Article

Synchronous replication initiation in novel Mycobacterium tuberculosis dna A cold-sensitive mutants

Biomedical Research, Department of Biochemistry, The University of Texas Health Science Center at Tyler, Tyler, TX 75708-3154, USA.
Molecular Microbiology (Impact Factor: 5.03). 12/2008; 71(2):291-304. DOI: 10.1111/j.1365-2958.2008.06523.x
Source: PubMed

ABSTRACT 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.

Download full-text

Full-text

Available from: Malini Rajagopalan, Dec 19, 2014
0 Followers
 · 
121 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The genetic and biochemical aspects of the essential Mycobacteriumtuberculosis MtrAB two-component regulatory signal transduction (2CRS) system have not been extensively investigated. We show by bacterial two-hybrid assay that the response regulator (RR) MtrA and the sensor kinase MtrB interact. We further demonstrate that divalent metal ions [Mg²+, Ca²+ or both] promote MtrB kinase autophosphorylation activity, but only Mg²+ promotes phosphotransfer to MtrA. Replacement of the conserved aspartic acid residues at positions 13 and 56 with alanine (D13A), glutamine (D56E) or asparagine (D56N) prevented MtrA phosphorylation, indicating that these residues are important for phosphorylation. The MtrA(D56E) and MtrA(D13A) proteins bound to the promoter of fbpB, the gene encoding antigen 85B protein, efficiently in the absence of phosphorylation, whereas MtrA(D56N) did not. We also show that M.tuberculosismtrA merodiploids overproducing MtrA(D13A), unlike cells overproducing wild-type MtrA, grow poorly in nutrient broth and show reduced expression of fbpB. These latter findings are reminiscent of a phenotype associated with MtrA overproduction during intramacrophage growth. Our results suggest that MtrA(D13A) behaves like a constitutively active response regulator and that further characterization of mtrA merodiploid strains will provide valuable clues to the MtrAB system.
    Plasmid 02/2011; 65(3):210-8. DOI:10.1016/j.plasmid.2011.01.002 · 1.76 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The design of a 5 Volts fully integrated magnetic sensor able to operate up to 270°C is presented. Fabricated in a Partially Depleted (PD) 1 μm SOI process, this monolithic sensor comprises a resistive Hall plate, an amplifier stage and an A/D converter delivering a temperature stabilized 8-bit digital readout of the magnetic field. This circuit uses analog techniques for continuous compensation of temperature. Design issues inherent to partially depleted SOI, as well as constraints due to high temperature, are discussed.
    Custom Integrated Circuits Conference, 2002. Proceedings of the IEEE 2002; 02/2002
  • [Show abstract] [Hide abstract]
    ABSTRACT: A 500-dpi cellular-logic processing array performs all fingerprint identification steps on one chip, from image acquisition, enhancement, to verification. Morphological functions for executing these steps are implemented in a 30×50-μm<sup>2</sup> processing unit. A single-cycle datapath and a shared logic structure enable compact implementation of the processing unit. A fabricated 224×256-pixel fingerprint identification LSI demonstrates fully-functional image processing and practical accuracy of identification.
    Custom Integrated Circuits Conference, 2002. Proceedings of the IEEE 2002; 02/2002