Transcriptional inactivation of a regulatory site for replication of Vibrio cholerae chromosome II

Abstract

The oriCII region that includes the initiator gene, rctB, can function as a plasmid in E. coli. Here we show that RctB suffices for the oriCII-based plasmid replication, and rctA in cis or trans reduces the plasmid copy number, thereby serving as a negative regulator. The inhibitory activity could be overcome by increasing the concentration of RctB, suggesting that rctA titrates the initiator. Purified RctB bound to a DNA fragment carrying rctA, confirming that the two can interact. Although rctA apparently works as a titrating site, it is nonetheless transcribed. We find that the transcription attenuates the inhibitory activity of the gene, presumably by interfering with RctB binding. RctB, in turn, repressed the rctA promoter and, thereby, could control its own titration by modulating the transcription of rctA. This control circuit appears to be a putative novel mechanism for homeostasis of initiator availability.O ur knowledge of DNA replication control in bacteria comes largely from studying the chromosome and plasmids of Escherichia coli (1, 2). In general, controlling proteins that initiate replication, initiator proteins, seems to be the central mechanism to regulate replication. A variety of mechanisms have been found to control the initiator.Transcriptional autoregulation of the initiator gene is common in plasmids with iterated initiator-binding sites (iterons), where the initiator serves as the repressor of its own synthesis (3). The E. coli initiator, DnaA, also is autoregulated (4). In addition, the dnaA promoter is inactivated by sequestration to the cell membrane for approximately one-third of the cell generation (5). More traditional modes of transcriptional control using repressors and activators also have been found in phage and plasmid RK2 (6, 7).Initiator translation is regulated by antisense RNAs in a variety of plasmids, the classic examples being plasmids R1 and pT181 (8, 9). The antisense RNA either blocks the ribosomebinding site of the initiator gene directly or of a regulatory peptide for the initiator gene (10).Most other mechanisms operate posttranslationally either by titrating or inactivating the initiator protein. In E. coli, there are Ϸ300 DnaA-binding sites scattered all over the chromosome (11,12). Titration of the initiator to these sites is believed to reduce availability of the protein after replication initiation, because new sites are created during replication elongation.Initiator inactivation is the most widely used mechanism to prevent premature replication reinitiation. The pT181 initiator is inactivated by covalent modification at the end of one round of replication (13). E. coli DnaA is converted from an active ATP bound form to an inactive ADP form after replication (14-16). A similar mechanism also operates in Bacillus subtilis (17). The initiators of iteron-carrying plasmids are inactivated by dimerization, the active form being monomers (18). Dimerization not only reduces monomer concentration, the dimers also participate in inactivating origins (19)(20)(21). Euk...

Transition from a plasmid to a chromosomal mode of replication entails additional regulators

“…These results support initiator titration as a mechanism to inhibit replication by both kinds of sites because increasing the initiator could reverse the inhibition. When multiple binding sites were present in the incII fragment, the copy number decreased further, particularly when the 39-mer was included (e.g., fragments [8][9][10][11][12][13][14]. The combined effect of the two kinds of sites was more than additive, suggesting that the sites cooperate to inhibit oriII (fragments 1 and 7 vs. 9).…”

“…1). Except for the 14-mer locus of incII (defined as a regulatory sequence conserved among sequenced Vibrio genomes), other elements of incII appear to be binding sites of RctB (4,(9)(10)(11). The mechanism of the 14-mer function was unknown.…”

“…EMSA showed that the 39-mer binds RctB (10). Compared with the iterons, RctB bound to the 39-mer with a slightly higher affinity (Fig.…”

“…These features are conserved in other Vibrio species. We have been studying these features for their likely role in revealing how the chrII system might have diverged from a simpler plasmid system (10).…”

ATP negatively regulates the initiator protein of Vibrio cholerae chromosome II replication

“…We constructed a linear DNA molecule that consisted only of rctA-oriCIIvc-rctB and a kanamycin resistance gene. After in vitro ligation, attempts to recover a circular form of this DNA molecule after its transformation into E. coli DH5␣ routinely failed, presumably because rctA is a negative regulator of RctB (20). On the rare occasions when colonies arose after this transformation, they usually contained deletions in rctA (as in pYB289) or mutations in rctB.…”