Novel insertion and deletion mutants of RpoB that render Mycobacterium smegmatis RNA polymerase resistant to rifampicin-mediated inhibition of transcription.
The startling increase in the occurrence of rifampicin (Rif) resistance in the clinical isolates of Mycobacterium tuberculosis worldwide is posing a serious concern to tuberculosis management. The majority of Rif resistance in bacteria arises from mutations in the RpoB subunit of the RNA polymerase. We isolated M. smegmatis strains harbouring either an insertion (6 aa) or a deletion (10 aa) in their RpoB proteins. Although these strains showed a compromised fitness for growth in 7H9 Middlebrook medium, their resistance to Rif was remarkably high. The attenuated growth of the strains correlated with decreased specific activities of the RNA polymerases from the mutants. While the RNA polymerases from the parent or a mutant strain (harbouring a frequently occurring mutation, H442Y, in RpoB) were susceptible to Rif-mediated inhibition of transcription from calf thymus DNA, those from the insertion and deletion mutants were essentially refractory to such inhibition. Three-dimensional structure modelling revealed that the RpoB amino acids that interact with Rif are either deleted or unable to interact with Rif due to their unsuitable spatial positioning in these mutants. We discuss possible uses of the RpoB mutants in studying transcriptional regulation in mycobacteria and as potential targets for drug design.
Malshetty
Vidyasagar
V
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Kurthkoti
Krishna
K
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
China
Arnab
A
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Mallick
Bratati
B
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Yamunadevi
Subburaj
S
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
Sang
Pau Biak
PB
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Srinivasan
Narayanaswamy
N
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
Nagaraja
Valakunja
V
Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India.|Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Varshney
Umesh
U
Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India.|Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
D000595:Amino Acid Sequence; D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D020102:Mycobacterium smegmatis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011487:Protein Conformation; D012293:Rifampin / Q000494:pharmacology*; D017384:Sequence Deletion; D014158:Transcription, Genetic
D000904:Antibiotics, Antitubercular; D019384:Nucleic Acid Synthesis Inhibitors; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D012293:Rifampin