A broad-range of recombination cloning vectors in mycobacteria

The increased incidence of tuberculosis (TB) gave impetus for the increased interest in the study of mycobacterial genetics, which culminated in the publication of the full genome sequence of many mycobacterial strains. Since then, many genes and open reading frames of unknown function have been described and the expression of their encoded proteins is critical toward understanding the pathogenesis of TB and developing therapeutic and preventive strategies. Therefore there is an increased need for highly efficient methods for cloning of mycobacterial genes, as the limited cloning flexibility of current Escherichia coli–mycobacteria shuttle vectors remains a frequent impediment in genetic manipulation of mycobacteria. In order to overcome this limitation, we have converted representative extrachromosomal and integrative vectors into multiple destination mycobacterial vectors for one-step and restriction enzyme-free recombination cloning methodology that uses in vitro site-specific recombination. We provide several examples that highlight the potential of recombination cloning for gene expression in slow and fast-growing mycobacteria. Thus, a gene of interest can be transferred by simple recombination into our mycobacterial destination vectors, which serve a multitude of functional genomic studies.     

The genetics ofMycobacterium tuberculosis

Gene manipulation inMycobacterium tuberculosis has been slow in coming of age owing to the inherent difficulties associated with working on this aerosol-transmitted pathogen, in addition to the paucity of molecular tools such as plasmids and transposons. One of the early approaches to overcome these difficulties was the development of phasmids, which combined the properties of phages and plasmids and allowed introduction of recombinant genes into mycobacteria. The lone plasmid pAL5000 of mycobacteria has been exploited to its fullest potential in the construction of a plethora of vectors. Above all, the single most important achievement has been the development of elegant and innovative approaches to overcome the problem of illegitimate recombination which threatened the success of allelic-exchange mutagenesis in the slow-growing pathogenic mycobacterial species. In this review I discuss the current status of conditionally replicating plasmid and transposon vectors and their application in generating targeted mutations in mycobacteria.     

Transformation of cultured cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulence region of pTiBo542

Summary A 15.2-kb KpnI fragment from the virulence region of pTiBo542, the Ti plasmid harbored by Agrobacterium tumefaciens strain A281, was introduced into binary vectors. The fragment contained the virB, virC and virG genes, and it is known to have the ability to increase the virulence of strains of A. tumefaciens. The strains of A. tumefaciens that carried the resulting plasmids were able to transform cells in a suspension culture of Chenopodium quinoa Willd cells which were not transformable by common vectors. Although the sizes of the plasmids was very large, a foreign segment of DNA was introduced into one of the plasmids by homologous recombination in A. tumefaciens cells, and the segment was subsequently transferred to plant cells.Abbreviations NPT neomycin phosphotransferase - SPT streptomycin/spectinomycin phosphotransferase     

Isolation of symbiotically defective mutants in Rhizobium leguminosarum by insertion of the transposon Tn5 into a transmissible plasmid

Summary Selection was made for the transposition of the kanamycin resistance transposon Tn5 from a location on the chromosome of R. leguminosarum into a transmissible, bacteriocinogenic plasmid that also carries genes required for the induction of nitrogen-fixing nodules on peas.One hundred and sixty independent insertions into transmissible plasmids were isolated. When these plasmids were transferred by conjugation into a non-nodulating strain, which carries a deletion in one of its resident plasmids, of the 160 isolates tested 14 yielded transconjugants that formed nodules that did not fix nitrogen (Fix^-) and in a further 15 cases the transconjugants were unable to form nodules (were Nod^-). When transferred to a symbiotically proficient strain (i.e. Nod⁺ Fix⁺) none of the transconjugants was symbiotically defective; thus the mutations were not dominant.When kan was transduced from the clones that generated Fix^- transconjugants into a Fix⁺ recipient the majority of transductants inherited Fix^- indicating that the insertion of Tn5 had induced the symbiotic mutations. Transduction of kan, from the clones that failed to donate Nod⁺ by conjugation to strain 6015, occurred at barely detectable frequencies and it was not possible to demonstrate transduction of Nod^-. kan was co-transduced with Nod⁺ from some of the clones and some of these transductants also inherited the ability to produce medium bacteriocin and to transfer at high frequency by conjugation. Thus the genes for all these characters are closely linked.     

Electro-transformation of Clostridium beijerinckii NRRL B-592 with shuttle plasmid pHR106 and recombinant derivatives

Conditions for transformation of the solventogenic anaerobe Clostridium beijerinckii NRRL B-592 with plasmid DNA via electroporation are described. Shuttle plasmid pHR106 and two derivatives constructed in this study were transferred and were expressed in this organism. One recombinant derivative of pHR106 was constructed by separately subcloning the clostridial tetracycline (tetP) resistance genes into pHR106. The second vector conferring erythromycin resistance was obtained via in-vivo recombination. The new constructs, termed pRZL and pRZE respectively, were then transferred to C. beijerinckii in order to evaluate their potential as shuttle vectors. The recombinant plasmids were shown to transfer to C. beijerinckii and were expressed as autonomously replicating vectors. The use of these plasmids as cloning and shuttle vectors for C. beijerinckii is discussed.Correspondence to: R. M. Zsigray     

Manipulation of the genes for poly-β-hydroxybutyric acid synthesis in Alcaligenes eutrophus

Summary In order to establish the molecular breeding system in Alcaligenes eutrophus producing poly--hydroxybutyric acid (PHB), phbCAB genes from A. eutrophus were recombined into the E. coli-A. eutrophus shuttle vector and directly transferred into A. eutrophus by the electroporation. In A. eutrophus transformants, recombinant plasmids were stably maintained and enzyme activities for PHB biosyntheses were elevated 1.4–2.7 fold by the cloned genes.     

Use of transmissible plasmids as cloning vectors in Caulobacter crescentus

Cloning vectors for studies of Caulobacter crescentus genes should be transferable between Escherichia coli and C. crescentus since a transformation system has not been developed for C. crescentus. We have tested a large number of vectors containing IncP or IncQ replicons and found that many of the vectors containing IncQ replicons, and all but one of the vectors containing IncP replicons, are readily transferred by conjugation into C. crescentus. All of the plasmids tested were maintained in C. crescentus at 1 to 5 copies per cell, but plasmids containing IncP replicons were more stable than plasmids containing IncQ replicons. Further studies with a derivative of the IncQ plasmid R300B showed that when a promoterless kanamycin (Km)-resistance gene (npt2) was inserted into the intercistronic region of the sul-aphC (Su^R-Sm^R) operon, Km resistance was expressed only when the npt2 gene was inserted such that it would be transcribed from the sul promoter. These data indicate that R300B does not contain sequences which would provide promoter function in C. crescentus in the orientation opposite to that of the sul operon and that any genes cloned in this orientation would require native promoters for expression. To provide greater versatility for cloning into R300B, additional vectors were constructed by the addition of multiple cloning sites in the intercistronic region of the sul-aphC operon. In addition, chromosomal DNA libraries were constructed in R300B and in the cosmid vector pLAFR1-7. Specific clones from these libraries containing genes of interest were identified by complementation of the appropriate C. crescentus mutants.     

A disarmed binary vector from Agrobacterium tumefaciens functions in Agrobacterium rhizogenes

Summary Binary Ti plasmid vector systems consist of two plasmids in Agrobacterium, where one plasmid contains the DNA that can be transferred to plant cells and the other contains the virulence (vir) genes which are necessary for the DNA transfer but are not themselves stably transferred. We have constructed two nononcogenic vectors (pARC4 and pARC8) based on the binary Ti plasmid system of Agrobacterium tumefaciens for plant transformation. Each vector contains the left and right termini sequences from pTiT37. These sequences, which determine the extent of DNA transferred to plant cells, flank unique restriction enzyme sites and a marker gene that functions in the plant (nopaline synthase in pARC4 or neomycin phosphotransferase in pARC8). After construction in vitro, the vectors can be conjugatively transferred from E. coli to any of several Agrobacterium strains containing vir genes. Using A. rhizogenes strain A4 containing the resident Ri plasmid plus a vector with the nopaline synthase marker, we found that up to 50% of the hairy roots resulting from the infection of alfalfa or tomato synthesized nopaline. Thus, vector DNA encoding an unselected marker was frequently co-transferred with Ri plasmid DNA to an alfalfa or a tomato cell. In contrast, the frequency of co-transfer to soybean cells was difficult to estimate because we encountered a high background of non-transformed roots using this species. Up to five copies of the vector DNA between the termini sequences were faithfully transferred and maintained in most cases suggesting that the termini sequences and the vir genes from the Ri and Ti plasmids are functionally equivalent.     

Linkage of genes for nitrogenase and nodulation ability on plasmids in Rhizobium leguminosarum and R. phaseoli

Summary The ability to identify genes that specify nitrogenase (nif genes) in Rhizobium depends on the close homology between then and the corresponding nif genes of Klebsiella pneumoniae (Nuti et al. 1979; Ruvkun and Ausubel 1980). Rhizobium plasmids of high molecular weight (>100 Md) were separated on agarose gels, transferred to nitrocellulose filters and tested for their ability to hybridise with radioactively labelled pSA30, containing the nifKDH region of K. pneumoniae. Five large plasmids, each present in different strains of R. leguminosarum or R. phaseoli, were found to hybridise. Each of these plasmids had previously been shown to determine other symbiotic functions such as nodulation ability. The nif genes on three different plasmids appeared to be in conserved DNA regions since they were within an EcoRI restriction fragment of the same size.     

Horizontal gene transfer of stress resistance genes through plasmid transport

The horizontal gene transfer of plasmid-determined stress tolerance was achieved under lab conditions. Bacterial isolates, Enterobacter cloacae (DGE50) and Escherichia coli (DGE57) were used throughout the study. Samples were collected from contaminated marine water and soil to isolate bacterial strains having tolerance against heavy metals and antimicrobial agents. We have demonstrated plasmid transfer, from Amp⁺Cu⁺Zn⁻ strain (DGE50) to Amp⁻Cu⁻Zn⁺ strain (DGE57), producing Amp⁺Cu⁺Zn⁺ transconjugants (DGE_TC50→57) and Amp⁺Cu⁻Zn⁺ transformants (DGE_TF50→57). DGE57 did not carry any plasmid, therefore, it can be speculated that zinc tolerance gene in DGE57 is located on chromosome. DGE50 was found to carry three plasmids, out of which two were transferred through conjugation into DGE57, and only one was transferred through transformation. Plasmid transferred through transformation was one out of the two transferred through conjugation. Through the results of transformation it was revealed that the genes of copper and ampicillin tolerance in DGE50 were located on separate plasmids, since only ampicillin tolerance genes were transferred through transformation as a result of one plasmid transfer. By showing transfer of plasmids under lab conditions and monitoring retention of respective phenotype via conjugation and transformation, it is very well demonstrated how multiple stress tolerant strains are generated in nature.     

Identification and initial utilization of a portion of the smaller plasmid of Anabaena variabilis ATCC 29413 capable of replication in Anabaena sp. strain M-131

Summary Anabaena variabilis ATCC 29413 contains two cryptic plasmids. Clones of the smaller (41 kb) plasmid, designated pRDS1, in cosmid vectors were used to construct a physical map. A clone bank of pRDS1 constructed by ligating fragments from aXhoII digest of a pRDS1 cosmid clone into a mobilizable plasmid was used to locate an origin of replication of pRDS1. Because we were unable to cureA. variabilis of pRDS1, the clone bank was transferred by conjugation to another strain ofAnabaena sp., strain M-131. A 5.3 kb fragment of pRDS1 contained all of the sequences necessary for replication inAnabaena sp. strain M-131 as judged by the ability to rescue the hybrid vector from exconjugants in unchanged form after many generations. Hybrid plasmids derived from pRDS1, one bearing genes for luciferase, were also transferred by conjugation toA. variabilis, where they appeared to recombine with pRDS1.     

Conjugational transfer systems of Rhodopseudomonas capsulata mediated by R plasmids

Plasmids RP1, R68.45 and RP4::Mu cts 61 were transferred into Rhodopseudomonas capsulata from Escherichia coli. The frequency of intraspecies transfer of these plasmids in R. capsulata was 10^-4–10^-5 per donor. The plasmids also mobilized chromosomal genes at a low frequency. Phototrophic recombinants from matings between recipient strains defective in the photosynthetic-apparatus and wild type donors were obtained at a frequency of 10^-7–10^-8 per donor.     

Construction and Properties of Cloning Vectors Based on a 7.2-kb Rhizobium meliloti Cryptic Plasmid

Cloning vectors (pFD1001, pFD1192, pFD1194, and pFD1212) were constructed by extension of the host range of a 7.2-kb Rhizobium meliloti cryptic plasmid (pRm1132f) with the ColE1-based plasmids, pBR322, pACYC177, pACYC 184, pSUP301, or pHC179; mobilization was facilitated by introduction of the ori T region from pRK2, a broad-host-range plasmid. The vector plasmids transferred readily into a wide range of gram-negative bacteria and had relatively low copy number in R. meliloti; two constructs, pFD1001 and pFD1212, were completely stable in R. meliloti isolated from nodules of alfalfa (Medicago sativa). A representative of the vector constructs (pFD1001) could be maintained in R. meliloti in the presence of the broad-host-range shuttle plasmid pRK290. These two vector plasmids could be introduced into R. meliloti, either simultaneously or singly when pRK290 was the resident plasmid; however, entry of pRK290 was blocked when pFD1001 was the resident plasmid. The cloning vectors constructed in this study should prove to be useful for the genetic manipulation of Rhizobium.     

Evidence of abortive recombination in ruv mutants of Escherichia coli K12

Summary Genetic recombination in Escherichia coli was investigated by measuring the effect of mutations in ruv and rec genes on F-prime transfer and mobilization of nonconjugative plasmids. Mutation of ruv was found to reduce the recovery of F-prime transconjugants in crosses with recB recC sbcA strains by about 30-fold and with recB recC sbcB sbcC strains by more than 300-fold. Conjugative plasmids lacking any significant homology with the chromosome were transferred normally to these ruv mutants. Mobilization of the plasmid cloning vectors pHSG415, pBR322, pACYC184 and pUC18 were reduced by 20- to 100-fold in crosses with ruv rec ⁺ sbc ⁺ strains, depending on the plasmid used. Recombinant plasmids carrying ruv ⁺ were transferred efficiently. With both F-prime transfer and F-prime cointegrate mobilization, the effect of ruv was suppressed by inactivating recA. It is proposed that the failure to recover transconjugants in ruv recA ⁺strains is due to abortive recombination and that the ruv genes define activities which function late in recombination to help convert recombination intermediates into viable products.     

Killer toxin producing strains of the yeasts Hanseniaspora uvarum and Pichia kluyveri

By heat treatment killer strains of the type K1 of Saccharomyces cerevisiae that are known to harbour dsRNA plasmids were completely cured, whereas only a small fraction of the clones of the killer type K2 had lost the dsRNA dependent killer character. The K2 killers but not the strains of killer type K1 were easily cured by cycloheximide. Killer strains of Hanseniaspora uvarum were not curable by heat treatment. Curing was successfull with cycloheximide or 5-fluorouracil. Two double-stranded RNA plasmids were detected in the killer strains of H. uvarum. The smaller dsRNA plasmid was absent in the strains that were cured of their killer character by 5-fluorouracil. The killer character of H. uvarum was transferred to S. cerevisiae by spheroplast fusion. The fusion products showing the killer character contained both dsRNA plasmids, obviously the smaller plasmid (M-dsRNA) carries the genes for killer toxin formation. Killer strains of Pichia kluyveri were not curable of their killer character, in these strains no dsRNA plasmids were detected.This paper was kindly supported by a grant from the Deutsche Forschungsgemeinschaft     

Plasmids in tributyltin-resistant bacteria from fresh and estuarine waters

Summary Twenty-six tributyltin (TBT)-resistant bacterial strains isolated from sediments were examined for the presence of plasmids. Plasmids of the size reported to carry metal resistance genes were not found in 15 of the strains, indicating that resistance does not have to be plasmid-mediated. Attempts to cure plasmid-containing strains using acridine organge, ethidium bromide, novobiocin or sodium dodecylsulfate, or by growth at elevated temperature were not successful, nor were plasmids transferred from TBT-resistant strains into TBT-sensitive organisms by electroporation. In a broth mating experiment however, plasmid pUM505, a conjugative plasmid known to encode chromium resistance inPseudomonas aeruginosa PAO1, was introduced into TBT-sensitiveBeijerinckia sp. MC-27 isolated from freshwater sediment. The TBT tolerance of theBeijerinckia sp. increased 100-fold, from 8.4 M TBT inBeijerinckia sp. MC-27 to 840 M TBT inBeijerinckia sp. MC-27 (pUM505) on solid medium. The plasmid was transferred at a frequency of approximately 6×10^–4. TBT-resistant transconjugants grew faster in media containing TBT and lost their enhanced TBT tolerance and the plasmid upon serial transfer in medium without TBT. Spontaneous mutants of the donorP. aeruginosa lost both TBT resistance and the plasmid. Therefore, TBT resistance in bacteria can be plasmid-mediated. To our knowledge, this is the first report that resistance to a tin compound can be plasmid-mediated.     

Conjugation in Archaea: Frequent Occurrence of Conjugative Plasmids inSulfolobus

We describe five novel conjugative plasmids (CPs) and two subfamilies, each comprising several closely related variants of CPs isolated from colony-cloned strains of the extremely thermophilic, heterotrophic archaeonSulfolobus islandicus,which were obtained by plating of samples from Icelandic solfataras after liquid enrichment. They are related to each other and to the previously described CP pNOB8 from a JapaneseSulfolobusstrain in that they share essential functions and limited similarity of genomes as demonstrated by DNA cross-hybridization and sequences. All these plasmids thus form a family of highly efficient self-spreading elements directly transferred from donor into recipient cells. Conjugation is initiated by pair formation, followed by selective transfer of the plasmids into the recipient and expression of transfer functions. Some of these CPs exclude superconjugation of the transcipients with closely related CPs. The novel CPs are stable upon conjugative transfer, but vary upon growth of transcipients. The stability of the CPs is higher in their original hosts or in relatedS. islandicusstrains, than inSulfolobus solfataricusstrain PH1 as recipient. The deletion variant pING3 has lost the ability to transfer itself but is still subject to being transferred by the transfer apparatus of its complete relative, pING6. The dissection of genes and functions has been initiated by characterizing this incomplete variant.     

Agrobacterium as a tool for gene transfer in woody species: The state of the art and practical perspectives. A review

Abstract

The Agrobacterium-mediated ability to transfer genes into organisms without sexual crossing provides breeders with new opportunities to improve the efficiency of plant production. Gene transfer offers advantages over classical genetic manipulation since plants are improved without disruption of the integrity of their genomes. Several useful genes isolated from microrganisms and affecting pest resistance, rooting ability, hormonal metabolism etc., are now available. These genes can be easily cloned into suitable Ti and Ri derived plasmid vectors and transferred into woody species. The scarce ability of most fruit trees to regenerate the whole plant from in vitro-cultured cells remains the main obstacle to a wider use of gene transfer technology.     

Plasmid profiles ofMycobacterium fortuitum complex isolates

Six plasmids differing in molecular weights were found in isolates ofMycobacterium fortuitum, M. chelonae, and other nonclassified, nonchromogenic, rapidly growing mycobacteria. One of the plasmids of molecular weight of 32.0 Kb was present in all strains analyzed; a large plasmid of 112.0-Kb molecular weight was present only in strains identified asM. fortuitum var.peregrinum. Although the strains had distinct plasmid profiles, none of the 15 cultural and biochemical properties and susceptibility to 18 drugs that were tested could be attributed to the occurrence of these plasmids.     

Drug Resistance and R Plasmids in Vibrio anguillarum Isolated in Cultured Ayu (Plecoglossus altivelis)

Two hundred twenty-six strains of Vibrio anguillarum collected from cultured ayu (Plecoglossus altivelis) between 1978 and 1980 were studied for their sensitivities to 10 chemotherapeutics. In order to determine whether the drug-resistant strains possessed transferable R plasmids, they were conjugated with Escherichia coli. Almost all the strains isolated during the 3 years showed resistance to nalidixic acid (NA) and/or furazolidone (NF). NA and NF resistance were not transferred to Escherichia coli from any of the strains. Chloramphenicol-resistant strains were isolated in every year and almost all of them carried transferable R plasmids. Only one strain with tetracycline resistance was found among the strains tested. Strains resistant to sulfonamides, streptomycin, ampicillin (ABP), and trimethoprim (TMP) increased rapidly in 1980, and a large number of them carried transferable R plasmids. Transferable R plasmids encoded with resistance to ABP and TMP were detected for the first time in V. anguillarum strains. The R plasmids detected in the strains isolated in 1980 were classified into incompatibility groups E, A, and an untypable group. The R plasmid DNAs were cleaved by EcoRI to yield 11 to 13 fragments. The estimated molecular weights of the R plasmids from the five strains ranged from 97 to 104 M daltons.