Characterization of two genes encoding the Mycobacterium tuberculosis ribonucleotide reductase small subunit

Area of Science:

• Molecular Biology
• Biochemistry
• Microbiology

Background:

• Mycobacterium tuberculosis possesses two genes, nrdF1 and nrdF2, encoding the small subunit (R2) of ribonucleotide reductase (RR).
• These R2 subunits share 71% amino acid identity and homology with Salmonella typhimurium R2F.
• Both R2 subunits are expressed in vivo in M. tuberculosis extracts.

Purpose of the Study:

• To investigate the expression and function of the two nrdF genes (nrdF1 and nrdF2) encoding ribonucleotide reductase R2 subunits in Mycobacterium tuberculosis.
• To determine the genetic organization and complementation capabilities of these genes in relation to the large subunit (nrdE).

Main Methods:

• Western blot analysis to detect R2 expression in M. tuberculosis extracts.
• Enzymatic assays using recombinant R2-2 and R1 subunits.
• Bacterial complementation assays using an E. coli nrdA(Ts) strain (E101) with M. tuberculosis nrdE and nrdF genes.
• Site-directed mutagenesis of a critical tyrosine residue in nrdF2.

Main Results:

• Recombinant M. tuberculosis R2-2 subunit is enzymatically active with the R1 subunit, and its activity is modulated by ATP and dATP.
• The nrdF1 and nrdF2 genes are not genetically linked to each other or to the nrdE gene.
• Simultaneous transformation with M. tuberculosis nrdE and nrdF2 complemented the E. coli strain E101, whereas nrdF1 did not; a mutated nrdF2 also failed to complement.

Conclusions:

• The nrdEF system in M. tuberculosis appears to encode the biologically active ribonucleotide reductase.
• M. tuberculosis is unique among identified bacterial species in that its class I RR subunits are not arranged in an operon.
• The nrdF2 gene, in conjunction with nrdE, is essential for functional ribonucleotide reductase activity in M. tuberculosis.