Transposons are mobile DNA elements that mediate genome rearrangements by inserting themselves within and between genomes. These elements are the main cause of numerous biological phenomena, including the emergence of insertion mutations that often lead to inactivation of gene function, the spread of antibiotic resistance genes among bacterial populations, and the integration of retroviruses into their host genome (5, 11, 33). Recent findings also suggest that the immunoglobulin gene rearrangement machinery may be derived from an ancestral transposon system (1, 22).
Transposons are indispensable tools in modern genetics. The original in vivo transposition-based strategies (for reviews, see references 4 and 25) paved the way for further in vitro transposition-based developments that utilize the mechanisms of a variety of transposable elements, including Tn10, Ty1, Tn5, Tn552, Mu, Tn7, and mariner (6, 9, 13, 14, 17, 19, 41). Typical transposon applications include gene mapping and DNA sequencing strategies as well as insertion mutagenesis and functional genetic analysis methods (for recent microbial examples, see references 2, 10, 18, 20, 34, 36, and 38). DNA transposition-based strategies have also been employed successfully in functional analyses of proteins (21) and studies on protein-DNA complexes (43).
Bacteriophage Mu replicates its genome using DNA transposition machinery and is one of the best-characterized mobile genetic elements (8, 29). Its transposition reaction consists of DNA cleavage and joining steps and occurs within higher-order protein-DNA complexes called transpososomes (12, 39). While Mu transposition, both in vivo and with plasmid substrates in vitro, is complex and requires a variety of protein and DNA cofactors (8, 29), a substantially simplified version of the reaction can be reproduced in vitro by altering reaction conditions and critical DNA substrates (12, 19, 32).
In the simplest case, Mu transposition complexes can be assembled in vitro using MuA transposase protein and a transposon right-end (R-end) segment that includes two MuA binding sites. These complexes contain four molecules of MuA that synapse two molecules of the end segment (32). Analogously, when two R-end sequences are located as terminal inverted repeats in a longer DNA molecule, transposition complexes form by synapsing the transposon ends (12, 19) (Fig. 1A). The complexes remain inactive in the absence of metal ions but are activated for transposition chemistry upon addition of Mg2+. The majority of complexes subsequently execute two-ended transposon integration involving both transposon ends and generate a transposition DNA intermediate containing 5-nucleotide single-stranded regions flanking the transposon DNA. Within compatible cells (e.g., Escherichia coli), the intermediate can be repaired by host machinery, resulting in a 5-bp target site duplication that is a hallmark of Mu transposition (3, 12, 19, 24, 29).
To date, Mu in vitro transposition-based strategies have been utilized for a variety of molecular biology applications, including DNA sequencing (20), functional analysis of plasmid DNA and virus genome regions (19, 27), analysis of proteins by pentapeptide scanning mutagenesis (40), and generation of DNA constructs for gene targeting (42). To extend the scope of Mu technology, we examined whether preformed Mu transposition complexes can be utilized as delivery vehicles for gene integration into bacterial genomes. Here, we report the assembly of integration-proficient Mu transposition complexes that, after introduction into bacterial cells by electroporation, execute transposon integration into bacterial chromosomes with high efficiency and fidelity, generating an accurate 5-bp target site duplication. This strategy may be applicable to a variety of organisms in which efficient genetic manipulation systems are lacking or difficult to exploit.

RESULTS

In the absence of divalent metal ions, Mu transpososomes that assemble with precut transposon ends are stable but catalytically inert. The complexes withstand not only conventional (30) but also relatively harsh electrophoresis conditions and remain stable even when challenged with high concentrations of heparin embedded in an agarose gel (31, 32; this study). Therefore, it is conceivable that after electroporation into bacterial cells, these complexes remain functional and become activated for transposition chemistry upon encountering Mg2+ ions within the cells, potentially facilitating transposon integration into host chromosomal DNA. This hypothesis was tested experimentally.
The assembly of Mu transpososomes was initially examined by incubating the MuA protein with the Cat-Mu transposon and analyzing the formation of stable protein-DNA complexes by agarose gel electrophoresis (Fig. 2). The reaction with Cat-Mu transposon generated several bands of protein-DNA complexes (lane 5) that disappeared when the same sample was loaded in the presence of SDS (lane 6), thus indicating that covalent protein-DNA interactions are not involved in complex formation. The migration pattern of these complexes is consistent with Mu transpososomes connecting the ends of a single or several transposon molecules (see the legend to Fig. 2). A control DNA fragment lacking Mu transposon end sequences failed to produce detectable complexes (Fig. 2, lane 2). Next, an aliquot of each assembly reaction mixture was electroporated into E. coli cells, and bacterial clones were selected for chloramphenicol resistance. Only the sample containing detectable protein-DNA complexes yielded chloramphenicol-resistant (Cmr) colonies (with a frequency of 105 CFU/μg of input transposon DNA). A similar frequency was obtained with the Kan-Mu transposon after selection for kanamycin resistance (Table 1).

TABLE 1.

TABLE 1. Number of antibiotic resistant colonies detected following electroporation into E. coli MC1061a
    No. of antibiotic-resistant colonies (CFU/μg of DNA) after:
Substrate DNAb Selected resistance No preincubation with MuA Preincubation with MuA
Cat Chloramphenicol 0 0
Cat-Mu Chloramphenicol 0 1 × 105
Kan-Mu Kanamycin 0 6 × 104

a

Twelve nanograms of DNA (1 μl) was used for electroporation. The competence status of these cells was 5 × 108 CFU/μg of pBR322-Cm DNA.

b

Substrate DNA molecules are depicted diagrammatically in Fig. 1B.
Accumulation of the complexes and their relationship to the capacity for colony formation was then examined in a time course experiment (Fig. 3). Formation of the complexes correlated with the appearance of resistant colonies. Collectively, these results suggest that the detected complexes (or a fraction of them) are responsible for the appearance of resistant colonies. Transposon integration by transpososomes in vivo is consistent with these findings.

FIG. 3.

FIG. 3. Time course of complex and colony formation. (A) A 2% agarose gel. Analysis was performed with Cat-Mu substrate as for Fig. 2 with no SDS in the sample loading step. Lanes 1 to 8, reaction time course; lane C, Cat-Mu transposon DNA as a control. (B) Quantitation of complex formation. The C1 complexes formed were quantified (see Materials and Methods) from the ethidium bromide-stained gel shown in panel A. ODU, arbitrary optical density units. (C) Formation of chloramphenicol-resistant colonies after electroporation into E. coli strain MC1061.
Other laboratory strains of E. coli were tested for comparison with the above-described results (Table 2). The competence status of each strain was evaluated in parallel by electroporation of a control plasmid. The most efficient strain, MC1061, produced approximately 106 antibiotic-resistant colonies/μg of input transposon DNA (12 ng of DNA electroporated), while the least efficient strain, HB101, displayed an efficiency of less than 105 CFU/μg of input transposon DNA. No colonies were detected in any of the corresponding control reaction mixtures for the E. coli strains (no added MuA protein, 12 ng of transposon DNA electroporated). However, control experiments in which greater amounts of transposon DNA (500 ng) were electroporated into these E. coli strains yielded detectable colonies, although with a low frequency (≈101 CFU/μg of DNA). These colonies most likely represent spontaneous resistance-generating mutations, or possibly recombination events involving a mechanism(s) other than DNA transposition, and were not studied further.

TABLE 2.

TABLE 2. Number of chloramphenicol-resistant colonies detected following electroporation into different bacterial strains
      No. of chloramphenicol-resistant colonies (CFU/μg of DNA)
      E. coli K-12        
Reaction DNA electroporated (amt, ng)a Preincubation with MuA MC10161 DH10B HB101 E. coli (BL21) S. enterica serovar Typhimurium L2 SL5676 E. carotovora LMG2404 Y. enterocolitica serotype O:3 (6471/76-C)
Standard Cat-Mu (12) Yes 9 × 105 2 × 105 6 × 104 3 × 104 3 × 104 1 × 105 2 × 104
  Cat-Mu (12) No c
Control Cat-Mu (500) No 3 × 101 2 × 101 3 × 101 2 × 101
  pBR322-Cmb (1) No 2 × 109 3 × 108 3 × 108 4 × 108 NDd ND ND

a

One microliter of DNA solution was electroporated. For standard reactions, the standard assembly reaction solution was diluted 1:4 with water. For control reactions, the DNA was in solution in water.

b

Electroporation of plasmid pBR322-Cm DNA served as a control for competence status.

c

—, no colonies detected.

As might be expected, the capacity for colony formation correlated with the competence status of each strain. Furthermore, different batches of electrocompetent cells of a given strain exhibited variable capacities for colony formation that correlated with their competence status (data not shown). The assembled transpososomes were stable for several months when stored at −80°C, since no reduction in colony formation capacity was detected after prolonged storage (data not shown).

We next examined whether transposon DNA was inserted into the chromosomal DNA of Cmr clones and, moreover, the number of transposon copies present. Chromosomal DNAs from 17 potential transposon integration clones were isolated, digested with PstI (which does not cut the transposon sequence), and analyzed by Southern hybridization with a Cat-Mu transposon probe. Each isolate generated a single band with a discrete gel mobility (Fig. 4A). A parallel analysis was performed using NcoI, which cleaves the transposon sequence once. Two bands of different gel mobility were generated for each isolate (Fig. 4B). Control chromosomal DNA from the strain that we initially used for electroporation did not generate bands in the analyses. These data indicate that a single copy of transposon DNA was integrated into the chromosome of each isolate.

FIG. 4.

FIG. 4. Southern blot analysis of insertions into the bacterial chromosome. Genomic DNAs of 17 chloramphenicol-resistant E. coli DH10B clones were digested with PstI (A) or NcoI (B) and probed with Cat-Mu transposon DNA. Lanes: 1 to 17, transposon insertion mutants; M, molecular size marker; C, genomic DNA of original E. coli DH10B recipient strain as a negative control; P, linearized plasmid DNA containing Cat-Mu transposon sequence as a positive control.
Genuine Mu transposition produces a 5-bp target site duplication flanking the integrated transposon (3, 19, 24). To investigate whether the integration events detected were indeed caused by transposition, we determined the DNA sequences (see Materials and Methods) on both sides of the transposon in nine randomly selected E. coli clones. In each case, the integrated transposon was flanked by a target site duplication, thus confirming that integrations were generated by DNA transposition chemistry (Table 3).

TABLE 3.

TABLE 3. Transposon integration sites and target site duplications
        Genetic locationc
Strain Clone Sequencea Transposon orientationb Gene Coordinates Section
E. coli K-12 C1 tttcaatataTTGCT(Cat-Mu)TTGCTggagtttgag + mrdA 5173-5177 58
DH10B C2 ggcgacacctACGGA(Cat-Mu)ACGGAcgcgttttta + yhdX 10168-10172 295
  C4 tgacgatgccGTTGC(Cat-Mu)GTTGCggtagcaccg + hrpB 2039-2043 14
  C5 ccaggtcataTTCAG(Cat-Mu)TTCAGgccatcatct bglX 6810-6814 192
  C6 gtcgctaatgCCGGA(Cat-Mu)CCGGAgacaatacca ycjJ 10126-10130 117
  C7 tcactccagcGCAGC(Cat-Mu)GCAGCaccatcaccg lacZ 8143-8147 31
  C9 caaagtatgcCCGTC(Cat-Mu)CCGTCtggccagtgc fes 10020-10024 53
  K2 tgtttaattgCCGGA(Kan-Mu)CCGGAtgtcagacat + yhjG 1748-1752 319
  K3 aggtgataccCTGGC(Kan-Mu)CTGGCggcctgcctg yrfI 7397-7401 305
S. enterica S5 tcaacatcaaGCGGC(Cat-Mu)GCGGCaggaaagagg + Unknown    
serovar S6 cgaca-caacAGCAA(Cat-Mu)AGCAAcctggtacag + Unknown    
Typhimurium S7 gctc-t-acgCAGAC(Cat-Mu)CAGACgatgtaacgt + Unknown    
LT2 SL5676 S8 gtattgcagcGCAGG(Cat-Mu)GCAGGcgctggtgaa + Unknown    
E. carotovora E1 gagctccggcGTAGG(Cat-Mu)GTAGGcgagtccacc + Unknown    
type strain E2 ctgcgtgctgCTGCC(Cat-Mu)CTGCCgattctgttt + Unknown    
LMG2402 E3 atcgatcacgCTATC(Cat-Mu)CTATCgataaagcta + Unknown    
  E4 attcgt-tctGTCTG(Cat-Mu)GTCTGggttcaccaa + Unknown    
Y. enterocolitica Y9 cggca-tattTTGGG(Cat-Mu)TTGGGggctaaattt + Unknown    
serotype Y10 aaatgagtatCCGGC(Cat-Mu)CCGGCatctgaatat + Unknown    
O:3 (6471/76-c) Y11 acggatactgGCAGG(Cat-Mu)GCAGGtaaagaaatc + Unknown    
  Y12 gcg-aattatGCCGC(Cat-Mu)GCCGCtgcatcagtg + Unknown    

a

Nucleotides that were difficult to interpret in the sequencing analysis are indicated by hyphens. Target site duplications are in boldface capital letters.

b

Compared to the genomic sequences shown, the transcription from the transposon proceeds from left to right (+) or from right to left (−).

c

Genetic locations of E. coli insertions were determined by comparison to the K-12 strain MG1655 complete genome (GenBank accession numbers AE000111to AE000510).
The protocol was extended to other bacterial species (Table 2). The three additional species tested, S. enterica serovar Typhimurium, E. carotovora, and Y. enterocolitica, produced Cmr colonies at efficiencies comparable to those obtained with the E. coli strains. Control reactions lacking MuA produced no colonies. Similar to the E. coli studies, each clone that was analyzed from these bacterial species contained a target site duplication flanking the transposon DNA (Table 3).

DISCUSSION

We developed a gene delivery methodology for bacterial cells that results in the integration of artificial transposons into the bacterial chromosome. The system is based on the in vitro assembly of the bacteriophage Mu DNA transposition complexes and their subsequent electroporation into bacterial cells (Fig. 1A). The strategy utilizes mini-Mu transposons that contain a pair of MuA binding sites as inverted terminal repeats at each end (Fig. 1B). Upon MuA binding, these sites promote the assembly of a tetrameric Mu transposition complex that functions as a molecular machine to splice the transposon into target DNA in a divalent metal ion (e.g., Mg2+)-dependent manner (12, 19, 32).
The precut mini-Mu transposons used in this study have exposed 3" ends and contain several extra 5"-flanking nucleotides at each end. These features facilitate the formation of stable complexes that become activated for strand transfer after encountering Mg2+ ions (31, 32). Our data indicate that this activation also takes place within bacterial cells after electroporation. We obtained up to ≈106 integrants/μg of input transposon DNA with standard E. coli strains used for high-efficiency plasmid transformation. In general, the efficiency of integration per microgram of input transposon DNA was about three orders of magnitude lower than the plasmid electroporation efficiency. The competence status of electrocompetent cells was a major variable affecting integration frequency, and consistent differences were observed both between bacterial strains and between different batches of a particular strain.

Importantly, mini-Mu transposon insertions are stable in cells that do not express MuA. However, at least in E. coli cells, it is likely that the inserted transposons can be further mobilized by expressing MuA (e.g., using plasmid expression systems).

We were able to detect only a limited number of antibiotic-resistant colonies when the transposon DNA was electroporated alone (with no added MuA) into the E. coli strains used in this study. Furthermore, similar control experiments for the other bacterial species studied produced no resistant colonies (Table 2). These results were not unexpected and are consistent with the general assumption that the bacterial species tested do not contain an efficient machinery for the recombination of incoming nonhomologous DNA.
Transposons represent the mutagenesis system of choice for genetic studies that require gene inactivation. They provide primer binding sites that can be used to retrieve DNA sequence information flanking the transposon insertion site, thereby allowing direct access to a particular gene of interest. Thus, the approach described here for the efficient mutagenesis of various bacterial genomes should facilitate molecular analyses of diverse biological processes. While it is clear that different mutants may be analyzed separately, an opportunity for the development of remarkably efficient strategies lies in the analysis of pools of insertion mutants. For example, recently developed bacterial genomic footprinting techniques (2, 44) may be directly applicable to Mu-generated mutant banks.
We have established that DNA transposition complexes assembled with artificial mini-Mu transposons can be introduced into bacterial cells, whereby transposons become integrated into the genome. Since the molecular machinery functioned well also when complexes were introduced into cells other than those of E. coli (the natural host of phage Mu), we conclude that Mu integration in these cases occurred without the aid of E. coli host proteins. This is in contrast to standard Mu DNA transposition in vivo, which involves a number of protein and DNA cofactors (8, 29). However, our results are not entirely unexpected given that the cofactors involved in standard Mu DNA transposition are required specifically for steps leading to transposition complex formation and are not essential thereafter. We have yet to define the upper limit for the length of mini-Mu transposons that can be utilized for efficient chromosomal integration. To date, the longest transposons utilized by our group in standard in vitro transposition experiments (into plasmid targets) are 6.8 kb in length. These transposons function almost as efficiently as 1- to 2-kb transposons, and thus it is probable that they function in chromosomal integration as well.

The present study describes mini-Mu transposon integration events in gram-negative bacteria. However, since the transposition machinery is fully functional on encounter with DNA and Mg2+ ions, in principle the strategy could also be applicable to gram-positive bacteria and perhaps to some eukaryotic organisms (such as yeast) as well. While host-encoded restriction systems may create an impediment to efficient functioning of the system in some organisms, this problem may be circumvented by custom designing transposons that do not contain critical recognition sites or by using mini-Mu transposons isolated from a compatible bacterial strain. Host proteases in some organisms may cause additional problems by destroying incoming transpososomes prior to chromosomal integration. However, given the success of our approach in different bacteria, it may be that transpososome function is activated immediately upon binding of divalent metal ions within the cell. Thus, the subsequent series of reactions may actually occur very rapidly, thereby preventing proteases from locating and destroying transpososomes inside cells prior to integration.

Thus far, a strategy similar to that presented here for Mu has been described only for Tn 5-derived minitransposons (15, 23). Nevertheless, since a common DNA transposition mechanism is shared among a variety of mobile elements (11), it is likely that other in vitro DNA transposition systems, such as Tn10, Ty1, Tn552, Tn7, and mariner (6, 9, 13, 17, 41), may also be utilized analogously.



Source link