In vivo random mutagenesis of streptomycetes using mariner-based transposon Himar1

We report here the in vivo expression of the synthetic transposase gene himar1(a) in Streptomyces coelicolor M145 and Streptomyces albus. Using the synthetic himar1(a) gene adapted for Streptomyces codon usage, we showed random insertion of the transposon into the streptomycetes genome. The insertion frequency for the Himar1-derived minitransposons is nearly 100 % of transformed Streptomyces cells, and insertions are stably inherited in the absence of an antibiotic selection. The minitransposons contain different antibiotic resistance selection markers (apramycin, hygromycin, and spectinomycin), site-specific recombinase target sites (rox and/or loxP), I-SceI meganuclease target sites, and an R6Kγ origin of replication for transposon rescue. We identified transposon insertion loci by random sequencing of more than 100 rescue plasmids. The majority of insertions were mapped to putative open-reading frames on the S. coelicolor M145 and S. albus chromosomes. These insertions included several new regulatory genes affecting S. coelicolor M145 growth and actinorhodin biosynthesis.     

In vivo Tn<Emphasis Type="Italic">5</Emphasis>-based transposon mutagenesis of Streptomycetes

This paper reports the in vivo expression of the synthetic transposase gene tnp(a) from a hyperactive Tn5 tnp gene mutant in Streptomyces coelicolor. Using the synthetic tnp(a) gene adapted for Streptomyces codon usage, we showed random insertion of the transposon into the Streptomycetes genome. The insertion frequency for the hyperactive Tn5 derivative is 98% of transformed S. coelicolor cells. The random transposition has been confirmed by the recovery of ~1.1% of auxotrophs. The Tn5 insertions are stably inherited in the absence of apramycin selection. The transposon contains an apramycin resistance selection marker and an R6Kγ origin of replication for transposon rescue. We identified the transposon insertion loci by random sequencing of 14 rescue plasmids. The majority of insertions (12 of 14) were mapped to putative open-reading frames on the S. coelicolor chromosome. These included two new regulatory genes affecting S. coelicolor growth and actinorhodin biosynthesis.     

piggyBac转座子介导的转基因叉尾斗鱼的构建

为探讨piggyBac转座子在鱼类动物中应用的可能性,以包含家蚕(Bombyx mori)肌动蛋白3启动子驱动的增强型绿色荧光蛋白(enhance green fluorescent protein,EGFP)基因的piggyBac质粒为载体,以及一个包含piggyBac转座酶的辅助质粒,采用显微注射的方法将其导入叉尾斗鱼(Macropodusopercularis)受精卵中,利用PCR技术证实了piggyBac转座子能够介导EGFP基因进入叉尾斗鱼基因组,并能够稳定遗传到下一代,符合孟德尔遗传规律。EGFP基因遗传到G1代的阳性鱼占交配鱼比率,即外源基因整合率为12.30%。实验证明,piggyBac质粒有可能成为水产动物转基因实验的新型载体。     

A Product of the Tn5 Transposase Gene Inhibits Transposition

The bacterial transposon Tn5 possesses a regulatory mechanism that allows it to move with higher efficiency when it is first introduced into a cell than after it is established. Tn5 is a composite transposable element containing inverted repeats of two nearly identical elements, IS 50R, which encodes the transposase protein necessary for Tn5 movement, and IS50L which contains an ochre mutant allele of the transposase gene. Data presented here show that Tn5 transposition is inhibited about 50-fold in cells of Escherichia coli which already carry IS 50R in the multicopy plasmid pBR322. If the cells contain a plasmid carrying either IS50L instead of IS50R, or derivatives of IS 50R in which the transposase gene has been mutated, little if any inhibition of Tn5 transposition is found. Although inhibition had previously been hypothesized to require interaction between the products of IS50 L and IS50R, our results show that IS50R alone is sufficient to mediate inhibition and suggest that the inhibitor is a product of the transposase gene itself.     

The goldfish hAT-family transposon Tgf2 is capable of autonomous excision in zebrafish embryos

The goldfish (Carassius auratus) Tgf2 transposon is a vertebrate DNA transposon that belongs to the hAT transposon family. In this study, we constructed plasmids containing either the full-length Tgf2 transposon (pTgf2 plasmid) or a partially-deleted Tgf2 transposon (ΔpTgf2 plasmid), and microinjected these plasmids into fertilized zebrafish (Danio rerio) eggs at the one- to two-cell stage. DNA extracted from the embryos was analyzed by PCR to assess transient excision, if any, of the exogenous plasmid and to verify whether Tgf2 is an autonomous transposon. The results showed that excision-specific bands were not detected in embryos injected with the ΔpTgf2 plasmid, while bands of 300–500 bp were detected in embryos injected with pTgf2, which indicated that the full-length Tgf2-containing plasmid could undergo autonomous excision in zebrafish embryos. DNA cloned from 24 embryos injected with pTgf2 was sequenced, and the results suggested that Tgf2 underwent self-excision in zebrafish embryos. Cloning and PCR analysis of DNA extracted from embryos co-injected with ΔpTgf2 and in vitro-transcribed transposase mRNA indicated that partially-deleted-Tgf2-containing ΔpTgf2 plasmid also underwent excision, in the presence of functional transposase mRNA. DNA cloned from 25 embryos co-injected with ΔpTgf2 and transposase mRNA was sequenced, and the results suggested that partially-deleted Tgf2 transposons plasmids were excised. These results demonstrated that excisions of Tgf2 transposons were mediated by the Tgf2 transposase, which in turn confirmed that Tgf2 is an autonomous transposon.     

Characterization of the transposon carrying the STII gene of enterotoxigenic Escherichia coli

Summary The Escherichia coli enterotoxin STII gene is carried by Tn4521. The terminal repeats of Tn4521 are composed of IS2 sequences; however, neither repeat is a complete IS2. In order to determine how this seemingly defective transposon could transpose, mutations were generated within Tn4521 to determine the regions essential for transposition. The left terminal repeat region was found to be non-essential, but the right terminal repeat area was demonstrated to be crucial for transposition. Within the right terminal repeat area is an open reading frame (ORF), capable of encoding a 159 amino acid protein, which was shown by frameshift mutation analysis to be required for transposition. This protein may be the transposase of Tn4521. A pair of 11 bp repeat sequences flanking the ORF was also found to be important. The right 11 bp repeat is part of the left IS2 terminal sequence, and the left 11 bp repeat is located about 300 bp upstream from the right IS2 terminal sequence located within the right terminal repeat region. The results of this study suggest that Tn4521 is a functional transposon and that the sequence including this pair of 11 bp sequences plus the intervening sequence is a transposable element which may be responsible for Tn4521 transposition.     

IS<Emphasis Type="Italic">Rm31</Emphasis>, a new insertion sequence of the IS<Emphasis Type="Italic">66</Emphasis> family in<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti pSymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, ISRm31. ISRm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that ISRm31 is a member of the IS66 family. ISRm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying ISRm31 suggested that ISRm31 inserts randomly into S. meliloti genomes. Moreover, analysis of ISRm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, ISRm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.     

Low target site specificity of an IS6100-based mini-transposon, Tn1792, developed for transposon mutagenesis of antibiotic-producing Streptomyces

To improve transposon mutagenesis of antibiotic-producing Streptomyces, a mini-transposon, Tn1792, was constructed, based on IS6100, originally isolated from Mycobacterium fortuitum. Easily manageable transposition assays were developed to demonstrate inducible transposition of Tn1792 into the Streptomyces genome from a temperature-sensitive delivery plasmid. Introduction of the selectable aac1 gene between the inverted repeats in Tn1792 allowed for both reliable identification of transposition events in Streptomyces, and also subsequent cloning of transposon-tagged sequences in Escherichia coli. This enabled the target site specificity of Tn1792 to be determined at nucleotide resolution, revealing no significant shared homology between different target sites. Consequently, Tn1792 is well suited for random mutagenesis of Streptomyces.     

DNA inversion mediated by the r-determinant of plasmid NR1: evidence for the intramolecular replicative transposition of a 23 kb IS1-flanked transposon?

Summary The r-determinant (r-det) of the R plasmid NR1-Basel is a 23 kb, IS1-flanked transposon, called Tn2671, which has been shown to transpose to the genome of bacteriophage P7. Among the derivatives of phage P7::r-det we found one which carried two copies of the r-det as inverted repeats and which also contained the P7 genome segment between them in inverted orientation. Its generation is best explained by assuming that the entire 23 kb Tn2671 transposon has undergone intramolecular replicative transposition.     

Cointegration and resolution mediated by IS101 present in plasmid pSC101

Summary A certain class of cointegrate plasmids was found to occur between a pSC101 derivative and a second plasmid pBV320 in E. coli F^- cells. Cleavage analysis and DNA sequencing showed that the cointegrate plasmid contained direct repeats of an insertion sequence IS101 at the recombination junctions, indicating that formation of cointegrates was mediated by IS101, which is a natural constitutent of pSC101. These cointegrates were formed only in cells which contained the transposon gamma-delta, suggesting that the gamma-delta sequence, which provides transposase, is responsible for cointegration. Whenever the cointegrate plasmids were present in cells containing gamma-delta or its related transposon Tn3, the cointegrates were dissolved to give pBV320::IS101 due to recombination at duplicated IS101 sequences in the cointegrates, suggesting that both gamma-delta and Tn3, which provide a resolvase, are responsible for the resolution of the cointegrates. Comparison between the nucleotide sequence of IS101 and those of gamma-delta and Tn3 shows a high degree of homology in the regions that have been shown to be the binding sites of resolvases, as well as in the terminal inverted repeats. However, there is no homology between IS101 and the other element, gamma-delta or Tn3, in the internal resolution site, at which the resolution event may occur.Abbreviations Tc tetracycline - Cm chloramphenicol - Ap ampicillin - bp base pairs - kb kilobase pairs     

Whole genome sequence of Klebsiella pneumoniae U25, a hypermucoviscous,multidrug resistant,biofilm producing isolate from India

Klebsiella pneumoniae U25 is a multidrug resistant strain isolated from a tertiary care hospital in Chennai, India. Here, we report the complete annotated genome sequence of strain U25 obtained using PacBio RSII. This is the first report of the whole genome of K. pneumoniaespecies from Chennai. It consists of a single circular chromosome of size 5,491,870-bp and two plasmids of size 211,813 and 172,619-bp. The genes associated with multidrug resistance were identified. The chromosome of U25 was found to have eight antibiotic resistant genes [blaOXA-1,blaSHV-28, aac(6’)1b-cr,catB3, oqxAB, dfrA1]. The plasmid pMGRU25-001 was found to have only one resistant gene (catA1) while plasmid pMGRU25-002 had 20 resistant genes [strAB, aadA1,aac(6’)-Ib, aac(3)-IId,sul1,2, blaTEM-1A,1B,blaOXA-9, blaCTX-M-15,blaSHV-11, cmlA1, erm(B),mph(A)]. A mutation in the porin OmpK36 was identified which is likely to be associated with the intermediate resistance to carbapenems in the absence of carbapenemase genes. U25 is one of the few K. pneumoniaestrains to harbour clustered regularly interspaced short palindromic repeats (CRISPR) systems. Two CRISPR arrays corresponding to Cas3 family helicase were identified in the genome. When compared to K. pneumoniaeNTUHK2044, a transposase gene InsH of IS5-13 was found inserted.     

Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats

Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity.     

Identification of Novel Non-autonomous CemaT Transposable Elements and Evidence of their Mobility within the C. elegans Genome

We describe here two new transposable elements, CemaT4 and CemaT5, that were identified within the sequenced genome of Caenorhabditis elegans using homology based searches. Five variants of CemaT4 were found, all non-autonomous and sharing 26 bp inverted terminal repeats (ITRs) and segments (152–367 bp) of sequence with similarity to the CemaT1 transposon of C. elegans. Sixteen copies of a short, 30 bp repetitive sequence, comprised entirely of an inverted repeat of the first 15 bp of CemaT4’s ITR, were also found, each flanked by TA dinucleotide duplications, which are hallmarks of target site duplications of mariner-Tc transposon transpositions. The CemaT5 transposable element had no similarity to maT elements, except for sharing identical ITR sequences with CemaT3. We provide evidence that CemaT5 and CemaT3 are capable of excising from the C. elegans genome, despite neither transposon being capable of encoding a functional transposase enzyme. Presumably, these two transposons are cross-mobilised by an autonomous transposon that recognises their shared ITRs. The excisions of these and other non-autonomous elements may provide opportunities for abortive gap repair to create internal deletions and/or insert novel sequence within these transposons. The influence of non-autonomous element mobility and structural diversity on genome variation is discussed.     

Transposition and transduction of plasmid DNA inStreptomyces spp.

Summary To expand the application of molecular genetics to many different streptomycete species, we have been developing two potentially widely applicable methodologies: transposon mutagenesis and plasmid transduction. We constructed three transposons from theStreptomyces lividans insertion sequence IS493. Tn5096 and Tn5097 contain an apramycin resistance gene inserted in different orientations between the two open reading frames of IS493. These transposons transpose from different plasmids into many different sites in theStreptomyces griseofuscus chromosome and into its resident linear plasmids. Tn5099 contains a promoterlessxylE gene and a hygromycin-resistance gene inserted in IS493 close to one end. Tn5099 transposes inS. griseofuscus giving operon fusions in some cases that drive expression of thexylE gene product, catechol deoxygenase, giving yellow colonies in the presence of catechol. We have also developed plasmid vectors that can be transduced into many streptomycete species by bacteriophage FP43. We describe the characterization of FP43 and mapping of several bacteriophage functions. The region of cloned FP43 DNA essential for plasmid transduction includes the origin for headful packaging.     

Mutator Transposon in Maize and MULEs in the Plant Genome

Mutator (Mu) is by far the most mutagenic plant transposon. The high frequency of transposition and the tendency to insert into low copy sequences for such transposon have made it the primary means by which genes are mutagenized in maize (Zea mays L.). Mus like elements (MULEs) are widespread among angiosperms and multiple-diverged functional variants can be present in a single genome. MULEs often capture genetic sequences. These Pack-MuLEs can mobilize thousands of gene fragments, which may have had a significant impact on host genome evolution. There is also evidence that MULEs can move between reproductively isolated species. Here we present an overview of the discovery, features and utility of Mu transposon. Classification of Mu elements and future directions of related research are also discussed. Understanding Mu will help us elucidate the dynamic genome.     

MiniUIB,a Novel Minitransposon-Based System for Stable Insertion of Foreign DNA into the Genomes of Gram-Negative and Gram-Positive Bacteria

Transposition of the insertion sequence (IS) ISPpu12 is actively induced after conjugative interaction. The transposase of this IS can act in trans on structures flanked by inverted repeats similar to those of the transposon. Based on that fact, an ISPpu12-based minitransposon, miniUIB, has been constructed in order to biotechnologically exploit the self-regulation of ISPpu12 and its increased activity after conjugative interaction. Mobilization of the miniUIB structure into the genome of Pseudomonas stutzeri AN10 after conjugative interaction was demonstrated. A single gene, i.e., the kanamycin resistance determinant, or large genetic structures of >12 kb, i.e., alkBFGHJKL and alkST operons of Pseudomonas putida TF4-1L (GPo1), have been easily integrated in P. stutzeri AN10 by an RP4-based delivery system. Therefore, the integration of the alk determinants by use of the miniUIB system has extended the biodegradation capabilities of this strain. Plasmid pJOC100, containing the transposase and regulator genes of ISPpu12 adjacent to the miniUIB structure, was constructed in order to extend the host range of this biotechnologically useful genetic tool to other model and real-world bacteria. The effectiveness of the system for random mutagenesis in a phylogenetic wide range of bacteria and for the insertion of novel functions has been demonstrated, even in successive steps.     

ISD1, an Insertion Element from the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough: Structure, Transposition, and Distribution

Insertion element ISD1, discovered when its transposition caused the insertional inactivation of an introduced sacB gene, is present in two copies in the genome of Desulfovibrio vulgaris Hildenborough. Southern blot analysis indicated at least two insertion sites in the sacB gene. Cloning and sequencing of a transposed copy of ISD1 indicated a length of 1,200 bp with a pair of 44-bp imperfect inverted repeats at the ends, flanked by a direct repeat of the 4-bp target sequence. AAGG and AATT were found to function as target sequences. ISD1 encodes a transposase from two overlapping open reading frames by programmed translational frameshifting at an A₆G shifty codon motif. Sequence comparison showed that ISD1 belongs to the IS3 family. Isolation and analysis of the chromosomal copies, ISD1-A and ISD1-B, by PCR and sequencing indicated that these are not flanked by direct repeats. ISD1-A is inserted in a region of the chromosome containing the gapdh-pgk genes (encoding glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase). Active transposition to other loci in the genome was demonstrated, offering the potential of a new tool for gene cloning and mutagenesis. ISD1 is the first transposable element described for the sulfate reducers, a large and environmentally important group of bacteria. The distribution of ISD1 in genomes of sulfate-reducing bacteria is limited. A single copy is present in the genome of D. desulfuricans Norway.     

IS1-encoded proteins,InsA and the InsA-B′-InsB transframe protein (transposase): Functions deduced from their DNA-binding ability

Insertion sequence IS1 encodes a transframe protein, InsA-B′-InsB, which is produced from two out-of-phase reading frames, insA and B′-insB, by translational frameshifting at a run of adenines. Unless the frameshifting event occurs, the InsA protein is produced from IS1. We found that cells harboring a plasmid carrying an IS1 mutant with a single adenine insertion in the run of adenines contained miniplasmids. Cloning and DNA sequencing analyses of the miniplasmids revealed that they had a deletion extending from an inverted repeat (IR) at the left end of IS1. This indicates that they were generated by IS1-mediated deletion due to efficient production of the InsA-B′-InsB transframe protein that is IS1 transposase. Both the InsA protein and transposase were partially purified as a fusion protein with collagen-LacZ by LacZ-specific affinity column chromatography. The InsA¹ and the collagenolyzed InsA¹ were found to bind specifically to a 24-bp region within each of the IRs at the ends of IS1. The transposase Tnp¹ and the collagenolyzed Tnp¹ were found to bind to the sequence with or without IR, but preferentially to that with IR. The nonspecific DNA-binding ability of transposase may be involved in recognition of the target DNA, an important process of transposition of IS1. Both InsA and transposase have the IR-specific DNA binding ability and a common polypeptide segment containing the α-helix-turn-α-helix motif, supporting the previous indication that InsA competes with transposase to bind to IRs and thus becomes a transposition inhibitor. Based on the observations described in this article, we speculate that transposase of IS1 consists of at least two domains, the N-terminal half, which almost entirely overlaps InsA, and the C-terminal half, which almost entirely overlaps B′-InsB. The frameshifting event adds the latter domain to the former to give the transposase activity recognizing IRs and the target sequence to initiate the transposition reaction.     

Generation of trangenic Xenopus laevis using the Sleeping Beauty transposon system

Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. Transposase-dependent reporter gene expression was observed in cell clones and in hemi-transgenic animals. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. The transgene is integrated into the genome and may be transmitted to the F1 offspring depending on the germline mosaicism. Although the transposase is necessary for efficient generation of transgenic Xenopus, the integration of the transgene occurred by an non-canonical transposition process. This was observed for two transgenic lines analysed. The transposon-based technique leads to a high transgenesis rate and is simple to handle. For these reasons, it could present an attractive alternative to the classical Restriction Enzyme Mediated Integration (REMI) procedure.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .