The ptsH gene from Bacillus thuringiensis israelensis characterization of a new phosphorylation site on the protein HPr

Sharik R. Khan, Josef Deutscher, Ram A. Vishwakarma, Vicente Monedero, Nirupama B. Bhatnagar

Research output: Contribution to journalArticlepeer-review


The ptsH gene from Bacillus thuringiensis israelensis (Bti), coding for the phosphocarrier protein HPr of the phosphotransferase system has been cloned and overexpressed in Escherichia coli. Comparison of its primary sequence with other HPr sequences revealed that the conserved His15 and Ser46 residues were shifted by one amino acid and located at positions 14 and 45, respectively. The biological activity of the protein was not affected by this change. When expressed in a Bacillus subtilis ptsH deletion strain, Bti HPr was able to complement the functions of HPr in sugar uptake and glucose catabolite repression of the gnt and iol operons. A modified form of HPr was detected in Bti cells, and also when Bti ptsH was expressed in E. coli or B. subtilis. This modification was identified as phosphorylation, because alkaline phosphatase treatment converted the modified form to unmodified HPr. The phosphoryl bond in the new form of in vivo phosphorylated HPr was resistant to alkali treatment but sensitive to acid treatment, suggesting phosphorylation at a histidine residue. Replacement of His14 with alanine in Bti HPr prevented formation of the new form of phosphorylated HPr. The phosphorylated HPr was stable at 60 °C, in contrast with HPr phosphorylated at the Nδ1 position of His14 with phosphoenolpyruvate and enzyme I. 31P-NMR spectroscopy was used to show that the new form of P-HPr carried the phosphoryl group bound to the Nε2 position of His14 of Bti HPr. Phosphorylation of HPr at the novel site did not occur when Bti HPr was expressed in an enzyme I-deficient B. subtilis strain. In addition, P-(Nε2)His-HPr did not transfer its phosphoryl group to the purified glucose-specific enzyme IIA domain of B. subtilis.

Original languageEnglish (US)
Pages (from-to)521-530
Number of pages10
JournalEuropean Journal of Biochemistry
Issue number3
StatePublished - 2001
Externally publishedYes


  • Bacillus thuringiensis israelensis
  • HPr
  • Histidine phosphorylation
  • Phosphorylation
  • Phosphotransferase system

ASJC Scopus subject areas

  • Biochemistry


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