A Brief History of Plasmids.

In the late 1950s, a number of laboratories took up the study of plasmids once the discovery was made that extrachromosomal antibiotic resistance (R) factors are the responsible agents for the transmissibility of multiple antibiotic resistance among the enterobacteria. The use of incompatibility for the classification of plasmids is now widespread. It seems clear now on the basis of the limited studies to date that the number of incompatibility groups of plasmids will likely be extremely large when one includes plasmids obtained from bacteria that are normal inhabitants of poorly studied natural environments. The presence of both linear chromosomes and linear plasmids is now established for several Streptomyces species. One of the more fascinating developments in plasmid biology was the discovery of linear plasmids in the 1980s. A remarkable feature of the Ti plasmids of Agrobacterium tumefaciens is the presence of two DNA transfer systems. A definitive demonstration that plasmids consisted of duplex DNA came from interspecies conjugal transfer of plasmids followed by separation of plasmid DNA from chromosomal DNA by equilibrium buoyant density centrifugation. The formation of channels for DNA movement and the actual steps involved in DNA transport offer many opportunities for the discovery of proteins with novel activities and for establishing fundamentally new concepts of macromolecular interactions between DNA and specific proteins, membranes, and the peptidoglycan matrix.

The incC korB region of RK2 repositions a mini-RK2 replicon in Escherichia coli.

Analysis by fluorescence microscopy has established that plasmid RK2 in Escherichia coli and other gram-negative bacteria is present as discrete clusters that are located inside the nucleoid at the mid- or quarter-cell positions. A mini-RK2 replicon containing an array of tetO repeats was visualized in E. coli cells that express a TetR-EYFP fusion protein. Unlike intact RK2, the RK2 mini-replicon (pCV1) was localized as a cluster at the cell poles outside of the nucleoid. Insertion of the O(B1)incC korB partitioning (par) region of RK2 into pCV1 resulted in a shift of the mini-replicon to within the nucleoid region at the mid- and quarter-cell positions. Despite the repositioning of the mini-RK2 replicon to the cellular positions where intact RK2 is normally located, the insertion of the intact O(B1) incC korB region did not significantly stabilize the mini-RK2 plasmid during cell growth. Deletions within the O(B1)incC or the korB region resulted in a failure of this par region to move pCV1 out of its polar position. The insertion of the par system of plasmid F into pCV1 resulted in a similar shift in the location of pCV1 to the nucleoid region. Unlike O(B1)incC korB, the insertion of the RK2 parABC resolvase system into pCV1 did not affect the polar positioning of pCV1. This effect of O(B1)incC korB on the location of pCV1 provides additional evidence for a partitioning role of this region of plasmid RK2. However, the failure of this region to significantly increase the stability of the mini-RK2 plasmid indicates that the localization of the plasmid to the mid- and quarter cell positions in E. coli is not in itself sufficient for the stable maintenance of plasmid RK2.

Localization of the naturally occurring plasmid ColE1 at the cell pole.

The naturally occurring plasmid ColE1 was found to localize as a cluster in one or both of the cell poles of Escherichia coli. In addition to the polar localization of ColE1 in most cells, movement of the plasmid to the midcell position was observed in time-lapse studies. ColE1 could be displaced from its polar location by the p15A replicon, pBAD33, but not by plasmid RK2. The displacement of ColE1 by pBAD33 resulted in an almost random positioning of ColE1 foci in the cell and also in a loss of segregational stability, as evidenced by the large number of cells carrying pBAD33 with no visible ColE1 focus and as confirmed by ColE1 stability studies. The addition of the active partitioning systems of the F plasmid (sopABC) or RK2 (O(B1) incC korB) resulted in movement of the ColE1 replicon from the cell pole to within the nucleoid region. This repositioning did not result in destabilization but did result in an increase in the number of plasmid foci, most likely due to partial declustering. These results are consistent with the importance of par regions to the localization of plasmids to specific regions of the cell and demonstrate both localization and dynamic movement for a naturally occurring plasmid that does not encode a replication initiation protein or a partitioning system that is required for plasmid stability.

Inhibition of protein and RNA synthesis in Escherichia coli results in declustering of plasmid RK2.

Multi-copy plasmids in Escherichia coli are not randomly distributed throughout the cell but are present as clusters of plasmid molecules that are localized at preferred cellular locations. A plasmid RK2 derivative (pZZ15) that can be tagged with a green fluorescent protein-LacI fusion protein normally exists as clusters that are localized at the mid- and quarter-cell positions. In this study the effect of the protein synthesis inhibitor, chloramphenicol, and the RNA synthesis inhibitor, rifampicin, on RK2 clustering and localization was examined. The addition of either inhibitor to exponentially growing E. coli cells carrying pZZ15 results in a displacement of the position and a declustering of this multi-copy plasmid indicating that continued protein synthesis and RNA synthesis are required for clustering and localization of this plasmid. It is likely that it is not just the process of transcription or translation that is important for clustering but rather some host or plasmid encoded factor(s) that is required.

Functional analysis of two putative chromosomal replication origins from Pseudomonas aeruginosa.

Two autonomously replicating elements previously isolated from Pseudomonas aeruginosa were characterized in vitro for pre-priming complex formation using combinations of replication proteins from P. aeruginosa and Escherichia coli. The results of these studies showed that the P. aeruginosa DnaA and DnaB proteins could form a pre-priming complex on plasmid templates containing either of the two autonomously replicating elements of P. aeruginosa, pYJ50 (containing oriCI), and pYJ52 (containing oriCII), or the E. coli chromosomal origin (plasmid pYJ2). The E. coli DnaA, DnaB, and DnaC proteins were also able to form a pre-priming complex on pYJ2, pYJ50, and pYJ52. Neither pYJ50 nor pYJ52 could be established in E. coli, suggesting a block in steps subsequent to the formation of the pre-priming complex. Similarly, pYJ2 could not be established in P. aeruginosa. Since pYJ50 and pYJ52 could be established in P. aeruginosa and both putative origins form a pre-priming complex in vitro, attempts were made to delete each of these two putative origins. The results indicate that the oriCI sequence is essential for cell viability under typical laboratory growth conditions but that oriCII is not.

Gyrase inhibitors and thymine starvation disrupt the normal pattern of plasmid RK2 localization in Escherichia coli.

Multicopy plasmids in Escherichia coli are not randomly distributed throughout the cell but exist as defined clusters that are localized at the mid-cell, or at the 1/4 and 3/4 cell length positions. To explore the factors that contribute to plasmid clustering and localization, E. coli cells carrying a plasmid RK2 derivative that can be tagged with a green fluorescent protein-LacI fusion protein were subjected to various conditions that interfere with plasmid superhelicity and/or DNA replication. The various treatments included thymine starvation and the addition of the gyrase inhibitors nalidixic acid and novobiocin. In each case, localization of plasmid clusters at the preferred positions was disrupted but the plasmids remained in clusters, suggesting that normal plasmid superhelicity and DNA synthesis in elongating cells are not required for the clustering of individual plasmid molecules. It was also observed that the inhibition of DNA replication by these treatments produced filaments in which the plasmid clusters were confined to one or two nucleoid bodies, which were located near the midline of the filament and were not evenly spaced throughout the filament, as is found in cells treated with cephalexin. Finally, the enhanced yellow fluorescent protein-RarA fusion protein was used to localize the replication complex in individual E. coli cells. Novobiocin and nalidixic acid treatment both resulted in rapid loss of RarA foci. Under these conditions the RK2 plasmid clusters were not disassembled, suggesting that a completely intact replication complex is not required for plasmid clustering.

Plasmid host-range: restrictions to F replication in Pseudomonas.

Host-range, a fundamental property of a bacterial plasmid, is primarily determined by the plasmid replication system. To investigate the basis of the restricted host-range of the well-studied F-plasmid of Escherichia coli, we characterized in vitro the interactions of the host DnaA initiation protein and DnaB helicase from Pseudomonas aeruginosa and Pseudomonas putida with the replication origin, oriS, and initiation protein, RepE, of the RepFIA replicon. The results presented here show that a pre-priming complex can form at the F-origin with the replication proteins from the non-native hosts in the presence of RepE. However, RepE cannot form a stable complex with DnaB of P. aeruginosa or P. putida but does stably interact with E. coli DnaB. This unstable association may affect the ability of F to replicate in Pseudomonas. In addition, replication studies in vivo suggest that inefficient expression of the RepE initiation protein from its native promoter in Pseudomonas is a factor in restricting its host-range. This, however, is not the only barrier to F replication, as mini-F derivatives with an alternative promoter for RepE expression do not replicate in P. putida and are not stably maintained in P. aeruginosa.

A specific region in the N terminus of a replication initiation protein of plasmid RK2 is required for recruitment of Pseudomonas aeruginosa DnaB helicase to the plasmid origin.

Broad host range plasmid RK2 encodes two versions of its essential replication initiation protein, TrfA, using in-frame translational starts spaced 97 amino acids apart. The smaller protein, TrfA-33, is sufficient for plasmid replication in many bacterial hosts. Efficient replication in Pseudomonas aeruginosa, however, specifically requires the larger TrfA-44 protein. With the aim of identifying sequences of TrfA-44 required for stable replication of RK2 in P. aeruginosa, specific deletions and a substitution mutant within the N terminus sequence unique to TrfA-44 were constructed, and the mutant proteins were tested for activity. Deletion mutants were targeted to three of the four predicted helical regions in the first 97 amino acids of TrfA-44. Deletion of TrfA-44 amino acids 21-32 yielded a mutant protein, TrfA-44Delta2, that had lost the ability to bind and load the DnaB helicase of P. aeruginosa or Pseudomonas putida onto the RK2 origin in vitro and did not support stable replication of an RK2 mini-replicon in P. aeruginosa in vivo. A substitution of amino acid 22 within this essential region resulted in a protein, TrfA-44E22A, with reduced activity in vitro, particularly with the P. putida helicase. Deletion of amino acids 37-55 (TrfA-44Delta3) slightly affected protein activity in vitro with the P. aeruginosa helicase and significantly with the P. putida helicase, whereas deletion of amino acids 71-88 (TrfA-44Delta4) had no effect on TrfA activity in vitro with either helicase. These results identify regions of the TrfA-44 protein that are required for recruitment of the Pseudomonas DnaB helicases in the initiation of RK2 replication.

A multifunctional plasmid-encoded replication initiation protein both recruits and positions an active helicase at the replication origin.

The DnaA replication initiation protein has been shown to be essential for DNA strand opening at the AT-rich region of the replication origin of the Escherichia coli chromosome as well as serving to recruit and position the DnaB replicative helicase at this open region. Homologues of the dnaA gene of E. coli have been found in most bacterial species, and the DnaA protein has been shown to be required for the initiation of replication of both chromosomal and plasmid DNA. For several plasmid elements it has been found that a plasmid-encoded initiation protein is required along with the DnaA protein to bring about opening of the AT-rich region at the replication origin. The broad host range plasmid RK2 encodes two forms of its replication initiation protein (TrfA-33 and TrfA-44) that differ by an additional 98 aa at the N terminus of the larger (TrfA-44) form. Both forms initiate replication of RK2 in E. coli in vitro by a DnaA-dependent mechanism. However, as shown in this study, TrfA-44 specifically interacts with the DnaB replicative helicase of Pseudomonas putida and Pseudomonas aeruginosa and initiates the formation of a prepriming open complex in the absence of DnaA protein. Thus, the TrfA-44 initiation protein has the multifunctional properties of recruiting and positioning an active form of the DnaB helicase at the RK2 replication origin by a DnaA-independent process. This unique property for a replication initiation protein undoubtedly plays an important role in extending the host range of the RK2 antibiotic resistance plasmid.

Nucleotide sequence based characterizations of two cryptic plasmids from the marine bacterium Ruegeria isolate PR1b.

Two plasmids, 76 and 148 kb in size, isolated from Ruegeria strain PR1b were entirely sequenced. These are the first plasmids to be characterized from this genus of marine bacteria. Sequence analysis revealed a biased distribution of function among the putative proteins encoded on the two plasmids. The smaller plasmid, designated pSD20, encodes a large number of putative proteins involved in polysaccharide biosynthesis and export. The larger plasmid, designated pSD25, primarily encodes putative proteins involved in the transport of small molecules and in DNA mobilization. Sequence analysis revealed uncommon potential replication systems on both plasmids. pSD25, the first repABC-type replicon isolated from the marine environment, actually contains two repABC-type replicons. pSD20 contains a complex replication region, including a replication origin and initiation protein similar to iteron-containing plasmids (such as pSW500 from the plant pathogen Erwinia stewartii) linked to putative RepA and RepB stabilization proteins of a repABC-type replicon and is highly homologous to a plasmid from the phototrophic bacterium Rhodobacter sphaeroides. Given the nature of the putative proteins encoded by both plasmids it is possible that these plasmids enhance the metabolic and physiological flexibility of the host bacterium, and thus its adaptation to the marine sediment environment.

ParE toxin encoded by the broad-host-range plasmid RK2 is an inhibitor of Escherichia coli gyrase.

Broad-host-range plasmid RK2 encodes a post-segregational killing system, parDE, which contributes to the stable maintenance of this plasmid in Escherichia coli and many distantly related bacteria. The ParE protein is a toxin that inhibits cell growth, causes cell filamentation and eventually cell death. The ParD protein is a specific ParE antitoxin. In this work, the in vitro activities of these two proteins were examined. The ParE protein was found to inhibit DNA synthesis using an E. coli oriC supercoiled template and a replication-proficient E. coli extract. Moreover, ParE inhibited the early stages of both chromosomal and plasmid DNA replication, as measured by the DnaB helicase- and gyrase-dependent formation of FI*, a highly unwound form of supercoiled DNA. The presence of ParD prevented these inhibitory activities of ParE. We also observed that the addition of ParE to supercoiled DNA plus gyrase alone resulted in the formation of a cleavable gyrase-DNA complex that was converted to a linear DNA form upon addition of sodium dodecyl sulphate (SDS). Adding ParD before or after the addition of ParE prevented the formation of this cleavable complex. These results demonstrate that the target of ParE toxin activity in vitro is E. coli gyrase.

A 50-kb plasmid rich in mobile gene sequences isolated from a marine micrococcus.

A 50,709-bp cryptic plasmid isolated from a marine Micrococcus has been sequenced and found to contain a number of putative mobile genetic elements. The coding regions for 11 putative transposases comprise approximately 17% of the total plasmid sequence. The majority of these transposases are located within a 13-kb cluster which includes a 1553-bp direct repeat consisting of a duplicated pair of transposase genes. The remaining putative ORFs showed similarity to a variety of proteins, the most notable being spider silk.

Regulation of the switch from early to late bacteriophage lambda DNA replication.

There are two modes of bacteriophage lambda DNA replication following infection of its host, Escherichia coli. Early after infection, replication occurs according to the theta (theta or circle-to-circle) mode, and is later switched to the sigma (sigma or rolling-circle) mode. It is not known how this switch, occurring at a specific time in the infection cycle, is regulated. Here it is demonstrated that in wild-type cells the replication starting from orilambda proceeds both bidirectionally and unidirectionally, whereas in bacteria devoid of a functional DnaA protein, replication from orilambda is predominantly unidirectional. The regulation of directionality of replication from orilambda is mediated by positive control of lambda p(R) promoter activity by DnaA, since the mode of replication of an artificial lambda replicon bearing the p(tet) promoter instead of p(R) was found to be independent of DnaA function. These findings and results of density-shift experiments suggest that in dnaA mutants infected with lambda, phage DNA replication proceeds predominantly according to the unidirectional theta mechanism and is switched early after infection to the sigma mode. It is proposed that in wild-type E. coli cells infected with lambda, phage DNA replication proceeds according to a bidirectional theta mechanism early after infection due to efficient transcriptional activation of orilambda, stimulated by the host DnaA protein. After a few rounds of this type of replication, the resulting increased copy number of lambda genomic DNA may cause a depletion of free DnaA protein because of its interaction with the multiple DnaA-binding sites in lambda DNA. It is proposed that this may lead to inefficient transcriptional activation of orilambda resulting in unidirectional theta replication followed by sigma type replication.