of bacteriology.

[48]  D. White,et al.  HEMIN BIOSYNTHESIS IN HAEMOPHILUS , 1963, Journal of bacteriology.

[49]  Philip S. Humphrey.  Evolution by Natural Selection , 1958, The Yale Journal of Biology and Medicine.

[50]  S. Granick,et al.  THE PORPHYRIN REQUIREMENTS OF HAEMOPHILUS INFLUENZAE AND SOME FUNCTIONS OF THE VINYL AND PROPIONIC ACID SIDE CHAINS OF HEME , 1946, The Journal of general physiology.

[51]  D. Maclaren,et al.  Transferrins and heme-compounds as iron sources for pathogenic bacteria. , 1992, Critical reviews in microbiology.

[52]  J. Tomb,et al.  Genetic systems in Haemophilus influenzae. , 1991, Methods in enzymology.

[53]  S. Payne,et al.  Molecular Studies on Iron Acquisition by Non-Escherichia coli Species , 1990 .

[54]  G von Heijne,et al.  Signal sequences. The limits of variation. , 1985, Journal of molecular biology.

[55]  L. Hood,et al.  Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. , 1983, Methods in enzymology.

[56]  T. Close,et al.  M13 bacteriophage and pUC plasmids containing DNA inserts but still capable of beta-galactosidase alpha-complementation. , 1983, Gene.