1887

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Volume 148, Issue 12

Conditional expression of , an essential DNA replication gene Free

Abstract

To begin to understand the role of in DNA replication, the gene was characterized at the genetic level. Western analyses revealed that DnaA accounts for approximately 018% of the total cellular protein during both the active and stationary growth periods. Expression of antisense RNA reduced viability, indicating that is an essential gene in replication. To further understand the role(s) of in replication, a conditionally expressing strain was constructed in which expression of was controlled by acetamide. Growth in the presence of 02% 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 are linked, and it is proposed that DnaA has a role in both of these processes.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): d.t., doubling time(s) , DCO, double crossover , DNA synthesis , filamentation , gene replacement , kan, kanamycin , mycobacteria , oriC , origin of replication , regulated expression and SCO, single crossover
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2002-12-01
2024-04-20
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Conditional expression of Mycobacterium smegmatis dnaA, an essential DNA replication gene
Microbiology 148, 3887 (2002); https://doi.org/10.1099/00221287-148-12-3887
/content/journal/micro/10.1099/00221287-148-12-3887
/content/journal/micro/10.1099/00221287-148-12-3887
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