Synchronous replication initiation in novel Mycobacterium tuberculosis dnaA cold-sensitive mutants.
The genetic aspects of oriC replication initiation in Mycobacterium tuberculosis are largely unknown. A two-step genetic screen was utilized for isolating M. tuberculosis dnaA cold-sensitive (cos) mutants. First, a resident plasmid expressing functional dnaA integrated at the attB locus in dnaA null background was exchanged with an incoming plasmid bearing a mutagenized dnaA gene. Next, the mutants that were defective for growth at 30 degrees C, a non-permissive temperature, but resumed growth and DNA synthesis when shifted to 37 degrees C, a permissive temperature, were subsequently selected. Nucleotide sequencing analysis located mutations to different regions of the dnaA gene. Modulation of the growth temperatures led to synchronized DNA synthesis. The dnaA expression under synchronized DNA replication conditions continued to increase during the replication period, but decreased thereafter reflecting autoregulation. The dnaAcos mutants at 30 degrees 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 degrees C, but not at 37 degrees C. Our results suggest that the optimal cell cycle progression and replication initiation in M. tuberculosis requires that the dnaA promoter remains active during the replication period and that the DnaA protein is able to interact with ATP.
Nair
Naveen
N
Biomedical Research, Department of Biochemistry, The University of Texas Health Science Center at Tyler, Tyler, TX 75708-3154, USA.
Dziedzic
Renata
R
Greendyke
Rebecca
R
Muniruzzaman
Syed
S
Rajagopalan
Malini
M
Madiraju
Murty V
MV
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle; D003080:Cold Temperature; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000166:cytology / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D000255:Adenosine Triphosphate