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. 1993 May;175(10):3161–3173. doi: 10.1128/jb.175.10.3161-3173.1993

Suppression of replication-deficient mutants of IncFII plasmid NR1 can occur by two different mechanisms that increase expression of the repA1 gene.

R Wu 1, X Wang 1, D D Womble 1, R H Rownd 1
PMCID: PMC204639  PMID: 8491730

Abstract

Replication-proficient (Rep+) revertants were isolated from mutants of IncFII plasmid NR1 that were replication defective (Rep-). The parental Rep- plasmids contained a mutation that inactivated promoter PE for transcription of RNA-E, a trans-acting repressor of translation of the essential RepA1 replication initiation protein of NR1. The PE mutation also introduced a nonsense codon into a leader peptide gene that precedes and slightly overlaps the repA1 translation initiation site in the mRNA. This reduced the rate of synthesis of RepA1 by uncoupling its translation from that of the leader peptide. The reduced rate of RepA1 synthesis was responsible for the Rep- phenotype. All Rep+ revertants retained the PE mutation and contained second-site mutations responsible for suppression of the Rep- phenotype. One Rep+ revertant contained a second mutation adjacent to the Shine-Dalgarno sequence of repA1. Another Rep+ revertant contained a mutation in the repA2 gene, which encodes the trans-acting repressor of transcription of repA1. By using translational lacZ gene fusions, it was found that both kinds of suppressor mutation increased the expression of repA1 to a level sufficient to support replication. In both cases, the synthesis of RepA1 remained uncoupled from that of the leader peptide. The Shine-Dalgarno mutation increased the rate of leader peptide-independent translation of repA1 mRNA and also reduced the sensitivity of repA1 mRNA to inhibition by RNA-E. The repA2 mutation inactivated the RepA2 repressor and increased the rate of transcription of repA1 mRNA. The translational lacZ gene fusions were used to assess the range of regulation of expression of repA1 provided by each of the RNA-E and RepA2 regulatory circuits. By constructing miniplasmids that contained various combinations of the mutations, the contributions of the RNA-E and RepA2 regulatory circuits were assessed with respect to control of plasmid copy number and stable inheritance. Plasmids that lacked either circuit were less stable than wild-type plasmids.

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Selected References

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