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Molecular cloning and functional characterization of fumarases C in Neisseria species

Liuh Ling Goh1, Timothy Barkham, Tiow Suan Sim

  • 1Department of Microbiology, Faculty of Medicine, National University of Singapore, MD4A, 5 Science Drive 2, 117597, Singapore.

Antonie Van Leeuwenhoek
|April 2, 2005
PubMed

Insights

Researchers identified and characterized the fumarase C (FUMC) enzyme in Neisseria species. This enzyme is crucial for the TCA cycle and may play a role in bacterial survival and virulence.

Area of Science:

  • Microbiology
  • Enzymology
  • Molecular Biology

Background:

  • Fumarase (FUMC) is a key enzyme in the tricarboxylic acid (TCA) cycle.
  • FUMC is implicated in microbial virulence and survival under stress conditions.

Purpose of the Study:

  • To identify, clone, and characterize the fumC genes encoding FUMC in Neisseria meningitidis, N. gonorrhoeae, and N. subflava.
  • To analyze the conservation, phylogenetic relationship, and enzymatic activity of neisserial FUMCs.

Main Methods:

  • Homology-based analysis for gene identification.
  • Polymerase chain reaction (PCR) for gene cloning and sequencing.
  • Gene expression in Escherichia coli and protein purification via affinity chromatography.
  • Enzymatic activity assays.

Main Results:

  • Identified and sequenced fumC genes from three Neisseria species.
  • Neisserial FUMCs exhibit high amino acid sequence conservation (97.8-98.7%).
  • Phylogenetic analysis shows divergence from other microbial, rat, and human Class II fumarases.
  • Purified recombinant FUMCs displayed high catalytic activity (120-180 U/mg).

Conclusions:

  • The cloned genes encode functional fumarase C enzymes in Neisseria species.
  • Neisserial FUMCs are distinct from Class II fumarases found in other organisms.
  • Further studies involving gene disruption are needed to elucidate the physiological role of FUMC in Neisseria pathogenesis.

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