Electrotransformation of three pathovars of Xanthomonas campestris

Procedures for electrotransformation have been adapted for three pathovars of Xanthomonas campestris: campestris, vesicatoria and manihotis. Three differently sized plasmids (51, 9.3 and 3.3 kb) at different concentrations (10, 30 and 50 ng/sample) and different field strengths (10, 12, 14 and 18 kV/cm) were used. The efficiency of transformation was dependent on the recipient strain and the plasmid introduced. In general, a field strength of 14 kV/cm as well as a concentration of 30 ng plasmid DNA/sample seemed to be adequate for most conditions. Only X. campestris pv. vesicatoria strain 479 required a higher field strength for better efficiency. The plasmid size was inversely related to the efficiency of transformation; up to 1.6 × 10¹⁰, 5.3 × 10⁷ and 3.7 × 10⁵ transformants/g DNA were obtained using the pXG31 (3.3 kb), pUFR027 (9.3 kb) and RP4 (51 kb) plasmids, respectively.     

Use of cloned DNA methylase genes to increase the frequency of transfer of foreign genes into Xanthomonas campestris pv. malvacearum.

In vitro-packaged cosmid libraries of DNA from the bacterium Xanthomonas campestris pv. malvacearum were restricted 200- to 1,000-fold when introduced into Mcr+ strains of Escherichia coli compared with restriction in the Mcr- strain HB101. Restriction was predominantly associated with the mcrBC+ gene in E. coli. A plasmid (pUFR052) encoding the XmaI and XmaIII DNA methylases was isolated from an X. campestris pv. malvacearum library by a screening procedure utilizing Mcr+ and Mcr- E. coli strains. Transfer of plasmids from E. coli strains to X. campestris pv. malvacearum by conjugation was enhanced by up to five orders of magnitude when the donor cells contained pUFR052 as well as the plasmid to be transferred. Subcloning of pUFR052 revealed that at least two regions of the plasmid were required for full modification activity. Use of such modifier plasmids is a simple, novel method that may allow the efficient introduction of genes into any organism in which restriction systems provide a potent barrier to such gene transfer.     

Novel plasmid vectors for gene cloning in Pseudomonas.

Novel host-vector systems have been developed for gene cloning in the metabolically versatile bacterial genus Pseudomonas. We found that a new Pseudomonas strain, Pseudomonas flavida IF-4, isolated from soil, carried two small cryptic plasmids, named pNI10 and pNI20. They were multi-copy, but not self-transmissible, and the genome size was 3.7 kb for pNI10 and 2.9 kb for pNI20. Several types of cloning vectors containing a kanamycin or streptomycin resistance (Kmr or Smr) gene were constructed from pNI10 and pNI20. These plasmid vectors were efficiently transformed into several strains of Pseudomonas at a frequency up to 4 x 10(5) transformants per 1 microgram plasmid DNA by the usual competent cell method. The vectors derived from pNI10 replicated not only in Pseudomonas but also in some other Gram-negative enteric bacteria such as Escherichia coli, Enterobacter aerogenes, and Proteus mirabilis.     

Construction and characterization of plasmid vectors for cloning in Staphylococcus aureus and Staphylococcus carnosus

Several plasmid vectors for cloning in Staphylococcus aureus and S. carnosus have been constructed and characterized. The chimeric plasmids are composed of parts of the following parental plasmids: The chloramphenicol-resistance plasmid, pC194, the tetracycline-resistance plasmid, pMK148, and the erythromycin-resistance plasmid, pE12. All the chimeric plasmids confer two selectable antibiotic-resistance markers on host cells. Insertional inactivation of the various antibiotic-resistance markers occurred at the BclI site of pE12, and the Sau96- or AvaII-site of pMK148; only a slight inactivation of the chloramphenicol-resistance marker occurred at the HaeIII-site of pC194. The chimeric plasmids pCT20 and pCE10 are both stable in S. aureus and S. carnosus. In addition, the hybrid plasmids of pCT20 and pCE10, containing lambda-DNA fragments in various restriction sites between 0.4 and 1.2 kb, are stably maintained. The inserted lambda-DNA fragments appear unchanged.     

Localization and restriction maps of the replication origin regions of the plasmids of Agrobacterium rhizogenes strain A4

Summary Banks of cosmids of the plasmids of the agropine-type Agrobacterium rhizogenes strain A₄ were used to transform Agrobacterium tumefaciens strain C58C1, which lacks a Ti plasmid. Hybrid cosmids able to be maintained in this strain were subcloned to localize precisely the origin of replication regions. These regions were mapped with restriction enzymes and compared by hybridization with those of Agrobacterium tumefaciens nopaline pTiC58 and octopine pTiAch5. This led to the characterization of three new plasmids suitable as cloning vectors in Escherichia coli and A. tumefaciens. They are compatible with pTi and pAt plasmids of A. tumefaciens and are maintained stably, even without selection pressure.     

Characterization of the IncW cryptic plasmid pXV2 from Xanthomonas campestris pv. vesicatoria

The gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria strain Xv2 harbors an indigenous, cryptic plasmid pXV2 of 14.6 kb. This plasmid can only be maintained in Xanthomonas and is incapable of self-transmission. However, incompatibility testing classified it in IncW, a group containing the smallest number of naturally occurring, broad-host-range, conjugative plasmids. A pXV2 derivative containing only a 5.5-kb PstI fragment is stably maintained. Deletion of a 3.0-kb region from the PstI fragment causes a loss of plasmid stability. Nucleotide sequencing of the 2. 1-kb region essential for autonomous replication revealed a repA gene and a downstream noncoding region containing four iterons, two 17- and two 19-nt direct repeats, and an AT-rich region lying between the two sets of iterons. The sequence of the deduced RepA and the iterons shows homology to the RepA (39% identity) and the iterons, respectively, of the IncW plasmid pSa. Maxicell expression of the repA gene produced a protein of 35 kDa, a size similar to that deduced from the nucleotide sequence. Trans-complementation test confirmed that the repA gene and the iterons are indeed the essential elements for pXV2 replication.     

Improved cloning vectors for bifidobacteria, based on the Bifidobacterium catenulatum pBC1 replicon

This study reports the development of several cloning vectors for bifidobacteria based on the replicon of pBC1, a cryptic plasmid from Bifidobacterium catenulatum L48 thought to replicate via the theta mode. These vectors, in which antibiotic resistance genes encoding either erythromycin or tetracycline resistance acted as selection markers, were able to replicate in a series of eight Bifidobacterium species at frequencies ranging from 4.0 x 10(1) to 1.0 x 10(5) transformants microg(-1) but not in Lactococcus lactis or Lactobacillus casei. They showed a relative copy number of around 30 molecules per chromosome equivalent and a good segregational stability, with more than 95% of the cells retaining the vectors after 80 to 100 generations in the absence of selection. Vectors contain multiple cloning sites of different lengths, and the lacZalpha peptide gene was introduced into one of the molecules, thus allowing the easy selection of colonies harboring recombinant plasmids in Escherichia coli. The functionality of the vectors for engineering Bifidobacterium strains was assessed by cloning and examining the expression of an alpha-l-arabinofuranosidase gene belonging to Bifidobacterium longum. E. coli and Bifidobacterium pseudocatenulatum recombinant clones were stable and showed an increase in alpha-arabinofuranosidase activity of over 100-fold compared to that of the untransformed hosts.     

Stabilization of a miniderivative of the broad-host-range IncW plasmid pSa by insertion of plasmid R1parB region

The plasmid vector pEM100 (13.5 kb) constructed from pGV1106, a miniderivative of the broad-host-range IncW pSa plasmid, and the pAM330 plasmid ofBrevibacterium lactofermentum is not stably maintained inEscherichia coli host cells under nonselective growth conditions. By insertion of a 0.9 kb DNA fragment containing theparB locus (responsible for the maintenance of plasmid R1 inE. coli cells) to plasmid pEM100, plasmid pEM110 was prepared which is maintained in a population ofE. coli cells growing without a selection pressure very stably. Translated by Č. Novotny     

Multimerization of high copy number plasmids causes instability: CoIE1 encodes a determinant essential for plasmid monomerization and stability

Although the natural multicopy plasmid CoIE1 is maintained stably under most growth conditions, plasmid cloning vectors related to it are relatively unstable, being lost at frequencies of 10(-2)-10(-5) per cell per generation. Evidence suggests that CoIE1 and related plasmids are partitioned randomly at cell division and that plasmid stability is correlated inversely with plasmid multimerization; factors or conditions that reduce multimerization increase stability. Cells containing plasmid multimers segregate plasmid-free cells because the multimers are maintained at lower copy numbers than monomers, as predicted by origin-counting models for copy number control. CoIE1 is stable because it encodes a determinant, cer, that is necessary for recA-, recF-, and recE-independent recombination events that efficiently convert any multimers to monomers. We have localized monomerizing and stability determinants of CoIE1 to within a 0.38 kb region that, when cloned into plasmid vectors, greatly increases their stability.     

Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas

Host-vector systems have been developed for gene cloning in the metabolically versatile bacterial genus Pseudomonas. They comprise restriction-negative host strains of Pseudomonas aeruginosa and P. putida and new cloning vectors derived from the high-copy-number, broad-host-range plasmid RSF1010, which are stably maintained in a wide range of Gram-negative bacteria. These plasmids contain EcoRI, SstI, HindIII, XmaI, XhoI, SalI, BamHI, and ClaI insertion sites. All cloning sites, except for BamHI and ClaI, are located within antibiotic-resistance genes' insertional inactivation of these genes during hybrid plasmid formation provides a readily scored phenotypic change for the rapid identification of bacterial clones carrying such hybrids. One of the new vector plasmids is a cosmid that may be used for the selective cloning of large DNA fragments by in vitro lambda packaging. An analogous series of vectors that are defective in their plasmid-mobilization function, and that exhibit a degree of biological containment comparable to that of current Escherichia coli vector plasmids, are also described.     

Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria.

A simple procedure for cloning and stable insertion of foreign genes into the chromosomes of gram-negative eubacteria was developed by combining in two sets of plasmids (i) the transposition features of Tn10 and Tn5; (ii) the resistances to the herbicide bialaphos, to mercuric salts and organomercurial compounds, and to arsenite, and (iii) the suicide delivery properties of the R6K-based plasmid pGP704. The resulting constructions contained unique NotI or SfiI sites internal to either the Tn10 or the Tn5 inverted repeats. These sites were readily used for cloning DNA fragments with the help of two additional specialized cloning plasmids, pUC18Not and pUC18Sfi. The newly derived constructions could be maintained only in donor host strains that produce the R6K-specified pi protein, which is an essential replication protein for R6K and plasmids derived therefrom. Donor plasmids containing hybrid transposons were transformed into a specialized lambda pir lysogenic Escherichia coli strain with a chromosomally integrated RP4 that provided broad-host-range conjugal transfer functions. Delivery of the donor plasmids into selected host bacteria was accomplished through mating with the target strain. Transposition of the hybrid transposon from the delivered suicide plasmid to a replicon in the target cell was mediated by the cognate transposase encoded on the plasmid at a site external to the transposon. Since the transposase function was not maintained in target cells, such cells were not immune to further transposition rounds. Multiple insertions in the same strain are therefore only limited by the availability of distinct selection markers. The utility of the system was demonstrated with a kanamycin resistance gene as a model foreign insert into Pseudomonas putida and a melanin gene from Streptomyces antibioticus into Klebsiella pneumoniae. Because of the modular nature of the functional parts of the cloning vectors, they can be easily modified and further selection markers can be incorporated. The cloning system described here will be particularly useful for the construction of hybrid bacteria that stably maintain inserted genes, perhaps in competitive situations (e.g., in open systems and natural environments), and that do not carry antibiotic resistance markers characteristic of most available cloning vectors (as is currently required of live bacterial vaccines).     

Transfer and expression of broad host range plasmids in Zymomonas mobilis

Summary The broad host range vectors, pKT210, pKT212, pKT248, pKT240, pGSS33 were mobilized into Zymomonas mobilis with the help of conjugative plasmids belonging to various incompatibility groups. The vectors pKT210, pKT212, pKT248, pGSS33 were stably maintained in Zymomonas mobilis in contrast to the conjugative plasmids. Based on these results we suggest the potential usefulness of these vectors as cloning vehicles in Zymomonas mobilis.     

Broad host range and promoter selection vectors for bacteria that interact with plants.

A plasmid vector, pGV910, and a derived cosmid, pRG930, have been constructed. Both contain the ColE1 and pVS1 origins of replication and are stably maintained in Escherichia coli, Agrobacterium tumefaciens, and Azorhizobium caulinodans ORS571. They are compatible with commonly used IncP cloning vectors, although pVS1 was classified as an IncP plasmid, unable to replicate in E. coli (Y. Itoh, J.M. Watson, D. Haas, and T. Leisinger, Plasmid 11:206-220, 1984). Promoter selection vectors were derived from both of these plasmids by using a promoterless beta-glucuronidase and/or beta-galactosidase gene. These vectors facilitate the study of gene expression in bacteria under particular environmental conditions. This is illustrated by the expression of the gusA gene under the control of a nod promoter in A. caulinodans nodulating stem-located infection sites on Sesbania rostrata.     

Natural transformation of Pseudomonas stutzeri by plasmids that contain cloned fragments of chromosomal deoxyribonucleic acid

Natural transformation of plasmids by Pseudomonas stutzeri was found to depend on their bearing inserts of chromosomal DNA. A set of plasmids derived from the nonconjugative broad host range plasmid pSa151 was constructed by integrating various chromosomal DNA fragments into the single EcoR1 site of pSa151. Selection for the kanamycin resistance determined by pSa151 demonstrated that the derivative plasmids were taken into the cells by natural transformation and stably maintained; each could be reisolated unchanged. Thirty-two different derivative plasmids, 14 to 31 kbase pairs in size, all transformed. The frequency of transformation increased with the size of the chromosomal insert over a twenty fold range. These results suggest that the mechanism of transformation of plasmids by the Gram-negative P. stutzeri is similar to those proposed to operate in Gram-positive bacteria.Dedicated to Prof. Dr. H.-G. Schlegel on the occasion of his 60 th birthday     

Nuclear plasmids in the Dictyostelium slime molds

Cellular slime molds are one of only three types of eukaryotes known to contain circular nuclear plasmids. Unlike the 2-microns circle in Saccharomyces, different strains of Dictyostelium can carry different, nonhomologous plasmids. Covalently closed, circular DNA plasmids have been identified in D. discoideum, D. mucoroides, D. giganteum, and D. purpureum. These plasmids range in size from 1.3-27 kb and in copy number from 50-300 molecules per cell. Plasmids have been identified in approximately one-fifth of all isolates examined. The organization of their DNA in nucleosomes establishes their presence in the nucleus. We have successfully cotransformed endogenous Dictyostelium plasmids into D. discoideum using the G418 resistance shuttle vector B10S. Transformants carrying D. discoideum plasmids are recovered at much higher frequency than those carrying plasmids from the other Dictyostelium species. We have constructed recombinant plasmids based on the D. discoideum plasmid Ddp2 and the G418 resistance gene. With these extrachromosomal vectors, transformed cells are recovered at frequencies of up to 10(-4) per input cell, the vectors are stably maintained at high copy number in the absence of selection, and the vectors can be used to introduce foreign DNA sequences into D. discoideum cells.     

基于XcmⅠ酶切的高效TA克隆载体的构建

目的：制备基于XcmⅠ酶切的高效TA克隆载体,并检测其克隆PCR产物的效果。方法：设计一对互补配对的寡核苷酸,经过变性及退火后插入质粒pUC19的多克隆位点,从而在该多克隆位点中引入2个XcmⅠ酶切位点,用XcmⅠ酶切后即获得含有3’突出T碱基的T载体;为了提高该T载体的克隆效率,优化了2个XcmⅠ酶切位点之间的碱基数目,排除了载体自连产生白色克隆的可能性,使假阳性大大减少;此外,为了便于完全酶切与未完全酶切载体的分离,在2个XcmⅠ之间插入了一段无关DNA片段。结果：改进得到的T载体可以有效克隆PCR产物,其阳性克隆率可达95％。结论：构建了基于XcmⅠ酶切的TA克隆载体,经过改进的T载体具有很高的克隆效率。     

Plasmid cloning vectors replicating in Escherichia coli,amino acid-producing coryneform bacteria and Methylobacillus sp.

Summary Four hybrid plasmids were constructed from the cryptic plasmid pAM330 (from Brevibacterium lactofermentum; 4.5 kb) and the broadhost-range plasmid pGV1106 (9.0 kb; Km^r Sm^r) isolated from Escherichia coli. All of them were mobilized from E. coli into the Gram-negative methylotrophic bacterium Methylobacillus sp. and two of these constructs (pCEM300 and pCEM400) were transferred by transformation into B. flavum and Corynebacterium glutamicum. Their kanamycin-resistance determinant coming from Gram-negative hosts was expressed in these Gram-positive bacteria. Both pCEM300 and pCEM400 are very stably maintained in B. flavum and represent suitable vectors for gene cloning in coryneform producers of amino acids.     

Genetic and physical analyses of a cluster of genes essential for xanthan gum biosynthesis in Xanthomonas campestris.

Xanthomonas campestris produces copious amounts of a complex exopolysaccharide, xanthan gum. Nonmucoid mutants, defective in synthesis of xanthan polysaccharide, were isolated after nitrosoguanidine mutagenesis. To isolate genes essential for xanthan polysaccharide synthesis (xps), a genomic library of X. campestris DNA, partially digested with SalI and ligated into the broad-host-range cloning vector pRK293, was constructed in Escherichia coli. The pooled clone bank was conjugated en masse from E. coli into three nonmucoid mutants by using pRK2013, which provides plasmid transfer functions. Kanamycin-resistant exconjugants were then screened for the ability to form mucoid colonies. Analysis of plasmids from several mucoid exconjugants indicated that overlapping segments of DNA had been cloned. These plasmids were tested for complementation of eight additional nonmucoid mutants. A 22-kilobase (kb) region of DNA was defined physically by restriction enzyme analysis and genetically by ability to restore mucoid phenotype to 10 of the 11 nonmucoid mutants tested. This region was further defined by subcloning and by transposon mutagenesis with mini-Mu(Tetr), with subsequent analysis of genetic complementation of nonmucoid mutants. A region of 13.5 kb of DNA was determined to contain at least five complementation groups. The effect of plasmids containing cloned xps genes on xanthan gum synthesis was evaluated. One plasmid, pCHC3, containing a 12.4-kb insert and at least four linked xanthan biosynthetic genes, increased the production of xanthan gum by 10% and increased the extent of pyruvylation of the xanthan side chains by about 45%. This indicates that a gene affecting pyruvylation of xanthan gum is linked to this cluster of xps genes.