Arresting chromosome replication upon energy starvation in Escherichia coli

Most organisms possess several cell cycle checkpoints to preserve genome stability in periods of stress. Upon starvation, the absence of chromosomal duplication in the bacterium Escherichia coli is ensured by holding off commencement of replication. During normal growth, accumulation of the initiator protein DnaA along with cell cycle changes in its activity, ensure that DNA replication starts only once per cell cycle. Upon nutrient starvation, the prevailing model is that an arrest in DnaA protein synthesis is responsible for the absence of initiation. Recent indications now suggest that DnaA degradation may also play a role. Here we comment on the implications of this potential new layer of regulation.

Phage T5 two-step injection

Escherichia coli bacteriophage T5 differs from most phages in that it injects its genome in two steps: First Step Transfer, FST, corresponding to leftmost 7.9% of the genome and Second Step Transfer, SST, corresponding to the remainder of the genome. Expression of genes A1 and A2 is required for SST. DNA injection stops at a site known as the injection stop signal (iss) which is a cis acting site located in the large untranslated region of the Left Terminal Repeat (LTR). The iss region is extremely complicated with many repeats, inverted repeats and palindromes. This report compares the iss regions of 25 T5-like phages and shows that all have a common conserved structure including a series of 8 DnaA boxes arranged in a highly specific manner; reminiscent of the origin of replication (oriC) of E. coli. DnaA protein, which binds to DnaA boxes, is a mostly membrane bound. A new, radically different, mechanism of T5 2-step injection is proposed whereby injecting T5 DNA stops at the plasma membrane due to the binding of the iss DnaA boxes to membrane-bound DnaA protein. Injection of the SST continues later via the combined action of the A1 and A2 proteins which cleave the FST DNA at a site upstream (right) of the iss region, thereby liberating it. They also cleave the incoming SST DNA at the same site on the RTR thus facilitating circularisation of one complete genome via the cohesive ends. Circle formation protects the T5 DNA from the degradative action of the RecBCD nuclease and eventually leads to rolling circle DNA replication.

The Caulobacter crescentus Homolog of DnaA (HdaA) Also Regulates the Proteolysis of the Replication Initiator Protein DnaA

ABSTRACTIt is not known how diverse bacteria regulate chromosome replication. Based onEscherichia colistudies, DnaA initiates replication and the homolog of DnaA (Hda) inactivates DnaA using the RIDA (regulatoryinactivation ofDnaA) mechanism that thereby prevents extra chromosome replication cycles. RIDA may be widespread, because the distantly relatedCaulobacter crescentushomolog 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 aC. crescentusstrain that shuts offhdaAtranscription 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 establishedE. colimechanism whereby Hda converts active DnaA-ATP to inactive DnaA-ADP. However, unlikeE. coliDnaA,C. crescentusDnaA is also regulated by selective proteolysis.C. crescentuscells lacking HdaA reduce DnaA proteolysis in logarithmically growing cells, thereby implicating HdaA in this selective DnaA turnover mechanism. Also, wild-typeC. crescentuscells 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 freshwaterCaulobacterspecies also remove DnaA during entry to stationary phase, implying a wider role for HdaA as a novel component of programed proteolysis.IMPORTANCEBacteria must regulate chromosome replication, and yet the mechanisms are not completely understood and not fully exploited for antibiotic development. Based onEscherichia colistudies, DnaA initiates replication, and the homolog of DnaA (Hda) inactivates DnaA to prevent extra replication. The distantly relatedCaulobacter crescentushomolog HdaA also regulates chromosome replication. Here we unexpectedly discovered that unlike theE. coliHda, theC. crescentusHdaA 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.