1887

Abstract

A urease-negative serotype A strain of (B-4587) was isolated from the cerebrospinal fluid of an immunocompetent patient with a central nervous system infection. The gene encoding urease failed to complement the mutant phenotype. Urease-positive clones of B-4587 obtained by complementing with a genomic library of strain H99 harboured an episomal plasmid containing DNA inserts with homology to the gene of . The gene harboured by these plasmids was named since it enabled the transformants to grow on media containing urea as the sole nitrogen source while the transformants with an empty vector failed to grow. Transformation of strain B-4587 with a plasmid construct containing a truncated version of the gene failed to complement the urease-negative phenotype. Disruption of the native gene in a wild-type serotype A strain H99 and a serotype D strain LP1 of resulted in the inability of the strains to grow on media containing urea as the sole nitrogen source, suggesting that the gene product is involved in the utilization of urea by the organism. Virulence in mice of the urease-negative isolate B-4587, the urease-positive transformants containing the wild-type copy of the gene, and the urease-negative vector-only transformants was comparable to that of the H99 strain of regardless of the infection route. Virulence of the disruption stain of H99 was slightly reduced compared to the wild-type strain in the intravenous model but was significantly attenuated in the inhalation model. These results indicate that the importance of urease activity in pathogenicity varies depending on the strains of used and/or the route of infection. Furthermore, this study shows that complementation cloning can serve as a useful tool to functionally identify genes such as that have otherwise been annotated as hypothetical proteins in genomic databases.

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2006-12-01
2019-10-19
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Protein sequence alignment between the gene of and the gene of . The third row reflects the consensus protein sequence between the two strains. [ PDF] (35 kb) Transcriptional analysis of urease ( ) and genes by RT-PCR in the wild type strain H99, the urease-negative clinical isolate B-4587, the disruption strain, and the -complemented strain B-4587C. Oligonucleotides used as primers for the analysis were designed from DNA sequences of the urease gene , the gene, and the constitutively expressed gene as control. [ PDF] (126 kb)

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Protein sequence alignment between the gene of and the gene of . The third row reflects the consensus protein sequence between the two strains. [ PDF] (35 kb) Transcriptional analysis of urease ( ) and genes by RT-PCR in the wild type strain H99, the urease-negative clinical isolate B-4587, the disruption strain, and the -complemented strain B-4587C. Oligonucleotides used as primers for the analysis were designed from DNA sequences of the urease gene , the gene, and the constitutively expressed gene as control. [ PDF] (126 kb)

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