Evidence towards the role of arylamine N-acetyltransferase in Mycobacterium smegmatis and development of a specific antiserum against the homologous enzyme of Mycobacterium tuberculosis.
Arylamine N-acetyltransferase (NAT) in humans inactivates the anti-tubercular drug isoniazid (INH). Homologues of human NAT are present in Mycobacterium tuberculosis and Mycobacterium smegmatis, where they can acetylate, and hence inactivate, INH. The in vivo role of mycobacterial NAT is not known but heterologous expression of the M. tuberculosis gene increases the INH resistance. The 0.85 kb nat gene is part of a gene cluster in M. smegmatis. The gene is transcribed as a large, 7.5 kb mRNA as demonstrated by Northern analysis. A nat knockout strain of M. smegmatis was generated by targeted disruption. The new strain was confirmed to be devoid of NAT activity. The growth of the knockout strain is considerably delayed compared with the wild-type, due to an extended lag phase. The knockout mutant has an increased sensitivity to INH as would be predicted. The NATs from M. smegmatis and M. tuberculosis have a high degree of homology, except in the region of the C terminus. A specific polyclonal antiserum raised against recombinant NAT protein from M. tuberculosis is described that recognizes a stretch of about twenty residues within the C terminus of M. tuberculosis NAT. This highly specific antiserum will enable comparison of nat expression between isolates of M. tuberculosis.
Payton
M
M
University Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK.
Gifford
C
C
Schartau
P
P
Hagemeier
C
C
Mushtaq
A
A
Lucas
S
S
Pinter
K
K
Sim
E
E
D000070:Acebutolol; D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology*; D000918:Antibody Specificity; D000995:Antitubercular Agents / Q000494:pharmacology; D001191:Arylamine N-Acetyltransferase / Q000235:genetics / Q000276:immunology*; D024881:Drug Resistance, Bacterial; D007538:Isoniazid / Q000494:pharmacology; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000276:immunology; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D017386:Sequence Homology, Amino Acid
D000907:Antibodies, Bacterial; D000995:Antitubercular Agents; D011994:Recombinant Proteins; D000070:Acebutolol; D001191:Arylamine N-Acetyltransferase; D007538:Isoniazid