The construction and preliminary analysis of a Tn5 transposon based random mutant library of baculovirus

A transposon-based random mutation library of AcMNPV, the type species of baculovirus, was constructed using a Tn5 transposon. The green fluorescence protein gene under the control of the Drosophila hsp 70 promoter was inserted into the transposon for easy tracking in insect cells. In vitro transposition was carried out using the transposon and AcMNPV genomic DNA to allow the random insertion of the transposon into the virus genome. The transposed genome was then used to transfect Sf21 insect cells, and a library of mutant viruses capable of expressing green fluorescence protein was obtained. Two mutant viruses, B9F and Li6A were isolated, and the sites of transposon insertion were determined to be within the coding regions of the 94k and p10 genes, respectively. Both genes were determined to be nonessential in viral replication and infection. This technique will be very useful in the functional study of baculovirus genes. __________ Translated from Journal of Fudan University(Natural Science), 2005,44(4) [译自: 复旦学报(自然科学版),2005,44(4)]     

Tn5 transposase-assisted transformation of indica rice

Here, we describe experiments on Tn5 transposase-assisted transformation of indica rice. Transposomes were formed in vitro as a result of hyperactive Tn5 transposase complexing with a transposon that contained a 19-bp tetracycline operator (tetO) sequence. To form modified projectiles for transformation, the Tn10-derived prokaryotic tetracycline repressor (TetR) proteins, which can bind transposomes via the high affinity of TetR for tetO, were immobilized onto the surface of bare gold microscopic particles. These projectiles were introduced into cells of the indica rice cultivar Zhuxian B by particle bombardment. Once projectiles were inside the cell, tetracycline induced an allosteric conformational change in TetR that resulted in the dissociation of TetR from tetO, and thus generated free transposomes. Molecular evidence of transposition was obtained by the cloning of insertion sites from many transgenic plants. We also demonstrated that the introduced foreign DNA was inherited stably over several generations. This technique is a promising transformation method for other plant species as it is species independent.     

Structural basis of transposon end recognition explains central features of Tn7 transposition systems

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利用Tn5转座子介导突变提高大肠杆菌丁醇生产水平

目前,对于构建高产丁醇大肠杆菌工程菌株的工作,主要是对丁醇通路和相关途径的基因进行理性改造。为进一步提升菌株的丁醇生产能力,需要发掘基因组上可影响丁醇生产能力的基因,但这很难通过已有认识或计算机模型进行预测。本工作以一株实验室前期构建的产丁醇大肠杆菌工程菌株为研究对象,利用Tn5转座子构建了一个含有1 196个菌株的突变文库。丙酮酸是丁醇的前体,并且在发酵终产物中,副产物丙酮酸的含量与丁醇的含量呈反相关,因此,可以利用丙酮酸的含量来间接反映丁醇的含量,而丙酮酸可用二硝基苯肼显色法进行快速测定,基于此,建立了96孔板——酶标仪快速筛选方法。利用该方法成功筛选到了比对照菌株丁醇产量提高了29%、49%、56%的3个突变体菌株。利用反向PCR及测序的方法,确定了其转座子插入位置分别为:pyk A、tdk、cad C基因。这些基因可以作为进一步提高菌株丁醇产量的靶点,同时这种利用Tn5转座子筛选基因靶标的策略也为构建其他微生物细胞工厂提供了新思路。     

A practical random mutagenesis system for probiotic Lactobacillus casei using Tn5 transposition complexes

Aims: Despite the fact that the entire genome sequence of probiotic Lactobacillus casei has recently been available, their mechanisms of beneficial effects are poorly clarified, probably because of the lack of an efficient mutagenesis system. The aim of this study was to establish a practical random mutagenesis system of L. casei using the Tn5 transposome complexes. Methods and Results: We optimized the conditions for transformation using a plasmid pUCYIT356‐1‐Not2 and then transposition reaction using Tn5 transposome system for L. casei ATCC 27139. Tn5 insertion library of this strain being consisted of 9408 mutants was constructed by repeating the mutagenesis procedure. To examine the utility of this mutagenesis system, we screened a panel of insertion mutants for nutrient requirements. Six auxotrophic mutants were isolated and their Tn5 insertion sites were determined by inverse PCR, which demonstrated that insertions occur randomly throughout the whole bacterial genome. Conclusions: Tn5 transposome system functioned efficiently to generate transposon insertion mutants of L. casei and enabled to construct useful L. casei Tn5 insertion library at optimized conditions for transformation and transposition. Significance and Impact of the Study: The availability of this system facilitates the study of the mechanisms of beneficial effects of L. casei for human health.     

途径工程及Tn5转座子介导突变提高大肠杆菌丙酮酸生产

为了开发丙酮酸高产菌株,以大肠杆菌MG1655为出发菌株,通过基因敲除阻断副产物途径构建了产丙酮酸大肠杆菌工程菌KLPP。进一步利用p UT Mini-Tn5载体进行转座子随机突变,构建了含有7 197个单克隆的突变体文库。使用基于丙酮酸的二硝基苯肼显色法,建立了96孔板-酶标仪快速筛选方法,经过两轮的筛选,成功筛选到了6个突变体菌株,比KLPP丙酮酸产量提高了38%、31%、19%、28%、44%和14%。利用全基因组重测序确定了其转座子插入的位置,进而确定了可能影响丙酮酸产量的基因位点,为后续菌株改造工作奠定了基础。     

Introduction of the mercury transposon Tn501 into Rhizobium japonicum strains 31 and 110

Abstract Transposon Tn 501 , which encodes resistance to mercuric ions, was introduced into Rhizobium japonicum 110 and 31 by conjugal transfer. The transposon donor plasmid (pMD100) was able to mobilize into R. japonicum , but could not be maintained. Hg²⁺-resistant colonies were recovered at a frequency of 1.9 × 10⁻⁸/recipient for strain 110, and 1.7 × 10⁻⁷/recipient for strain 31. Presence of Tn 501 in Hg-resistant isolates was verified by Southern analysis and demonstrating transposition of Hg resistance. Transposon mutagenesis has been used to generate auxotrophic mutations at low frequency.     

Spread and evolution of natural plasmids harboring transposon Tn5

Abstract: The presence of transposon Tn 5 was studied in 730 Enterobacteriaceae strains from clinical and sewage origin. From these strains, twenty-five conjugative plasmids harboring transposon Tn 5 were isolated. These plasmids were compared with pJR67 and pRYC119, the only previously studied plasmids harboring Tn 5 . A phylogenetic tree of the evolution of all different plasmids was proposed. Irrespective of their bacterial host and geographical place of isolation, some of the plasmids were shown to be identical. All of them can be included in only eight different prototypical plasmid species. Twenty-two plasmids (88%) carried an IncI1 incompatibility determinant as judged form DNA hybridization experiments. The presence of some other common resistance genes suggested that these plasmids are descendants of a common ancestor. These IncI1 plasmids could be grouped in six prototypical species. The results presented here suggest that Tn 5 spread in nature may be dependent on the conjugative ability of the IncI plasmids harboring the transposon, rather than on the efficiency of Tn 5 transposition between different replicons.     

Transfer of a conjugative transposon, Tn5397 in a model oral biofilm

A tetracycline resistance profile was established from a microcosm dental plaque in a constant depth film fermenter. The fermenter was inoculated with a Bacillus subtilis strain which contained the conjugative transposon, Tn5397, which confers tetracycline resistance upon its host. After 6 hour and 24 hour the tetracycline resistance profile of the biofilm was redetermined and a tetracycline resistant Streptococcus species was isolated. A molecular analysis of this strain confirmed that Tn5397 was present in the genomic DNA of the isolate. These data represent the first report, to our knowledge, of intergeneric transfer of a conjugative transposon in a mixed species biofilm and demonstrates the ability of conjugative transposons to disseminate antibiotic resistance genes in a mixed species environment.     

Mutagenesis of Alcaligenes eutrophus by insertion of the drug-resistance transposon Tn5

Drug-resistance element Tn5 coding for kanamycin resistance was used for mutagenesis of Alcaligenes eutrophus strain H16. The vehicle for introducing Tn5 into A. eutrophus was plasmid pJB4JI harboured by Escherichia coli. Kanamycin-resistant transconjugants occurred at a frequency of approximately 5×10^-8. One third of the transconjugants exhibited other plasmid-coded resistances such as gentamycin and spectinomycin. However, the latter markers were not stably maintained in the new host. Among the kanamycin-resistant transconjugants three classes of mutants were found: (i) Auxotrophic mutants occurred at a frequency of 0.8% and showed requirements for histidine, methionine, aspartate orisoleucine. Out of eleven auxotrophic mutants examined eight reverted to prototrophy. However, none of the revertants was kanamycin-sensitive. (ii) Mutants unable to grow with fructose as the carbon source occurred at a frequency of almost 10%. (iii) Mutants which had lost the ability to grow autotrophically with hydrogen and carbon dioxide were found at a frequency of 1%. Further analyses revealed that this class of mutants was either defective in carbon dioxide fixation or impaired in hydrogen metabolism.     

A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900 CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens.     

Tn5转座突变技术在革兰氏阴性细菌分子遗传研究中的应用

随着广宿主载体系统的发展,Tn5及其衍生载体已经广泛应用于革兰氏阴性细菌的分子遗传学研究。主要综述了Tn5转座突变技术在生防细菌生防机理研究、细菌必需基因的鉴定、病原细菌毒力相关基因研究、代谢调控基因研究和菌株的遗传改良方面的应用研究进展。     

Isolation and transposon mutagenesis of a Pseudomonas putida KT2442 toluene-resistant variant: involvement of an efflux system in solvent resistance

A toluene-resistant variant of Pseudomonas putida KT2442, strain TOL, was isolated after liquid cultivation under xylene followed by toluene for 1 month in each condition. Almost all the populations of the variant strain formed small but readily visible colonies under toluene within 24 h at 30°C. The toluene-resistant strain also showed an increase in resistance to some unrelated antibiotics. Several toluene-sensitive Tn5 mutants have been isolated from the toluene-resistant strain and showed various levels of sensitivity. Most of these mutations did not cause significant changes in antibiotic resistance; however, one of the mutants (TOL-4) was highly susceptible to both organic solvents and various antibiotics, especially β-lactams. Sequencing analysis revealed that the mutation in TOL-4 had been introduced into a gene that may encode a transporter protein of an efflux system. This efflux system is very similar to one of the multidrug efflux systems of Pseudomonas aeruginosa. These observations indicate that a multidrug efflux system plays a major role in the organic solvent resistance of P. putida TOL. However, several other genes may also be involved. Received: December 18, 1997 / Accepted: March 16, 1998     

The identification a novel gene required for lipopolysaccharide biosynthesis by Haemophilus influenzae RM7004, using transposon Tn916 mutagenesis

Abstract Mutagenesis with the transposon Tn916 was used as a strategy to identify genes required for synthesis of the Galα(1–4)βGal component of Haemophilus influenzae strain RM7004 lipopolysaccharide. Insertion of Tn916 into an open reading frame (ORF) encoding a protein with 75% homology to the Escherichia coli methionine related protein (Mrp) is described. Mutations in mrp resulted in loss of reactivity with monoclonal antibody (mAb) 4C4, which recognises Galα(1–4)βGal, and expression of LPS with a different electrophoretic profile to that of wild-type RM7004. An unexpected feature of this mutation was that it appeared to influence the number of copies of 5'-CAAT-3' present in lic2A , a gene which is also required for biosynthesis and phase variable expression of the Galα(1–4)βGal LPS epitope.     

Tn5介导的致病性副溶血弧菌溶血能力差异突变株表型分析

【目的】构建致病性副溶血弧菌(Vibrio parahaemolyticus,Vp)突变体库,分析溶血能力差异突变株表型特征,为深入挖掘和认识tdh的调控机制提供研究基础。【方法】利用双亲本接合法,将Mini-Tn5-Km2片段随机插入致病性Vp (ATCC 33846)基因组,并以含有卡那霉素(Km)和氨苄青霉素(Amp)的TCBS选择性培养基进行突变株(KmRAmpS)筛选;结合PCR方法对突变菌株进行Km基因筛查,构建在不同基因位点随机插入突变的Vp突变体库。以我妻氏血平板筛选溶血表型变化菌株,并对其生长曲线、菌膜形成能力和运动能力进行测定。【结果】采用双亲本接合法,成功建立包含490株Vp突变株的突变体库,并获得5株溶血表型变化稳定的菌株(2株为溶血能力上调,3株为下调)。5株突变株在生长速率、菌膜形成能力及运动能力方面与亲本株有显著性差异。其中,2株溶血能力上调菌株及1株溶血能力下调菌株在运动能力、生长速率和菌膜形成能力方面较亲本株显著降低(P<0.05);另2株溶血能力下调菌株菌膜形成能力较亲本株显著提高(P<0.05)。【结论】Tn5转座子可用于建立Vp突变体库;Vp溶血能力与其表型特征具有相关性;研究所获得的5株溶血表型突变株为进一步探讨Vp tdh的调控机制奠定基础。     

Characterization of an Azospirillum brasilense Tn5 mutant with enhanced N(2) fixation: the effect of ORF280 on nifH expression

Disruption of an open reading frame (ORF) of 840 bp (280 amino acids; ORF280) in an Azospirillum brasilense Tn5 mutant resulted in a pleiotrophic phenotype. Besides an enhanced N(2)-fixing capacity and altered expression pattern of a nifH-gusA fusion, growth on the charged polar amino acids glutamate and arginine was severely affected. ORF280, similar to previously identified ORFs present in Bradyrhizobium japonicum (ORF277), Paracoccus denitrificans (ORF278) and Rhodobacter capsulatus (ORF277), exhibits in its C-terminus a significant similarity with the recently defined family of universal stress proteins.     

Characterization of Tn5-induced symbiotically defective mutants of cowpea rhizobia

Symbiotically defective mutants of cowpea rhizobia strain IRC256 were isolated by random Tn5 mutagenesis and characterized. One auxotroph (MS1) requiring adenine and thiamine was a non-nodulating mutant (Nod⁻) and three prototrophic mutants were Nod⁺ Fix⁻ which formed small and ineffective nodules on cowpeas (Vigna unguiculata). Acetylene reduction activity of the Nod⁺ Fix⁻ mutants was reduced to 80–94% of that of the wild-type strain. The non-nodulating mutant (MS1) induced root-hair curling but did not show any nodule initiation or nodule development. Ultrastructural examination of nodules formed by Fix⁻ mutants showed that these contained few bacteroids, indicating either early senescence or a reduction in bacterial release into the cytoplasm of the host cell. DNA hybridization of total DNAs from a representative number of Tn5 mutants showed that each of them had one copy of the transposon Tn5 which was randomly inserted into the genome of cowpea rhizobia.     

Mechanism of F factor-enhanced excision of transposon Tn5

The reversion of lac:: Tn5 insertion mutations was used to examine the control of excision of the kanamycin-resistance transposon Tn5 in Escherichia coli. Earlier work which showed that the fertility factor F enhances Tn5 excision had led another group to suggest that this is due to the product of a putative transposable element-specific "recombination" gene in the F factor which can act on Tn5 located anywhere in the genome. We show, however, that Tn5 is excised from sites in the lac operon of F'lac plasmids several orders of magnitude more efficiently than from the same sites in the chromosomes of F-, F+ or homozygous lac:: Tn5[F'lac:: Tn5] strains. Thus F enhances Tn5 excision, but only if F and Tn5 are in cis in the same DNA molecule. Bacterial crosses showed that transfer of F'lac:: Tn5 plasmids by conjugation stimulates Tn5 excision, and that transfer is frequent even within F' populations. These results suggest that the ability of F to enhance excision is the consequence of DNA transfer in conjugation.