Transposon Tn5 confers streptomycin resistance to Myxococcus xanthus

Abstract In addition to resistance to kanamycin, transposon Tn 5 confers resistance to streptomycin in Myxococcus xanthus . The streptomycin determinant is located within the Bgl II fragment of Tn 5 . The level of resistance varies among strains bearing Tn 5 insertions in different chromosomal loci and there is a correlation between the levels of resistance to streptomycin and to kanamycin.     

Tn5Map, a transposon for the rapid mapping of restriction sites in plasmids

Abstract A transposon was constructed allowing the rapid restriction mapping of plasmids. This transporon, Tn5Map, contains a cleavage site for the I- Sce I endonuclease which recognizes an 18-mer. After iivo transposition of Tn5Map into the plasmid of interest, the plasmid is isolated and linearized with I- Sce I. Splinkers labelled with digoxygenin and complementary to the left and right end of the linearized molecule are added and ligated. After partial digestion of the splinkered molecules with the restriction enzyme of interest, separation of the cleavage products in an agarose gel, and Southern transfer, the labelled fragments are visualized by the addition of the chemiluminescent substrate AMPPD and alkaline phosphatase. The restriction map can be directly read from the bottom to the top of the gel.     

Pseudo-transposition of a Tn5 derivative in Neisseria gonorrhoeae

Abstract We constructed a Tn5 derivative for potential use in transposon mutagenesis of Neisseria gonorrhoeae . It was incorporated into the chromosome apparently at random following transformation, but the insertion events were dependent on a functional RecA and independent of a functional transposase. Furthermore, in most cases there was an incomplete transposon inserted with little or no IS50 insertion sequence. These observations suggest that TnJ transposition may not be possible in N. gonorrhoeae and that this organism may have an unexplored illegitimate recombination system.     

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.     

高温胁迫对根瘤菌Tn5在土壤中的存活及其表型表达的影响

研究了３株弗氏中华根瘤菌（Ｒｈｉｚｏｂｉｕｍｆｒｅｄｉｉ）Ｔｎｓ突变株于适宜温度和高温胁迫两种条件下在土壤中的存活和Ｔｎｓ表型的表达．在适宜温度（２８℃）条件下的灭菌和未灭菌土壤中的存活研究表明生物因素抑制了突变株和野生型的生长．但野生型和突变株的存活种群密度之间无显著差异（Ｐ＝０．０１）．在高温胁迫（４０℃）条件下,土壤中野生型和突变株的种群密度迅速下降,其中部分ＯＮ－２和ＯＮ－３细胞丢失了Ｔｎｓ表型,说明部分细菌的Ｔｎ５表型在高温胁迫条件下不能表达．     

Transposon mutagenesis in Staphylococcus epidermidis using the Enterococcus faecalis transposon Tn917

We transformed a clinical Staphylococcus epidermidis isolate with the Enterococcus faecalis transposon Tn917-carrying plasmid pTV1. Loss of plasmid replication was observed at 47 degrees C. Tn917 transposes efficiently and apparently randomly. The transposition frequency could be stimulated by erythromycin. Transposon mutagenesis in S. epidermidis provides a means for genetic study of the various virulence factors of this pathogen.     

Precise excision of bacterial transposon Tn5 in yeast

Summary We have demonstrated that precise excision of bacterial transposon Tn5 can occur in the yeast, Saccharomyces cerevisiae. Tn5 insertions in the yeast gene LYS2 were generated by transposon mutagenesis made in Escherichia coli by means of a ::Tn5 vector. Nine insertions of Tn5 into the structural part of the yeast LYS2 gene situated in a shuttle epsiomal plasmid were selected. All the plasmids with a Tn5 insertion were used to transform yeast strains carrying a deletion of the entire LYS2 gene or a deletion of the part of LYS2 overlapping the point of insertion.All insertions inactivated the LYS2 gene and were able to revert with low (about 10^-8) frequencies to lysine prototrophy. Restriction analysis of revertant plasmids revealed them to be indistinguishable from the original plasmid without Tn5 insertion. DNA sequencing of the regions containing the points of insertions, made for two revertants, proved that Tn5 excision was completely precise.     

以转座子Tn5作弗氏中华根瘤菌的可识别生态学标记的研究──Tn5的水平转移及其对R．fredii Tn5突变株运动的影响

将一株弗氏中华根瘤菌（Ｒ．ｆｒｅｄｉｉ）ＱＢ１１３０的Ｔｎ５插入突变株ＯＮ－２用于生态学研究,以评估Ｔｎ５在自然环境中的水平转移以及各种水势下Ｔｎ５对突变株ＯＮ－２在土壤中运动的影响．试验表明,在自然潮湿的土壤中,Ｔｎ５本身的水平转移频率很低,且与Ｔｎ５插入相关的突变株卡那霉素抗性表型标记在非选择性平板上连续传４０代后仍然稳定．突变株ＯＮ－２与相对应的野生型菌株ＱＢ１１３０在各种相同水势的土壤中的运动无明显差异（Ｐ＝０．０１）,表明Ｔｎ５的插入不影响突变株的运动．因此,Ｔｎ５可作为研究Ｒ．ｆｒｅｄｉｉ基因工程菌大回应用的一个稳定有效的生态学标记．     

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。     

Transposon Tn5 mutagenesis in Rhodopseudomonas palustris

Abstract The potential of the antibiotic resistance transposon Tn5 for random insertion mutagenesis in Rhodopseudomonas palustris was assessed. The Tn5 containing suicide vector plasmid pSUP2021, was transferred from Escherichia coli to Rhodopseudomonas palustris and kanamycin-resistant transconjugants arose at a frequency of 2.7×10⁻⁷ per recipient. In the majority of transconjugants tested, Tn5 was found to have successfully transposed to yield a single chromosomal insertion, with the concomitant loss of the vector plasmid through segregation. Two Tn5 mutants, one defective in carotenoid synthesis, and one exhibiting a reduced anaerobic growth rate on aromatic acids, were partially characterised. This is the first study to show that Tn5 mutagenesis can be applied successfully to isolate mutants of Rhodopseudomonas palustris .     

Transposon Tn5 mutagenesis of Pseudomonas fluorescens to isolate mutants deficient in antibacterial activity

Abstract Pseudomonas fluorescens was subjected to insertion mutagenesis studies using the transposon Tn5-GM to generate mutants deficient in antibacterial activity minus mutants. The transposon located on the temperature-sensitive plasmid pCHR84 was conjugally transferred into the non-pathogenic pseudomonad using the triparental mating procedure. Random integration of Tn 5 -GM into the chromosome of P. fluorescens was achieved by heat ttreatment of the transformed cells at 42°C. Approximately 2% of transconjugants revealed an auxotrophic phenotype indicating efficient integration of the employed transposon into the chromosome of P. fluorescens . One transposon insertion mutant was obtained showing an antibacterial activity minus phenotype. This mutant (MM-7) was found to be defective in the production of an unidentified antibacterial compound against B. subtilis . These results introduce Tn 5 transposon mutagenesis as a new useful tool for the molecular analysis of P. fluorescens .     

转座子Tn5-Mob对菜豆根瘤菌共生基因的诱变、转移和初步定位

转座子Tn5-Mob在质粒RP4-4配合下能诱动(Mobilization)菜豆根瘤菌RCR3622内源质粒的诱动转移。在种间根瘤菌杂交过程中,二个巨型质粒的转移频率均大于10~(-3);分子量约为285kb的psym3622是带有结瘤(nod)和产黑素(mel)基因的共生质粒(Symbiotic plasmid);这二个基因的最大距离不超过70kb左右。另一个分子量约为135kb的质粒在试验中为不具结瘤功能的隐蔽质粒。将psym3622共生质粒导入不结瘤(Nod-)的豌豆根瘤菌菌株B151,能够使后者在菜豆植物上表达结瘤的特性,形成无效根瘤。将psym3622共生质粒导入不结瘤的菜豆根瘤菌菌株JI8400,能够在菜豆植物上形成正常发育的有效根瘤。     

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)]     

Diversity, biology and evolution of IncQ-family plasmids

Plasmids of IncQ-family are distinguished by having a unique strand-displacement mechanism of replication that is capable of functioning in a wide variety of bacterial hosts. In addition, these plasmids are highly mobilizable and therefore very promiscuous. Common features of the replicons have been used to identify IncQ-family plasmids in DNA sequence databases and in this way several unstudied plasmids have been compared to more well-studied IncQ plasmids. We propose that IncQ plasmids can be divided into four subgroups based on a number of mutually supportive criteria. The most important of these are the amino acid sequences of their three essential replication proteins and the observation that the replicon of each subgroup has become fused to four different lineages of mobilization genes. This review of IncQ-family plasmid diversity has highlighted several events in the evolution of these plasmids and raised several questions for further research.     

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 hsp70 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.     

Isolation and characterization of transposon Tn4001-generated, cytadherence-deficient transformants of Mycoplasma pneumoniae and Mycoplasma genitalium

Abstract Cytadherence and subsequent parasitism of host cells by the human pathogens, Mycoplasma pneumoniae and Mycoplasma genitalium , are mediated by adhesins and adherence-related accessory proteins. In this report we demonstrate the use of transposon Tn 4001 to generate Tn-induced transformants displaying cytadherence-deficient characteristics. Mycoplasma pneumoniae Tn-generated transformant, designated 8R, lacked the high-molecular weight adherence-accessory proteins HMW1/4 and was deficient in hemadsorption and cytadherence capabilities. In transformant 8R, Tn 4001 was not localized in or near the hmw 1 gene or in the upstream adhesin (p30/hmw3) locus, suggesting an alternate site associated with the regulation of hmw 1 gene expression. Sequence analysis identified the transposon insertion site at the crl locus previously reported, although the protein characteristics of transformant 8R differed from the earlier described transformants. The M. genitalium Tn-transformant, designated G26, was also defective in hemadsorption and cytadherence. However, transformant G26 synthesized adhesins P140 and P32 suggesting that Tn 4001 transposed into a new gene or site previously unlinked to cytadherence, namely ORF MG032. This study demonstrates the utility of Tn 4001 mutagenesis for both M. pneumoniae and M. genitalium which, in the latter case, has special relevance in light of the recent complete characterization of its continuous total genomic sequence.     

Isolation and characterization of transposon Tn5 mutants of Rhodobacter sphaeroides deficient in both nitrate and chlorate reduction.

Abstract The wild-type strain Rhodobacter sphaeroides DSM 158 is a nitrate-reducing bacterium with a periplasmic nitrate reductase. Addition of chlorate to the culture medium causes a stimulation of the phototrophic growth, indicating that this strain is able to use chlorate as an ancillary oxidant. Several mutant strains of R. sphaeroides deficient in nitrate reductase activity were obtained by transposon Tn5 mutagenesis. Mutant strain NR45 exhibited high constitutive nitrate and chlorate reductase activities and phototrophic growth was also increased by the presence of chlorate. In contrast, the stimulation of growth by chlorate was not observed in mutant strains NR8 and NR13, in which transposon Tn5 insertion causes the simultaneous loss of both nitrate and chlorate reductase activities. Tn5 insertion probably does not affect molybdenum metabolism since NR8 and NR13 mutants exhibit both xanthine dehydrogenase and nitrogenase activities. These results that a single enzyme could reduce both nitrate and chlorate in R. sphaeroides DSM 158.     

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。     

The use of Tn5 transposable elements in a gene trapping strategy for the protozoan Leishmania

The use of transposable elements as a gene-trapping strategy is a powerful tool for gene discovery. Herein we describe the development of a transposable system, based on the bacterial Tn5 transposon, which has been used successfully in Leishmania braziliensis. The transposon carries the neomycin phosphotransferase gene, which is expressed only when inserted in-frame with a Leishmania gene present in the target DNA. Four cosmid clones from a L. braziliensis genomic library were used as targets in transposition reactions and four insertional libraries were constructed and transfected in L. braziliensis. Clones resistant to G418 were selected and analysed by immunofluorescence in order to identify the subcellular localisation of the protein coded by the trapped gene. A definitive subcellular localisation for neomycin phosphotransferase/targeted protein fusion was not obtained in any of the four Leishmania clones investigated. However, the constructed transposable element is highly efficient considering the frequency of insertion in large targets and is therefore a useful tool for functional genetic studies in Leishmania. Our data confirm the utility of the Tn5 transposon system for insertion of sequencing priming sites into target DNA. Furthermore, the high frequency of insertion and even distribution are important in studying genomic regions bearing long and polymorphic repetitive sequences.     

Transposon mutagenesis in halophilic eubacteria: conjugal transfer and insertion of transposon Tn5 and Tn1732 in Halomonas elongata

Abstract Molecular genetic studies of halophilic eubacteria have been limited by the lack of a suitable method for mutagenesis. To overcome this, we established a transposon mutagenesis procedure for the ectoine-producing, halophilic bacterium Halomonas elongata . We used suicide plasmids pSUP101 and pSUP102-Gm to introduce the transposons Tn5 and Tn7732 respectively into H. elongata via Escherichia coli SM10 mediated conjugation. Our finding that H. elongata is sensitive to aminoglycoside antibiotics at low salinity enabled us to apply transposons that mediate kanamycin resistance. The insertions of transposon In 1732 occurred at different sites in the chromosome of H. elongata , as proved by Southern hybridization analysis. Phenotypic analysis revealed that different auxotrophic and salt sensitive mutants were generated by mutagenesis with transposon Tn 1732 . To our knowledge this is the first report of a successful application of a transposon for direct generalized mutagenesis in a halophilic eubacterium.