Log₁₀ of the CFU per milliliter (with standard deviations) recovered from the spinal fluid of rabbits infected with H99 (solid circles) and the ure1 strain (open circles) plotted against time.

(a) Number of surviving mice that were infected via intravenous injection with the ure1 strain (open circles), H99 (solid circles), and the ure1+URE1-1 strain (inverted triangles) plotted against time. (b) Number of surviving mice that were infected via the inhalational route with the ure1 strain (open circles), H99 (solid circles), the ure1+URE1-1 strain (solid inverted triangles), and the ure1+URE1-2 strain (open inverted triangles) plotted against time.

Comparison of portions of the putative urease amino acid sequences from C. neoformans, C. immitis, S. pombe, and H. pylori. The sequences correspond to amino acids 395 to 408 and 507 to 519 of the C. neoformans urease. Conserved histidine residues thought to be important in binding to the nickel metallocenter (amino acid 400), binding to the substrate (amino acid 402), and catalysis (amino acid 512) are in boldface and marked with a star.

(A) Restriction map of the genomic fragment containing the urease gene (URE1). Pertinent restriction enzyme sites are shown. The small bar above URE1 denotes the area that was originally amplified using primers derived from conserved areas of bacterial urease coding sequences. (B) The SalI genomic fragment containing URE1 that was subcloned to a plasmid and used to create the disruption construct. This fragment was digested with BglII, and the deleted fragment containing most of URE1 was replaced with ADE2. The resulting construct (C) was used in the gene disruption studies. The solid arrows flanking the BglII sites designate the sites of the PCR primers that were used to screen for disruption of the native gene, and the expected sizes of the amplicons with the native and disrupted genes as templates are noted (B and C, respectively). The ADE2 fragment that was used to replace the deleted portion of URE1 is shaded in panel C. (D) Construct used to complement ure1 strain to urease positivity. A hygromycin B resistance cassette (Hyg) was ligated into the plasmid containing the SalI fragment and used as a selectable marker.

(a) Agarose gel of PCR products using primers Bgl5 and Bgl3 (Fig. 1) and genomic DNA from strain H99 and the ure1 and ure1+URE1-1 (Rec) strains as the template. The amplicon of the native URE1 fragment from H99 is 1,954 bp, whereas the amplified fragment from the ure1 strain is 3,090 bp. The displacement of the ure1 band suggests that the native URE1 was disrupted. The amplicons from the ure1+URE1-1 strain demonstrate the disrupted native locus as well as a band corresponding to the wild-type copy of URE1 used to reconstitute the strain to urease positivity. Size standards in base pairs are indicated to the left of the gel. (b) Southern blot of BamHI/EcoRI-digested genomic DNA from H99 (A), the ure1 strain (B), the ure1+URE1-1 strain (C), and the ure1+URE1-2 strain (D) and of BamHI-digested genomic DNA from the ure1+URE1-1 strain (E) and the ure1+URE1-2 strain (F). The blot was probed with a labeled EcoRI-BamHI genomic fragment containing a portion of URE1 (Fig. 1A). The hybridizing band seen in the ure1 strain is displaced compared to that of H99 suggesting replacement of the native urease gene in the ure1 strain with the disruption construct. The hybridization patterns in the two reconstituted strains demonstrate the presence of the disrupted urease gene as well as copies of the reconstitution construct that have integrated into the genome at different locations. Size standards in base pairs are indicated to the left of the gel. (c) Northern blots of total RNA obtained from H99, the ure1 strain, and the ure1+URE1-1 strain (Rec) probed with URE1 (left) and the actin gene (right).