Synchronous replication initiation in novel Mycobacterium tuberculosis dnaA cold‐sensitive mutants

Abstract

SummaryThe 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°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 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°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 dnaA promoter remains active during the replication period and that the DnaA protein is able to interact with ATP.

A Parallel Adder Coordinates Mycobacterial Cell-Cycle Progression and Cell-Size Homeostasis in the Context of Asymmetric Growth and Organization

“…B period was disproportionatly extended in slow growth, particularly in small cells (Figures 4A&S3D). Additionally, BCG spent an average of 9.4 hours in C, similar to previous measurements in Mycobacterium tuberculosis (Figure S3D)[29]. BCG and carbon limited M. smegmatis birth lengths were more variable than M. smegmatis growing in rich medium, with CVs of 20% and 22%, respectively, compared to 19% for M. smegmatis in rich medium (Figures 3B, 4B, S3A&S3E), and 12% for E. coli [8].…”

Intracellular Locations of Replication Proteins and the Origin of Replication during Chromosome Duplication in the Slowly Growing Human Pathogen Helicobacter pylori

“…A low frequency of cells showing replication foci may be the result of an unusually long (ϳ4.8-h) generation time with a short C period but an extended D period; this implies that the replication rate for the H. pylori chromosome is compa- rable to that of E. coli notwithstanding its long generation time. This is in contrast to the long C period (estimated to be 11 h) (67) for the slowly growing human pathogen Mycobacterium tuberculosis (doubling time, 24 h).…”

A DinB Ortholog Enables Mycobacterial Growth under dTTP-Limiting Conditions Induced by the Expression of a Mycobacteriophage-Derived Ribonucleotide Reductase Gene

“…A similar effect could be produced by treatment of cells with rifampin, a transcriptional inhibitor that can inhibit replication by preventing initiation (29,33,44). Given the similarity in the phenotypes observed between gene 50 expression and rifampin treatment, it is tempting to speculate that under dTTP-deficient conditions such as those described here, replication initiation in mycobacteria is inhibited.…”

“…On the contrary, a minor dip was observed. For the sake of control, a similar experiment was repeated with M. smegmatis mc 2 155 cells in which growth inhibition was induced with rifampin, a well-known replication initiation inhibitor (29,33), at a concentration of 60 g/ml (Fig. 2C, red trace).…”