Date of Award

2005

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Abstract

Many bacteria store energy and phosphate in the form of polyphosphate. Polyphosphate is involved in mRNA processing and degradation and is a regulator for stress responses in certain bacteria. The synthesis of polyphosphate is mediated by the reversible action of polyphosphate kinase (PPK). In H. pylori , accumulation of polyphosphate has been observed under anaerobic conditions and during formation of unculturable, coccoid forms at stationary phase. Previous observations in our laboratory showed that H. pylori significantly reduced the rate of phosphate uptake under acidic conditions, indicating that polyphosphates may be an alternative phosphate source during growth in acid. This study focuses on the role of polyphosphates and PPK of H. pylori with respect to acidic adaptation and survival. A knock-out mutation was performed by homologous recombination. A suicide vector (pBCα3) carrying a 700 bp fragment of polyphosphate kinase gene ( ppk ) and a kanamycin resistant cassette was transformed into H. pylori . Homologous recombination with the chromosomal copy of the ppk gene results in the insertion of the pBCα3 vector into the ppk gene. This insertion creates a functional disruption of the ppk gene. The transformed cells were selected based on resistance to kanamycin. Mutation was verified using PCR and plasmid rescue strategy, followed by sequencing. Polyphosphates were extracted from wild type and ppk mutant strains grown at various pHs. Cells were taken periodically and polyphosphate extraction performed using CTAB and BaCl 2 , followed by metachromatic measurement with toluidine blue. Mutant cells were not culturable, but remained viable under neutral and acidic pH. Wild type cells were culturable at neutral pH and pH 5.0; though the number of culturable cells was low under acidic conditions, they appeared metabolically active. Polyphosphate content in mutant cells was significantly higher than wild type after 24 h in pH 4.5 media. Longer incubation in acidic media resulted in higher polyphosphate accumulation in the wild type cells, but still significantly higher in mutant cells. The presence of polyphosphate in H. pylori lacking ppk indicates that other polyphosphate-synthesizing enzymes occur. Accumulation of polyphosphate in mutant cells after 24 h at pH 4.5 may be due to impaired reverse function of PPK or regulation of other phosphatase activities. Results from growth experiments indicated that polyphosphate may be necessary for survival in an acidic environment. Our results are consistent with the observation that polyphosphates accumulate in unculturable cells, however results from the PPK mutant show that ppk may not be the source of the accumulation.

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