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Published online before print
March 12, 2003, 10.1101/gr.611403
METHODS Chromosomal Deletion Formation System Based on Tn5 Double Transposition: Use For Making Minimal Genomes and Essential Gene AnalysisDepartment of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA
In this communication, we describe the use of specialized transposons (Tn5 derivatives) to create deletions in the Escherichia coli K-12 chromosome. These transposons are essentially rearranged composite transposons that have been assembled to promote the use of the internal transposon ends, resulting in intramolecular transposition events. Two similar transposons were developed. The first deletion transposon was utilized to create a consecutive set of deletions in the E. coli chromosome. The deletion procedure has been repeated 20 serial times to reduce the genome an average of 200 kb (averaging 10 kb per deletion). The second deletion transposon contains a conditional origin of replication that allows deleted chromosomal DNA to be captured as a complementary plasmid. By plating cells on media that do not support plasmid replication, the deleted chromosomal material is lost and if it is essential, the cells do not survive. This methodology was used to analyze 15 chromosomal regions and more than 100 open reading frames (ORFs). This provides a robust technology for identifying essential and dispensable genes.
[Supplemental material is available
online at www.genome.org and is supplied as an extended table
enumerating genes lost in two multiple round deletion strains (
Transposons are powerful tools for performing genomic structure/function studies. They have long been used in the generation of knockout mutations. With the advent of in vitro transposition systems (Devine and Boeke 1994  ; Gwinn et al. 1997;
Akerley et al. 1998; Goryshin and Reznikoff 1998; Griffin IV et al.
1999; Haapa et al. 1999), the use of transposons in genome analysis has
been greatly expanded to include, for instance, their being applied as
mobile primer binding sites in high-throughput sequencing efforts
(Butterfield et al. 2002; Shevchenko et al. 2002). In this
communication, we will describe another powerful application of DNA
transposition that combines in vitro and in vivo Tn5-based
technologies to generate random deletions in the Escherichia
coli K12 genome. As will be described, the Tn5 deletion
formation system can be used in essentially any bacterial species to
define essential genes, to generate minimal essential genomes, and to
clone genes.
The entire DNA sequences of many bacterial genomes have been
determined. However, the experimentalist is still faced with the
challenge of determining the role of various putative genes.
Transposition-based strategies have been developed recently for
identifying essential genes (for review, see Judson and Mekalanos
2000a In addition to insertions, transposons are also capable of promoting other types of DNA rearrangements. Deletion (and inversion) formation is a natural feature of composite transposons. Composite transposons can create deletions or inversions by an intramolecular transposition mechanism (Fig. 1). Internal transposon ends are oriented in such a way that DNA between the ends can be considered as the donor DNA, which is released by transposase-catalyzed cleavage. The rest of the DNA (a plasmid or a chromosome) is recognized as a transposon that can undergo self-integration, leading to a deletion or inversion event. During this process, two deletions are formed, removal of the internal part of the transposon by double-ended cleavage at the transposon ends and deletion of a portion of the chromosome by the integration event.
The system that we will describe makes use of both types of deletions associated with intramolecular transposition (Fig. 1A). Double-ended cleavage eliminates the transposase gene and the selectable marker used for the prior transposon insertion selection. The self-integration reaction results in formation of the second deletion that starts at the point of transposon location and extends to the point on the chromosome or plasmid defined by the second transposition event. This deletion is the deletion of interest. An important feature of this scenario is that the protocol eliminates all selectable markers and the transposase gene and, thus, can be performed repetitively for an accumulation of deletions. In this communication, we will describe the use of the transposon-based deletion strategy as a tool for both defining essential genes and for removing nonessential genes from the chromosome.
Deletion Modules: Composition and Strategy The transposons we used exploit the observation that composite transposons make deletions by use of internal transposon ends. The structures of transposons used in this study are shown in Figure 2. The transposon Tn5Del7 is designed for immediate deletion of chromosomal DNA and does not contain an origin of replication. The Tn5Del8 transposon contains a conditional origin of replication that allows the capture of deleted chromosomal DNA in the cell as a complementary plasmid. Elimination of this plasmid is triggered by the removal of IPTG from the medium.
The particular features of the transposons used in these experiments are as follows. Both transposons are flanked by 19-bp inside ends (IEs) and can be separated from the donor DNA component of the vector by digestion with PshAI. The Tn5 IE ends are capable of participating in synaptic complex formation in the presence of mutant Tnp protein Tnp sC7v2.0 (Naumann and Reznikoff 2002 ). The synaptic complexes are delivered into cells by
electroporation (Goryshin et. al. 2000). The second set of 19-bp end
sequences in the deletion modules are artificially constructed mosaic
ends (MEs) (Zhou et. al. 1998). We use MEs in combination
with Tnp EK/LP for the second transposition step, as this was the most
efficient Tnp:DNA end combination available when these experiments were
performed. A high efficiency of in vivo transposition events is
absolutely critical for their identification by screening. The deletion formation protocol is as follows (also see Fig. 1). Cells are electroporated with preformed transposome complexes, and transposon inserts are selected for by plating on LB medium containing both kanamycin and chloramphenicol. Then, either a single colony or a collection of colonies is used to start a liquid culture. At early exponential phase, arabinose is added to induce Tnp EK/LP synthesis. In the case of Tn5Del8, IPTG and chloramphenicol are added to activate the transposon origin of replication and to select for the presence of the excised plasmid, respectively. After a few hours, cells are subcultured in the same medium with a 500-fold dilution and grown overnight with shaking. Cells are then diluted and plated on agar medium containing the same components to obtain single colonies for replica plating. In the case of Tn5Del7, colonies that are sensitive to both kanamycin and chloramphenicol are picked as deletions. In the case of Tn5Del8, cells are checked for sensitivity to kanamycin on plates that contain IPTG. Plasmid DNA is then isolated for further analysis.
Deletions in the Lactose Operon Region
Transposition events were stimulated by inducing Tnp EK/LP synthesis.
The resulting colonies were tested for transposition-associated events
by replica plating as described above and in the Methods section. As
was typical of experiments using Tn5Del7, transposition events
had occurred in A total of nine independent deletions were selected and analyzed by DNA sequencing either directly from chromosomal DNA or by the use of inverse PCR. Two deletions ended within the Cam gene. The other seven deletions are described in Figure 3. The deletion sizes varied from 4–23 kb, with most deletion sizes being around 20 kb. This large deletion size makes it feasible to create a minimal genome in a reasonable amount of time by using our method recursively.
Recursive Deletion Formation An important feature of this transposition/deletion system is that after the deletion is generated, all components of the transposon are lost except for a short linker (64 bp). The loss of the transposase gene ensures the stability of the chromosome (in terms of transposition). The loss of all selectable markers provides an opportunity for recursive deletion formation using transposon insertion and transposase-mediated deletion formation, repetitively. We performed 20 rounds of recursive deletion formation in strain MG1655. Rather than focusing on one deletion at a time, we isolated and mixed at least 10 colonies following each round. This was then used as the starting material for the subsequent rounds. It was assumed that strains with debilitating deletions would be lost because other deletion strains with unimpaired growth would out compete them during growth of the mixed culture. We obtained 10 final strains after 20 rounds that had a growth rate equal to that of the parental strain (data not shown). Pulsed-field gel electrophoresis analysis of NotI-digested chromosomal DNA was used to analyze the diversity among the 10 deletion strains and to estimate the average deletion size that occurred. An example of such a gel is shown in Figure 4. The differences and similarities between samples are evident by inspection of the gel. In some cases, bands disappear from their original position (in the MG1655 DNA digestion) and are substituted by shorter fragments. In other cases, longer fragments appear, presumably due to the loss of NotI site(s), which results in the formation of a large band coupled with the loss of two or more smaller bands. We calculated the total amount of DNA deleted for 4 strains that had undergone 20 rounds of deletion by estimating the size of each chromosomal band compared with marker DNAs. From several gels that were run under various conditions to resolve different regions in the DNA pattern, we calculated that the four strains whose DNAs were analyzed in Figure 4 contain deletions of 250, 262, 100, and 247 kb. This indicates that the average deletion size per round is 11 kb, which is in reasonable agreement with the results obtained for the lactose operon region in the previous experiment.
Microarray Mapping of Deletions The locations of deletions found in strains  20-1 (as judged by
the pulsed-field gel electrophoresis to be missing  250 kb) and
20-4 (missing 262 kb) were mapped by microarray hybridization
experiments versus the progenitor strain. This technique provided an
excellent means to determine which genes were removed by the
transposition/deletion procedure. Figure
5 presents the results schematically and
Table 1 describes the results in more detail. The two
strains are apparently each lacking four common sets of genes. 20-1
has seven unique deletions and 20-4 has five unique deletions. The
data suggests that the two strains diverged after the fourth common
deletion was generated. The two strains contain a wide range of
deletion sizes. The deletion sizes presumably reflect the locations of
the essential genes nearest the different transposon inserts and/or
differences in localized DNA condensation near various inserts (see
below). The mapped deletions are consistent with both the pulsed-field
gel electrophoresis pattern and the total amount of DNA missing, as
determined by the pulsed-field gel electrophoresis patterns.
There are a few surprises. First, we found positive hybridization results interrupting what would otherwise appear to be a contiguous set of deleted genes. This observation is likely due to a phenomenon observed by Richmond et al. (1999) that cross hybridization occurs
between IS or paralog-containing sequences. The majority of these
positive hybridization signals do represent IS sequences found
elsewhere in the genome or genes that have known paralogs in the
genome. Another surprising result is that there are fewer apparent
deletions in each of the strains than might be expected from 20 cycles
(9 and 11 deletions were found in the two strains). It is possible that
some deletions may have ended within the transposon as found in our
lac insert-deletion analysis and, thus, they would not be
detected. Alternatively, some deletions may be too small to have been
picked up by the microarray hybridization technique. In some cases,
what looks like one deletion by the array data may really represent two
or more adjacent deletions. Finally, both of the strains each have one
extremely large deletion (125,000 and 146,000-bp long) in
NotI fragment G, near the chromosome replication terminus.
These two deletions have one end in common (near the NotI I/G
fragment boundary), which suggests that they arose from the same
insertion event, but had separate deletion events. Thus, it is likely
that the two strains diverged with the formation of these deletions.
The observation that these deletions were both relatively large
indicates that the transposition/deletion system is capable of
generating extremely large deletions, that there are no essential genes
in this region, and that the chromosome condensation of this region of
the chromosome may favor large deletion formation.
Coupled Chromosomal Deletion/Plasmid Formation System The protocol used for the Tn5Del8 experiments is described above, in the Methods section, and in Figure 1B. The critical feature is that IPTG is present during all steps, commencing with the induction of transposase in order to support replication of plasmids formed during deletion formation. In addition to supporting replication of the newly formed plasmids, addition of IPTG activates origins of replication that remain on the chromosome in cells that have not undergone deletion formation. This activation presumably depresses cell growth and creates selection against cells that had not undergone excision of the transposon-encoded origin of replication, as in these experiments, >95% of the resulting colonies contained the desired class of deletions. We used a coupled deletion/plasmid system for the search of essential genes. A total of 15 insertion events were treated independently as above to isolate deletion events. Final induced cultures for each were diluted and plated on agar medium containing chloramphenicol and IPTG. For each insert, a total of 20 colonies were analyzed by isolation of plasmid DNA, followed by an estimation of plasmid size by agarose gel electrophoresis. In each case, the largest plasmid isolated was analyzed by DNA sequencing of the two transposon/chromosomal DNA junctions to determine the portion of the chromosome that was deleted. Cells from the colony were then streaked onto plates containing only LB (no IPTG) to determine whether cells could survive without the deleted chromosomal material. The colonies were also streaked on LB plates with chloramphenicol and no IPTG to confirm loss of plasmid without integration into the chromosome. Growth of cells on this medium was not detected in any case, indicating that reintegration of the plasmid did not occur. Results of this analysis are shown in Table 2. Of 15 insertion strains, 11 yielded viable cells following plasmid loss from the largest isolated deletion. This indicated that these regions of DNA are dispensable. In the other four cases, cells were unable to grow without the deleted plasmid DNA, indicating the presence of at least one essential gene in the deleted material. An example of this is deletion 4.9. In this case, a smaller deletion, 4.6, was analyzed and found to survive in the absence of the plasmid (Fig. 6). In this case, the result indicates that genes glyS and/or glyQ are essential.
In the case of insert 8, only a short chromosomal DNA deletion was isolated. This transposon insert resides between open reading frames (ORFs), and the deletion does not contain any predicted ORFs. The inability to isolate larger deletions from this insert could be due to the fact that the area contains a large region of essential DNA that cannot be bypassed by typical deletion lengths. Alternatively, the localized condensation of the DNA may not be amenable to the formation of large deletions. A final possibility is that the capture of DNA immediately next to the insert, when transferred to the moderate copy number plasmid, may be lethal to the cell. We cannot distinguish between these options, but this area contains a stretch of essential genes, including gyrB and dnaA.
The data shown in Table 2 were compared with known information about
the essentiality of E. coli ORFs by using the PEC database
(www.shigen.nig.ac.jp/ecoli/pec/). Of the four deletions that we
determined to contain essential DNA, all but one contained an ORF(s)
that had been reported previously to be essential. In the remaining
case, the deletion occurs in a single ORF, dnaK. Mutations in
dnaK give rise to a conditionally essential phenotype at
37°C (Bukau and Walker 1989
In this communication, we describe a simple and reliable system for making chromosomal deletions in E. coli K-12. This system should also be of use in any other bacterial species for which the Tn5 transposome/electroporation strategy can be utilized. We used transposition driven by Tn5 transposase derivatives in such a way that we can separate two transposition events, each using different Tnp:transposon end combinations (Naumann and Reznikoff 2002 ).
The first transposition event delivers components for the second
transposition event into the chromosome via transposase–DNA complex
codelivery (Goryshin et al. 2000). The second transposition event
generates a deletion or inversion event. By simple screening for loss
of antibiotic resistance, deletions with only a small portion of the
transposon left in the chromosome can be chosen. As a variation of this
technique, deleted material can be saved in the same cell as a
conditional plasmid.
Determining the minimal genome content is a topic of interest for many
laboratories. One approach used for determining the minimal genome is
to assemble a theoretical minimal genome in silico by the comparison of
a variety of different microbial genomes. This method assumes that all
essential DNA exists in homologous form in all genomes, and that all
nonessential DNA will be absent in one or more cases. Alternatively,
the smallest genome among existing genomes (mycoplasma) has been
analyzed by transposon knockout mutagenesis and large-scale sequencing
(Hutchison III et al. 1999 E. coli K-12 is an ideal subject for the minimal genome construction. It has a short generation time, extensive genetic tools, and a wealth of knowledge of its genome and physiology. It therefore seems attractive to try to generate a minimal or significantly reduced E. coli K-12 genome. In addition to the knowledge gained by the creation of such a strain, the strain itself could be useful. Two examples of the usefulness of such a strain are (1) overproduction of recombinant proteins with fewer E. coli proteins that need to be removed, and (2) as a simplified metabolic model that could be used for mathematical modeling of whole cell metabolism. We are currently going beyond the 20 rounds presented here in an effort to create such a strain. The average size of deletions observed in our experiments for the lac operon area and random deletions gives us hope that we will be able to reduce the size of the E. coli chromosome significantly in a reasonable time frame.
The large average deletion size is striking, because if the genomic DNA
were a random coil, the intramolecular transposition site selection
would be strongly biased toward distances within a few hundred base
pairs of the transposon end sequences. We observed this bias in vitro,
in which case, the DNA was free from packing (York at al. 1998
Recently, two strategies for repetitive reduction of the E.
coli chromosome have been described (Kolisnychenko et al. 2002 In this communication, we started a list of essential/nonessential genes for E. coli by using the Tn5Del8 system. In the future, it is technically possible to create a representative library of deletions with complementary plasmids that would cover the entire E. coli genome multiple times. Systematic sequencing of deletion borders coupled with survival tests could be used to determine the essentiality of all of the genes in the entire chromosome. This type of analysis could also be adapted, through the use of an appropriate origin of replication, for analyzing the chromosomes of other bacteria.
Medium For all experiments, we used Luria-Bertanii (LB) broth liquid or agar medium or Lactose MacConkey agar medium (Sambrook et. al. 1989 )
modified to contain the following antibiotics when indicated:
chloramphenicol (20 mg/L), kanamycin (40 mg/L), ampicillin (100mg/L).
Bacterial Strains
Plasmids
Plasmid pGT8 was the source of transposon Tn5Del8 (Fig. 2).
The two KpnI–BssSI fragments of pGT7 were ligated
with an AatII–BamHI fragment of pAM34 (ATCC 77185,
Hare et al. 2001
Transposase Purification
Transposome Complex Preparation Transposome complexes were assembled by incubation of precut transposon with TnpsC7v2.0 in 20 mM Tris Acetate (pH 7.5), 100 mM K Glutamate for 1 h at 37°C. The DNA:protein molar ratio was 1:5, with a DNA concentration of 0.1µg/µl.
Selection for Initial Transposition Events After electroporation, cells were recovered by incubation for 1 h in LB broth, and then plated on LB agar containing kanamycin and chloramphenicol.
Induction of Deletions (Secondary Transposition Events) Screening for deletions with Tn5Del7 was done by replica plating colonies from LB agar onto agar containing LB, LB-kanamycin, and LB-chloramphenicol. Cells sensitive to kanamycin were considered to have undergone transposition, and cells sensitive to both drugs were considered to have a deletion of adjacent DNA and the correct portion of the transposon with only a small transposon linker being left on the chromosome. In the case of Tn5Del8, the cells of interest were kanamycin sensitive and chloramphenicol resistant, as the replication of excised DNA circles was supported by the presence of 1 mM IPTG in the medium.
Microarray Analysis
DNA Sequence Analysis
Pulsed-Field Gel Electrophoresis
http://www.shigen.nig.ac.jp/ecoli/pec/; Genetic Resource Committee of Japan. http://www.gcow.wisc.edu/Gec/index.htm; Gene Expression Center, University of Wisconsin-Madison.
We thank Barb Schriver for providing all of the medium and reagent preparations for this work. We thank Laura Vanderploeg and the Department of Biochemistry Media Lab for their valuable assistance with the figures and tables. We also thank Kelly Winterberg for her helpful discussion of the manuscript and the other members of our research laboratory for their support. This research was supported in part by NSF grant MCB-0084089, NIH grant GM50692, and a grant from the Robert Draper Technology Fund, Wisconsin Alumni Research Foundation. The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
1 These authors contributed equally to this work.  E-MAIL Reznikoff{at}biochem.wisc.edu; FAX (608) 265-2603. Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.611403. Article published online before print in March 2003.
Received July 10, 2002; accepted in revised format December 23, 2002. 13:644-653 © by 2003 Cold Spring Harbor Laboratory Press ISSN 1088-9051/03 $5.00  CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
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ABSTRACT
RESULTS
red recombination system.
This approach is different from ours in several fundamental ways.
First, the approach described by Kolisnychenko et al. (2002)
(Sambrook et al. 1989
