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).
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