Plasmid Complementation Research Articles

The pCLIP plasmids: versatile cloning vectors based on the bacteriophage λ origin of replication

A series of general-purpose plasmid vectors based on the phage λ origin of replication (ori) has been constructed. Each vector consists of a backbone plasmid encoding chloramphenicol resistance (Cm R) and containing a unique HaeII site into which the lacZα-complementing multiple cloning site (MCS) region of an established vector was inserted. To increase the cloning potential of the inserted MCS, superfluous restriction sites in the backbone were removed by a variety of techniques. The vectors, designated pCLIP (for Cm R λ ori integration proficient) plasmids, are of medium copy number and are compatible with most other vectors in common use. A total of 17 unique restriction sites in pCLIP8, pCLIP9, pCLIP18, pCLIP19 and pCLIP23 are available for cloning. As well as possessing the usual properties of vectors, the pCLIP plasmids are able to integrate reversibly into λ prophage by homologous recombination. Thus, cloned DNA can be maintained in single or multiple copy at will. By integrating recombinant plasmids into appropriate deletion prophages followed by induction, phage::plasmid hybrids are produced which can be manipulated as phage. These properties are demonstrated using a test recombinant plasmid, pCLIPLEU2. The pCLIP vectors are therefore useful for routine plasmid cloning, complementation analysis and applications where the ability to manipulate recombinants in plasmid, phage or prophage forms is advantageous.

Limitations of plasmid complementation test for determination of quinolone resistance due to changes in the gyrase A protein and identification of conditional quinolone resistance locus.

Plasmid pJSW101 derived from pUC19 and carrying the wild-type gyrA gene was found to be unstable in HM72, a quinolone-resistant (QR) clinical isolate of Escherichia coli, and resulted in no change in quinolone MICs. MICs determined in the presence of ampicillin to ensure plasmid presence, however, resulted in complementation. HM72 was proved to have a gyrA mutation based on the DNA sequence of a 418-bp fragment of gyrA. DNA sequencing identified a common mutation encoding Leu-83 as the cause of QR. To identify loci other than gyrA and nfxB contributing to QR in KF111b, zgh-3075::Tn10 (67 min) in CAG12152 was transduced into KF111b. Sixteen percent of the transductants had a fourfold decrease in norfloxacin MIC, indicating the presence of a locus, nfxD, which contributes to QR. Outcross of nfxD from DH151 (gyrA nfxB nfxD zgh-3075::Tn10) resulted in 8% of the KF130 gyrA, 2% of the EN226-3 gyrA, and none of the KL16 (wild-type) transductants, with a four- to eightfold increase in norfloxacin MIC. In the presence of ampicillin, the resistance of a gyrA nfxD double mutant, DH161 nfxD gyrA (from EN226-3), was fully complemented by gyrA+. Thus, gyrA+ plasmid complementation tests for QR may be falsely negative with plasmid instability, a difficulty which may be circumvented by maintenance of plasmid selection. In addition, if nfxD-like mutations occur in gyrA clinical isolates, a positive test may overestimate the level of resistance attributable to gyrA alone.

The Salmonella typhimurium virulence plasmid complement resistance gene rck is homologous to a family of virulence-related outer membrane protein genes, including pagC and ail

A fragment of the Salmonella typhimurium virulence plasmid containing the rck locus, when cloned in the recombinant cosmid pADE016, was shown previously to confer high-level complement resistance on both rough and smooth Escherichia coli, Salmonella minnesota, and S. typhimurium and was associated with the production of an outer membrane protein. We determined the nucleotide sequence of the fragment containing the rck locus. Mutations in the two major open reading frames confirmed that the complement resistance mediated by pADE016 was due to a single 555-bp rck gene encoding a 17-kDa outer membrane protein. Analysis of the rck gene revealed that the Rck outer membrane protein consisted of 185 amino acid residues, with a calculated postcleavage molecular mass of 17.4 kDa. Rck is homologous to a family of outer membrane proteins expressed in gram-negative bacteria, two of which have been associated with virulence-related phenotypes: PagC, required by S. typhimurium for survival in macrophages and for virulence in mice; and Ail, a product of the Yersinia enterocolitica chromosome capable of mediating bacterial adherence to and invasion of epithelial cell lines. Rck, most closely related to PagC, represents the third outer membrane protein in this five-member family with a distinct virulence-associated phenotype.

Organization of genes encoding membrane proteins of the Escherichia coli ferrienterobactin permease

Transposon mutagenesis and plasmid complementation studies have identified two genes, fepD and fepG, which are essential for ferrienterobactin transport in Escherichia coli. These genes mapped in the enterobactin gene cluster and genetic evidence indicated that they are transcribed as part of an operon (fepD, fepG, fepC). The nucleotide sequence of fepD was determine; it could encode a hydrophobic 33.8 kDa protein with sequence homologies to other iron and vitamin B12 transport proteins. Also identified, between fepD and fepB, was an open reading frame (ORF43) with no detectable function; its 43 kDa protein product (P43) was seen on polyacrylamide gels. The fepD-C operon and ORF43 were divergently transcribed from a 110bp region containing a binding site for the repressor protein Fur.

Characterization of plasmids and plasmid-borne macrolide resistance from Lactobacillus sp. strain 100-33

Lactobacillus sp. strain 100-33 is resistant to macrolides, lincosamides, and streptogramin B-type antibiotics (MLS R) and appears to contain several major and minor plasmids. One of these plasmids, pLAR33, is approximately 18 kbp in size. When cells of strain 100-33 were protoplasted and regenerated, an MLS S isolate was derived. The derivative, designated strain ES1, contained a unique plasmid complement in which it had apparently lost the major plasmids of the parental strain, including pLAR33, and retained only a minor plasmid seen in low concentrations in strain 100-33. The MLS R determinant was cloned from plasmid DNA of strain 100-33 on a 3-kbp EcoRV fragment into pBR322 and localized to pLAR33. The determinant expressed macrolide and lincosamide resistance in Escherichia coli HB101, was localized to approximately 1 kbp on the cloned sequence, and is apparently under the control of its own promoter. MLS R electroporants were derived from strain ES1 electroporated with plasmid DNA from strain 100-33; these MLS R isolates had acquired a plasmid complement similar to that of strain 100-33, including pLAR33. Endonuclease digestion and Southern analysis of plasmid DNA from both strains indicated that the major plasmids are multimeric and deleted forms of one archetypal extrachromosomal element.

Role of ruvAB genes in UV- and γ-radiation and chemical mutagenesis in Escherichia coli

Escherichia coli umuC122:: Tn5 was mutagenized with N-methyl- N′-nitro- N-nitrosoguanidine to isolate mutations that block the residual γ-radiation mutagenesis observed in umuC strains. Two of these mutations were shown by transductional mapping and plasmid complementation to map in the ruvA and ruvB genes (i.e., ruvA200 and ruvB201). Whereas ruvA200 was complemented by ruvA + plasmids, the only other known ruvA mutation, ruvA59:: Tn 10 required both the ruvA + and ruvB + genes to show complementation. The ruvA200, ruvB201, ruvA59:: Tn 10 and ruvB60:: Tn 10 mutations all reduced γ-radiation-induced ochre reversion [ argE3(Oc) → Arg + ]] to about 30% of the wild-type level, and they all reduced UV-radiation-induced ochre reversion to about 15% of the wild-type level. The ruvA200 and ruvB201 mutants also showed reduced γ- and UV-radiation mutagenesis with two other assays [ hisG4(Oc) → His + and Rif s → Rif r]. Streptozotocin mutagenesis (Rif r was reduced to about half of the wild-type level in ruv strains, but ethyl methanesulfonate mutagenesis was normal. While the umuC strain did not show the oxygen enhancement of γ-radiation mutagenesis, the ruvA200 strain showed an oxygen effect that was similar to that shown by the wild-type strain. When the ruvA200 mutation was combined with the umuC mutation, γ-radiation mutagenesis was further reduced to 5% of the wild-type level and cells showed a synergistic sensitization to UV- and γ-radiation-induced killing. A mutational spectrum analysis indicates a general depression of both umuC-dependent and umuC-independent γ-radiation mutagenesis in the ruvA strain, which is in contrast with the site-specific reduction in γ-radiation mutagenesis that is observed in the umuC mutant. The reduced radiation mutagenesis in the ruvA strain could not be correlated with a reduction in transcription of the recA or umuC genes.

Possibilities of the use of recombinant-DNA techniques with rumen micro-organisms

Possible ways in which modification of the rumen gene pool could result in increased efficiency of ruminant production are outlined. Current knowledge of basic genetics of anaerobic and facultatively anaerobic rumen bacteria is described in relation to the requirements of recombinant-DNA technology, including gene cloning, plasmid complements, possible vector systems and transformation methods. Problems of monitoring specific strains of bacteria containing heterologous genes, and their survival in the rumen, are considered.

Why do unrelated insertion sequences occur together in the genome of Escherichia coli?

Natural isolates of Escherichia coli are polymorphic for the presence or absence of insertion sequences. Among the ECOR reference collection of 71 natural isolates studied for the number of copies of the insertion sequences IS1, IS2, IS3, IS4, IS5 and IS30, the number of strains containing no copies of the insertion sequences were 11, 28, 23, 43, 46 and 36, respectively. Significant correlations occur in the ECOR strains in the presence or absence of unrelated insertion sequences in the chromosome and plasmid complements. Strains containing any insertion sequence are more likely to contain additional, unrelated insertion sequences than would be expected by chance. We suggest that the positive correlations result from horizontal transfer mediated by plasmids. A branching-process model for the plasmid-mediated transmission of insertion sequences among hosts yields such a correlation, even in the absence of interactions affecting transposition or fitness. The predictions of the model are quantitatively in agreement with the observed correlations among insertion sequences.

Comparative Complementation Mapping of Methylophilus spp. Using Cosmid Gene Libraries and Prime Plasmids

Cosmid gene libraries of the obligate methylotrophs Methylophilus viscogenes and Methylophilus methylotrophus AS1 were generated by ligating Sau3A partial digests of chromosomal DNA into the broad-host-range cosmid vector pLA2917. Genetic linkage groups were identified for each organism by complementation following mobilization of the recombinant cosmids to a wide range of Pseudomonas aeruginosa and Escherichia coli auxotrophs. The linkage data thus obtained confirmed and extended those previously established by prime plasmid complementation, and the two methylotrophs showed very similar arrangements for four of the gene clusters studied. However, comparison of DNA sequences which complemented the same auxotrophic markers failed to demonstrate restriction fragment identity or substantial DNA-DNA homology between the two methylotrophs.

Comparison of the Toxicity., Parasporal Body Protein Composition, and Plasmid Complements of Nine Isolates of Bacillus thuringiensis subsp. israelensis

Toxicity, parasporal body composition, and plasmid complements of nine isolates of the mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis (H-14) obtained from international standards (Institute Pasteur Standard 78, IPS-80, IPS-82), field isolations (2013-9, 2013-9+), and experimental or commercial formulations (R-153-78, Bactimos, Teknar, Vectobac) were compared. For all isolates, LC50’s for fourth-instar Aedes aegypti (L.) were in the range of 9–13 ng/ml, except for R-153-78 (392 ng/ml) and 2013-9 (2,767 ng/ml). Toxicity of the latter two isolates was low because most colonies produced low levels of parasporal bodies, a situation corrected by selecting for colonies that were high in numbers of parasporal bodies. LC50’s for the purified parasporal bodies of all isolates were 3–4 ng/ml; no significant differences were obtained among the isolates. Plasmid complements and parasporal body composition of all isolates were also similar. However, isolates IPS-80, IPS-82, Bactimos, and Vectobac lacked a small plasmid of 4.9 megadaltons (mDa). The marked similarity of the nine isolates suggests that most, if not all, isolates of B. thuringiensis subsp. israelensis under study or development as insecticides are closely related to ONR 60A, the original isolate from Israel.

Separation of temporal control and trans-acting modulation of flagellin and chemotaxis genes in Caulobacter.

The genes involved in the biogenesis of the flagellum and the chemotaxis machinery are temporally regulated during the Caulobacter crescentus cell cycle. Using plasmid complementation, we have mapped the extent of the flaY and flaE genes. These genes function in trans to regulate the expression of the flagellin genes and the chemotaxis genes. We have found that the trans regulation that modulates the amount of the flagellins and the chemotaxis proteins can be separated from the temporal control of fla and che gene expression. This conclusion is based on two observations: the low level of synthesis of flagellins and chemotaxis proteins in flaY and flaE mutant strains occurred at the correct time in the cell cycle, and complementation with plasmids containing intact flaY and flaE genes resulted in the synthesis of normal levels of flagellins and chemotaxis gene products with the maintenance of temporal cell cycle control.

DNA-DNA homology between plasmids from Streptococcus thermophilus

Summary Fift Y strains of Streptococcus thermophilus isolated from raw milk, industrial starters or issued from collections were studied for their plasmid complement. Most of them did not contain plasmid DNA. Six strains only harboured one plasmid and three strains two plasmids. Ali these plasmids were small sized: 2.9 to 7.6 kilobases. Restriction analysis and DNA-DNA hybridization were used to demonstrate similarity between plasmids of these strains and to distinguish several homology groups among them.

Identification of lactose fermentation plasmids of streptococcal dairy starter strains by Southern hybridization

Twenty-two strains of Streptococcus cremoris, seven strains of Streptococcus lactis and three strains of Streptococcus lactis subsp. diacetilactis, each with a different plasmid complement, were isolated from a starter culture used in a Finnish dairy plant. By using DNA-DNA hybridization, with cloned 6-P-s-galactosidase gene of the Strep, lactis plasmid pLP712 as a probe, the lactose fermentation genes were located, in each strain, in the large ( 30 MD) plasmid.

The evolution of DNA sequences in Escherichia coli.

It is proposed that certain families of transposable elements originally evolved in plasmids and functioned in forming replicon fusions to aid in the horizontal transmission of non-conjugational plasmids. This hypothesis is supported by the finding that the transposable elements Tn3 and gamma delta are found almost exclusively in plasmids, and also by the distribution of the unrelated insertion sequences IS4 and IS5 among a reference collection of 67 natural isolates of Escherichia coli. Each insertion sequence was found to be present in only about one-third of the strains. Among the ten strains found to contain both insertion sequences, the number of copies of the elements was negatively correlated. With respect to IS5, approximately half of the strains containing a chromosomal copy of the insertion element also contained copies within the plasmid complement of the strain.

Regulation of guaC expression in Escherichia coli.

The guaC gene encodes GMP reductase, which converts GMP to inosine monophosphate. Regulation of guaC expression was examined by use of guaC-lac fusions created by Mu d1(lac). In these strains, beta-galactosidase is induced by guanine derivatives, and this induction is prevented by adenine. Our previous implication that glutamine acts as a negative effector of transcription was confirmed by showing that glutamine analogs (diazo-oxo-norleucine and methionine sulfoximine) can also induce beta-galactosidase. GMP was implicated as a likely candidate for the in vivo inducer by introducing a gpt block to prevent the conversion of guanine to GMP and a deoD block to prevent the interconversion of guanine and guanosine. Regulatory mutants were isolated by growth on lactose plus adenine. Though these showed high constitutive levels of beta-galactosidase, they were normal for the regulation of GMP reductase when the fusion was corrected by transduction to guaC+ or when guaC+ was introduced by plasmid complementation. The regulatory mutants were linked to guaC.

Bacteriophage T4 DNA replication protein 41. Cloning of the gene and purification of the expressed protein.

The bacteriophage T4 primase, composed of the T4 proteins 41 and 61, synthesizes pentaribonucleotides used to prime DNA synthesis on single-stranded DNA in vitro. 41 protein is also a DNA helicase that opens DNA in the same direction as the growing replication fork. Previously, Mattson et al. (Mattson, T., Van Houwe, G., Bolle, A., Selzer, G., and Epstein, R. (1977) Mol. Gen. Genet. 154, 319-326) located part of gene 41 on a 3400-base pair EcoRI fragment of T4 DNA (map units 24.3 to 21.15). In this paper, we report the cloning of T4 DNA representing map units 24.3 to 20.06 in a multicopy plasmid vector. Extracts of cells containing this plasmid complement gene 41- extracts in a DNA synthesis assay, indicating that this region contains all the information necessary for the expression of active 41 protein. We located gene 41 more precisely between T4 map units 22.01 to 20.06 since our cloning of this region downstream of the strong lambda promoter PL results in the production of active 41 protein at a level 100-fold greater than after T4 infection. We have purified 133 mg of homogeneous 41 protein from 27 g of these cells. Like the 41 protein from T4 infected cells, the purified 41 protein in conjunction with the T4 gene 61 priming protein catalyzes primer formation (assayed by RNA primer-dependent DNA synthesis with T4 polymerase, the genes 44/62 and 45 polymerase accessory proteins, and the gene 32 helix-destabilizing protein) and is a helicase whose activity is stimulated by T4 61 protein.

Bacteriophage P4 DNA replication: Location of the P4 origin

An electron microscopic examination of replicating bacteriophage P4 DNA molecules has revealed theta-type structures that replicate bidirectionally from a single origin. Many replicating P4 DNA molecules also contain long (2000 bases) single-strand DNA regions at the growing fork that are deployed in a trans configuration, which supports the concept of continuous leading strand and discontinuous lagging strand syntheses. The position of the P4 origin was localized by the use of a plasmid complementation test for replication in vivo, as well as by labeling of DNA replicating in vitro in the presence of a chain-terminating inhibitor. During this study we discovered a second site on the P4 genome which is essential for replication, and we have named it crr ( cis region required for replication). This site is located at least 3300 bases from the origin but appears to be required for the initiation of DNA replication in vivo as well as in vitro.

Chromosomal deletion and plasmid complementation of the photosynthetic reaction center and light-harvesting genes from Rhodopseudomonas capsulata

Using in vitro interposon mutagenesis, Rhodopseudomonas capsulata strains have been constructed wherein all or part of the reaction center (RC), light-harvesting I (LHI), and light-harvesting II (LHII) structural genes have been deleted. In one series of strains, the 2778-bp ApaI fragment bearing more than 90% of the rxcA operon (promoter and structural genes coding for LHIβ, LHIα, RC-L and RC-M) has been deleted from the chromosome. When the rxcA operon is deleted, resultant strains possess only LHII and are photosynthetically defective. The rxcA deletion in an LHII − background results in a strain lacking all LH antennae and RC subunits. As expected this strain has no near-infrared absorption characteristic of LH or RC bacteriochlorophyll. The rxcA deletion may be complemented by a pBR322 derivative carrying the entire rxcA operon (pU21). In a second series of deletion mutants, the 2500-bp BstEII- StuI fragment, including the β and α structural genes coding for LHII has been deleted from the chromosome. In the wild-type background, functional RC and LHI are synthesized. LHII may be restored in the deletion strain by conjugal transfer of the plasmid pU2 which carries the LHII operon.

Analysis of the pleiotropic regulation of flagellar and chemotaxis gene expression in Caulobacter crescentus by using plasmid complementation.

The biosynthesis of the single polar flagellum and the proteins that comprise the chemotaxis methylation machinery are both temporally and spacially regulated during the Caulobacter crescentus cell-division cycle. The genes involved in these processes are widely separated on the chromosome. The region of the chromosome defined by flaE mutations contains at least one flagellin structural gene and appears to regulate flagellin synthesis and flagellar assembly. The protein product of the adjacent flaY gene was found to be required to regulate the expression of several flagellin proteins and the assembly of a functional flagellum. We demonstrate here that each of these genes is also required for the expression of chemotaxis methylation genes known to map elsewhere on the chromosome. In order to study the regulation of these genes, plasmids were constructed that contain either an intact flaYE region or deletions in the region of flaY. These plasmids were mated into a wild-type strain and into strains containing various Tn5 insertion and deletion mutations and a temperature-sensitive mutation in the flaYE region. The presence of a plasmid containing the flaYE region allowed the mutant strains to swim and to exhibit chemotaxis, to synthesize increased amounts of the flagellins, to methylate their "methyl-accepting chemotaxis proteins" (MCPs), and to regain wild-type levels of methyltransferase activity. Chromosomal deletions that extend beyond the cloned region were not complemented by this plasmid. Plasmids containing small deletions in the flaY region failed to restore to any flaY or flaE mutants the ability to swim or to assemble a flagellar filament. When mated into a wild-type strain, plasmids bearing deletions in the flaY region were found to be recessive. The pleiotropic regulation of flagellin synthesis, assembly, and chemotaxis methylation functions exhibited by both the flaY and flaE genes suggest that their gene products function in a regulatory hierarchy that controls both flagellar and chemotaxis gene expression.

The effect of canavanine on the maintenance in yeast of chimeric plasmids containing portions of the 2-�m DNA plasmid

We have found that the application of the amino acid analog canavanine to a culture of yeast cells transformed with chimeric plasmids based on the yeast 2-µm DNA plasmid increases the percentage of cells which have lost the transforming plasmid. This effect is found whether the plasmid carries the CAN1 sensitive allele and the yeast strain carries a can1 mutation confering resistance, or the plasmid contains no CAN1 allele and the yeast strain carries the wild-type CAN1 sensitive allele. Canavanine exerts this effect on yeast strains transformed with chimeric plasmids containing either a portion or the entire 2-µm DNA plasmid, yet canavanine does not appear to effect the maintenance of the native 2-µm DNA plasmid complement within the cell. The effect of canavanine on strains transformed with chimeric plasmids is the same whether or not the yeast strain also contains native 2-µm plasmid DNA. Neither the amino acid analog ethionine, the protein synthesis inhibitor cycloheximide, nor the DNA replication inhibitor hydroxyurea exhibit this effect. Some of the experimental results suggest that canavanine may be a curing agent rather than an agent which selects for spontaneous plasmid loss.