Clustering of genes involved in replication, copy number control, incompatibility, and stable maintenance of the resistance plasmid R1drd-19

SummaryThe expression of dnaA is autoregulated, in that transcription of the gene increases when DnaA is inactivated (and initiation of replication prevented) and decreases when DnaA is supplied in excess. However, the inactivation of DnaA does not necessarily lead to increased DnaA production, as dnaA(Ts; temperature sensitive) strains which are integratively suppressed by derivatives of the plasmid R1 do not show temperatureinduced derepression. Several possible explanations for this unanticipated behaviour were considered and ruied out. We suggest here that the completion of a critical step in initiation may prevent dnaA derepression: although DnaA would be required t o complete this step at oriC, DnaA(Ts) would be sufficient at the R1 origin. Autoregulation of dnaA has been attributed to the binding of DnaA at a consensus binding site in the dnaA promoter region. We show here, using reporter systems, that this DnaA-binding site is not required for the autoregulatory response. We find, further, that replacement of the chromosomal dnaA gene with one containing a mutated binding site causes no demonstrable phenotypic change: cells with the mutant gene show no disadvantage in competition with dnaAf cells.

Section: Resultsmentioning

confidence: 99%

Autoregulation of the Escherichia coli replication initiator protein, DnaA, is indirect

Smith

McAteer

Masters

1997

“…Plasmid pOU47 also carries the parA region of R1. Plasmid pKN501 (Molin et al, 1979) is a small Par ¹ derivative of R1 Fig. 1A.…”

Section: Methodsmentioning

confidence: 99%

Inactivation of the replication‐termination system affects the replication mode and causes unstable maintenance of plasmid R1

Krabbe

Zabielski

Bernander

et al. 1997

SummaryTwo so-called Ter sites, which bind the Escherichia coli Tus protein, are located near the replication origin of plasmid R1. Inactivation of the tus gene caused a large decrease in the stability of maintenance of the R1 mini-derivative pOU47 despite the presence of a functional partition system on the plasmid. Deletion of the right Ter site caused a drop in stability similar to that observed after inactivation of the tus gene. Substitution of 2 bp required for Tus binding also caused unstable plasmid maintenance, whereas no effects on stability were observed when the left Ter site was deleted. Inactivation of the tus gene was coupled to an increased occurrence of multimeric plasmid forms as shown by gel electrophoresis of pOU47 DNA. Inactivation of the recA gene did not increase plasmid stability, suggesting that the multimerization was not mediated by RecA. Plasmid DNA was isolated from the tus strain carrying plasmid pOU47 and from a wild-type strain carrying pOU47 in which the right Ter site had been inactivated; in both cases, electron microscopy revealed the presence of multimers as well as rolling-circle structures with double-stranded tails. Thus, the right Ter site in plasmid R1 appears to stabilize the plasmid by preventing multimerization and shifts from theta to rolling-circle replication.

“…Integratively suppressed strains: increased fts promoter activity is correlated with initiation defects and reduced growth rate In integratively suppressed strains, an integrated, DnaA independent, plasmid-replication origin can replace oriC, allowing the transcriptional consequences of DnaA deprivation to be studied while replication continues. dnaA46 strains integratively suppressed by the 23.7 kb R1-derived plasmid, pKN500 (Molin et al, 1979), grow at normal rates at 428C, and are presumed to initiate at normal frequency despite the inability of DnaA46 to promote initiation at oriC. As integratively suppressed dnaA46 l JFL100 lysogens fail to show derepression of ftsZ 3p4p at 428C (Masters et al, 1989), and because we were then attributing ftsZ derepression in dnaA46 strains to inactivation of DnaA, we were puzzled by these results and suspected that pKN500 might encode a previously undocumented DnaA-like function.…”

Section: Overproduction Of Dnaa Protein Leads To Reduced Expression Fmentioning

confidence: 99%

“…Plasmids pLSK5 contains dnaA under the control of Ptac and specifies ampicillin resistance (Kücherer et al (1986). pGW 0 IS was constructed by cloning the 3 kb Hin dIII fragment carrying the IS1 sequence from pKN500 (Molin et al, 1979) into pGW71, an R1 minimal replicon (Bernander et al, 1989 (Ward and Lutkenhaus, 1985) was ligated into the Pst I and Cla I sites in the polylinker of the vector pT7-3, yielding pT7/ZAQ. The cloned fragment contains the complete ftsQ, ftsA and ftsZ genes.…”

Section: Introduction Of Fts Mutations Onto the Chromosomementioning

confidence: 99%

**The coupling between ftsZ transcription and initiation of DNA replication is not mediated by the DnaA‐boxes upstream of ftsZ or by DnaA**

Smith

McAteer

Masters

1996

The DnaA protein of Escherichia coli is a multi-functional protein which, In addition to promoting initiation of replication, can regulate the initiation or termination of transcription of a variety of genes. It acts by binding to DNA at a defined sequence, termed a DnaA-box. Three candidate DnaA-boxes which occur within the essential cell-division genes, ftsQ and ftsA, have been hypothesized to mediate the response of the downstream ftsZ gene to intracellular levels of DnaA, and thus to couple the processes of initiation and cell division. We show here that, although transcription from promoters upstream of ftsZ is increased when initiation of chromosome replication is blocked by DnaA inactivation, this response is not mediated by the DnaA-boxes near these promoters, nor is it specific to DnaA. We show, furthermore, that mutational inactivation of the putative DnaA-binding sites in the fts region of the chromosome does not lead to impaired growth or reduced survival of cells.