Detection of phase variation in expression of proteins involved in hemoglobin and hemoglobin-haptoglobin binding by nontypeable Haemophilus influenzae.
Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study (I. Maciver, J. L. Latimer, H. H. Liem, U. Muller-Eberhard, Z. Hrkal, and E. J. Hansen. Infect. Immun. 64:3703-3712, 1996) indicated that nontypeable H. influenzae (NTHI) strain TN106 expressed a protein that bound hemoglobin-haptoglobin and was encoded by an open reading frame (ORF) that contained a CCAA nucleotide repeat. Southern blot analysis revealed that several NTHI strains contained between three and five chromosomal DNA fragments that bound an oligonucleotide probe for CCAA repeats. Three ORFs containing CCAA repeats were identified in NTHI strain N182; two of these ORFs were arranged in tandem. The use of translational fusions involving these three ORFs and the beta-lactamase gene from pBR322 revealed that these three ORFs, designated hgbA, hgbB, and hgbC, encoded proteins that could bind hemoglobin, hemoglobin-haptoglobin, or both compounds. Monoclonal antibodies (MAbs) specific for the HgbA, HgbB, and HgbC proteins were produced by immunizing mice with synthetic peptides unique to each protein. Both HgbA and HgbB were readily detected by Western blot analysis in N182 cells grown in the presence of hemoglobin as the sole source of heme, whereas expression of HgbC was found to be much less abundant than that of HgbA and HgbB. The use of these MAbs in a colony blot radioimmunoassay analysis revealed that expression of both HgbA and HgbB was subject to phase variation. PCR and nucleotide sequence analysis were used in conjunction with Western blot analyses to demonstrate that this phase variation involved the CCAA repeats in the hgbA and hgbB ORFs.
Cope
L D
LD
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9048, USA.
Hrkal
Z
Z
Hansen
E J
EJ
D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006193:Haemophilus influenzae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D006242:Haptoglobins / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D007501:Iron / Q000378:metabolism; D051379:Mice; D018895:Microsatellite Repeats; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D006242:Haptoglobins; D006454:Hemoglobins; C101107:HhuA protein, Haemophilus influenzae; D011993:Recombinant Fusion Proteins; C017206:hemoglobin-binding protein, bacteria; D006418:Heme; D007501:Iron