Physiological consequences of blocked Caulobacter crescentus dnaA expression, an essential DNA replication gene.

Caulobacter crescentus chromosome replication is precisely coupled to a developmental cell cycle. Like most eubacteria, C. crescentus has a DnaA homologue that is presumed to initiate chromosome replication. However, the C. crescentus replication origin (Cori) lacks perfect consensus Escherichia coli DnaA boxes. Instead, the Cori strong transcription promoter (Ps) may regulate chromosome replication through the CtrA cell cycle response regulator. We therefore created a conditional dnaA C. crescentus strain. Blocking dnaA expression immediately decreased DNA synthesis, which stopped after approximately one doubling period. Fluorescent flow cytometry confirmed that DNA synthesis is blocked at the initiation stage. Cell division also stopped, but not swarmer to stalked cell differentiation. All cells became stalked cells that grew as long filaments. Therefore, general transcription and protein synthesis continued, whereas DNA synthesis stopped. However, transcription was selectively blocked from the flagellar fliQ and fliL and methyltransferase ccrM promoters, which require CtrA and are blocked by different DNA synthesis inhibitors. Interestingly, transcription from Cori Ps continued unaltered. Therefore, Ps transcription is not sufficient for chromosome replication. Approximately 6-8 h after blocked dnaA expression, cells lost viability exponentially. Coincidentally, beta-galactosidase was induced from one transcription reporter, suggesting an altered physiology. We conclude that C. crescentus DnaA is essential for chromosome replication initiation, and perhaps also has a wider role in cell homeostasis.

Selective cell cycle transcription requires membrane synthesis in Caulobacter.

Caulobacter crescentus divides asymmetrically and creates distinct polar membrane surfaces that partition during the cell cycle to distinct cell progeny. Blocking membrane synthesis prevented transcription from selective promoters involved in asymmetric cell division. Transcription from sigma-54-dependent flagellar promoters was blocked completely; however, transcription from the CtrA response regulator-dependent flagellar promoters was activated but reduced. Transcription from the ccrM (DNA methylation) promoter and the che (chemosensory) promoter was also blocked completely. Transcription from a strong promoter at the chromosome replication origin was first stopped then induced by blocked membrane synthesis. We propose a feedback control coupling membrane synthesis to transcription that selectively supports membrane-associated processes such as flagellar assembly, chemosensory biogenesis and chromosome replication.