Negative control of replication initiation by a novel chromosomal locus exhibiting exceptional affinity for Escherichia coli DnaA protein

Abstract

Replication of the Escherichia coli chromosome is initiated at a unique site, oriC. Concurrent initiation occurs at all oriC sites present in a cell once, and only once, per cell cycle. A mechanism to ensure cyclic initiation events was found operating through the chromosomal site, datA, a 1-kb segment located at 94.7 min on the genetic map that titrates exceptionally large amounts of the bacterial initiator protein, DnaA. A strain lacking datA grew normally but exhibited an asynchronous initiation phenotype as a result of extra initiation events. This mutant phenotype was suppressed by DnaA-titrating plasmids. Furthermore, mutations in a 9-bp DnaA-binding sequence (the DnaA box) in datA were enough to induce the mutant phenotype. Thus, datA is a novel chromosomal element that appears to adjust a balance between free and bound DnaA for a single initiation event at a fixed time in the bacterial cell cycle. Titration of DnaA to newly duplicated datA during oriC sequestration, which is mediated by hemimethylated GATC sequences in oriC and the SeqA protein, would contribute to prevention of reinitiations when oriC is desequestered.

Figure 1

Location of datA and ΔdatA::kan mutation on the E. coli chromosome. ORFs are indicated with molecular masses (kD) of their products. In the enlarged map, the extent of the ORFs are indicated by bars; those above the line read to the right and the one below the line to the left. Bent arrows are initiation sites of transcription. Five arrowheads denote DnaA boxes. The second DnaA box, which was inactivated by the datA mutation is gray. The datA region is indicated by a thick striped bar. The region substituted by the gene conferring kanamycin resistance (kan) is also shown.

Figure 2

Asynchronous and extra initiations in the ΔdatA:: kan strain. Cells growing exponentially in the indicated medium were treated with rifampicin and cephalexin for six generations, and run-out DNA histograms (A) or light-scatter histograms (B) were obtained by flow cytometry. Both W3110 (wild type) and RSD448 (ΔdatA::kan) had the same doubling time of 72 min in M9 medium, 50 min in M9CAA medium, and 28 min in L broth. Run-out DNA histograms measure the distribution of chromosome numbers per cell, which is equal to the number of origins per cell present at the time of addition of rifampicin and cephalexin. Light-scatter histograms measure cell size distribution.

Figure 3

Fluorescence (DNA) histograms of strains disturbed in DnaA titration. (A) The datA1 mutation. (B) Run-out DNA histogram of RSD561 (W3110datA1) grown in M9 medium. The experiment was performed as described in Figure 2. (C) Run-out DNA histogram of RSD448 (pBR322), RSD448 (pTOA502), W3110 (pBR322), and W3110 (pTOA502). Cells grown in L broth supplemented with 40 μg/ml ampicillin were treated as described in Fig. 2. pTOA502 is a pBR322 derivative carrying the DnaA boxes of oriC region (see text).

Figure 4

Fluorescence (DNA) and light-scatter histograms of RSD448 carrying various plasmids. RSD448 (ΔdatA::kan) cells transformed with the indicated plasmids were grown to log phase in M9 medium supplemented with 20 μg/ml ampicillin and analyzed as described in Fig. 2. pKV713 and pMW119 are mini-F- and pSC101-based vectors, repectively. Fragments EX and EX2 are EcoNI–XhoI fragments (Fig. 1), carrying wild-type datA and the datA1 mutation, respectively.

Figure 5

Change of origin number per cell during the cell cycle. Newborn cells of ON338 (B/rF26ΔdatA::kan) in M9 medium were collected using the baby machine technique and grown as batch cultures at 37°C. At the indicated times, samples were treated with rifampicin and cephalexin, and run-out DNA histograms were obtained by flow cytometry. The frequency of origin numbers was determined by measuring the area of each peak. Origin numbers are indicated on the graph.

Figure 6

Effect of the chromosomal location of datA on the frequency and synchrony of initiation. Derivatives of the wild-type strain MG1655 devoid of datA at the original locus and instead carrying Tn10 or Tn10datA cat at indicated chromosomal loci were grown in M9 medium or L broth and analyzed as described in Fig. 2.

Figure 7

Model for the control of DNA replication initiation in E. coli. Periods of oriC sequestration and transcription of the indicated genes are shown by horizontal bars. Relative concentrations of free DnaA molecules are expressed by the degree of shading. See text for details. Cell cycle-dependent transcription from the gid and mioC promoters is accommodated, inasmuch as they are implicated in the control of initiation of replication of minichromosomes (Theisen et al. 1993; Ogawa and Okazaki 1994). Involvement of transcription from these promoters for efficient replication from the chromosomal oriC is suggested only for replication that takes place under certain suboptimal conditions (de Wind et al. 1987; Bates et al. 1997).