Purification and biochemical characterization of DnaK and its transcriptional activator RpoH from Neisseria gonorrhoeae

Shalini Narayanan, Simone A. Beckham, John K. Davies, Anna Roujeinikova

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DnaK plays a central role in stress response in the important human pathogen Neisseria gonorrhoeae. The genes encoding the DnaK chaperone machine (DnaK/DnaJ/GrpE) in N. gonorrhoeae are transcribed from RpoH (σ32)-dependent promoters. In this study, we cloned, purified and biochemically characterised N. gonorrhoeae DnaK (NgDnaK) and RpoH. The NgDnaK and RpoH sequences are 73 and 50 % identical to the sequences of their respective E. coli counterparts. Similar to EcDnaK, nucleotide-free NgDnaK exists as a mix of monomers, dimers and higher oligomeric species in solution, and dissociates into monomers on addition of ATP. Like E. coli σ32, RpoH of N. gonorrhoeae is monomeric in solution. Kinetic analysis of the basal ATPase activity of purified NgDnaK revealed a Vmax of 193 pmol phosphate released per minute per microgram DnaK (which is significantly higher than reported basal ATPase activity of EcDnaK), and the turnover number against ATP was 0.4 min−1 under our assay conditions. Nucleotide-free NgDnaK bound a short model substrate, NR-peptide, with micromolar affinity close to that reported for EcDnaK. Our analysis showed that interaction between N. gonorrhoeae RpoH and DnaK appears to be ATP-dependent and non-specific, in stark contrast to the E. coli DnaK system where σ32 and DnaK interact as monomers even in the absence of ATP. Sequence comparison showed that the DnaK-binding site of σ32 is not conserved in RpoH. Our findings suggest that the mechanism of DnaK/RpoH recognition in N. gonorrhoeae is different from that in E. coli.
Original languageEnglish
Pages (from-to)7945-7953
Number of pages9
JournalMolecular Biology Reports
Issue number12
Publication statusPublished - Dec 2014


  • neisseria gonorrhoeae
  • stress response
  • DnaK
  • RpoH

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