Genes involved in Haemophilus influenzae type b capsule expression are part of an 18-kilobase tandem duplication.

Abstract

Encapsulated Haemophilus influenzae type b produce nonencapsulated variants at high frequency (0.1-0.3%). Cosmid cloning was used to investigate the genetic mechanism responsible for this instability. Analysis of three independently derived cosmid clones showed that the b+ parental strain contains an 18-kilobase tandem duplication of genes involved in type b capsule expression. Loss of one complete copy of the 18-kilobase tandem duplication occurred following transformation of the cosmid clones into Rec', but not Rec , Escherichia coli, and inH. influenzae strains that had spontaneously lost capsule expression. These results suggest that high-frequency loss of type b capsule expression is due to rec-dependent recombination between the two copies of the 18-kilobase tandem repeat. This is further supported by our finding that introduction of the H. influenzae rec-) mutation METHODSBacterial Strains. The source of DNA for the H. influenzae cosmid library was the prototypic type b strain Eagan (6). The Rec-E. coli strain HB101 and the Rec+ E. coli strain C600 are described elsewhere (7). A rec-J streptomycin-resistant (SmR) H. influenzae strain (8) was kindly provided by J.Setlow.Cosmid Cloning. High molecular weight H. influenzae chromosomal DNA was isolated essentially as described by Smith (9), but the EDTA concentration of the resuspension buffer was reduced to 5 mM to prevent premature lysis. This procedure employs gentle phenol extraction, and there is no ethanol precipitation before the Sau3A cleavage reaction. Residual phenol was removed by dialysis, and the DNA was partially cleaved with Sau3A (Boehringer Mannheim). The DNA was phenol-extracted, butanol-extracted, and ethanolprecipitated prior to size fractionation in a 5-20% NaCl gradient (SW41 rotor, 35,000 rpm, 4.5 hr, 40C). Salt was removed by dialysis, and fractions containing DNA in the 30-to 50-kb size range were identified by electrophoresis in a 0.3% agarose gel. DNA from such fractions was mixed with BamHI-cleaved pHC79 (10) vector DNA at an insert/vector molar ratio of approximately 2:1. The DNAs were ligated using T4 DNA ligase (Bethesda Research Laboratories) and the ligated DNA was packaged using X DNA packaging extracts (also from Bethesda Research Laboratories). Recombinant clones were isolated on LB plates (7) containing ampicillin at 50 pug/ml. Colony Hybridization. Recombinant clones were prongreplicated to nitrocellulose filters, and the filters were incubated for 6-18 hr on LB plates containing ampicillin at 50 ,ug/ml. The colonies were lysed and neutralized according to Hill and Payne (11). The filters were prewashed, prehybridized, and hybridized as described (7). DNA Probes. 32P-labeled probes were prepared using a nick-translation kit (Bethesda Research Laboratories) and [a-32P] for cotransformation of rec-J using UV-sensitivity. PurificaAbbreviation: kb, kilobase(s). 1106The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accord...

Pharyngeal Colonization Dynamics of Haemophilus influenzae and Haemophilus haemolyticus in Healthy Adult Carriers

“…Furthermore, type b strains are more clonal than NTHI (44,53). Capsule-deficient, or b Ϫ , H. influenzae mutants carry the b-type cap region but do not have the bexA gene required for exportation of the capsule to the cell surface (13,24). To our knowledge, long-term colonization with b Ϫ mutants has not been documented, although b Ϫ mutants have been isolated from the throats of patients with type b infections (23).…”

Capsulation in distantly related strains of Haemophilus influenzae type b: genetic drift and gene transfer at the capsulation locus

“…Total cellular DNA was prepared from RM926 as previously described (11) and partially digested with restriction endonuclease Sau3AI. The resulting fragments were size fractionated by centrifugation through a sucrose density gradient, and the fractions containing 12-to 25-kilobase (kb) fragments were selected.…”

Capsulation gene loss and 'rescue' mutations during the Cap+ to Cap- transition in Haemophilus influenzae type b

“…Otherwise the probing patterns are identical. This change in probe hybridization has been shown to correspond to reduction of cap to a single copythe residual cap locus -through recombination between the direct repeats (Hoiseth et al, 1986). In this rearrangement, the 9 kb EcoRI fragment spanning the junction between the cap repeats is lost, and with it the 1.2 kb bridge region (Fig.…”

“…This locus contains two directly repeated 17 kb DNA segAbbrwiations : b+, type b capsulate; (s)BHI, (supplemented) brainheart infusion; cap, capsulation locus; FITC, fluorescein isothiocyanate; PRP, ply-ribosyl ribitol phosphate. ments separated by a unique region of approximately 1.2 kb -the bridge region (Hoiseth et al, 1986;. When EcoRI digests of total genomic DNA of either class I or class I1 variants are probed with pSKH2, containing DNA cloned from the cap locus (Hoiseth et al, 1985), a 9 kb band seen in the capsulate (b+) parent strain is lost.…”