Site-directed mutations in the repA C-terminal region of plasmid Rts1: pleiotropic effects on the replication and autorepressor functions.

We induced site-directed mutations near the 3' terminus of the gene repA, which encodes the protein of 288 amino acid residues essential for plasmid Rts1 replication, and obtained seven repA mutants. Three of them contained small deletions at the 3' terminus. Mutant repAz delta C4, which encodes a RepA protein that lacks the C-terminal four amino acids, expressed a high-copy-number phenotype and had lost both autorepressor and incompatibility functions. Deletion of one additional amino acid residue to form the RepAz delta C5 protein caused restoration of the wild-type copy number and strong incompatibility. Studies of the remaining four repA mutants, each of which contained a single amino acid substitution near the RepA C terminus, suggested that Lys-268 is involved in both ori(Rts1) activation and autorepressor-incompatibility activities and that Arg-279 contributes to ori(Rts1) activation but not to incompatibility. Lys-268 is part of a dual-lysine sequence with Lys-267 and is located 21 amino acids upstream of the RepA C terminus. A dual-lysine sequence is also found at a similar position in both mini-F RepE and mini-P1 RepA proteins.     

Control of replication and segregation of R plasmid Rts1.

A mutant plasmid, pTW2, which was derived from the integrated Rst1 genome in the Escherichia coli chromosome, was studied as to its mode of replication at 30 degrees C. When Proteus mirabilis Pm17 harboring pTW2 was grown in broth at 30 degrees C, a considerable number of R- segregants (approximately 40%) were consistently observed. This indicates that pTW2 is unstable even at the permissive temperature for the replication of Rts1. The pTW2+ cells in a culture were heterogeneous with respect to the level of kanamycin resistance, ranging from 500 to 4,000 mug of the drug per ml. The amount of pTW2 deoxyribonucleic acid (DNA) relative to the Pm17 chromosomal DNA was about fivefold as large as that of Rts1 DNA in an exponentially growing culture. In addition, pTW2 in P. mirabilis continued to replicate after the chromosome had ceased to replicate, which was shown in the study of the inhibition of protein synthesis. Contrary to pTW2, the parent plasmid Rts1 is highly stable, and the relative percent Rts1 DNA is maintained at approximately 7% in any cultural conditions at a permissive temperature. These results suggest that copies of pTW2 may not segregate evenly into the host progeny upon cell division and that the replication of pTW2 does not coordinate with that of the chromosome. A remarkable instability of pTW2 as well as an increase in the relative percent pTW2 DNA was also shown when E. coli were used as the host cells. These results suggest the possibility that there is a gene or a gene cluster on the Rst1 genome responsible for the control of both replication and segregation of Rts1.     

The repA2 gene of the plasmid R100.1 encodes a represser of plasmid replication

We have constructed two miniplasmids, derived from the resistance plasmid R100.1. In one of these plasmids 400 bp of R100.1 DNA have been replaced by DNA from the transposon Tn1000 (gamma-delta). This substitution removes the amino-terminal end of the repA2 coding sequence of R100.1 and results in an increased copy number of the plasmid carrying the substitution. The copy number of the substituted plasmid is reduced to normal levels in the presence of R100.1. The repA2 gene thus encodes a trans-acting repressor function involved in the control of plasmid replication.     

The region controlling the thermosensitive effect of plasmid Rts1 on host growth is separate from the Rts1 replication region.

Rts1 is a high-molecular-weight (126 x 10(6)) plasmid encoding resistance to kanamycin. It expresses unusual temperature-sensitive phenotypes, which affect plasmid maintenance and replication, as well as host cell growth. We have cloned the essential replication region of Rts1 from pAK8, a smaller derivative which is phenotypically similar to Rts1. Restriction endonuclease digests of isolated pAK8 deoxyribonucleic acid were allowed to "self-ligate" (ligation without an additional cloning vector) and subsequently were used to transform Escherichia coli strain 20SO to kanamycin resistance. Screening of these strains for the phenotypes of thermosensitive host growth and temperature-dependent plasmid elimination demonstrated that these two properties were expressed independently. Furthermore, it was shown that the Rts1 replication locus per se is not necessarily responsible for altered host growth at the nonpermissive temperature. The kanamycin resistance fragment of pAK8 was also cloned into pBR322. Electrophoretic analysis of BamHI restriction enzyme digests of this plasmid and similar digests of an Rts1 miniplasmid has allowed the identification of an 18.6-megadalton fragment carrying the replication locus and a 14.1-megadalton fragment carrying the kanamycin resistance gene.     

Importance of the C terminus of plasmid Rts1 RepA protein for replication and incompatibility of the plasmid.

RepA protein, essential for replication of plasmid Rts1, was found to bind in vivo immediately upstream of the repA promoter in studies with mini-Rts1 derivatives with deletions in the upstream region of repA. We constructed another series of repA mutants that would encode RepA derivatives containing oligopeptide substitutions in place of the carboxyl-terminal six amino acids. These modified RepA proteins could not activate ori (Rts1) at all and showed various degrees of incompatibility, or no incompatibility, toward a mini-Rts1 plasmid. These results suggest that the carboxyl-terminal six (or fewer) amino acids of RepA are important for exerting replication and incompatibility functions. One of the RepA derivatives, which showed an evident incompatibility without initiating replication, was examined for its ability to repress the repA gene.     

Further characterization of the R plasmid Rts1 and its mutant pTW2: replication and incompatibility of the plasmid.

Incompatibility of the R plasmid Rts1 and its replication mutant pTW2 was studied in recA host cells of Escherichia coli. When the R plasmid R401, belonging to the same incompatibility group as Rts1, was used as a test plasmid, R401 was eliminated preferentially from (Rts-R401)+ cells irrespective of the direction of transfer. In contrast, pTW2 and R401 were mutually excluded. The decreased incompatibility of pTW2 was confirmed by a direct incompatibility test in which a derivative of Rts1 expelled pTW2 exclusively. Alkaline sucrose gradients of pTW2 and Rts1 DNA indicated that approximately one-fourth of the Rts1 genome was deleted in pTW2. In addition, both the various temperature-dependent properties of Rts1 and the inhibitory effect on phage T4 development were also lost in pTW2. A possible mechanism that regulates the stringent replication of Rts1 is discussed.     

Cloning of the replication and incompatibility regions of a plasmid derived from Rts1

A replication region, consisting of a 1.1-megadalton (Md) EcoRI/HindIII fragment, was isolated from an Rts1 derivative plasmid. This 1.1-Md fragment, designated as mini-Rts1, was ligated to either pBR322 or a nonreplicating DNA fragment specifying a drug resistance, and its replication properties were investigated. The mini-Rts1 plasmid was cured at a high frequency at 42 °C, while it was maintained stably at 37 °C despite it existed in low copy number. These behaviors are quite similar to those of Rts1. By dissecting the pBR322:mini-Rts1 chimeric plasmid with AccI endonuclease, an inc region of 0.34 Md in size was cloned, which expressed incompatibility toward Rts1. Proteins encoded on the mini-Rts1 genome were examined in the minicell system, and one specific product of 35,000 daltons in molecular weight was identified. Any polypeptides specific for the 0.34-Md inc⁺ region within mini-Rts1 were not detected.     

Control of replication of plasmid R1: the intergenic region between copA and repA modulates the level of expression of repA

The RepA protein of plasmid R1 is rate-limiting for initiation of R1 replication. Its synthesis is mainly regulated by interactions of the antisense RNA, CopA, with the leader region of the RepA mRNA, CopT. This work describes the characterization of several mutants with sequence alterations in the intergenic region between the copA gene and the repA reading frame. The analysis showed that most of the mutations led both to a decrease in stability of maintenance of mini-R1 derivatives and to lowered repA expression assayed in translational repA-lacZ fusion constructs. Destruction of the copA gene and replacement of the upstream region by the tac promoter in the latter constructs indicated that these mutations per se alter the expression of repA. In addition, we show that particular mutations in this region can directly affect CopA-mediated control, either by changing the kinetics of interaction of CopA RNA with the RepA mRNA and/or by modifying the activity of the copA promoter. These data indicate the importance of the region analysed in the process that controls R1 replication.     

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.     

Essential DNA sequence for the replication of Rts1.

The promoter sequence of the mini-Rts1 repA gene encoding the 33,000-dalton RepA protein that is essential for replication was defined by RNA polymerase protection experiments and by analyzing RepA protein synthesized in maxicells harboring mini-Rts1 derivatives deleted upstream of or within the presumptive promoter region. The -10 region of the promoter which shows homology to the incII repeat sequences overlaps two inverted repeats. One of the repeats forms a pair with a sequence in the -35 region, and the other forms a pair with the translation initiation region. The replication origin region, ori(Rts1), which was determined by supplying RepA protein in trans, was localized within 188 base pairs in a region containing three incII repeats and four GATC sequences. Dyad dnaA boxes that exist upstream from the GATC sequences appeared to be dispensable for the origin function, but deletion of both dnaA boxes from ori(Rts1) resulted in reduced replication frequency, suggesting that host-encoded DnaA protein is involved in the replication of Rts1 as a stimulatory element. Combination of the minimal repA and ori(Rts1) segments, even in the reverse orientation compared with the natural sequence, resulted in reconstitution of an autonomously replicating molecule.     

Escherichia coli dnaT gene function is required for pBR322 plasmid replication but not for R1 plasmid replication.

Plasmid pBR322 was unable to replicate in a temperature-sensitive dnaT1 strain at a nonpermissive temperature, whereas a pBR322-derived plasmid carrying the wild-type dnaT+ gene was able to replicate under the same conditions. In contrast to pBR322, plasmid R1 could replicate in the dnaT1 strain at a nonpermissive temperature. In keeping with this finding, in vitro replication of plasmid R1 did not require DnaT protein.     

Plasmid copy number mutation in repA gene encoding RepA replication initiator of cryptic plasmid pHM1519 in Corynebacterium glutamicum

ABSTRACT

Cryptic plasmid pHM1519 is a rolling-circular replication mode plasmid of the pCG1 plasmid family in coryneform bacteria. The derived shuttle vector pPK4 is maintained at about 40–50 copies per chromosome in Corynebacterium glutamicum 2256 (ATCC 13869). We found that a mutation (designated copA1) within the repA gene encoding essential initiator protein RepA of the pHM1519-replicon increased the copy number of the mutant plasmid to about 800 copies per chromosome. The mutation was a single G to A base transition, which changed Gly to Glu at position 429 of the amino acid sequence of RepA. In silico secondary structure prediction of RepA suggested that Gly429 is situated in a disordered region in a helix-turn-helix motif, which is a typical DNA-binding domain. This study shows the first example of a high copy number of a C. glutamicum cryptic plasmid caused by an altered replication initiator protein.     

Analysis of mutations in trfA, the replication initiation gene of the broad-host-range plasmid RK2.

Plasmids with mutations in trfA, the gene encoding the replication initiation protein of the broad-host-range plasmid RK2, were isolated and characterized. Mutants identified from a nitrosoguanidine bank were defective in supporting the replication of a wild-type RK2 origin in Escherichia coli. Most of the mutations were clustered in a region of trfA corresponding to the carboxy-terminal quarter of the TrfA protein. 5' and 3' deletion mutants of trfA were also constructed. A C-terminal deletion of three amino acids of the Tr A protein was completely nonfunctional for RK2 replication. However, a deletion of 25 amino acids from the start of the 33-kDa TrfA protein was still competent for replication. Further characterization of the point and deletion trfA mutants in vivo revealed that a subset was capable of supporting RK2 replication in other gram-negative bacteria, including Pseudomonas putida, Agrobacterium tumefaciens, and Azotobacter vinelandii. Selected mutant TrfA proteins were partially purified and characterized in vitro. Velocity sedimentation analysis of these partially purified TrfA proteins indicated that the wild-type protein and all mutant TrfA proteins examined exist as dimers in solution. Results from in vitro replication assays corroborated the experimental findings in vivo. Gel retardation results clearly indicated that the point mutant TrfA-33:151S, which was completely defective in replication of an RK2 origin in all of the bacterial hosts tested in vivo, and a carboxy-terminal deletion mutant, TrfA-33:C delta 305, were not able to bind iterons in vitro. In addition to the partially defective or could not be distinguished from the wild-type protein in binding to the origin region. The mutant proteins with apparently normal DNA-binding activity in vitro either were inactive in all four gram-negative bacteria tested or exhibited differences in functionality depending on the host organism. These mutant TrfA proteins may be altered in the ability to interact with the replication proteins of the specific host bacterium.     

Replication of thermosensitive Rts1 plasmid deoxyribonucleic acid at the nonpermissive temperature.

Replication of the thermosensitive drug resistance factor Rts1 was studied at the nonpermissive temperature (42 degrees C). It was concluded from the following observations that replication of this plasmid takes place at 42 degrees C without involving the covalently closed circular (CCC) form of deoxyribonucleic acid (DNA). (i) DNA-DNA- reassociation kinetics studies with purified Rts1 DNA showed that Rts1 DNA increased several-fold during cell growth at 42 degrees C while very little, if any, CCC DNA was synthesized. (ii) When Escherichia coli 20S0(Rts1) was labeled with [3H]thymidine at 42 degrees C, a significant amount of radioactive DNA hybridizable to Rts1 DNA was formed. This DNA was found in a fraction where DNA other than CCC DNA was expected in alkaline sucrose density gradient centrifugation analysis. When E. coli 20S0(Rts1) was labeled at 32 degrees C, the labeled CCC DNA did not disappear during a chase period at 42 degrees C. This indicates that preformed CCC DNA does not participate in replication at the nonpermissive temperature. These results are consistent with the hypothesis that there are two modes of replication of Rts1 DNA, one involving a CCC molecule and the other not involving this form, and that only the latter mode takes place at the nonpermissive temperature.     

Specific protein-DNA and protein-protein interaction in the hig gene system, a plasmid-borne proteic killer gene system of plasmid Rts1

The hig (host inhibition of growth) gene system of plasmid Rts1 belongs to the plasmid-encoded proteic killer gene family. Among the proteic killer genes described so far, hig is unique in that the toxin gene (higB) exists upstream of the antidote gene (higA). There are two promoters in the hig locus, Phig and PhigA, and only the former, which expresses both higB and higA genes, is negatively controlled by HigA and HigB proteins. In this study, we purified HigA protein by means of GST fusion. The electrophoretic mobility shift assay using the purified protein revealed that HigA specifically bound to the Phig region, but not to PhigA. The HigA-binding sequence was determined by DNase I footprinting assay to be a 56-bp sequence that completely covered the -35 and -10 boxes of Phig. The presence of two inverted repeats in the binding sequence and the identification of a dimer form of HigA by cross-linking experiment suggested that the protein bound to the Phig region as a dimer. HigB was purified as a GST fusion protein as well, though it was achieved only in the presence of HigA. HigA and GST-HigB formed a highly stable complex where the two proteins were present in an equimolar ratio.