Use of flow cytometry to identify a Caulobacter 4.5 S RNA temperature-sensitive mutant defective in the cell cycle

Abstract

Flow cytometry was used to screen a collection of temperature-sensitive mutants for those blocked at discrete points in the cell cycle with respect to the replicative status of the chromosome. At the non-permissive temperature, one such mutant, LS439, could not initiate new rounds of DNA replication and arrested primarily as cells with two completed chromosomes Extended incubation at the restrictive temperature resulted in filament formation. Following the shift to the restrictive temperature protein synthesis continued, but at a reduced rate. A 0.2 kb fragment of DNA located immediately upstream of the Caulobacter homolog of the Escherichia coli dnaX gene was able to completely rescue the temperature-sensitive phenotype of LS439. The 0.2 kb fragment contained a homolog of the bacterial gene encoding 4.5 S RNA. The original point mutation is predicted to disrupt the stem structure in the 4.5 S RNA thus providing a rationale for the genetic basis of the LS439 phenotype.