Donald R. Helinski

Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression

Derivatives of plasmid pRK290 that are useful for cloning and for analyzing gene expression in a wide variety of Gram-negative bacteria are described. A smaller broad host range plasmid derived from RK2, with properties similar to that of pRK290, is also described.

[17] Plasmid cloning vehicles derived from plasmids ColE1, F, R6K, and RK2

Publisher Summary This chapter discusses the cloning vehicles related to plasmids ColE1, F, R6K, and RK2. Because these replicons have different properties, cloning vehicles derived from one of them may be more suitable for a particular application than vehicles derived from another. Also, because derivatives of a single plasmid do not stably coexist, it is often desirable to have available cloning vehicles from a variety of incompatibility groups to study the interactions among cloned fragments. The ColE1 vehicles pMK20, pMK16, and pMK2004 are very useful for general-purpose cloning because they are maintained in high copy number and provide good selective markers for transformation. Like the ColE1 vehicles, pRK353, a derivative of the antibiotic resistance plasmid R6K, is present in a large number of copies per cell and can be used as a high-copy-number cloning vehicle that can coexist with ColE1. In addition, the R6K replicon has been separated into two components that make up a functional replicon. Plasmid RK2 is unusual because it is maintained in a wide variety of gram-negative bacteria, and therefore RK2 derivatives may be of use as cloning vehicles in gram-negative bacteria other than Escherichia coli ( E. coli ).

Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K

A non-self-replicating segment (1370 base pairs) of plasmid R6K was cloned in E. coli and shown to trans-complement temperature-sensitive replication mutants of this plasmid. This segment contains the gene which codes for a protein required for initiation of replication of the plasmid, and was used as a helper in a functional assay for an origin of replication in R6K derivatives. A 420 bp fragment, derived from R6K DNA, was shown to carry a functional origin since it was capable of replicating as a plasmid in E. coli cells carrying the helper segment either on the host chromosome or on a plasmid Col E1 derivative. The copy number of the origin fragment in cells carrying the helper segment on the chromosome is essentially the same as the copy number of R6K. A model for the positive regulation of plasmid R6K replication is presented.

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.

Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda pL promoter.

Two multiple-copy, ColE1-type, plasmid cloning vehicles, pHUB2 and pHUB4, have been constructed that carry four different single restriction sites down-stream from the phage lambda promoter pL. The promoting activity of pL is switched off at low temperature in the presence of a cIts gene that specifies a temperature-sensitive repressor but could be activated by heat induction. cIts was located either on the host chromosome, or on a second plasmid pRK248 that is compatible with the cloning vehicle, or on the vehicle itself. Three different restriction fragments, each carrying the gene trpA of Salmonella typhimurium or Shigella dysenteriae, have been inserted into the EcoRI, BamHI and SalI sites, respectively, of these plasmids and pL dependent expression of the inserted gene in Escherichia coli was determined by measuring the enzymatic activity of the trpA gene product. Heat induction resulted in a level of expression of trpA corresponding to 1 to 6.6% of the total soluble cell protein as trpA protein. The level of trpA protein production depended on the particular insert and the plasmid used.

DNA polymerase as a requirement for the maintenance of the bacterial plasmid colicinogenic factor E1

Abstract The extrachromosomal elements Col V, Col I, F ′14, R1 , and R 64 can be transferred by conjugation into and maintained in Escherichia coli cells carrying the pol A1 mutation. The Col E2 factor can be transferred into pol A1 cells but is poorly maintained. The Col E2 cells show a segregation to Col − at a rate of about 50% per generation. The Col E1 factor cannot be transferred into pol A1 cells and is lost when the pol A1 mutation is tranduced into Col E1 containing cells. Evidence is provided that DNA polymerase is essential to the maintenance of the Col E1 factor.

Structural relationships among rhizobium meliloti symbiotic promoters

Symbiotic nitrogen fixation by Rhizobium meliloti requires the developmentally specific expression of certain bacterial genes. One set of these genes encodes the subunits of nitrogenase, the enzyme responsible for the reduction of atmospheric dinitrogen to ammonia, and another set consists of closely linked genes also essential for nitrogen fixation. Examination of promoter and probable regulatory regions for these gene sets has revealed extensive DNA sequence conservation for more than 160 bp upstream of the respective transcription start points. Three such promoter regions have been identified in the nitrogen fixation (nif) gene cluster of R. meliloti strain 102F34. Using one of these promoter regions as a hybridization probe, three additional sequences were found in the genome of this strain. The DNA of other R. meliloti strains and Rhizobium species were also examined for homology to the symbiotically regulated promoters of R. meliloti 102F34. DNA sequences homologous to these R. meliloti promoters were found among diverse rhizobia, and in at least some cases were associated with nif genes.

Multicopy plasmids are clustered and localized in Escherichia coli

We localized the multicopy plasmid RK2 in Escherichia coli and found that the number of fluorescent foci observed in each cell was substantially less than the copy number of the plasmid, suggesting that many copies of RK2 are grouped into a few multiplasmid clusters. In minimal glucose media, the majority of cells had one or two foci, with a single focus localized near midcell, and two foci near the 1/4 and 3/4 cell positions. The number of foci per cell increased with cell length and with growth rate, and decreased upon entering stationary phase, suggesting a coordination of RK2 replication or segregation with the bacterial cell cycle. Time-lapse microscopy demonstrated that partitioning of RK2 foci is achieved by the splitting of a single focus into two or three smaller foci, which are capable of separating with rapid kinetics. A derivative of the high-copy-number plasmid pUC19 containing the lacO array was also localized by tagging with GFP-LacI. Whereas many of the cells contained numerous, randomly diffusing foci, most cells exhibited one or two plasmid clusters located at midcell or the cell quarter positions. Our results suggest a model in which multicopy plasmids are not always randomly diffusing throughout the cell as previously thought, but can be replicated and partitioned in clusters targeted to specific locations.

Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2.

SummaryA DNA sequence cosisting of 617 base pairs (bp) from the region of the origin of replication of the broad-host range plasmid RK2 has been determined. Included within this sequence is a 393 bp HpaII restriction fragment that provides a functional origin or replication when other essential RK2 specified functions are provided in trans. Also contained in this sequence is a region, distinguished functionally from the replication origin, which is involved in the expression of inc2 incompatibility, i.e., the ability of derivatives of RK2 to eliminate a resident RK2 plasmid. The 617 bp sequence includes eight 17 base pair direct repeats with 5 located within the region required for a functional replication origin and 3 within the region involved in inc2 incompatibility. In addition, a 40 bp region rich in A-T followed by a 60 bp stretch having a high G+C content is present. Deletion evidence indicates that the A-T rich and possibly the G+C regions are required for a functional replication origin. Based on the evidence contained in this and the preceding paper (Thomas et al. 1980 b) a model will be presented for the involvement of these specific sequences in the initiation of RK2 DNA replication, plasmid maintenance and plasmid incompatibility.

Plasmids in bacteria

Structure and Evolution.- Plasmids as Organisms.- Report on a Workshop: Structure and Function.- Evolutionary Relevance of Genetic Rearrangements Involving Plasmids.- Mechanisms of Transposition in Bacteria.- Insertion of Transcriptional Elements Outside the Replication Region Can Interfere with Replication, Maintenance, and Stability of ColE1-Derived Plasmids.- Studies on the Transposition of IS1.- On the Transposition and Evolution of Tn1721 and its Relatives.- Repeated DNA Sequences Recombine 1,000 Times More Frequently in a Plasmid Than in the Chromosome of BacillusSubtilis.- Mercury Resistance Transposon Tn813 Mediates Chromosome Transfer in Rhodopseudomonas sphaeroides and Intergeneric Transfer of pBR322.- Report on a Workshop: Plasmids in Unusual Systems.- Replication, Incompatibility, and Partition.- Chairmans Introduction: Replication, Incompatibility, and Partition.- Role of the ? Initiation Protein and Direct Nucleotide Sequence Repeats in the Regulation of Plasmid R6K Replication.- Initiation of Replication of the EscherichiaColi Chromosomal Origin Reconstituted with Purified Enzymes.- Origin and Initiation Sites of ?dv DNA Replication In Vitro.- Broad Host-Range Plasmid R1162: Replication, Incompatibility, and Copy-Number Control.- Control of Plasmid Replication: Theoretical Considerations and Practical Solutions.- The Partition Functions of PI, P7, and F Miniplasmids.- Genetic Interactions of Broad Host-Range Plasmid RK2: Evidence for a Complex Replication Regulon.- Replication Determinants of the Broad Host Range Plasmid RSF1010.- Regulation of Replication and Maintenance Functions of Broad Host-Range Plasmid RK2.- Control of Chromosome Replication in Bacteria.- Stable Maintenance of Plasmid CLO DF13: Structural and Functional Relationships Between Replication, Control, Partitioning, and Incompatibility.- Replication Control for PT181, An Indirectly Regulated Plasmid.- Construction of ColE1 RNA1 Mutants and Analysis of their Function In Vivo.- Incompatibility and INCFII Plasmid Replication Control.- P1 Plasmid Maintenance: A Paradigm of Precise Control.- Partitioning of the pSC101 Plasmid During Cell Division.- DNA-Protein Interaction at the Replication Origins of Plasmid Chromosomes.- Positive Regulation and Transcription Initiation of xy1 Operons on TOL Plasmid.- Plasmid Transfer.- Chairmens Introduction: Plasmid Transfer.- Linear Plasmids with Terminal Inverted Repeats Obtained from Streptomycesrochei and Kluyveromyceslactis.- Conjugal Plasmid Transfer in BacillusThuringiensis.- Mechanisms Essential for Stable Inheritance of Mini-F Plasmid.- Sex Pheromones and Plasmid Transfer in StreptococcusFaecalis: A Pheromone, cAM373, Which is Also Excreted by StaphylococcusAureus.- General Genetic Recombination of Bacterial Plasmids.- The Origin of Plasmid DNA Transfer During Bacterial Conjugation.- Genes and Gene Products Involved in the Synthesis of F-PILI.- Genetics of Clindamycin Resistance in Bacteroides.- Effect of Chromosome Homology of Plasmid Transformation and Plasmid Conjugal Transfer in Haemophilus Influenzae.- Plasmid DNA Primases and Their Role in Bacterial Conjugation.- Promoters in the Transfer Region of Plasmid F.- Conjugative Sex Plasmids of Streptomyces.- Specialized Functions, Structure, and Evolution.- The Structure and Source of Plasmid DNA Determine the Cloning Properties of Vectors for BacillusSubtilis.- Shuttle Vector for EscherichiaColi, PseudomonasPutida, and PseudomonasAeruginosa.- Report on a Workshop: Plasmid Cloning Vehicles.- Plasmids in the Degradation of Chlorinated Aromatic Compounds.- Notes on Metabolic Plasmid Organization and Expression.- The Structure of the mer Operon.- Analysis and Manipulation of Plasmid-Encoded Pathways for the Catabolism of Aromatic Compounds by Soil Bacteria.- The Plasmid-Specified Aerobactin Iron Uptake System of EscherichiaColi.- Plasmid-Mediated Iron Sequestering Systems in Pathogenic Strains of Vibrioanguillarum and EscherichiaColi.- Chairmans Introduction: Specialized Functions.- Cloning of an Enzymatically Active Segment of the Exotoxin-A Gene of PseudomonasAeruginosa Ill.- Structure, Function, and Regulation of the Plasmid-Encoded Hemolysin Determinant of EscherichiaColi.- pIAA, A Virulence Plasmid in PseudomonasSavastanoi.- The Molecular Basis of Plant Cell Transformation by AgrobacteriumTumefaciens.- Post-Transcriptional Regulation of Chloramphenicol Acetyl Transferase.- Plasmid and Larval Toxin of Bacilluslaterosporus.- Posters (Poster abstracts have been reproduced as submitted).- Replication, Incompatibility, Partition.- Specialized Functions.- Structure and Evolution.- Transfer.- Vectors.- Rosters.- Speakers.- Participants.- Index of Poster Authors.