Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis.
Two genes with sequence homology to those encoding protein tyrosine phosphatases were cloned from genomic DNA of Mycobacterium tuberculosis H(37)Rv. The calculated molecular masses of these two putative tyrosine phosphatases, designated MPtpA and MPtpB, were 17. 5 and 30 kDa, respectively. MPtpA and MPtpB were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The affinity-purified proteins dephosphorylated the phosphotyrosine residue of myelin basic protein (MBP), but they failed to dephosphorylate serine/threonine residues of MBP. The activity of these phosphatases was inhibited by sodium orthovanadate, a specific inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine/threonine phosphatases. Mutations at the catalytic site motif, cysteine 11 of MPtpA and cysteine 160 of MPtpB, abolished enzyme activity. Southern blot analysis revealed that, while mptpA is present in slow-growing mycobacterial species as well as fast-growing saprophytes, mptpB was restricted to members of the M. tuberculosis complex. These phosphatases were present in both whole-cell lysates and culture filtrates of M. tuberculosis, suggesting that these proteins are secreted into the extracellular medium. Since tyrosine phosphatases are essential for the virulence of several pathogenic bacteria, the restricted distribution of mptpB makes it a good candidate for a virulence gene of M. tuberculosis.
Koul
A
A
Department of Molecular Biology, Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany.
Choidas
A
A
Treder
M
M
Tyagi
A K
AK
Drlica
K
K
Singh
Y
Y
Ullrich
A
A
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D015153:Blotting, Western / Q000379:methods; D020134:Catalytic Domain; D003001:Cloning, Molecular; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000201:enzymology; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D019000:Phosphotyrosine / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; C414465:MptpA protein, Mycobacterium tuberculosis; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases