The pAMβ1 CopF repressor regulates plasmid copy number by controlling transcription of the repE gene

Ehrlich, S. Dusko; Janniere, Laurent

doi:10.1111/j.1365-2958.1994.tb02181.x

Cited by 39 publications

(38 citation statements)

References 36 publications

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“…Based on the concerted action of these two regulatory elements, the stable inheritance of pIP501 can be accounted for. Plasmids pSM19035 (2) and pAM␤1 (14), which share the overall organization of pIP501 (inc18 plasmid family [8]) as well as the same mode of regulation (e.g., 22,23), may employ the same strategy to secure their stable maintenance.…”

Section: Discussionmentioning

confidence: 99%

Dual function of the copR gene product of plasmid pIP501

Brantl

Wagner

1997

J Bacteriol

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Replication of plasmid pIP501 is regulated at a step subsequent to transcription initiation by an antisense RNA (RNAIII) and transcriptionally by a repressor protein, CopR. Previously, it had been shown that CopR binds to a 44-bp DNA fragment upstream of and overlapping the repR promoter pII. Subsequently, we found that high-copy-number pIP501 derivatives lacking copR and low-copy-number derivatives containing copR produced the same intracellular amounts of RNAIII. This suggested a second, hitherto-unknown function of CopR. In this report, we show that CopR does not affect the half-life of RNAIII. Instead, we demonstrate in vivo that, in the presence of both pII and pIII, CopR provided in cis or in trans causes an increase in the intracellular concentration of RNAIII and that this effect is due to the function of the protein rather than its mRNA. We suggest that, in the absence of CopR, the increased (derepressed) RNAII transcription interferes, in cis, with initiation of transcription of RNAIII (convergent transcription), resulting in a lower RNAIII/plasmid ratio. When CopR is present, the pII promoter is repressed to >90%, so that convergent transcription is mostly abolished and RNAIII/plasmid ratios are high. The hypothesis that RNAII transcription influences promoter pIII through induced changes in DNA supercoiling is supported by the finding that the gyrase inhibitor novobiocin affects the accumulation of both sense and antisense RNA. The dual role of CopR in repression of RNAII transcription and in prevention of convergent transcription is discussed in the context of replication control of pIP501.Replication of the broad-host-range plasmid pIP501 (19) is controlled at a step subsequent to transcription initiation by CopR (7) and posttranscriptionally by an antisense RNA (RNAIII) (11). RNAIII induces transcriptional attenuation of RNAII within the leader region of the repR mRNA (11,12). CopR binds to a 44-bp sequence upstream of and partially overlapping the repR promoter pII, thereby repressing RNAII synthesis about 10-fold (7). Deletion of copR resulted in a 10-to 20-fold increase of pIP501 copy number (9), consistent with the elevated synthesis of RNAII, which encodes the rate-limiting RepR protein (10). Recently, we measured the steady-state concentrations of RNAIII in Bacillus subtilis cells in high-copynumber and low-copy-number pIP501 derivatives. Since RNAIII is synthesized constitutively, its concentration should be directly correlated with plasmid copy number. Surprisingly, we found that this was not the case: both pPR1 (rnaIII ϩ copR; 50 to 100 copies/cell) and pCOP4 (rnaIII ϩ copR ϩ ; 5 to 10 copies/ cell) expressed about 1,000 to 2,000 molecules of RNAIII/cell (1.4 to 2.8 M [13]). This suggested that, in addition to its function as a repressor of RNAII transcription, CopR is required for RNAIII accumulation. Previous analyses using transcriptional promoter-lacZ fusions ruled out the idea that CopR activates transcription from pIII (7). Hence, CopR must act either by affecting the stability of R...

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Section: Discussionmentioning

confidence: 99%

Dual function of the copR gene product of plasmid pIP501

Brantl

Wagner

1997

J Bacteriol

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“…For this, we used the pAM␤1 CopF repressor that was shown to reduce about 10 times the copy number of pIP501 derivatives by decreasing transcription of the repR gene (Brantl, 1994;Le Chatelier et al, 1994). A Polþ strain carrying pVA798 and pTB19F, a compatible erythromycin-resistant plasmid encoding the CopF protein under the control of the IPTGinducible spac-I promoter (Le Chatelier et al, 1994), was thus constructed and grown at 37ЊC in liquid medium supplemented with various concentrations of IPTG and with antibiotics selecting for the two plasmids. After approximately five generations, the cells were harvested and the total DNA was extracted and analysed by Southern blotting.…”

Section: Characterization Of Inhibitory Elementsmentioning

confidence: 99%

“…Plasmid replication is controlled by two independent systems each of which represses Ϸ10 times transcription of the repR gene. The first system involves the CopR repressor protein, which inhibits initiation of transcription at the repR promoter by binding to an operator (Brantl, 1994;Le Chatelier et al, 1994). The second system involves a small RNA complementary to the leader region of the repR mRNA (countertranscript RNA).…”

Section: Introductionmentioning

confidence: 99%

Inhibition of a naturally occurring rolling‐circle replicon in derivatives of the theta‐replicating plasmid pIP501

Pujol

Chédin

Ehrlich

et al. 1998

Molecular Microbiology

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SummaryThe mechanisms ensuring regulation of DNA replication in genomes containing multiple replicons are poorly understood. In this report, we addressed this question by analysing in Bacillus subtilis the replication of a derivative of the promiscuous plasmid pIP501 that carries a rolling-circle and a theta replicon. Genetic analyses revealed that the rolling-circle replicon is strongly inhibited in the derivative and that inhibition requires three elements involved in theta replication: the replication origin, the initiator RepR protein and strong transcription of the repR gene. Inhibition is, however, independent of DNA synthesis at the theta origin. We conclude that rolling-circle inhibition is caused by an inhibitor y signal encoded by the theta replicon and propose that the signal is composed, at least, of the RepR protein bound to its cognate origin.

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“…The second involves the 10-kDa products of the copF and copR genes. The two proteins bind to an operator sequence located just upstream of the Rep promoters, P DE in pAM␤1 and PII in pIP501, and repress Rep mRNA synthesis approximately 10-fold (2,22). CopF shares up to 95% identity with CopR of pIP501 and CopS, the corresponding protein of plasmid pSM19035.…”

mentioning

confidence: 99%

“…The DNA binding motifs of the CopF, CopR, and CopS proteins have not been characterized. However, there appears to be a significant probability that a helix-turn-helix (HTH) motif can form within the region of the CopF protein delimited by residues at positions 16 and 37 (22) (Fig. 1).…”

mentioning

confidence: 99%