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Synchronous replication initiation in novel Mycobacterium tuberculosis dna A cold‐sensitive mutants
Author(s) -
Nair Naveen,
Dziedzic Renata,
Greendyke Rebecca,
Muniruzzaman Syed,
Rajagopalan Malini,
Madiraju Murty V.
Publication year - 2009
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2008.06523.x
Subject(s) - biology , mycobacterium tuberculosis , mutant , dna replication , replication (statistics) , dna , genetics , virology , microbiology and biotechnology , computational biology , tuberculosis , gene , medicine , pathology
Summary The genetic aspects of ori C replication initiation in Mycobacterium tuberculosis are largely unknown. A two‐step genetic screen was utilized for isolating M. tuberculosis dna A cold‐sensitive (cos) mutants. First, a resident plasmid expressing functional dna A integrated at the att B locus in dna A null background was exchanged with an incoming plasmid bearing a mutagenized dna A gene. Next, the mutants that were defective for growth at 30°C, a non‐permissive temperature, but resumed growth and DNA synthesis when shifted to 37°C, a permissive temperature, were subsequently selected. Nucleotide sequencing analysis located mutations to different regions of the dna A gene. Modulation of the growth temperatures led to synchronized DNA synthesis. The dna A expression under synchronized DNA replication conditions continued to increase during the replication period, but decreased thereafter reflecting autoregulation. The dna Acos mutants at 30°C were elongated suggesting that they may possibly be blocked during the cell division. The DnaA115 protein is defective in its ability to interact with ATP at 30°C, but not at 37°C. Our results suggest that the optimal cell cycle progression and replication initiation in M. tuberculosis requires that the dna A promoter remains active during the replication period and that the DnaA protein is able to interact with ATP.