Conditional expression of Mycobacterium smegmatis dnaA, an essential DNA replication gene.

Abstract

To begin to understand the role of Mycobacterium smegmatis dnaA in DNA replication, the dnaA gene was characterized at the genetic level. Western analyses revealed that DnaA accounts for approximately 0.18% of the total cellular protein during both the active and stationary growth periods. Expression of antisense dnaA RNA reduced viability, indicating that dnaA is an essential gene in replication. To further understand the role(s) of dnaA in replication, a conditionally expressing strain was constructed in which expression of dnaA was controlled by acetamide. Growth in the presence of 0.2% acetamide elevated the intracellular levels of DnaA and increased cell length, but did not affect viability. Visualization of DNA by fluorescence microscopy revealed that DnaA-overproducing cells were multinucleoidal, indicating a loss of synchrony between the replication and cell-division cycles. Withdrawal of acetamide resulted in the depletion of the intracellular levels of DnaA, reduced viability and gradually blocked DNA synthesis. Acetamide-starved cells were very filamentous, several times the size of the parent cells and showed either abnormal or multi-nucleoid morphology, indicating a blockage in cell-division events. The addition of acetamide to the starved cells restored their viability and shortened the lengths of their filaments back to the size of the parent cells. Thus, both increasing and decreasing the levels of DnaA have an effect on the cells, indicating that the level of DnaA is critical to the maintenance of coordination between DNA replication and cell division. It is concluded that DNA replication and cell-division processes in M. smegmatis are linked, and it is proposed that DnaA has a role in both of these processes.