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Cloning and characterization of the meningococcal polyphosphate kinase gene: production of polyphosphate synthesis
1Laboratory of Bacterial Pathogenesis and Immunology, Rockefeller University, New York, New York 10021-6399, USA.
Abstract:
The pathogenic Neisseria species accumulate polyphosphate to levels between 10 and 20% of their total phosphate content. However, the significance of this compound for the growth and pathogenicity of these species is not understood. A previous report (C.R. Tinsley, B.N. Manjula, and E.C. Gotschlich, Infect. Immun. 61:3703-3710, 1993) describes the purification of polyphosphate kinase, the enzyme responsible for synthesis of polyphosphate, from Neisseria meningitidis BNCV. By use of probes based on the amino acid sequence of the purified enzyme, the structural gene ppk has been cloned and sequenced. The coding sequence is 2,055 bp long and codes for a protein of 77.2 kDa. The open reading frame of the cloned gene was interrupted by the insertion of a kanamycin resistance cassette, and ppk mutants were obtained in both Neisseria gonorrhoeae and N. meningitidis by transformation with the recombinant plasmid. Amounts of polyphosphate in the ppk mutants were reduced to between 2 and 10% of wild-type levels. The mutants grew less vigorously than wild-type organisms in vitro and showed a striking increase in sensitivity to killing by human serum.
Insights
Pathogenic Neisseria species accumulate polyphosphate, a compound whose role was unclear. Scientists cloned the polyphosphate kinase gene (ppk) and created mutants, revealing polyphosphate
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Pathogenic Neisseria species accumulate significant amounts of polyphosphate (10-20% of total phosphate).
- The functional role of polyphosphate in Neisseria growth and pathogenicity remains largely unknown.
- Polyphosphate kinase (ppk) is the enzyme responsible for polyphosphate synthesis in these bacteria.
Purpose of the Study:
- To investigate the significance of polyphosphate accumulation for Neisseria growth and pathogenicity.
- To clone and characterize the structural gene (ppk) encoding polyphosphate kinase.
- To generate and analyze polyphosphate kinase (ppk) mutants in pathogenic Neisseria species.
Main Methods:
- Probes based on purified polyphosphate kinase amino acid sequence were used to clone and sequence the ppk gene from Neisseria meningitidis.
- The ppk gene's open reading frame was disrupted by inserting a kanamycin resistance cassette.
- Neisseria gonorrhoeae and N. meningitidis ppk mutants were generated via transformation with the recombinant plasmid.
Main Results:
- The ppk gene sequence is 2,055 bp, encoding a 77.2 kDa protein.
- Polyphosphate levels in ppk mutants were reduced to 2-10% of wild-type levels.
- Mutants exhibited reduced in vitro growth vigor and significantly increased sensitivity to human serum killing.
Conclusions:
- Polyphosphate kinase is essential for normal polyphosphate levels in pathogenic Neisseria.
- Polyphosphate plays a crucial role in the in vitro growth and serum resistance of Neisseria gonorrhoeae and N. meningitidis.
- Disruption of polyphosphate synthesis impacts Neisseria virulence factors, suggesting therapeutic targets.