The product of the ferric uptake regulation (and genes (12, 19). pneumonia in patients with pre-existing pulmonary disease. More rarely, it also causes bacteremia and meningitis (10, 17, 23). Otitis media Lodoxamide affects approximately 70% of all children by the age of three, with many children experiencing recurrent disease (2). Chronic otitis media can lead to hearing, speech, and cognitive impairment in children, since it tends to occur at a time when language is usually developing. The incidence of is clearly needed. Iron restriction is usually a general host defense mechanism against microbial pathogens, and in the human host, iron is usually sequestered by transferrin, lactoferrin, hemoglobin, and other complex molecules. A number of bacterial species, including (22), (9), Lodoxamide (33), (1), (29, 33), (8), and spp. (34), have been shown to express outer membrane proteins which specifically bind human lactoferrin. utilize both transferrin and lactoferrin binding complexes, OBSCN and a single lactoferrin binding protein of 105 kDa was originally recognized in these organisms (33). The genes from and have been cloned and sequenced (1, 27), but until recently there was no evidence for the presence of an gene (3, 13, 25, 28). We statement here the cloning and sequencing of the lactoferrin binding protein genes and otitis media clinical isolates 4223 and 3 were kindly provided by T. Murphy (State University or college of New York, Buffalo, N.Y.), strain Q8 was a gift from M. Bergeron (University or college of Laval, Laval, Quebec, Canada), strain VH19 was provided by V. Howie (University or college of Texas, Galveston, Tex.), strain H-04 was from G. D. Campbell (Louisiana State University or college, Shreveport, La.), and strain LES-1 was obtained from L. E. Stenfors (University or college of Tromso, Tromso, Norway). strains were managed on Mueller-Hinton agar (Becton Dickinson, Cockeysville, Md.) or produced in brain heart infusion (BHI) medium (Difco, Detroit, Mich.) with or without the addition of ethylenediamine-di(strains were produced in YT medium supplemented with 50 g of ampicillin ml?1 Lodoxamide as required. Purification of LbpA and protein sequence determination. Native LbpA was purified by affinity chromatography under high-stringency conditions with immobilized lactoferrin (3). The purified LbpA protein was digested overnight with cyanogen bromide; then, fragments were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and submitted for sequence analysis on an ABI model 477A protein sequencer. A 13-kDa protein fragment was found to have the N-terminal sequence MVQYTYRKGKENKAH. Generation of a probe for screening libraries. A degenerate oligonucleotide primer was prepared based upon the Lodoxamide internal LbpA sequence 54223 and Q8. PCR amplification was performed in buffer made up of 10 mM Tris-HCl (pH 8.3), 50 mM potassium chloride, and 1.5 mM magnesium chloride. Each 100-l reaction mixture contained 1 g of chromosomal DNA, 0.1 g of each primer, 2.5 units of AmpliDNA polymerase (Perkin-Elmer Cetus, Foster City, Calif.), and 10 mM (each) deoxynucleoside triphosphate (Perkin-Elmer Cetus). The cycling conditions were 24 cycles at 94C for 1 min, 47C for 30 s, and 72C for 1 min. A specific band of 2.2 kb was amplified, and partial sequence analysis was done to ensure that the gene product was related to and was not (manuscript submitted). This 2.2-kb fragment was labelled with [-32P]dCTP (random-primed DNA labelling kit; Boehringer Mannheim) and used to screen genomic libraries. Construction and screening of genomic libraries. 4223 and Q8 EMBL3 libraries were prepared as explained previously (20). Briefly, chromosomal DNA was digested with LE392 cells were plated partially, and plaques had been raised onto nitrocellulose membranes for hybridization using the labelled 2.2-kb PCR fragment. Many putative phage clones had been from each collection, and phage DNA was.