- Methodology article
- Open Access
Generation of a restriction minus enteropathogenic Escherichia coli E2348/69 strain that is efficiently transformed with large, low copy plasmids
© Hobson et al; licensee BioMed Central Ltd. 2008
- Received: 05 March 2008
- Accepted: 05 August 2008
- Published: 05 August 2008
Many microbes possess restriction-modification systems that protect them from parasitic DNA molecules. Unfortunately, the presence of a restriction-modification system in a given microbe also hampers genetic analysis. Although plasmids can be successfully conjugated into the enteropathogenic Escherichia coli strain E2348/69 and optimized protocols for competent cell preparation have been developed, we found that a large, low copy (~15) bioluminescent reporter plasmid, pJW15, that we modified for use in EPEC, was exceedingly difficult to transform into E2348/69. We reasoned that a restriction-modification system could be responsible for the low transformation efficiency of E2348/69 and sought to identify and inactivate the responsible gene(s), with the goal of creating an easily transformable strain of EPEC that could complement existing protocols for genetic manipulation of this important pathogen.
Using bioinformatics, we identified genes in the unfinished enteropathogenic Escherichia coli (EPEC) strain E2348/69 genome whose predicted products bear homology to the HsdM methyltransferases, HsdS specificity subunits, and HsdR restriction endonucleases of type I restriction-modification systems. We constructed a strain carrying a deletion of the conserved enzymatic domain of the EPEC HsdR homologue, NH4, and showed that its transformation efficiency was up to four orders of magnitude higher than that of the parent strain. Further, the modification capacity of NH4 remained intact, since plasmids that were normally recalcitrant to transformation into E2348/69 could be transformed upon passage through NH4. NH4 was unaffected in virulence factor production, since bundle forming pilus (BFP) subunits and type III secreted (T3S) proteins were present at equivalent levels to those seen in E2348/69. Further, NH4 was indistinguishable from E2348/69 in tissue culture infection model assays of localized adherence and T3S.
We have shown that EPEC strain E2348/69 utilizes a type I restriction-modification system to limit entry of new DNA. This restriction-modification system does not appear to be involved in virulence determinant expression or infection phenotypes. The hsdR mutant strain should prove useful in genetic analysis of the important diarrheal pathogen EPEC.
- Transformation Efficiency
- Generalize Transduction
- Strain MC4100
- Localize Adherence
- EPEC Strain
Restriction-modification systems are wide-spread in eubacteria and archaea and are thought to protect the host from bacteriophages, facilitate the gain of new genetic information, and allow for the maintenance of selfish genetic elements [1, 2]. Type I restriction-modification systems were the first to be described and they are hetero-oligomeric enzymes consisting of a methyltransferase (HsdM), a specificity subunit (HsdS), and a restriction endonuclease (HsdR). The HsdR restriction endonuclease cleaves foreign DNA that has not been modified by the HsdM methyltransferase at a specific sequence recognized by the HsdS specificity subunit [1, 2]. While this is an effective mechanism for protecting a microbe from newly encountered bacteriophages, it severely limits genetic analysis in many organisms, since new DNA is difficult to introduce. Indeed, most commonly used non-pathogenic commercial and laboratory strains contain deletions of hsdR homologues or entire type I restriction systems. We suspected the EPEC type strain E2348/69 might possess a restriction-modification system, since we had great difficulty in obtaining transformants that carried a large, low copy (~15 copies/cell) bioluminescent reporter plasmid, pJW15, that we modified for use in EPEC  and also since this strain cannot be infected with the E. coli generalized transducing phage P1.
EPEC is a leading cause of infantile diarrhea in the developing world . Infection is thought to progress in three steps . Initially, a type IV bundle forming pilus (BFP) mediates adherence to intestinal epithelial cells [6, 7]. Following adhesion, a type III secretion system (T3SS) facilitates the transfer of translocator and effector proteins from the bacterial cytoplasm directly into the eukaryotic cytosol. One of these effectors, Tir, functions as a receptor in the eukaryotic cell membrane for the EPEC outer membrane protein intimin, fostering tight adherence between the microbe and the eukaryotic host cell . In addition Tir, and other effectors, disrupt eukaryotic cellular processes, leading to microvillus effacement, tight junction disruptions, and changes in signal transduction that ultimately cause diarrhea . Despite the health threat that EPEC poses, it remains relatively uncharacterized compared to its E. coli K-12 counterpart. One reason for this is likely due to the inability to efficiently introduce DNA through genetic techniques such as generalized transduction and transformation. Although a number of genetic techniques have been developed for use in EPEC based on conjugation [10, 11] and optimized competent cell preparation , we wished to determine if a restriction-modification system might be responsible for the genetic intractability of EPEC strain E2348/69. If so, we reasoned that inactivation of such a restriction-modification system would render an additional useful tool for the EPEC research community.
Identification of an hsdRhomologue in the E2348/69 unfinished genome
An E2348/69 hsdRmutant exhibits elevated transformation efficiency and maintains HsdM activity
Transformation efficiencies of E2348/69 vs. E2348/69 ΔhsdR
2.5 × 10-11
8.3 × 10-5
5.4 × 10-5
cosmid cloning vector
3.0 × 10-9
9.0 × 10-7
These data suggest that the introduced ΔhsdR allele does indeed make NH4 more competent for transformation with large, low copy number plasmids and argue that the HsdR endonuclease actively restricts incoming DNA in E2348/69. Further, this set of experiments shows that the E. coli K-12 strain MC4100 does not possess the E2348/69 HsdMSR restriction-modification system. Indeed, when the E2348/69 HsdR sequence was used in a BLAST search of the published E. coli K-12 genome http://genolist.pasteur.fr/Colibri, we detected only two proteins. A putative HsdR homologue shared only 23% identity over 181/1038 amino acids and the YejH protein of unknown function was 25% identical over 176/1038 amino acids. Both comparisons contained multiple, large gaps. Thus, E. coli K-12 does not contain the E2348/69 type I restriction-modification system identified here. As expected, this renders DNA isolated from E. coli K-12 (eg. MC4100) a poor substrate for transformation into EPEC (Figure 3).
Although type I restriction-modification systems consist of a hetero-oligomeric HsdMSR complex, it has been shown that a sub-complex consisting of HsdM and HsdS alone is competent for DNA modification . Since the hsdR homologue is found downstream of the putative hsdM and hsdS genes in E2348/69 (Figure 1), we predicted that the ΔhsdR allele in NH4 would not disrupt the modification activities of the remaining HsdMS complex. We tested this hypothesis by determining the transformation efficiencies for E2348/69 and NH4, as described above, using pJW15 plasmid isolated from E2348/69 or NH4. In contrast to what we observed with plasmid isolated from MC4100, both E2348/69- and NH4-isolated pJW15 permitted the isolation of large numbers of both E2348/69(pJW15) and NH4(pJW15) transformants (Figure 3). These data suggest that DNA isolated from NH4 has been modified such that it escapes restriction by the EPEC HsdMSR complex upon transformation. To determine if other large, low copy plasmids might be similarly modified upon transformation into NH4, we transformed the lux reporter plasmid pNLP10 (10 kb, pSC101 origin, copy number ~5) , and the cloning vector pLAFR1 (21.6 kb, RK2 origin, copy number 5–7)  into NH4, reisolated the plasmids and used them to transform E2348/69 in parallel with the same plasmids isolated from an E. coli K-12 laboratory strain (Table 1). As previously observed, transformation efficiencies for the pJW15 plasmid increased several orders of magnitude when this plasmid was isolated from NH4 as compared to an E. coli K-12 laboratory strain (Table 1). Similarly, pNLP10 and pLAFR1 could both be transformed into E2348/69 at least two orders of magnitude better after they had been passaged through NH4 (Table 1), although transformation efficiencies were very low for the large 21.6 kb cosmid pLAFR-1. Accordingly, we conclude that both E2348/69 and NH4 contain active modification systems that permit plasmids isolated from these strains to be transformed into restriction-competent (E2348/69) hosts. Thus, disruption of the hsdR allele in NH4 leaves the modification activity of the predicted HsdMS complex intact.
Mutation of hsdRdoes not render E2348/69 amenable to generalized transduction
In addition to being recalcitrant to transformation with large plasmids, E2348/69 is also resistant to infection with the E. coli generalized transducing phage P1. This is a serious drawback in genetic analysis of this organism, since the study of a given gene necessitates time consuming construction of mutant alleles and their recombination onto the E2348/69 chromosome by relatively cumbersome techniques. Creating strains carrying multiple mutant genes is even more tedious. Conversely, the movement of alleles between strain backgrounds by P1-mediated generalized transduction in E. coli K-12 can be accomplished in one day. In order to determine if the ΔhsdR mutation facilitated the movement of genetic material into E2348/69 by generalized transduction, we subjected NH4, E2348/69, and the E. coli K-12 strain MC4100 to P1 infection with phage lysates that had been grown on a strain carrying a nadA::Tn10 mutation. The nadA::Tn10 mutation confers tetracycline resistance as well as an inability to grow on unsupplemented minimal media. While we obtained hundreds of tetracycline resistant, minimal media deficient MC4100 nadA::Tn10 transductants, none were observed with E2348/69 or NH4. The same results were obtained with P1 lysates grown on strains carrying different mutant alleles that conferred various antibiotic resistant phenotypes. Thus, the HsdMSR restriction-modification system identified here is not responsible for the inability to infect E2348/69 with the P1 generalized transducing phage.
Abrogation of hsdRdoes not affect virulence factor production in NH4
The E2348/69 ΔhsdRmutant NH4 is a new tool for genetic analysis of EPEC infection
In this study we have identified an hsdMSR gene cluster in the E2348/69 genome and shown that mutation of the hsdR homologue produces a strain that can be transformed with large, low copy plasmids efficiently (Figure 3). Further, the production of the major EPEC virulence determinants in the ΔhsdR mutant, NH4, were unaffected (Figure 4) and we could discern no differences between E2348/69 and NH4 using tissue culture models of adherence and infection (Figure 5). These findings have important implications for the study of EPEC virulence. Although genetic techniques exist for conjugation of plasmids into E2348/69 [10, 11], some plasmids, which are not amenable to conjugation, such as the pJW15 plasmid we used , will be much easier to work with using our newly developed ΔhsdR strain. This is a unique genetic tool that we expect will complement existing optimized techniques for preparing EPEC cells that are competent for transformation .
Although it should be possible to study pathogenesis directly in NH4 since our experiments indicate virulence determinant production is unaffected, we cannot say at this time whether NH4 may have diminished fitness relative to E2348/69 in vivo. It has been suggested that restriction-modification systems may provide an advantage to the bacterium in new environments where unfamiliar bacteriophages may be encountered [1, 2]. Thus, it may be that the HsdMSR system identified here provides an advantage upon infection of the intestine. Even if this proved to be true, NH4 should still prove invaluable as a bridging strain. We have shown that the modification activity of the Hsd system identified here remains intact in the ΔhsdR NH4 mutant (Figure 3). Accordingly, exogenous DNA that is difficult to introduce into E2348/69 could first be introduced into NH4, where it would be modified, reisolated, and then moved into E2348/69. Indeed, we were able to use NH4 as an effective bridging strain for other large, low copy plasmids, including pLAFR1 and pNLP10. We are also hopeful that NH4 will improve the efficiency of other genetic techniques in EPEC that require the introduction of large, foreign DNA molecules, such as allelic exchange and transposon mutagenesis. We are currently testing these techniques in NH4. Thus, we hope that NH4 will be a useful tool to the EPEC research community.
Genetic techniques that are routinely performed in laboratory strains of E. coli, such as generalized transduction and transformation, are impossible or orders of magnitude less efficient in unmodified pathogenic isolates. Because of this, genetic and molecular biological analysis of such microbes does not occur as rapidly as it does with "domesticated" strains. We modified a very low copy luminescent reporter plasmid for use in the EPEC type strain E2348/69, to monitor expression of genes of interest [3, 13]. To our dismay, this plasmid, pJW15, was exceedingly difficult to transform into E2348/69, and we routinely had to do multiple transformations to acquire transformants. To determine if a restriction-modification system might be responsible for our troubles, we searched the E2348/69 genome for homologues of hsd restriction and/or modification enzymes . In this paper, we report the identification of an operon encoding three genes with high homology to HsdM, HsdS, and HsdR proteins involved in DNA modification, restriction site specificity and DNA restriction. We engineered an E2348/69 strain lacking the conserved enzymatic domain of the HsdR protein, and demonstrated that this strain could be transformed orders of magnitude better than the wild-type strain with pJW15. The E2348/69ΔhsdR strain could also be transformed with other large, low copy plasmids bearing different replication origins, suggesting that this is a general attribute of this strain. Thus, the type I restriction-modification system encoded by these genes is active in E2348/69 and limits the acquisition of foreign DNA. The HsdMS enzyme complex remains functional for DNA modification in our ΔhsdR strain, since it can act as a bridging strain – pJW15, pLAFR1, or pNLP10 DNA that were passed through this strain could be transformed into the wild-type E2348/69 strain with ease. Other types of DNA modification can influence gene expression . Thus, we examined virulence determinant expression in our ΔhsdR strain, since we desired to use this strain to study pathogenesis. We found no changes in growth or expression and function of two of the most important virulence determinants of EPEC; the type IV BFP which facilitates attachment to the intestine, and the T3SS, which mediates infection and intoxication of host cells. Thus, our strain will be useful for studying pathogenesis of EPEC, since it readily takes up large molecules of DNA and retains its key virulence properties – adherence to, and intoxication of, epithelial cells. No such strain currently exists, and so we regard this as a useful new tool for the EPEC research community.
Bacterial strains and plasmids
Strains and plasmids used in this study.
Wild-type EPEC strain
EPEC (O127:H6) isolated from an infant with gastroenteritis
Wild-type E. coli K-12 lab strain
F- araD139 Δ(argF-lac)U169 rpsL150(StrR) relA1 flbB5301 deoC1 ptsF25 rbsR
Commercially available competent cells for cloning
F- mcrA Δ(mrr-hsdRMS-mcfBC) φ80lacZΔM15 ΔlacX74 recA1 araΔ139 Δ(ara-leu) 7697 galU galK rpsL endA1 nupG (StrR)
Invitrogen Canada Inc.
Commercially available competent cells of E. coli λlysogen that provide all trans acting and mobilization factors required for the replication and mobilization of λPi dependent plasmids
mcrA Δ(mrr-hsdRMS-mcfBC) φ80lacZΔM15 ΔlacX74 recA1 araΔ139 Δ(ara-leu) 7697 galU galK rpsL endA1 nupG λpir
E. coli SM10 λlysogen that provide all trans acting and mobilization factors required for the replication and mobilization of λPi dependent plasmids
TOP10 (pUC19 ΔhsdR)
SM10 λpir (pCVD442 ΔhsdR)
5 kb low copy number cloning vector
Invitrogen Canada Inc.
pUC19 carrying ΔhsdR construct
Cloning vector requiring λ Pi protein to replicate, carries sacB for negative selection (AmpR)
pCVD442 carrying ΔhsdR construct
Broad host range promoterless lux reporter plasmid
Low copy number, broad host range plasmid
Low copy number lux reporter plasmid, pSC101 origin
Construction of a E2348/69 hsdRmutant
DNA fragments encoding the amino and carboxyl terminal portions of the E2348/69 HsdR homologue were amplified from the E2348/69 chromosome using the restriction site-tagged primer pairs HsdR-L1Ec (5'-GGGAATTCGTTAGTCTACCAATGGGCGAC-3', EcoRI tag) and HsdR-R1Nc (5'-CGCCATGGTGCCACTCGCTGTCATTAAAC-3', NcoI tag) or HsdR-L2Nc (5'-CGCCATGGATTTGATGAATGCCACCGCAG-3', NcoI tag) and HsdR-R2Xb (5'-GGTCTAGAGATTGCGGGTTTAACGGACTG-3', XbaI tag), respectively (restriction sites underlined). The PCR program used cycled the reaction at 95°C for 1 minute, 48°C for 1 minute, and 72°C for 2 minutes, 35 times and finished with a 72°C, 4 minute extension followed by a hold at 4°C. Using standard cloning procedures, equal amounts of the two purified PCR fragments were digested with NcoI and ligated to form a product encoding an N-terminal deletion of the predicted conserved helicase and ATP binding domains of HsdR. This fragment was digested with XbaI and EcoRI (Invitrogen Canada Inc.) and cloned into the same sites in pUC19. In order to recombine the ΔhsdR allele onto the E2348/69 chromosome, the pUC19:ΔhsdR construct was digested with EcoRI and the recessed ends were filled in using Klenow fragment. This product was digested with XbaI and the resulting ΔhsdR fragment was cloned into the XbaI and SmaI sites of the gene replacement vector pCVD442 . This construct was conjugated into E2348/69 and double recombinants that contained the ΔhsdR allele were sequentially selected for by antibiotic resistance and sucrose sensitivity as previously described . The resulting colonies were screened for the presence of the ΔhsdR allele via PCR using the primers HsdR-L1Ec and HsdR-R2Xb. One positive isolate was named NH4.
Transformation efficiency tests
Electroporation competent cells were prepared using standard techniques from equal numbers of E2348/69 and NH4 by normalizing culture volumes according to absorbance measured at 600 nm. Plasmid samples were prepared with the GenElute Plasmid Miniprep Kit (Sigma-Aldrich) and DNA concentrations determined by measuring the absorbance at 260 nm. Equal volumes of competent EPEC and NH4 cells were transformed with 1 uL of the same plasmid preparation using a BioRad MicroPulser electroporator set on the bacterial setting and 2 mm gap electroporation cuvettes. The transformed cells were serially diluted and 100 uL of each dilution were plated on LB plates containing the appropriate antibiotic and in some cases to LB plates lacking antibiotics to ascertain the number of viable cells. The transformation efficiency was calculated as the total number of transformants divided by the amount of plasmid used in the transformation (μg) or by dividing the total number of transformants obtained by the number of viable cells and then dividing this number by the amount of DNA used in the transformation. All transformation efficiencies were determined at least three times. Although overall numbers varied depending on the plasmid preparation or batch of competent cells used, the trends within experiments remained the same over multiple repetitions. In Table 1, one representative experiment is shown.
Assays of virulence determinant production and infection phenotypes
BfpA, and Tir levels were measured as previously described [19, 20]. BfpA and Tir were assessed by western blot analysis (α-BfpA courtesy of M. Donnenberg, U. Maryland, α-Tir courtesy of B. Finlay, UBC). As a loading control, a cross-reactive protein was included in Figure 2c. Assays for localized adherence were performed as previously described [20, 25]. The FAS assay was adapted from Knutton et al.  and DeVinney et al. . Briefly, HEp-2 cells were seeded on coverslips in a 24 well plate at a concentration of 2 × 105 cells/mL and grown overnight at 37°C/5% CO2. Bacterial strains were inoculated in LB and grown statically overnight at 37°C/5% CO2. The HEp-2 monolayers were infected with 5 μL of static bacterial culture for 2.5 h. Specimens were washed thoroughly (4 times) with phosphate buffered saline (PBS) and fixed with 2.5% paraformaldehyde for 10 min at 37°C. Samples were washed with PBS (4 times) then permeabilized with PBS/0.1% Triton X-100/10% Fetal Bovine Serum (FBS, Invitrogen) for 30 min at 37°C. Antisera for EPEC (1:300, R. DeVinney U. Calgary) was added to the specimens for 30 min at 37°C. After washing again with PBS (4 times), specimens were stained with anti-rabbit-Cy3 (1:400, R. DeVinney. U. Calgary) and Alexa 488 phalloidin (1:400, Molecular Probes) for 30 min at room temperature in the dark. Samples were washed with PBS once more and then mounted for viewing. Confocal images were obtained using a Leica fluorescence microscope (BioSci Microscopy Unit) at 60× objective.
Generalized transduction was performed using routine procedures as previously described .
We are grateful to Rebekah DeVinney for advice and reagents used to perform the FAS assay. The authors wish to thank the Alberta Heritage Foundation for Medical Research, The Canadian Institutes of Health Research, and the Natural Sciences and Engineering Research Council of Canada for support.
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