Order of gene replication in Caulobacter crescentus; use of in vivo labeled genomic DNA as a probe.

Two methods for determining the time of gene replication in Caulobacter crescentus using a temperature sensitive DNA synthesis mutant to synchronize chromosome replication are described. Swarmer cells, blocked before DNA initiation at 37 degrees C, initiate chromosome replication within 2 min after releasing the temperature block in 32P-orthophosphate medium, as indicated by the appearance of a small number of unique genomic DNA fragments. The time at which a given chromosome segment replicates was determined by isolating genomic DNA from cells labeled for progressively longer times during the S period of the cell cycle and hybridizing the probes to cloned C. crescentus genes. The time of replication of genetically mapped Tn5 insertions was determined by preparing DNA from the Tn5 insertion mutants that had been labeled with 32P in similar experiments and hybridizing it to lambda::Tn5 DNA. These results furnish the first correlation between the order of chromosome replication and the genetic map of C. crescentus. They also show that the times of replication and expression of the hook protein and the flagellin genes, which require DNA synthesis for their transcription, both occur near mid-S phase.