David D. Womble

Regulation of IncFII plasmid DNA replication

A quantitative model for the regulation of replication of the low copy number IncFII plasmid NR1 in the Escherichia coli cell division cycle has been developed. The initiation of NR1 replication requires a cis-acting initiator protein whose synthesis is regulated by several mechanisms. The NR1 regulatory processes include co-operative protein-protein interactions in the formation of an active transcription repressor, the interaction of repressor with a rightward operator site in the control of transcription of the initiator gene, and the interaction of an inhibitor RNA transcript with the initiator mRNA in the control of translation of the initiation protein. A statistical thermodynamic model was used to predict probable configurations of the regulatory processes in a single growing cell. These probabilities were coupled by a kinetic model to the events of the cell cycle, such as initiation of mRNA transcription and protein translation, and the initiation of plasmid DNA replication. Parameter values were chosen so that the simulated values for plasmid copy number and the intracellular concentrations of repressor protein and mRNA agreed with experimentally determined estimates. A number of different copy number mutants that have altered one or another of the regulatory processes were simulated by the model. The contributions of each of the regulatory processes toward the overall stability of inheritance of plasmid NR1 in a population of cells in culture were examined. These simulations predict a very stable pattern of inheritance for plasmid NR1 despite its low copy number, in agreement with experimental observation.

Genetic organization and nucleotide sequence of the stability locus of IncFII plasmid NR1

The stability (stb) locus of IncFII plasmid NR1 was mapped to a 1700 base-pair NaeI-TaqI restriction fragment. A series of unstable plasmids that contained insertion, deletion, and point mutations that inactivated the stability function was isolated. The unstable point mutants examined were all stabilized (complemented) in trans by a copy of the wild-type stb locus, suggesting that the mutations had inactivated diffusible gene products. The nucleotide sequence of the stb locus contained two tandem open reading frames, designated stbA and stbB, that encoded essential trans-acting protein products with predicted sizes of 36,000 Mr and 13,000 Mr, respectively. A third open reading frame, stbC, that could encode a peptide of 8000 Mr was contained within stbB in the complementary DNA strand. Plasmid-encoded proteins of 36,000 Mr and 13,000 Mr were identified in minicell experiments as the products of stbA and stbB, respectively. Unstable deletion mutants that retained the promoter proximal region of the stb locus upstream from stbA but had deleted both stbA and stbB were stabilized in trans by plasmids that could supply StbA and StbB. In contrast, deletion mutants that had lost the stbAB promoter region were not complemented in trans, indicating that this region contained an essential cis-acting site (or sites). Unlike some other loci that mediate stable plasmid inheritance, cloned copies of the wild-type stb locus of NR1 did not exert strong incompatibility (i.e. trans destabilization) against other stb+ derivatives of plasmid NR1 present in the same cell.

Transcription of the replication control region of the IncFII R-plasmid NR1 in Vitro and in Vivo☆

The minimal replicon of the 90,000 base-pair IncFII R plasmid NR1 consists of a 2700 base-pair region of the DNA. Minireplicator plasmids consisting of the 2700 base-pair minimal replicon plus a 2200 base-pair region coding for chloramphenicol acetyltransferase (cat) were used as templates for in vitro transcription. Six RNA transcripts were synthesized from these templates in vitro. We have determined the directions of transcription and the approximate sites of initiation and termination of each of the in vitro RNA transcripts. One RNA transcript was synthesized from the cat gene, while the other five were transcribed from the minimal replicon. Four of the RNA transcripts also were identified by quantitative hybridization of RNA synthesized in vivo from these minireplicator plasmids. The strengths of the promoters for the RNA transcripts were estimated by the relative rates of transcription both in vitro and in vivo. Transcription from convergent promoters reduced the rate of RNA synthesis in vivo in both directions. In vivo, a significant fraction of the cat mRNA was extended past its in vitro termination point. Transcription of mutants that have altered plasmid copy number and/or incompatibility properties also were examined. The possible roles of each of the transcripts as mRNA and their involvement in regulation of DNA replication are discussed.

In-vivo studies on the cis-acting replication initiator protein of IncFII plasmid NR1.

Using segment-directed mutagenesis, a temperature-sensitive mutant of the gene that encodes the cis-acting RepA1 initiation protein of the IncFII plasmid NR1 was isolated. The mutant protein was unable to promote initiation of plasmid replication in vivo at 42 degrees C. Both the wild-type and the mutant repA1 genes were cloned separately into the high-expression vector plasmid pAS1. In these pAS1-repA1 derivatives, the transcription of the repA1 gene was under the control of the lambda PL promoter, which was regulated by the temperature-sensitive lambda cI857 repressor protein. The translation initiation of the repA1 mRNA from these derivatives was mediated by the lambda cII Shine-Dalgarno sequence and initiation codon. The yield of 33,000 Mr RepA1 protein detected on SDS/polyacrylamide gels from Escherichia coli cells containing the pAS1-repA1 derivatives was dependent upon whether the newly synthesized RepA1 was capable of interacting in cis with the downstream NR1 replication origin on the cloned DNA fragment. Mutations in the repA1 gene or deletions of the cis origin region dramatically increased the detectable yield of RepA1 protein. Deletion of the NR1 origin region from the pAS1 derivative containing the wild-type repA1 gene enabled the cis-acting RepA1 protein to complement partially the temperature-sensitive repA1 mutant in trans, to increase the copy number in trans of plasmids that contained the NR1 replicon, and to help NR1 derivatives overcome plasmid incompatibility. The trans effects of RepA1 provided by the pAS1-repA1 derivatives that retained the origin in cis were much less significant. RepA1 provided in trans also stimulated the replication of plasmids carrying cloned copies of the NR1 replication origin region regardless of whether the origin was transcribed from an upstream promoter.

Incompatibility and INCFII Plasmid Replication Control

The DNA coding for replication control and incompatibility of the plasmid NR1 serves as a template in vivo and in vitro for RNA transcription in both directions. In the rightward direction, RNA synthesis begins from 2 different promoters, one of which is regulated and the other constitutive. In vivo, each of these transcripts is more than 1,000 nucleotides long, terminating near the estimated site for the origin of replication. These transcripts serve as messenger RNA for several proteins. One protein (repA1) is required for replication and another (repA2) serves as the repressor for the regulated rightward promoter. RNA synthesis in the leftward direction is constitutive and produces a single transcript of 91 nucleotides which is complementary in sequence to the rightward transcripts. This small transcript is the incompatibility product which regulates the replication of the plasmid. When the intracellular concentration of the small transcript is experimentally varied, the rate of translation of the rightward transcripts and the rate of initiation of replication (plasmid copy number) vary inversely to its concentration. The mode of action of this inhibitor RNA is likely to be formation of an RNA-RNA duplex with the rightward transcripts, thereby inhibiting the translation which would produce the required replication protein. The probability that a rightward transcript will escape interaction with the small RNA molecules and thus allow replication to initiate can be predicted from the laws of mass action based on base-stacking free energies for the likely sequences of initial contact. The estimated intracellular RNA concentrations, based on quantitative hybridization experiments, are agreement with those predicted from the calculated equilibrium constants for duplex formation.

Complementation of mutants of the stability locus of IncFII plasmid NR1: Essential functions of the trans-acting stbA and stbB gene products☆

A series of unstable mutants of the stability (stb) locus of IncFII plasmid NR1 was subjected to a complementation analysis. The mutant collection included plasmids with point, insertion and deletion mutations in stb. These mutations affected the tandem genes stbA and stbB, which encode stability proteins StbA and StbB, or the PAB transcription promoter, which is located upstream from stbA in a region that contains an essential cis-acting site. Deletion mutants that lacked the region containing promoter PAB could not be complemented (stabilized) by providing StbA and StbB in trans. Deletion mutants that lacked stbA and stbB but retained the PAB region were complemented in trans but required both StbA and StbB, indicating that both proteins were essential for stable inheritance. stbA- point mutants were complemented in trans by either wild-type or stbA+ stbB- clones of the stability region. However, mutants with insertions in stbA were complemented only by wild-type clones, which suggested the insertions were polar on expression of the downstream stbB gene. A plasmid with a stbB- point mutation was complemented in trans by wild-type but not by stbA- stbB+ clones. In addition, plasmid clones that expressed StbB in the absence of StbA caused destabilization of (were incompatible with) stb+ derivatives of NR1 in trans, whereas clones that expressed only wild-type StbA or both StbA plus StbB did not. Plasmid clones that contained only the essential cis-acting PAB region did not cause destabilization of stb+ plasmids in trans. These results suggest that an excess of StbB protein provided in trans may cause a depletion of the essential StbA protein. Therefore, these results may be consistent with the hypothesis that StbB is an autorepressor of the stbAB operon.