λ Red for PCR-mediated gene replacement in EHEC
Red-promoted gene replacement using PCR derived substrates is diagrammed in Figure . Typically, PCR is performed with two primers (60 mers) which contain 20 bases at the 3' ends to amplify a drug marker. In addition, the primers contain 40 bases at the 5' ends that are complimentary to either the upstream or downstream regions of the target gene (see Figure ). The PCR product, which has the drug marker flanked by 40 bases upstream and downstream of the targeted gene of interest, is simply electroporated into Red + Gam producing E. coli
This strategy has been shown to promote high efficiency gene replacement with such substrates in E. coli
], as well as Salmonella
species (see Background).
Figure 1 Schematic of λ Red-promoted PCR-mediated recombination. PCR products, containing a drug marker flanked by 40–60 bp of target DNA, are generated by primers designated 5KO and 3KO (see Table 6 in Additional file #2) and electroporated into (more ...)
We have previously shown that a Red + Gam-producing plasmid (pTP223) promotes gene replacement efficiently with substrates containing long regions of homology to the target gene [2
]. On the other hand, PCR substrates containing short regions of homology (40–60 bp) recombine at very low frequency when Red and Gam are supplied by multi-copy plasmid pTP223 [reference [5
]; unpublished observations]. We and others have noted that expression of red
from the chromosome (or low copy number plasmids) is better suited for Red-promoted recombination [2
]. It is assumed that since Red induces the rolling circle mode of replication in medium or high copy number plasmids [29
], linear multimers of the plasmid are generated that may compete with the electroporated substrates for the Red recombination functions. This reasoning explains why pTP223, while expressing high levels of Red and Gam, is not optimal for Red-promoted gene replacement in E. coli
K-12. In addition, studies in our lab with ΔrecBCD::gam-red
chromosomal substitutions (i.e.,
no resident plasmid), expressing red
from either Plac
or the stronger Ptac
promoter, have shown that Red-promoted recombination with short-homology substrates requires higher level expression of the red
functions relative to long-homology substrates [unpublished observations]. This observation explains why pTP223 works well with long homology substrates, but not well with short homology substrates: the limited amount of "free" Red functions when expressed from pTP223 is adequate for long homology substrates, but not for PCR (short homology) substrates.
A construct that meets both these requirements (high expression from a single or low copy number replisome) is plasmid pKD46, described by Datsenko and Wanner [5
] which uses the pBAD promoter to express red
from a low copy number temperature-sensitive replicon. A similar plasmid was constructed in our lab, except that gam
are driven by the Ptac promoter (pKM201). A variation of pKM201 was constructed which expresses the lacI
repressor gene (pKM208) in order to keep expression of red
under tight control prior to IPTG induction. In anticipation of the requirement to easily remove these plasmids after gene replacement, both plasmids contain temperature-sensitive origins of replication.
EHEC containing pTP223, pKM201, or pKM208 were electroporated with a PCR substrate containing the kan
gene flanked by 40 bases from the 5' and 3' regions of the lacZ
gene, using an optimized protocol scheme for preparation of electrocompetence cells as described in the Methods section. Recombinants were detected as white KanR
colonies on LB-kan plates containing X-gal and IPTG, as previously described [2
]. As expected, plasmid pTP223 was unable to efficiently promote short-homology recombination in EHEC (data not shown; however, see results with EPEC below). On the contrary, low copy number plasmids pKM201 and pKM208 were able to promote short homology recombination in EHEC at the lacZ
locus. Cells harboring LacI-expressing pKM208 required prior induction with IPTG for efficient recombination (see below); cells containing pKM201 did not require IPTG addition (due to Ptac
leakiness in the absence of over-expressed LacI). In five separate experiments, Red expression from pKM208 produced gene replacements at a rate between 70–600 recombinants per 108
cell survivor (total number of recombinants varied from 750–3000). From one of these experiments, ten out of ten white KanR
transformants tested positive for gene replacement by PCR analysis (data not shown). In addition, a PCR fragment containing a lacZ
deletion with a cat
insertion worked as well as the one described above using kan
. It is noteworthy, however, that while the ΔlacZ::kan
allele yielded recombinants at a frequency of 0.7–6 × 10-6
per survivor in EHEC containing pKM208, the same PCR fragment in E. coli
K-12 containing pKM208 yielded recombinants at a frequency of 10-4
per survivor (data not shown). The sequences used in targeting ΔlacZ
to the E. coli
K-12 and EHEC chromosomes are identical. The reason for this lower frequency is not known, but may be due to lower expression of red
in EHEC, an EHEC-specific restriction system(s), or lower rates of DNA uptake following electroporation.
One other possibility for the reduction in efficiency of Red + Gam-promoted PCR-mediated gene replacement in EHEC relative to that seen in E. coli K-12 is that the λ Gam protein might not be as active on EHEC RecBCD as it is for E. coli K-12 RecBCD. This seemed unlikely given the conservation of the recBCD genes between these two species (97–98% conservation). Nonetheless, we made a EHEC Δ recC::cat knockout and tested its ability to perform Red-promoted PCR-mediated recombination. Deactivating RecBCD function by deletion of recC did not stimulate λ Red recombination in EHEC at the lacZ locus (data not shown). Thus, to a first approximation, λ Gam works as efficiently with EHEC RecBCD as it does with E. coli K-12 RecBCD. Another possibility is that the red functions from the endogenous lambdoid EHEC prophage 933 W would be better suited than λ red for gene replacement in EHEC. This seems unlikely given the high degree of conservation between λ and 933 W red genes (99.6% identity), and was not tested.
Gene replacements using short homology substrates were also performed in EHEC containing pKM201 at the tir and espF loci within the locus of enterocyte effacement (LEE), though frequencies of gene replacement at these sites (in repeated experiments) were lower than that seen with the lacZ substrate described above, and usually ranged from 0–20 recombinants per 108 survivor. However, this lower frequency of gene replacement at alternative loci relative to lacZ was also observed in E. coli K-12 [unpublished observations]. Thus, the lacZ region may be a hotspot for gene replacement, perhaps the result of stable expression of the drug marker following integration at this particular locus (see below). Nonetheless, Red-promoted PCR-mediated gene replacement was successful with both tir and espF.
To assess the overall usefulness of Red-promoted PCR-mediated gene replacement in EHEC, we targeted five O157-specific islands (O-islands) in the EHEC chromosome for deletion; these O-islands are not present in E. coli
]. PCR substrates containing the kan
gene flanked by 40 base pairs of DNA bordering O-islands #12, #77, #103, #130–131 and #169 (Table ) were electroporated into EHEC containing pKM208. These islands were targeted because they occupy different regions of the chromosome, are of moderate size (733–4253 base pairs in length), and encode either putative virulence factors or unknown proteins. In the first attempt, all five islands were successfully deleted (see Table ), though there was variability in the frequency of island replacement. Deletion of O-islands #130–131 (one replacement) occurred at a frequency similar to that seen with lacZ
gene (~100 KanR
transformants per 108
cell survivors), while the others showed rates ranging from 10–50 fold lower. Thus, λ Red is able to promote efficient short homology recombination with the EHEC chromosome, but with variability according to the target site.
λ Red-promoted PCR-mediated replacement of five targeted EHEC O-islandsa
To determine if any restrictions could be placed on the amount of DNA deleted by Red-promoted recombination, we generated a PCR product containing the cat gene flanked by regions upstream and downstream of an internal section of O-island #148, which contains the locus of enterocyte effacement (LEE). Electroporation with this PCR fragment, designed to delete 9 kb of genes encoding the type III secretion apparatus (see Table , strain KC30) generated recombinants at a relatively low frequency of 20 per 108 survivors, similar to the deletion frequencies seen with island #169 above. Thus, both small and large regions of the EHEC chromosome can be deleted in one step using Red-mediated recombination (as has been seen with E. coli K-12). Indeed, an additional 15 islands of EHEC of various sizes up to 45 kb have subsequently been deleted in EHEC by Red-promoted PCR-mediated island replacement using pKM201 [Campellone and Leong, unpublished].
Strains used and constructed in this study
Drug marker context dependency affects the efficiency of gene replacement
EHEC strains containing the kan
substitutions shown in Table were further purified by streaking on LB plates containing 40 μg/ml kanamycin. Interestingly, kan
substituted islands #12, #77 and #169, while selected on LB plates containing kanamycin at 20 μg/ml, did not grow at this higher kanamycin concentration (but did restreak well at 20 μg/ml). The other two substitutions (Δisland #103::kan
and Δislands #130–131::kan
), which consistently gave higher frequencies of gene replacement relative to the others, grew well on LB containing 40 μg/ml kanamycin. These results suggest that the position and/or orientation of the drug cassette within the chromosome alters its expression levels. Thus, the low frequency of O-island #169 replacement (see Table ) might be due to the influence of neighboring transcripts reading into the kan
gene following integration of the Δisland 169::kan
PCR substrate into the chromosome, or instability of the kan
transcript due to sequences fused to its 3' end. To test this hypothesis, deletion of O-island #169 was repeated using a PCR product that reversed the direction of kan
transcription within this chromosomal region. This PCR product was generated with primers Δisland169E & Δisland169F; see Table 6 (Additional file #2
). In three separate experiments, we found (on average) 10-fold higher KanR
transformants when kan
was reading leftward from the position of O-island #169 (according to the numbering in the sequence file) instead of rightward. This leftward reading direction of the kan
gene places it colinear with other genes in this region (ytfB
) and supports the notion that proper positioning of the drug marker in the chromosome can influence the recovery of the recombinant.
Extended expression of Red and Gam is mutagenic
Unlike E. coli K-12 and Salmonella enterica Serovar Typhimurium, there are no phage transductional protocols for EHEC to place λ Red-generated deletion alleles into clean genetic backgrounds. Thus, it was important to consider the possible mutagenic profile of λ Red expression in EHEC. Somewhat surprisingly, overnight cultures of EHEC containing uncontrolled expression of red and gam from pKM201 (which does not express lacI) showed a 10-fold increase in the rate of spontaneous rifampicin resistance (Figure ). EHEC containing pKM208 (which expresses the lacI repressor as well as the Ptac-red-gam operon) showed a significant increase in rifampicin resistance only when incubated in the presence of IPTG overnight.
Figure 2 Uncontrolled λ Red expression is mutagenic. A single fresh colony of EHEC strain TUV93-0 (with indicated plasmids) was suspended in 1 ml LB containing 100 μg/ml ampicillin. The cell suspension was diluted with LB-ampicillin to a final (more ...)
In order to determine the minimum time of Red induction required to generate the hyper-rec phenotype, we measured the frequency of gene replacement as a function of IPTG induction. Figure shows that a 20 minute exposure to IPTG is sufficient to induce the hyper-rec phenotype in EHEC. For most of the experiments reported above, we used a 1 hour IPTG induction period. Thus, we examined if Red induction for 1 hour induced a mutagenic phenotype in EHEC. EHEC cells containing pKM208 were exposed to IPTG for 1 hour in a manner identical that used for the preparation of electrocompetent cells, and plated on LB plates containing rifampicin. No increase in RifR cells was seen in such preparations when compared to uninduced cultures (see Figure , insert). Thus, while uncontrolled expression of red and gam causes a 10-fold increase in mutagenesis, limited expression of red and gam required for establishment of the hyper-rec phenotype is not mutagenic, at least with regard to the generation of rifampicin resistant mutants.
Figure 3 Time course for promotion of hyper-rec phenotype. EHEC strain TUV93-0 containing pKM208 (five cultures, 20 ml each) was grown for electrocompetence as described in the Methods section. At various times prior to collection, IPTG was added to four of the (more ...)
Long homology recombination (LHR) substrates with 1–2 kb of flanking DNA
While λ Red-promoted recombination with PCR-generated substrates offers the simplest mechanism for generating gene knockouts, there are other times when using long homology-containing plasmid substrates are advantageous. For instance, when multiple mutant alleles of a target gene need to be crossed into the chromosome, it is desirable to have the substrate previously cloned, in order to not induce PCR errors into the allele prior to transfer to the chromosome. A dedicated plasmid containing sequenced regions upstream and downstream regions of the target gene is required. Also, long homology-containing substrates promote higher frequencies of gene replacement relative to short homology substrates, and offer higher success rates for Red-promoted gene replacement in pathogenic hosts that are not as electrocompetent as E. coli
K-12. Thus, we examined a variety of plasmid-derived substrates to test Red-promoted knockouts of eae
and the tir-cesT-eae
region in the EHEC chromosome. The product of the eae
gene, intimin, is an outer membrane protein that is necessary for attachment to the target cell. The receptor for intimin, Tir (translocated intimin receptor), is a bacterial protein that is translocated into the host cell membrane by the LEE-encoded type III secretion system. cesT
encodes a chaperone for Tir [see reference [21
] for review].
Plasmid substrates were constructed that contained the cat gene (conferring resistance to chloramphenicol) flanked by upstream and downstream regions of tir, eae, or the tir-cesT-eae operon. Linear DNA recombination substrates (i.e., the cat gene flanked by 1.5 kb of target DNA) were generated from these plasmids by digestion with restriction enzymes(see Tables & 5 for details). EHEC cells harboring pTP223 were electroporated with the restriction digests, or gel-purified linear fragments containing the marked deletions, and plated on LB plates containing chloramphenicol. Among the chloramphenicol resistant colonies, potential chromosomal substitutions were distinguished from simple plasmid transformants by their sensitivity to ampicillin (bla gene carried within the pUC19 plasmid backbone). Results of a number of these experiments are shown in Table .
Gene replacements in the tir-cesT-eae region of enterohemorrhagic E. coli 0157:H7 using long homology-containing substrates
In all cases, Δeae, Δtir, or Δeae-cesT-tir was easily generated with these plasmid substrates, generating as few as 2 and as many as 1000 gene replacements per experiment, depending on the nature and amount of the transforming DNA. These genes were replaced with either the cat drug marker (Table , lines 1, 2, 4–9) or a cat-sacB cassette (Table , lines 3, 10 and 11). The latter substitutions generated strains that were subsequently used to generate in-frame, precise deletions (see Table ). Gene replacements were verified by PCR analysis on all selected candidates (1–6) in each experiment, using oligo primers within and outside the regions of the linear DNA substrates (data not shown). Overall, the following observations were evident: Electroporation of simple plasmid digests resulted in a high number of non recombinogenic plasmid transformants (i.e., CamR-AmpR, see Table , lines 1–3), due to incomplete digestion or religation of the plasmid substrate in vivo. A higher number of potential gene replacement transformants (i.e., CamR-AmpS colonies) could be obtained by further digestion of the plasmid with a backbone-specific restriction enzyme, ApaLI (see Table , lines 4–7), or by gel purification of the linear DNA recombination substrate (Table , lines 8–11). Interestingly, the Red-producing plasmid pTP223 is spontaneously lost during this process (between 10–50% of the transformants are TetS). Thus, fortuitously, a separate step to cure the recombinant of the Red plasmid is not required. These experiments show that marked and precise deletions can be easily generated in EHEC without the need to form and resolve plasmid co-integrates. We have used this procedure recently to generate deletions at the recC and dam loci in EHEC (experiments to be described elsewhere).
Generation of EHEC 0157:H7 unmarked deletions of eae and tir
The drug marker context effect seen above with construction of the Δisland #169 deletion using PCR substrates was also seen with one of the long homology substrates. Initial attempts to generate the EHEC chromosomal replacement Δeae::cat using SacI-SphI fragment from pKM184 yielded no recombinants with repeated attempts. The orientation of the cat gene inserted within the eae flanking regions of pKM184 was determined, and found to be reading opposite to the direction of the endogenous eae gene. Thus, a version of pKM184 was constructed where the cat gene was inserted co-directionally with eae. Electroporation with this substrate readily yielded recombinants (see Table , lines 1, 4–5). Thus again, we find that the orientation of the drug marker within the target gene site can alter either its expression level (preventing the selection of the recombinant) or its ability to be stably incorporated into the chromosome. With difficult substitutions, both orientations of the drug marker (or the use of properly positioned transcription terminators within the plasmid construct) should be attempted. Context dependent marker expression may be one of the primary causes of the variation seen among Red-promoted gene replacements of the same drug marker placed at different loci along the E. coli K-12 and Salmonella enterica Serovar typhimurium chromosomes [unpublished observations; S. Maloy, personal communication].