Marker rescue from the Nicotiana tabacum plastid genome using a plastid/Escherichia coli shuttle vector

We recently reported an 868-bp plastid DNA minicircle, NICE1, that formed during transformation in a transplastomic Nicotiana tabacum line. Shuttle plasmids containing NICEI sequences were maintained extrachromosomally in plastids and shown to undergo recombination with NICE1 sequences on the plastid genome. To prove the general utility of the shuttle plasmids, we tested whether plastid genes outside the NICE1 region could be rescued in Escherichia coli. The NICE1-based rescue plasmid, pNICER1, carries NICE1 sequences for maintenance in plastids, the CoIE1 ori for maintenance in E. coli and a spectinomcyin resistance gene (aadA) for selection in both systems. In addition, pNICERl carries a defective kanamycin resistance gene, kan*, to target the rescue of a functional kanamycin resistance gene, kan, from the recipient plastid genome. pNICERl was introduced into plastids where recombination could occur between the homologous kan/kan* sequences, and subsequently rescued in E. coli to recover the products of recombination. Based on the expression of kanamycin resistance in E. coli and the analysis of three restriction fragment polymorphisms, recombinant kan genes were recovered at a high frequency. Efficient rescue of kan from the plastid genome in E. coli indicates that NICE 1-based plasmids are suitable for rescuing mutations from any part of the plastid genome, expanding the repertoire of genetic tools available for plastid biology.     

Kanamycin resistance as a selectable marker for plastid transformation in tobacco

We report on a novel chimeric gene that confers kanamycin resistance on tobacco plastids. The kan gene from the bacterial transposon Tn5, encoding neomycin phosphotransferase (NPTII), was placed under control of plastid expression signals and cloned between rbcL and ORF512 plastid gene sequences to target the insertion of the chimeric gene into the plastid genome. Transforming plasmid pTNH32 DNA was introduced into tobacco leaves by the biolistic procedure, and plastid transformants were selected by their resistance to 50 g/ml of kanamycin monosulfate. The regenerated plants uniformly transmitted the transplastome to the maternal progeny. Resistant clones resulting from incorporation of the chimeric gene into the nuclear genome were also obtained. However, most of these could be eliminated by screening for resistance to high levels of kanamycin (500 g/ml). Incorporation of kan into the plastid genome led to its amplification to a high copy number, about 10000 per leaf cell, and accumulation of NPTII to about 1% of total cellular protein.     

Next generation synthetic vectors for transformation of the plastid genome of higher plants

Plastid transformation vectors are E. coli plasmids carrying a plastid marker gene for selection, adjacent cloning sites and flanking plastid DNA to target insertions in the plastid genome by homologous recombination. We report here on a family of next generation plastid vectors carrying synthetic DNA vector arms targeting insertions in the rbcL-accD intergenic region of the tobacco (Nicotiana tabacum) plastid genome. The pSS22 plasmid carries only synthetic vector arms from which the undesirable restriction sites have been removed by point mutations. The pSS24 vector carries a c-Myc tagged spectinomycin resistance (aadA) marker gene whereas in vector pSS30 aadA is flanked with loxP sequences for post-transformation marker excision. The synthetic vectors will enable direct manipulation of passenger genes in the transformation vector targeting insertions in the rbcL-accD intergenic region that contains many commonly used restriction sites. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Retrofitting ampicillin resistant vectors by recombination for use in generating C. elegans transgenic animals by bombardment

Caenorhabditis elegans is an important model organism for modern biologic research. An essential aspect of C. elegans research is the production of transgenic animals for study. These are often generated via microinjection, but biolistic bombardment has become increasingly popular. However, many of the plasmids previously generated for use in microinjection are not readily used for bombardment due to the lack of a convenient marker. The unc-119 gene is often used as a marker since unc-119 rescue can be observed at low magnification, allowing rescued animals to be easily distinguished from the larger number of non-rescued animals. Here we report the use of homologous recombination in Escherichia coli as a method to insert a cassette containing the unc-119 gene into commonly used plasmids at the site of the ampicillin resistance gene which is simpler than other methods like subcloning. These cassettes are flanked by regions homologous to the 5′ and 3′ ends of the ampicillin resistance gene and contain either the unc-119 gene and the kanamycin resistance gene or a unc-119:mCherry fusion gene and the kanamycin resistance gene. The resulting plasmids may be used for biolistic bombardment to yield animals that display unc-119 rescue, and also express the recipient plasmid transgene.     

Streptomycin and lincomycin resistances are selective plastid markers in cultured Nicotiana cells

Summary Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.     

Functional expression of the genes of Escherichia coli in gram-positive Corynebacterium glutamicum

Summary Hybrid plasmids were constructed by combining in vitro the Escherichia coli plasmid pGA22, which carries the genes determining resistance to kanamycin, tetracycline, chloramphenicol and ampicillin, with the cryptic plasmids, pCG1 and pCG2, of Corynebacterium glutamicum. The hybrid plasmids were introduced into C. glutamicum and E. coli and replicated in both hosts. They expressed all the E. coli resistance phenotypes except ampicillin resistance in C. glutamicum. The levels of antibiotic inactivating enzymes encoded on these plasmids were about four to ten times lower in C. glutamicum than in E. coli. Despite the lack of expression of ampicillin resistance, -lactamase activity was detected in C. glutamicum carrying hybrid plasmids.     

Site-specific mutagenesis of the Ti plasmid by transformation of Agrobacterium tumefaciens with mutagenized T-DNA fragments cloned in E. coli plasmids

Summary A DNA fragment covering the complete T-region of the Ti plasmid from Agrobacterium tumefaciens strain C58 was cloned in the Escherichia coli cosmid pHC79. This fragment was mutagenized by insertion of transposon Tn5. The isolated DNA from hybrid plasmids was used to transform cells of A. tumefaciens strain C58 applying the freeze-thaw method. Although the E. coli plasmids with the mutagenized Ti plasmid fragment cannot replicate in these cells, they can be rescued by recombination with the homologous region of the Ti plasmid. The cointegrates formed were resolved in a second recobination event, which was detected by loss of the drug resistance marker of the E. coli plasmid. Subcloning of the Ti plasmid fragments labeled with Tn5 showed that the frequency of rescue of the hybrid plasmid as a cointegrate and its segregation in agrobacteria depend on the degree of homology with the Ti plasmid. We also applied the strategy for site-directed Tn5 mutagenesis to insert specifically the replication origin of bacteriophage fd and the thymidine kinase gene from Herpes virus into the T-DNA of Ti plasmid-C58.     

In vivo testing of a tobacco plastid DNA segment for guide RNA function in psbL editing

A C-to-U RNA editing event creates a functional initiation codon for translation of the psbL mRNA in tobacco plastids. Small trans-acting guide RNAs (gRNAs) have been shown to be involved in editing site selection in kinetoplastid mitochondria. A computer search of the tobacco plastid genome (ptDNA) identified such a putative gRNA, a 14-nucleotide sequence motif that is complementary to the psbL mRNA, including the A nucleotide required to direct the C-to-U change. The critical A nucleotide of the putative gRNA gene was changed to G by plastid transformation. We report here that the introduced mutation did not abolish psbL editing. Since no other region of the plastid genome contains significant complementarity to the psbL editing site we suggest that, if gRNAs serve as trans-acting factors for plastid psbL mRNA editing, they either have only a limited complementarity to the editing site, or are encoded in the nuclear genome.     

Plasmid from photosynthetic bacterium Ectothiorhodospira sp. carries a transposable streptomycin resistance gene

Centrifugation through a cesium chloride density gradient and agarose gel electrophoresis of the DNA from the purple non-sulfur photosynthetic bacterium Ectothiorhodospira sp. resolved a single extrachromosomal element, plasmid pDG1. Its size was estimated to be 13.2 kilobases by restriction endonuclease mapping. Plasmid pDG1 and two restriction fragments thereof were cloned in Escherichia coli C600 with plasmid pBR327 as a vector to form mixed plasmids pDGBR1, pDGBR2, and pDGBR3. The resistance to streptomycin and mercury found in Ectothiorhodospira sp. was transferred to E. coli C600 after transformation with pDGBR1 but not with pDGBR2 and pDGBR3. The replication origin of pDG1 was estimated to be within a 2-kilobase restriction fragment of pDG1 by monitoring its replication in E. coli HB101, using a kanamycin resistance reporter gene. High stringency molecular hybridization with ³²P-labeled pDG1 identified specific fragments of genomic DNA, suggesting the integration of some plasmid sequences. In accordance with the hypothesis that this integration is due to a transposon, we tested the transfer of streptomycin resistance from pDG1 into plasmid pVK 100 used as a target. For this test, we regrouped in the same cells of E. coli HB101, pDGBR1 and mobilizable plasmid pVK100 (tet^r, km^r). We used the conjugation capacity of the pVK100/pRK2013 system to rescue the target plasmid pVK100 into nalidixic acid-resistant E. coli DH1. The transfer frequency of streptomycin resistance into pVK100 was 10⁻⁵, compatible with a transposition event. In line with the existence of a transposon on pDG1, heteroduplex mapping indicated the presence of inverted repeats approximately 7.5 kb from one another.     

A novel approach to plastid transformation utilizes the phiC31 phage integrase

Thus far plastid transformation in higher plants has been based on incorporation of foreign DNA in the plastid genome by the plastid's homologous recombination machinery. We report here an alternative approach that relies on integration of foreign DNA by the phiC31 phage site-specific integrase (INT) mediating recombination between bacterial and phage attachment sites (attB and attP, respectively). Plastid transformation by the new approach depends on the availability of a recipient line in which an attB site has been incorporated in the plastid genome by homologous recombination. Plastid transformation involves insertion of an attP vector into the attB site by INT and selection of transplastomic clones by selection for antibiotic resistance carried in the attP plastid vector. INT function was provided by either expression from a nuclear gene, which encoded a plastid-targeted INT, or expressing INT transiently from a non-integrating plasmid in plastids. Transformation was successful with both approaches using attP vectors with kanamycin resistance or spectinomycin resistance as the selective marker. Transformation efficiency in some of the stable nuclear INT lines was as high as 17 independently transformed lines per bombarded sample. As this system does not rely on the plastid's homologous recombination machinery, we expect that INT-based vectors will make plastid transformation a routine in species in which homologous recombination rarely yields transplastomic clones.     

Site-specific recombination between ColE1 tcer and NTP16 nmr sites in vivo

The site-specific recombination system used by multicopy plasmids of the ColE1 family uses two identical plasmid-encoded recombination sites and four bacterial proteins to catalyze the recombination reaction. In the case of the Escherichia coli plasmid ColE1, the recombination site, cer, is a 280 by DNA sequence which is acted on by the products of the argR, pepA, xerC and xerD genes. We have constructed a model system to study this recombination system, using tandemly repeated recombination sites from the plasmids ColE1 and NTP16. These plasmids have allowed us precisely to define the region of strand exchange during site-specific recombination, and to derive a model for cer intramolecular site-specific recombination.     

通辽地区犊牛腹泻大肠杆菌耐药性检测及一株多重耐药菌全基因组测序分析

【背景】大肠杆菌(Escherichia coli)是引起犊牛腹泻的最主要病原菌,其耐药性菌株的不断出现引起广泛关注。【目的】了解内蒙古自治区通辽市犊牛腹泻大肠杆菌耐药性及耐药基因流行情况。【方法】从通辽市多个旗县采集犊牛腹泻样品40份,经细菌分离纯化及16S rRNA基因测序,最终鉴定出20株大肠杆菌。采用药敏试验和PCR方法对分离菌进行耐药性及耐药基因检测分析,并对其中1株多重耐药菌株进行全基因组测序。【结果】20株分离菌均具有多重耐药性,对链霉素、环丙沙星、恩诺沙星和复方新诺明的耐药率达80%以上。所检耐药基因中,aphA1、strB、TEM-1和qnrS检出率达100%。通过对代表性菌株TL-13全基因组测序发现,其基因组大小为4897185bp,GC含量为50.68%,同时携带2个质粒,大小分别为108288bp(pTL13-1)和64018bp(pTL13-2)。质粒中共携带18个可移动耐药基因。【结论】通辽地区犊牛腹泻大肠杆菌多重耐药性普遍存在,4种常见耐药基因普遍流行。     

The Erythromycin Resistance Gene of the Corynebacterium xerosis R-plasmid pTP10 Also Carrying Chloramphenicol, Kanamycin, and Tetracycline Resistances Is Capable of Transposition in Corynebacterium glutamicum

The clinical isolate Corynebacterium xerosis M82B carries the 50-kb R-plasmid pTP10 that confers resistance to the antibiotics chloramphenicol, kanamycin, erythromycin, and tetracycline. A detailed restriction map of pTP10 was constructed by cloning and analyzing restriction fragments of pTP10 in Escherichia coli . The resistance determinants of pTP10 were located by studying the phenotype of the recombinant plasmids in E. coli and Corynebacterium glutamicum . Restriction patterns of fragments encoding the kanamycin and erythromycin resistances revealed striking similarity to the kanamycin resistance of transposon Tn903 and the erythromycin resistance on plasmid pNG2 from Corynebacterium diphtheriae, respectively. Expression of the resistance determinants in E. coli and C. glutamicum ATCC 13032 led to high resistance levels in both strains, with the exception of the tetracycline resistance gene, which could be expressed only in C. glutamicum. Furthermore, the erythromycin resistance gene was found to be located on a transposable element which is functional in C. glutamicum strains.     

Impact of the stringency of cell selection on plastid segregation in protoplast fusion-derived Nicotiana regenerates

Summary Vegetative segregation of a mixed plastid population in protoplast fusion-derived cell lines can be directed by a selection favouring the multiplication of one of the parental plastid types. This report defines some of the critical conditions leading to a homogeneous plastid population in cybrid plants generated by protoplast fusion between Nicotiana plumbaginifolia and an albino and streptomycin-resistant N. tabacum plastid mutant. Light (1,500 lx) conferred a strong selective advantage to chloroplasts versus albino plastids, while the lack of this effect in dim light (300 lx) indicated that a sufficient light intensity is essential to the phenomenon. Selection on streptomycin-containing medium in the dark, however, led to the preferential multiplication of resistant plastids. Streptomycin selection of resistant chloroplasts in the light, consequently, results in a plastid selection of doubled stringency. In another experiment a definite, but leaky, selection for chloroplast recombination (selection for greening on streptomycin-containing medium in dim light) was used to reveal various recombination products. Protoplast fusion in fact resulted in cybrid plants showing only simple chimeric segregation of unchanged parental plastids. These results demonstrate the essential requirement for stringent plastid selection, as defined by cell culture conditions, to precede the formation of shoots expected to possess the desired plastid genetic composition.     

Plastid DNA in the mitochondrial genome of Oenothera: Intra- and interorganellar rearrangements involving part of the plastid ribosomal cistron

Summary Part of the plastid rRNA cistron is present in the mitochondrial genome of Oenothera. This sequence of 2081 nucleotides contains the 3 half of the plastid 23 S rRNA, the adjacent intergenic region and the 4.5 S rRNA. Secondary intramitochondrial sequence rearrangements involve this region of plastid origin and the gene encoding the putative mitochondrial small ribosomal protein S13. Sequence comparison suggests that the interorganellar transfer event occurred a long time ago. The mitochondrial sequence contains regions more homologous to the plastid DNA from tobacco than from Oenothera itself in the regions analysed, suggesting faster sequence evolution in plastids than in mitochondria of Oenothera.     

Transfer of plasmid pRD1 from Escherichia coli to Azospirillum brasilense

Summary Data are presented which indicate that plasmid pRD1 can be transferred from Escherichia coli to strains of Azospirillum brasilense with a frequency of about 10^-7. The reverse was also possible; in this case the frequency of transfer appeared to be much higher, about 5×10^-1. Transfer of the plasmid was also obtained between strains of A. brasilense; in this cross the transfer frequency was very high (about 10^-1). Moreover the pRD1 plasmid seems very stable in A. brasilense cells.Abbreviations ade, his, and trp are requirements for adenine, histidine, and tryptophan, respectively - carb, kan, rif, spc, and tet are resistance to carbenicillin, kanamycin, spectinomycin, and tetracycline, respectively - recA56 recombination deficiency - nif genes for nitrogen fixation     

Identification of functional lox sites in the plastid genome

Our objective was to test whether or not cyclization recombination (CRE), the P1 phage site-specific recombinase, induces genome rearrangements in plastids. Testing was carried out in tobacco plants in which a DNA sequence, located between two inversely oriented locus of X-over of P1 (loxP) sites, underwent repeated cycles of inversions as a means of monitoring CRE activity. We report here that CRE mediates deletions between loxP sites and plastid DNA sequences in the 3'rps12 gene leader (lox-rps12) or in the psbA promoter core (lox-psbA). We also observed deletions between two directly oriented lox-psbA sites, but not between lox-rps12 sites. Deletion via duplicated rRNA operon promoter (Prrn) sequences was also frequent in CRE-active plants. However, CRE-mediated recombination is probably not directly involved, as no recombination junction between loxP and Prrn could be observed. Tobacco plants carrying deleted genomes as a minor fraction of the plastid genome population were fertile and phenotypically normal, suggesting that the absence of deleted genome segments was compensated by gene expression from wild-type copies. The deleted plastid genomes disappeared in the seed progeny lacking CRE. Observed plastid genome rearrangements are specific to engineered plastid genomes, which contain at least one loxP site or duplicated psbA promoter sequences. The wild-type plastid genome is expected to be stable, even if CRE is present in the plastid.     

Kanamycin-resistant alfalfa has a point mutation in the 16S plastid rRNA

Genes conferring resistance to kanamycin are frequently used to obtain transgenic plants as spontaneous resistance to kanamycin is not known to exist in higher plants. Nevertheless, mutations conferring kanamycin resistance have been identified in Chlamydomonas reinhardtii, raising the question as to why kanamycin-resistant mutants have not been found in higher plants. While attempting plastid transformation of alfalfa, we obtained non-transgenic but kanamycin-resistant somatic embryos following 2 months of culture in the presence of 50 mg l^–1 kanamycin. Sequencing of the plastid DNA region corresponding to the decoding site of the 16S rRNA in ten independent resistant events revealed an A to C transversion at position 1357 of the 16S plastid rDNA, the same site at which an A to G conversion confers kanamycin resistance to C. reinhardtii by reducing the ability of the antibiotic to bind to its target site. All plants derived from the resistant embryos through additional cycles of somatic embryogenesis in the absence of kanamycin retained the mutant phenotype, suggesting that the mutation was homoplastomic. Resistant plants produced 85% less biomass than controls; their leaves were chlorotic during early development and over time slowly turned green. The absence of kanamycin- resistant mutants in higher plants might be explained by the requirement for a regeneration system capable of resulting in homoplastomic individuals, or it may be the result of the detrimental effect of the mutation on the phenotype.Communicated by C.F. Quiros     

Expression of Tn5-derived kanamycin resistance in the fungus Phycomyces blakesleeanus

Summary A plasmid, carrying the Tn5 gene for kanamycin resistance lacking its own promoter, has successfully been used in the selection of DNA sequences of the fungus Phycomyces blakesleeanus having promoter activity in Escherichia coli. Many of these sequences were also effective in promoting resistance to kanamycin when the corresponding chimeric plasmids were introduced in the fungus via spheroplast transformation. The selected phenotype was easily propagated through vegetative spores and behaved as a stable character since it was not appreciably lost in the absence of selection.     

Construction of a fused operon consisting of the recA and kan (Kanamycin resistance) genes and regulation of its expression by the lexA gene

Summary The kanamycin resistance gene (kan) of transposon Tn5 was cloned into a derivative of plasmid pBR322. A DNA fragment containing the promoter-operator region of the recA gene was inserted into the promoter region of the cloned kan gene to produce a fused operon, recA-kan. Plasmid pMCR685 carrying recA-kan expressed a low level of activity of the kan gene product (kanamycin phosphotransferase; KPT) in the wildtype cells of Escherichia coli, while the plasmid showed an increased level of the activity in the Spr^- mutant cells which produce the inactive lexA protein. The KPT activity in the wildtype cells harboring the plasmid increased 6-to 11-fold upon treatment of the cells with mitomycin C or nalidixic acid, both of which are known to induce synthesis of recA protein.Expression of the recA-kan operon fusion was remakably repressed by the lexA gene cloned into a plasmid carrying the operon fusion. Higher concentrations of mitomycin C were required for maximal induction of KPT activity in the cells harboring the resulting plasmid pMCR687. These results strongly suggest that the lexA gene product can by itself repress the recA gene, and that pMCR687 is a useful vector to clone genes whose expression is harmful to the host cell growth.