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At least 19 recordsLinked to original sources

PubMed1984

RepFIC, a basic replicon of IncFI plasmids that has homology with a basic replicon of IncFII plasmids.

It was found that a DNA segment containing genes for autonomous replication and its control (basic replicon) present in the IncFI plasmid P307 has homology with RepA, a basic replicon present in IncFII plasmids. The basic replicon in P307 is referred to as RepFIC and the homologous basic replicon in IncFII plasmids is referred to as RepFIIA. In 11 other IncFI plasmids studied a region that has homology with RepFIC and RepFIIA was demonstrated. Thus, of the several basic replicons present in IncFI plasmids, RepFIC is evolutionarily related to a basic replicon of IncFII plasmids.

Base Sequence↗

PubMed1992

Expression of the repA1 gene of IncFII plasmid NR1 is translationally coupled to expression of an overlapping leader peptide.

Examination of a group of mutants of plasmid NR1 that had lost the expression of IncFII plasmid incompatibility (Inc-) revealed a group that had also lost replication proficiency (Rep-). These mutants were obtained from plasmids in which the NR1 replication control region was present in a cointegrate with plasmid pBR322. Whereas the wild-type parental cointegrate plasmid was capable of replicating in a polA host owing to the PolA independence of NR1 replication, the mutants were not able to transform a polA host. Losses of both expression of IncFII plasmid incompatibility and replication proficiency were found to result from the same single base-pair substitution in four independently isolated Inc- Rep- mutants. The mutation inactivates promoter PE for the transcription of RNA-E, a trans-acting repressor of translation of the essential RepA1 replication initiation protein of NR1. Although the loss of RNA-E synthesis had been expected to increase the expression of repA1, the efficiency of translation of repA1 mRNA from these mutants was at least 100-fold lower than that from the wild type, as revealed by repA1-lacZ translational fusions. The PE mutation introduced a stop codon into a 24-amino-acid reading frame that precedes the repA1 gene and terminates just 2 bp downstream from the repA1 start codon. This putative leader peptide was also expressed in a lacZ translational fusion, and its expression was reduced by a factor of 10(4) by the PE mutation. The expression of the leader peptide and the expression of repA1 were regulated by RNA-E. These results suggest that the expression of repA1 is coupled to the translation of the leader peptide and that the repression of repA1 translation by RNA-E may occur via inhibition of the translation of the leader peptide.

Amino Acid Sequence↗

PubMed1992

Autoregulation of the stability operon of IncFII plasmid NR1.

The stb locus of IncFII plasmid NR1, which mediates stable inheritance of the plasmid, is composed of an essential cis-acting DNA site located upstream from two tandem genes that encode essential stability proteins. The two tandem genes, stbA and stbB, are transcribed as an operon from promoter PAB. Using PAB-lacZ gene fusions, it was found that the stb operon is autoregulated. A low-copy-number stb+ plasmid introduced into the same cell with the PAB-lacZ fusion plasmid repressed beta-galactosidase activity about 5-fold, whereas a high-copy-number stb+ plasmid repressed beta-galactosidase about 15-fold. The details of autoregulation were analyzed by varying the concentrations of StbA and StbB to examine their effects on expression from the PAB-lacZ fusion plasmid. StbB protein by itself had autorepressor activity. Although StbA protein by itself had no detectable repressor activity, plasmids that encoded both stbA and stbB repressed more effectively than did those that encoded stbB alone. Plasmids with a mutation in stbA had reduced repressor activity. One mutation in stbB that inactivated the stability function also reduced, but did not eliminate, repressor activity. Repressor activity of the mutant StbB protein was effectively enhanced by stbA. These results indicate that StbB serves two functions, one for stable inheritance and one for autoregulation of the stb operon, both of which may be influenced by StbA protein.

DNA Transposable Elements↗

PubMed1987

Nucleotide sequence analysis of RepFIC, a basic replicon present in IncFI plasmids P307 and F, and its relation to the RepA replicon of IncFII plasmids.

RepFIC is a basic replicon of IncFI plasmid P307 which is located within a 3.09-kilobase SmaI fragment. The nucleotide sequence of this region has been determined and shown to be homologous with the RepFIIA replicon of IncFII plasmids. The two replicons share three homologous regions, HRI, HRII, and HRIII, which are flanked by two nonhomologous regions, NHRI and NHRII. A comparison of coding regions reveals that the two replicons have several features in common. RepFIC, like RepFIIA, codes for a repA2 protein with its amino-terminal codons in HRI and its carboxy-terminal codons in NHRI. Although the codons for the repA1 proteins are located in NHRII, the DNA region containing a putative promoter, ribosomal binding site, and initiation codons is located in HRII. This region also codes for an inc RNA. There are nine base-pair differences between the inc RNA of RepFIIA and that of RepFIC, and as a result, RepFIC and RepFIIA replicons are compatible. An EcoRI fragment from the F plasmid which shows homology with RepFIC of P307 has also been sequenced. This fragment contains only a portion of RepFIC, including the genes for the putative repA2 protein and inc RNA. The region coding for a putative repA1 protein is interrupted by the transposon Tn1000 and shows no homology with the repA1 region of RepFIIA and RepFIC of P307. Our comparative and structural analyses suggest that RepFIC and RepFIIA, although different, have a similar replication mechanism and thus can be assigned to the same replicon family, which we designate the RepFIIA family.

Base Sequence↗

PubMed1988

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.

Bacterial Proteins↗

PubMed1985

Analysis of the individual regulatory components of the IncFII plasmid replication control system.

Replication of the IncFII plasmid NR1 is controlled by regulating the amount of synthesis of the repA1 initiator protein at both the transcriptional and translational levels. We have examined mutations which have altered each of these levels of regulation, resulting in different plasmid copy numbers. The genes which encode each of the individual wild-type or mutant regulatory components from the replication control region of NR1 have been cloned independently into pBR322 vectors, and their effects in trans, either individually or in various combinations, on plasmid incompatibility, stability, copy number, and repA1 gene expression have been defined.

Adenosine Triphosphatases↗

PubMed1991

Transcription of the stability operon of IncFII plasmid NR1.

The stability (stb) locus of IncFII plasmid NR1 is composed of an essential cis-acting DNA site located upstream from two tandem genes that encode essential stability proteins. The stb locus was found to be transcribed from a promoter site just upstream from the first gene, stbA. This promoter was active for transcription both in vivo and in vitro and was located within the region that includes the essential cis-acting site. Transcripts initiated from this site were approximately 1,500 to 1,600 nucleotides in length. Northern (RNA) blot analysis indicated that the transcripts traversed both stbA and the downstream gene, stbB. Mutants from which the promoter had been deleted failed to produce detectable transcripts from either stbA or stbB. Transcription of a third open reading frame, stbC, which is contained within the stbB gene in the opposite DNA strand, could not be detected. For a mutant in which a transposon had been inserted in stbA, no transcription of stbB was detected. After deletion of most of the transposon, which left behind a 35-bp frameshift insertion in stbA, transcription of stbB was restored, although the insertion still had a polar effect on stbB function. The rate of in vivo transcription of the stb locus was measured by pulse-labeling of RNA followed by quantitative RNA-DNA hybridization. Mutants deleted of stbB had an approximately 10-fold increase in the rate of transcription, whereas those deleted of the promoter region had at least a 10-fold reduction in transcription rate. The half-life of stb mRNA was approximately 2 min. These data suggest that stbA and stbB are cotranscribed as an operon that may be autoregulated.

Blotting, Northern↗

PubMed1993

Insertion and deletion mutations in the repA4 region of the IncFII plasmid NR1 cause unstable inheritance.

Mutants of IncFII plasmid NR1 that have transposons inserted in the repA4 open reading frame (ORF) are not inherited stably. The repA4 ORF is located immediately downstream from the replication origin (ori). The repA4 coding region contains inverted-repeat sequences that are homologous to the terC inverted repeats located in the replication terminus of the Escherichia coli chromosome. The site of initiation of leading-strand synthesis for replication of NR1 is also located in repA4 near its 3' end. Transposon insertions between ori and the right-hand terC repeat resulted in plasmid instability, whereas transposon insertions farther downstream did not. Derivatives that contained a 35-bp frameshift insertion in the repA4 ORF were all stable, even when the frameshift was located very near the 5' end of the coding region. This finding indicates that repA4 does not specify a protein product that is essential for plasmid stability. Examination of mutants having a nest of deletions with endpoints in or near repA4 indicated that the 3' end of the repA4 coding region and the site of leading-strand initiation could be deleted without appreciable effect on plasmid stability. Deletion of the pemI and pemK genes, located farther downstream from repA4 and reported to affect plasmid stability, also had no detectable effect. In contrast, mutants from which the right-hand terC repeat, or both right- and left-hand repeats, had been deleted were unstable. None of the insertion or deletion mutations in or near repA4 affected plasmid copy number. Alteration of the terC repeats by site-directed mutagenesis had little effect on plasmid stability. Plasmid stability was not affected by a fus mutation known to inactivate the termination function. Therefore, it appears that the overall integrity of the repA4 region is more important for stable maintenance of plasmid NR1 than are any of the individual known features found in this region.

Amino Acid Sequence↗

PubMed1989

DnaA protein is not essential for replication of IncFII plasmid NR1.

By transformation of dnaA null mutant host cells that are suppressed either by an rnh mutation or by chromosomal integration of a mini-R1 plasmid, it was shown that replication of miniplasmids composed of the NR1 minimal replicon had no absolute dependence upon DnaA protein. In addition, the suppression of the dnaA null mutation by the integrated mini-R1, which is an IncFII relative of NR1, was found to be sensitive to the expression of IncFII-specific plasmid incompatibility. This suggests that the integrative suppression by mini-R1 is under the control of the normal IncFII plasmid replication circuitry. Although NR1 replication had no absolute requirement for DnaA, the copy numbers of NR1-derived miniplasmids were lower in dnaA null mutants, and the plasmids exhibited a much reduced stability of inheritance during subculture in the absence of selection. This suggests that DnaA protein may participate in IncFII plasmid replication in some auxiliary way, such as by increasing the efficiency of formation of an open initiation complex at the plasmid replication origin. Such an auxiliary role for DnaA in IncFII replication would be different from that for replication of most other plasmids examined, for which DnaA has been found to be either essential or unimportant.

Bacterial Proteins↗

PubMed1985

Regulation of transcription of the repA1 gene in the replication control region of IncFII plasmid NR1 by gene dosage of the repA2 transcription repressor protein.

Transcription of the repA1 gene of the IncFII plasmid NR1 is initiated at two promoters in the replication control region. Transcription from the upstream promoter is constitutive at a low level, whereas transcription from the downstream promoter is regulated. The 5' end of the constitutively synthesized transcript also encodes the transcription repressor protein for the regulated downstream promoter. Therefore, the level of the repressor protein in the cell is gene dosage dependent. Using both lac gene fusions and quantitative hybridization methods, we have determined the in vivo relationship between the rate of transcription from the regulated promoter and the repressor protein concentration as a function of gene dosage. At the wild-type copy number of NR1, transcription from the regulated promoter is 96% repressed, but substantial derepression occurs when the copy number falls below the normal value. At or above the normal plasmid copy number, the basal level of repA1 mRNA is provided by transcription from the constitutive upstream promoter.

Adenosine Triphosphatases↗

PubMed1986

Regulation of IncFII plasmid DNA replication. A quantitative model for control of plasmid NR1 replication in the bacterial cell division cycle.

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.

Cell Cycle↗

PubMed1984

The DNA sequence of the mercury resistance operon of the IncFII plasmid NR1.

The DNA sequence has been determined for a 3.8-kilobase region which encodes the mercury resistance (mer) operon of the IncFII plasmid NR1. The sequence reveals four open reading frames which could encode proteins of 12,391, 9,429, 14,965, and 58,781 daltons. On the basis of their sizes, amino acid compositions, hydropathicities, and estimated isoelectric points, the peptides encoded by these open reading frames correspond to the four previously described Hg-inducible proteins detected in minicells carrying mer+ plasmids. The NR1 mer locus is 63.4% GC overall, and the Hg(II) reductase protein sequence is 90% homologous to that of Tn501. The region encoding the merR (positive regulatory) function has three open reading frames. The smallest of these possible merR peptides (6,457 daltons) begins approximately 280 bp to the right of the adjacent IS1b and reads towards the structural genes of the mer operon. The next largest reading frame (13,139 daltons) in the merR region begins 37 bp to the left of the beginning of the smallest peptide and also reads towards the structural genes. The largest reading frame (15,907 daltons) in the merR region lies on the complementary strand and reads away from the structural genes towards IS1b. Although attempts to visualize the merR gene product were not successful, in vitro mutagenesis allows us to eliminate the largest reading frame as a merR candidate. We were also able to show that approximately 50% of the smallest detectable mer peptide (9,429 daltons) is located in the periplasm.

Bacterial Proteins↗

PubMed1988

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.

Bacterial Proteins↗

PubMed1984

IncFII plasmid incompatibility product and its target are both RNA transcripts.

The region of DNA coding for incompatibility (inc) and copy number control (cop) of the IncFII plasmid NR1 is transcribed in both the rightward and leftward directions. The rightward transcripts serve as mRNA for the repA1 protein, which is required for replication. A small, 91-base leftward transcript is synthesized from the opposite DNA strand and is complementary to a portion of the rightward mRNA near its 5' end. A 262-base-pair Sau3A restriction fragment that encodes the small leftward transcript, but does not include the rightward transcription promoters, was cloned into the vector pBR322 or pUC8. The same fragment was cloned from an Inc- mutant of NR1 that does not make the small leftward transcript. Transcription through the cloned fragments in these derivatives was under control of the tetracycline resistance gene in pBR322 or the lac promoter-operator in pUC8. In one orientation of the inserted DNA, a hybrid transcript containing rightward NR1 RNA sequences was synthesized. In the other orientation, a hybrid transcript containing leftward NR1 RNA sequences was synthesized. These plasmids were used to vary the intracellular levels of the rightward or leftward NR1 RNA transcripts and to test their effects in trans on various coresident derivatives of NR1. An excess of rightward NR1 RNA in trans stimulated expression of the essential repA1 gene and caused an increase in the copy number of a coresident NR1 plasmid. An excess of leftward NR1 RNA in trans inhibited the expression of the repA1 gene and lowered the coresident NR1 copy number, thereby causing incompatibility. A pBR322 derivative with no transcription through the cloned NR1 DNA had no effect in trans. These results suggest that the small leftward transcript is the incompatibility inhibitor of NR1 and that its target is the complementary portion of the rightward mRNA.

Base Sequence↗

PubMed1993

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

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.

Amino Acid Sequence↗

PubMed1988

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.

Amino Acid Sequence↗

PubMed1998

Combinatorial selection of a small RNA that induces amplification of IncFII plasmids in Escherichia coli.

Cellular RNAs play fundamental roles as genetic messages, structural components and, in some cases, as catalytic agents. The ability to create vast combinatorial libraries of random RNA sequences has previously been exploited in vitro to identify RNA aptamers with desirable binding specificities, and to isolate RNAs with novel catalytic properties. Despite the advantages of in vitro selections from RNA libraries, there is no way to predict if the identified RNAs can function in living cells. We are therefore exploring random RNA expression libraries in Escherichia coli to search for small RNAs with novel functions. Here we describe selections that identified a small RNA (approximately 260 nucleotides) capable of altering the copy-number control circuitry of IncFII plasmids. The novel RNA appears to function by annealing to a region of the mRNA encoding the plasmid replicator protein. The resulting RNA-RNA hybrid permits translation of the replicator protein, but blocks base-pairing with a natural negative regulatory RNA. Implications of this in vivo selection strategy are discussed.

Anti-Bacterial Agents↗

PubMed1983

Further studies on the inviability of Escherichia coli K12 rho mutant strains carrying IncFII plasmids.

Previous results (Baumberg and Lovett 1977) showed that many conjugative plasmids, most notably those of the IncFII group, were only transferable to Escherichia coli K12 rho mutants at very low frequencies, and that this appeared to be due to a lethal interaction between plasmid and rho- allele. Experiments reported here were designed to examine this phenomenon further, and in particular to test the possibility that uncontrolled plasmid replication in the presence of the rho mutant allele occurs. The rifampicin resistance allele rpo203 antagonizes the effect of rho201 on conjugal plasmid transfer; since the former is known to counteract the latter's effect on transcription termination, this result indicates that the plasmid-rho interaction stems directly from the lack of transcription termination caused by a rho mutant allele. Direct estimation of Rldrd19 replication after conjugal transfer to rho+ and rho201 recipients showed no difference according to rho status. Also, cotransduction of rho101 and 201 with ilv::Tn5 into recipients carrying miniplasmid derivatives of R1, which retain a restriction fragment containing all the DNA known to be involved in plasmid replication, was normal and the rho- transductants retained the miniplasmids. It therefore appears that the lethal interaction between the Inc FII plasmids and rho mutant alleles does not involve plasmid replication functions.

Alleles↗