Cloning and characterization of the meningococcal polyphosphate kinase gene: production of polyphosphate synthesis mutants.
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.
Tinsley
C R
CR
Laboratory of Bacterial Pathogenesis and Immunology, Rockefeller University, New York, New York 10021-6399, USA.
Gotschlich
E C
EC
D000595:Amino Acid Sequence; D001483:Base Sequence; D001770:Blood Bactericidal Activity; D003001:Cloning, Molecular; D005798:Genes, Bacterial / Q000235:genetics*; D006801:Humans; D007501:Iron / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009343:Neisseria / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity; D009344:Neisseria gonorrhoeae / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity; D009345:Neisseria meningitidis / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000235:genetics* / Q000502:physiology; D011122:Polyphosphates / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014169:Transformation, Bacterial
D011122:Polyphosphates; D007501:Iron; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase