Cloning of two distinct hemolysin genes from Porphyromonas (Bacteroides) gingivalis in Escherichia coli

T. Karunakaran, S. C. Holt

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

Hemolysin is considered a potent virulence factor in a large number of Gram-positive and Gram-negative bacterial pathogens. The hemolysin produced by the oral pathogen Porphyromonas gingivalis functions to provide the cell with its required heme-containing molecules for growth in the periodontal pocket. Two distinct P. gingivalis genes, each of which confers a hemolytic phenotype in Escherichia coli, were isolated by screening genomic DNA libraries of P. gingivalis on sheep blood agar plates. The results obtained from physical maps and Southern blots indicated a considerable degree of divergence in the nucleotide sequences of these two genes. Maxicell analyses of the recombinant plasmids in E. coli suggested that plasmid pPGH5 encoded a polypeptide of molecular weight 48 kDa, while an 18-kDa polypeptide was obtained with pPGH1 and pPGH7. When E. coli harboring these hemolysin genes was subjected to iron starvation, the levels of hemolysin activity increased. Biochemical characterization of hemolytic activities indicated that the activity of both hemolysins was inhibited by Mg2+ and Ca2+; but not by EDTA. Elevated levels of hemolytic activity were obtained from the E. coli recombinant strains in the presence of glutathione, DTT and 2-mercaptoethanol. Cholesterol inhibited the activity.

Original languageEnglish (US)
Pages (from-to)37-49
Number of pages13
JournalMicrobial Pathogenesis
Volume15
Issue number1
DOIs
StatePublished - Jan 1 1993

Keywords

  • Cloning
  • Escherichia coli
  • Expression
  • Hemolysin
  • Porphyromonas gingivalis

ASJC Scopus subject areas

  • Microbiology
  • Infectious Diseases

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