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Infection and Immunity, July 2000, p. 4092-4101, Vol. 68, No. 7
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Detection of Phase Variation in Expression of Proteins Involved in Hemoglobin and Hemoglobin-Haptoglobin Binding by Nontypeable Haemophilus influenzae

Leslie D. Cope,1 Zbynek Hrkal,2 and Eric J. Hansen1,*

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9048,1 and Department of Cellular Biochemistry, Institute of Haemotology and Blood Transfusion, Prague, Czech Republic2

Received 14 January 2000/Returned for modification 14 February 2000/Accepted 23 March 2000

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.

* Corresponding author. Mailing address: Department of Microbiology, Hamon Biomedical Research Building, NA6.200, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX 75390-9048. Phone: (214) 648-5974. Fax: (214) 648-5905. E-mail: hansen01{at}utsw.swmed.edu.

Infection and Immunity, July 2000, p. 4092-4101, Vol. 68, No. 7
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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