The Caulobacter crescentus Homolog of DnaA (HdaA) Also Regulates the Proteolysis of the Replication Initiator Protein DnaA.
It is not known how diverse bacteria regulate chromosome replication. Based on Escherichia coli studies, DnaA initiates replication and the homolog of DnaA (Hda) inactivates DnaA using the RIDA (regulatory inactivation of DnaA) mechanism that thereby prevents extra chromosome replication cycles. RIDA may be widespread, because the distantly related Caulobacter crescentus homolog HdaA also prevents extra chromosome replication (J. Collier and L. Shapiro, J Bacteriol 191:5706-5715, 2009, http://dx.doi.org/10.1128/JB.00525-09). To further study the HdaA/RIDA mechanism, we created a C. crescentus strain that shuts off hdaA transcription and rapidly clears HdaA protein. We confirm that HdaA prevents extra replication, since cells lacking HdaA accumulate extra chromosome DNA. DnaA binds nucleotides ATP and ADP, and our results are consistent with the established E. coli mechanism whereby Hda converts active DnaA-ATP to inactive DnaA-ADP. However, unlike E. coli DnaA, C. crescentus DnaA is also regulated by selective proteolysis. C. crescentus cells lacking HdaA reduce DnaA proteolysis in logarithmically growing cells, thereby implicating HdaA in this selective DnaA turnover mechanism. Also, wild-type C. crescentus cells remove all DnaA protein when they enter stationary phase. However, cells lacking HdaA retain stable DnaA protein even when they stop growing in nutrient-depleted medium that induces complete DnaA proteolysis in wild-type cells. Additional experiments argue for a distinct HdaA-dependent mechanism that selectively removes DnaA prior to stationary phase. Related freshwater Caulobacter species also remove DnaA during entry to stationary phase, implying a wider role for HdaA as a novel component of programed proteolysis.


OBJECTIVE
Bacteria must regulate chromosome replication, and yet the mechanisms are not completely understood and not fully exploited for antibiotic development. Based on Escherichia coli studies, DnaA initiates replication, and the homolog of DnaA (Hda) inactivates DnaA to prevent extra replication. The distantly related Caulobacter crescentus homolog HdaA also regulates chromosome replication. Here we unexpectedly discovered that unlike the E. coli Hda, the C. crescentus HdaA also regulates DnaA proteolysis. Furthermore, this HdaA proteolysis acts in logarithmically growing and in stationary-phase cells and therefore in two very different physiological states. We argue that HdaA acts to help time chromosome replications in logarithmically growing cells and that it is an unexpected component of the programed entry into stationary phase.
McGill University, Department of Microbiology and Immunology, Montreal, Quebec, Canada.
Richard
R
Wargachuk
McGill University, Department of Microbiology and Immunology, Montreal, Quebec, Canada gregory.marczynski@mcgill.ca.
Gregory T
GT
Marczynski
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016935:Caulobacter crescentus / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015151:Immunoblotting; D059748:Proteolysis; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases