Molecular vehicle properties of the broad host range plasmid RK2.

The pladmid RK2 is stably maintained in a broad range of gram-negative bacteria. The RK2 DNA has a single Eco RI restriction site. The insertion of a DNA fragment into this site does not interfere with either plasmid maintenance or self-transmissibility. Because RK2 has a broad host range, it should be useful for the construction in vitro of hybrid plasmid molecules capable of being established by conjugal transfer or transformation into many genera of gram-negative organisms.

Gene cloning and containment properties of plasmid Col E1 and its derivatives.

Colicinogenic plasmid E1 (Col E1) and Col E1 derivatives offer advantages as plasmid cloning vehicles with regard to both utility and biological containment. The Col E1 derivative pCR1 does not alter those essential characteristics of the enfeebled Escherichia coli strain x1776 that make this strain particularly useful as a host-vehicle system for recombinant DNA research.

Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants.

The luciferase gene from the firefly, Photinus pyralis, was used as a reporter of gene expression by light production in transfected plant cells and transgenic plants. A complementary DNA clone of the firefly luciferase gene under the control of a plant virus promoter (cauliflower mosaic virus 35S RNA promoter) was introduced into plant protoplast cells (Daucus carota) by electroporation and into plants (Nicotiana tabacum) by use of the Agrobacterium tumefaciens tumor-inducing plasmid. Extracts from electroporated cells (24 hours after the introduction of DNA) and from transgenic plants produce light when mixed with the substrates luciferin and adenosine triphosphate. Light produced by the action of luciferase was also detected in undisrupted leaves or cells in culture from transgenic plants incubated in luciferin and in whole transgenic plants "watered" with luciferin. Although light was detected in most organs in intact, transgenic plants (leaves, stems, and roots), the pattern of luminescence appeared to reflect both the organ-specific distribution of luciferase and the pathway for uptake of luciferin through the vasculature of the plant.

Cloning and characterization of small circular DNA from Chinese hamster ovary cells.

Small polydisperse circular (spc) DNA was isolated and cloned, using BglII from Chinese hamster ovary (CHO) cells. The properties of 47 clones containing at least 43 different BglII fragments are reported. The majority of the clones probably contain entire sequences from individual spcDNA molecules. Most of the clones were homologous to sequences in CHO cell chromosomal DNA, and many were also homologous to mouse LMTK- cell chromosomal sequences. The majority of homologous CHO cell chromosomal sequences were repetitive, although a few may be single copy. Only a small fraction of cloned spcDNA molecules were present in every cell; most occurred less frequently than once in 15 cells. Localization studies indicated that at least a portion of spcDNA is associated with the nucleus in CHO cells.

Firefly luciferase gene: structure and expression in mammalian cells.

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.

Unidirectional replication of the P-group plasmid RK2.

The mode of replication of the broad host-range plasmid RK2 has been determined from examination of molecular replicative forms cleaved with the restriction endonucleases EcoRI and Hind III. Replication is unidirectional, and proceeds from a unique origin. The location of the origin and other evidence suggests that genes involved in plasmid maintenance are not tightly clustered.

Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K.

The three replication origins of the antibiotic resistance plasmid R6K require for their activity in Escherichia coli a DNA segment containing seven 22 base-pair direct repeats and a plasmid-encoded initiation protein (pi). The pi protein functions in the negative control of R6K replication, in addition to its requirement for the initiation of replication. Construction of a plasmid containing the pi structural gene (pir) downstream from the inducible pR promoter of bacteriophage lambda provided high levels of production of pi protein in E. coli. The pi protein was purified and shown to possess general DNA binding properties with a preference for DNA fragments containing the gamma origin of replication, the operator region of the pir gene and the R6K beta-origin region. Velocity sedimentation analysis indicates that the pi protein exists as a dimer in its native form. Agarose gel electrophoresis analysis of pi-gamma-origin complexes suggests that one pi dimer binds to each copy of the 22 base-pair direct repeats in the gamma origin region. Purified mutant pi protein obtained from a temperature-sensitive initiation mutant (pir 105-ts) exhibited temperature-sensitive binding activity to the gamma-origin region, whereas two mutant proteins exhibiting a high copy number phenotype were unaltered (pir104-cop) or slightly reduced (pir1-cop) in binding activity. The patterns of DNase I protection and enhancement were similar for the wild-type and mutant proteins examined.

The parDE operon of the broad-host-range plasmid RK2 specifies growth inhibition associated with plasmid loss.

Recently, a 0.8 kb region of the broad-host-range plasmid RK2 has been shown to be sufficient to stabilize plasmids in a vector-independent, broad-host-range manner under some but not all growth conditions (Roberts, R. C. & Helinski, D. R. (1992). J. Bacteriol. 174, 8119-8132). This region encompasses the parDE operon, which encodes the small proteins ParD and ParE, both of which are required for the plasmid stabilization. This paper demonstrates that the 0.8 kb region encodes the capacity to inhibit cell growth of Escherichia coli, presumably of those bacteria that have lost plasmids carrying this stabilization region, and this inhibition appears to be associated with cell killing and bacterial cell filamentation. A good correlation was observed between the capacity of wild-type and mutated 0.8 kb regions to promote stable maintenance of a temperature-sensitive RK2 replicon plasmid and to inhibit bacterial cell division under specified medium conditions. The properties of the wild-type and mutant 0.8 kb regions further indicate that the ParE protein is responsible for the growth inhibition and the ParD protein neutralizes the toxic activity of the ParE protein. This is consistent with the finding that the presence of the parD gene in trans destabilizes a temperature-sensitive RK2 replicon carrying a copy of the functional 0.8 kb region. This destabilization appears to be the result of ParD protein-mediated suppression of growth inhibition, thus allowing survival of cells that have lost the temperature-sensitive plasmid. These observations indicate that the 0.8 kb sequence of RK2 encodes a growth inhibition function that is likely to play a role in the plasmid stabilization.

Complete nucleotide sequence of Birmingham IncP alpha plasmids. Compilation and comparative analysis.

The IncP alpha promiscuous plasmid (R18, R68, RK2, RP1 and RP4) comprises 60,099 bp of nucleotide sequence, encoding at least 74 genes. About 40 kb of the genome, designated the IncP core and including all essential replication and transfer functions, can be aligned with equivalent sequences in the IncP beta plasmid R751. The compiled IncP alpha sequence revealed several previously unidentified reading frames that are potential genes. IncP alpha plasmids carry genetic information very efficiently: the coding sequences of the genes are closely packed but rarely overlap, and occupy almost 86% of the genome's nucleotide sequence. All of the 74 genes should be expressed, although there is as yet experimental evidence for expression of only 60 of them. Six examples of tandem-in-frame initiation sites specifying two gene products each are known. Two overlapping gene arrangements occupy different reading frames of the same region. Intergenic regions include most of the 25 promoters; transcripts are usually polycistronic. Translation of most of the open reading frames seems to be initiated independently, each from its own ribosomal binding and initiation site, although, a few cases of coupled translation have been reported. The most frequently used initiation codon is AUG but translation for a few open reading frames begins at GUG or UUG. The most common stop-codon is UGA followed by UAA and then UAG. Regulatory circuits are complex and largely dependent on two components of the central control operon. KorA and KorB are transcriptional repressors controlling at least seven operons. KorA and KorB act synergistically in several cases by recognizing and binding to conserved nucleotide sequences. Twelve KorB binding sites were found around the IncP alpha sequence and these are conserved in R751 (IncP beta) with respect to both sequence and location. Replication of IncP alpha plasmids requires oriV and the plasmid-encoded initiator protein TrfA in combination with the host-encoded replication machinery. Conjugative plasmid transfer depends on two separate regions occupying about half of the genome. The primary segregational stability system designated Par/Mrs consists of a putative site-specific recombinase, a possible partitioning apparatus and a post-segregational lethality mechanism, all encoded in two divergent operons. Proteins related to the products of F sop and P1 par partitioning genes are separately encoded in the central control operon.

Temperature-sensitive mutants for the replication of plasmids in Escherichia coli. I. Isolation and specificity of host and plasmid mutations.

Temperature-sensitive mutants of Escherichia coli defective in the replication of the plasmid colicinogenic factor E1 (ColE(1)) were isolated following mutagenesis of E. coli K12 strain carrying the ColE(1) factor. Following the mutagenic treatment an enrichment procedure utilizing the replacement of thymine with bromouracil in the ColE(1) DNA duplicated at the restrictive temperature was used. The mutants isolated following this enrichment step were the result of a mutation event either in the host chromosome or in the ColE(1) plasmid. The host mutants fell into three phenotypic classes based on the effect each mutation had on the maintenance of a variety of other extrachromosomal DNA elements. Phenotypic class I mutations affected all E. coli plasmids, both the I and F sex factor types as well as the ColE(1) factor. Phenotypic class II mutations affected the maintenance of the ColE(1) and the F sex factor type plasmids and not the I type, while phenotypic class III mutations affected only ColE(1) replication. None of these mutations was found to have a significant effect on the replication of the E. coli chromosome. The plasmid-linked mutations fell into two phenotypic classes on the basis of the ability of the Flac episome to complement the mutation in the ColE(1) plasmid.

Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria.

A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined.

Temperature-sensitive mutants for the replication of plasmids in Escherichia coli. II. Properties of host and plasmid mutations.

Host mutations in Escherichia coli K12 selected for the temperature-sensitive replication of the bacterial plasmid colicinogenic factor E(1) (ColE(1)) exhibit a pleiotropic effect with respect to the effect of the mutation on other extra-chromosomal elements. The mutations also vary with respect to the time of incubation of the cells at 43 degrees C required for complete cessation of ColE(1) DNA synthesis. While the synthesis of the bacterial chromosome appears unaffected, supercoiled ColE(1) DNA replication stops immediately in some mutants and gradually decreases during several generations of cell growth before stopping in others. Mutations isolated in the ColE(1) plasmid resulted in only a gradual cessation of ColE(1) DNA synthesis over several generations of cell growth at 43 degrees C. Conjugal transfer of the ColE(1) and ColV factors occurs normally in the host mutants when the transfer is carried out at the permissive temperature; however, the presence of a group I mutation in the donor cell prohibited conjugal transfer of either plasmid DNA at 43 degrees C to a normal recipient cell. Similarly, the presence of this mutation in the recipient prevented the establishment of ColE(1) or ColV in the mutant recipient cell upon conjugation with a normal donor at 43 degrees C. Various host ColE(1) replication mutants carrying either ColE(1) or ColE(2) were also defective in the mitomycin C-induced production of colicin E(1) or colicin E(2) at 43 degrees C. The majority of the host mutations examined exhibited a temperature sensitivity to growth in deoxycholate in addition to the inhibition of plasmid DNA replication, suggesting a membrane alteration in these mutants when grown at the restrictive temperature.

Involvement of the host initiator function dnaA in the replication of coliphage lambda.

We demonstrate that the initiation of coliphage lambda DNA replication is dependent on the host initiator function dnaA, provided that the lambdoid prophage Rac is absent. Presence of Rac compensated the absence of dnaA function, causing initiation of replication. In dnaAts rac+ cells at 43 degrees, most of parental phage DNA molecules, after one round of theta replication, switched to a replication with features of the sigma mode and produced progeny at high yield. Initiation of replication of the lambda Pts1 mutant at 43 degrees was blocked by dnaA function; however, under dnaA-rac+ conditions all parental phage DNA molecules, after one round of theta replication, switched to the sigma mode and produced progeny at high yield. Taking into account our recent finding that transcriptional activation of ori lambda seems to be dnaA-regulated (to be published elsewhere), we suggest that the DnaA-lambda Pts1 incompatibility occurs at the insertion of the ori lambda-bound lambda O-lambda P-DnaB preprimosome between the complementary lambda DNA strands. The role of Rac and the mechanism of the switch from theta to sigma mode of lambda phage DNA replication are discussed.

TrfA dimers play a role in copy-number control of RK2 replication.

Copy-number regulation of the broad-host-range plasmid RK2 is dependent on the plasmid-encoded initiator protein, TrfA, and the RK2 origin of replication. The handcuffing model for copy-number control proposes that TrfA-bound oris reversibly couple to prevent the further initiation of plasmid replication when the copy number in vivo is at or above the replicon-specific copy number. TrfA mutants have been isolated which allow for oriV replication at elevated copy numbers. To better understand the mechanism of 'handcuffing', the copy-up TrfA(G254D/S267L) mutant was characterized further. In the present study we show by size exclusion chromatography and native gel electrophoresis that unlike wt TrfA which is largely dimeric, purified His6-TrfA(G254D/S267L) is primarily monomeric. In vivo, TrfA33(G254D/S267L) supports replication of an RK2 ori plasmid in trans at a greatly elevated copy number, while in cis the plasmid exhibits runaway replication. However, expression of either of two previously isolated DNA-binding defective TrfA mutants, TrfA33(P151S) or TrfA33(S257F), in a cell transformed with a mini-RK2 replicon encoding TrfA33(G254D/S267L) results in suppression of the runaway phenotype. His6-TrfA(P151S) and His6-TrfA(S257F) purify as dimers, and when expressed in vivo are incapable of supporting RK2 plasmid replication. In contrast, combination of the trfA(P151S) or trfA(S257F) mutation with the trfA(G254D/S267L) mutations results in the expression of mutant TrfA proteins which are mainly monomers and which can no longer restore copy control to replication directed by TrfA33(G254D/S267L) in vivo. On the basis of these findings a handcuffing model is proposed, whereby oriV-bound TrfA monomers are coupled by dimeric TrfA molecules.

Overlapping transcription of the nifA regulatory gene in Rhizobium meliloti.

Gene fusions were used to demonstrate complex overlapping transcriptional controls of the regulatory gene nifA in Rhizobium meliloti bacteroids isolated from alfalfa root nodules. Gene nifA has previously been shown to be transcribed from promoter PnifA and to be required for activation of promoters P1 (nifHDK) and P2 (fixABC) during symbiotic nitrogen fixation with alfalfa. P2 is located approximately 4 kb upstream from nifA and is shown in this report to be responsible for at least one-half of the nifA expression observed in bacteroids. Substantial transcription of nifA occurs during symbiosis as evidenced by the fact that PnifA was found to be 53% as active as either P1 or P2. Together, the data indicate that more than half of the transcripts initiated at P2 fail to terminate before reaching nifA. Additional studies indicated that there may be weak promoter activity in the symbiotically essential region downstream from nifA.

Firefly luciferase as a reporter enzyme for measuring gene expression in vegetative and symbiotic Rhizobium meliloti and other gram-negative bacteria.

A DNA segment carrying a cDNA copy of the luciferase gene (luc) of the North American firefly Photinus pyralis, fused to the lambda PR promoter and expressed in Escherichia coli [de Wet et al., Proc. Natl. Acad. Sci. USA 82 (1985) 7870-7873], was inserted into a broad-host-range plasmid vector and established in a variety of Gram-negative bacteria. Luciferase activity, expressed from the lambda PR promoter, was detected in both intact cells and extracts prepared from cells of strains of Rhizobium meliloti, R. phaseoli, R. fredii, Pseudomonas aeruginosa, Agrobacterium tumefaciens, Acinetobacter calcoaceticus and Azotobacter vinelandii. The highest levels of activity, determined by measurements of both intact cells and extracts, were observed for P. aeruginosa and the three species of Rhizobium examined. Expression of luciferase activity also was relatively high in R. meliloti bacteroids of mature alfalfa nodules. This activity was readily detectable in intact nodules using x-ray film or in extracts prepared from purified bacteroids.

Mutants of the two-component regulatory protein FixJ of Rhizobium meliloti that have increased activity at the nifA promoter.

FixL and FixJ belong to a two-component regulatory system in Rhizobium meliloti that induces the expression of numerous nitrogen-fixation genes during symbiosis with alfalfa. FixJ is a positive activator required for transcription of the regulatory genes nifA and fixK, while FixL is an oxygen-binding hemoprotein capable of regulating the phosphorylation status of both itself and FixJ, in response to oxygen availability. In this study, we isolated four FixJ mutants that display increased activity at the nifA promoter (PnifA) in Escherichia coli. All four mutants possess amino acid changes in a domain of FixJ that is conserved in other response regulator proteins, and all exhibit increased activity at PnifA in R. meliloti that is dependent on the presence of FixL. One of the mutant proteins, while less efficient at accepting phosphate from a truncated derivative of FixL (FixL*), nevertheless has a phosphorylated form that is more stable than the phosphorylated form of wild-type (wt) FixJ and is more resistant to the phosphatase activity of FixL*. The wt FixJ-phosphate was found to have a half-life of approximately 4 h, which makes it an unusually long-lived response regulator protein. The exceptional stability of wt FixJ-phosphate and the altered phosphorylation properties observed for the mutant are discussed in relation to signal transduction in the FixLJ system.

Mutations in the gene encoding the replication-initiation protein of plasmid RK2 produce elevated copy numbers of RK2 derivatives in Escherichia coli and distantly related bacteria.

Mini-replicons of the broad-host-range plasmid RK2 with increased copy number (cn) due to mutations in the gene encoding the essential replication initiation protein TrfA are described. The cn of these derivatives have been determined in Escherichia coli, Pseudomonas aeruginosa and Agrobacterium tumefaciens and were found to be elevated in all three bacterial hosts. One of the cn mutations was introduced into the intact 60-kb RK2 plasmid by homologous recombination in vivo, resulting in an approximately twofold cn increase. The expression of trfA from this mutant RK2 plasmid did not respond to the cn change as predicted by a simple transcription rate-limitation, replication control model. Implications for the model of RK2 replication control and the potential use of mutant RK2 mini-replicons as high-copy broad-host-range gene cloning vectors are discussed.

Generation in vitro of deletions in the broad host range plasmid RK2 using phage Mu insertions and a restriction endonuclease.

Several non-lethal deletions of the broad host range plasmid RK2 (molecular weight of 37.6 . 10(6) have been produced in vitro. The method employed relied on the single HindIII restriction nuclease site in RK2 and the ability of phage Mu to insert and thereby add new HindIII restriction sites at various positions in the plasmid. The deleted plasmids have in each case lost kanamycin (Km) resistance, and in two cases are defective in self-transmissibility. The method used to reduce the size of the RK2 plasmid also results in the cloning of each of the two ends of the Mu phage DNA on the plasmid derivatives.

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.