Cloning of the nptII gene of Escherichia coli
and construction of a recombinant strain
harboring functional recA and nptII
antibiotic resistance
S. Ghanem
Botany and Microbiology Department, Faculty of Science, Helwan University, Ain Helwan, Cairo, Egypt Corresponding author: S. Ghanem Genet. Mol. Res. 10 (3): 1445-1454 (2011) Received February 17, 2011 Accepted May 5, 2011 Published July 19, 2011 ABSTRACT. In an attempt to clone the ORF of the nptII gene of Esche-
richia coli K12 (ATCC 10798), two degenerate primers were designed based on the nptII sequence of its Tn5 transposon. The nptII ORF was placed un- der the control of the E. coli hybrid trc promoter, in the pKK388-1 vector, transformed into E. coli DH5a ΔrecA (recombinant, deficient strain). Trans- ferred cells were tested for ampicillin, tetracycline, kanamycin, neomycin, geneticin, paromomycin, penicillin, and UV resistance. The neomycin phosphotransferase gene of E. coli was cloned successfully and conferred kanamycin, neomycin, geneticin, and paromomycin resistance to recombi- nant DH5a; this did not inhibit insertion of additional antibiotic resistance against ampicillin and tetracycline, meaning the trc promoter can express two different genes carried by two different plasmids harbored in the same cell. This resistance conferral process could be considered as an emulation of horizontal gene transfer occurring in nature and would be a useful tool for understanding mechanisms of evolution of multidrug-resistant strains.
Key words: Escherichia coli; Neomycin phosphotransferase gene (nptII);
Homologous recombination gene (recA); Aminoglycoside resistance Genetics and Molecular Research 10 (3): 1445-1454 (2011) The neomycin phosphotransferase gene (nptII) was initially isolated from the transposon Tn5 of the bacterium Escherichia coli K12. Transposon Tn5 is a movable DNA element of about 5.3 kbp that carries resistance to the aminoglycoside antibiot- ics (Berg et al., 1975) and that also seems to encode proteins that participate in the transposition reaction (Rothstein et al., 1981). Furthermore, the presence of transpo- son Tn5 was studied in 730 Enterobacteriaceae strains from clinical and sewage origin (Blázquez et al., 2006). The nptII gene encodes neomycin phosphotransferase II (NPTII; EC, also called aminoglycoside 3'-phosphotransferase II (APH(3')II), which inactivates by phosphorylation a range of aminoglycoside antibiotics such as kanamycin, neomycin, geneticin, and paromomycin (Berg et al., 1975; Auerswald et al., 1981; Beck et al., 1982; Genilloud et al., 1988). The neo gene of the Tn5 appears to be an excellent selec- tion marker for vectors in prokaryotic as well as in eukaryotic systems (Herrmann et al., 1978; Rao and Rogers, 1979; Jimenez and Davies, 1980; Colbere-Garapin et al., 1981; Southern and Berg, 1982). Besides its use as a selectable marker, the gene is also very attractive for use as a generally applicable indicator gene to examine gene expression and gene regulation (Reiss et al., 1984). For such studies, it would be of advantage if the NPTII could be fused onto a gene product of interest without losing enzymatic activity. The aim of the present study was to clone the nptII gene of the E. coli K12 strain, by placing its open reading frame (ORF) under the control of a powerful promoter, in an expres- sive plasmid in E. coli. E. coli DH5α (ΔrecA) strains were selected to be transferred by plasmid harboring the nptII gene because ΔrecA strains have the advantage of stably maintaining introduced plasmids.This study would permit the evaluation of the ability of trc promoter to express two different genes carried by two different plasmids harbored by the same cell. In addition, this study would help to evaluate the effect of the presence of functional recA in a bacterial cell on its efficiency in receiving and expressing another gene carried on another plasmid, such as the nptII gene.
Bacterial strains and growth conditions
Tables 1 and 2 show the bacterial strains and vectors used in this study. E. coli was grown aerobically at 37°C in Luria-Bertani (LB) medium (Difco). Growth was monitored by OD600 nm. Ampicillin (100 µg/mL), kanamycin (25 µg/mL), tetracycline (10 µg/mL), neomycin (25 µg/mL), geneticin (10 µg/mL), paromomycin (10 µg/mL), and penicillin (25 µg/mL) were added to the medium when needed. All chemicals were purchased from Sigma.
Standard molecular biology procedures were used according to Sambrook et al. Genetics and Molecular Research 10 (3): 1445-1454 (2011) E. coli nptII gene and a recombinant strain construction (1989). Enzymes were obtained from Boehringer Mannheim. Plasmid DNA was prepared with the Wizard kit from Promega. DNA fragments were isolated from agarose gels with the Jetsorb kit (Genomed). The colonies kept for further characterization were purified once by single-colony isolation in selective solid medium. Stationary-phase cultures, grown from a single colony, were added to 1 mL liquid medium, mixed with 1 mL 80% glycerol and stored at -20°C. Table 1. Bacterial strains used in this study.
Principal characteristics Source or reference Escherichia coli (Migula, 1895; Castellani and Chalmers, 1919), Escherichia coli F-, Δ (lac-argF)U169, recA1, endA1, hsdR17(r - (Woodcock et al., 1989), supE44, gyrA1, relA1, deoR, thi-1 (ф 80dlacZ Δ M15) ATCC = American Type Culture Collection, Rockville, MD, USA.
Table 2. Vectors used in this study.
Principal characteristics Source or reference E. coli cloning vectors for the PCR products, origin of replication ColE1, Ampr bla lacI orif1 (pGEM-T and pGEM-T Easy, differing by the restriction sites present around the insertion site) E. coli expression vector with trc promoter, Apr (Brosius, 1988), Clontech, Inc.
pGEM-T Easy vector overhanging nptII ORF Plasmid derived from pKK388-1, nptII ORF under ptrc control Plasmid derived from pKK388-1, recA ORF in the form (El Shafey et al. 2009) of fragment NcoI/SmaI under ptrc control Promega Corporation Clontech Laboratories Inc.
Analysis of the nucleotide sequences
The CLC Sequence Viewer Software, GeneJockey program, DNA Strider, Blast (NCBI), and CLUSTAL W allowed the analysis of the nucleotide and amino acid se- Polymerase chain reaction (PCR) amplification
The oligonucleotides described in Table 3 were used as primers. PCR was carried out with 2.5 U thermostable DNA polymerase (AmpliTaqGold from Perkin-Elmer) in a re- action mixture containing an E. coli plasmid DNA, 0.2 mM deoxynucleotide triphosphates (Promega), 0.5 µM of both primers, 2 mM MgCl and 1X AmpliTaq buffer in a final volume of 50 µL. For the amplification reaction, after 10 min at 94°C, 25 identical cycles (1 min of denaturation at 94°C, 1 min of hybridization at 50°C, 1 min of elongation at 72°C) were followed by a final elongation step of 5 min at 72°C. The amplified DNA fragment of the expected size was cloned into the pGEM-T vector (Promega).
Genetics and Molecular Research 10 (3): 1445-1454 (2011) Ultraviolet (UV) resistance assays
To measure UV resistance in E. coli, a fresh culture was initiated from an overnight culture and incubated for 3 h (logarithmic phase of growth). Next, 2.5 mL culture was placed on glass Petri dishes of 5.5 cm in diameter. Uncovered plates were exposed to 0.004, 0.008, 0.012, 0.016, 0.020, 0.024, 0.028, 0.032 J/cm2 UV light. Five replicates were carried out for UV exposure experiments. Precautions against exposure of Petri dishes to light after irradia- tion were taken to avoid a photoreactivation phenomenon. Relative survival at each dose was calculated by comparing the number of colony-forming units (CFU) in the irradiated sample to the number of CFU in the non-irradiated sample.
Analysis of the nptII gene sequence
The nptII sequence of Tn5 transposon of E. coli K12 ATCC 10798 (Figure 1) was analyzed in silico and found to have a molecular mass of 257.378 kDa. Restriction sites were searched using the RestrictionMapper software. The sequence statistics of the nptII gene were calculated with the help of the CLC Sequence Viewer Software (Table 3).
Table 3. Base distribution.
Designing of primers
Two degenerate primers were designed based on the nptII sequence (Beck et al., 1982) of Tn5 transposon of E. coli (Figure 1). The Primer3 web designer tool (University of Massachu- setts Medical School, USA) was used for designing the two primers. Two primers, each with a length of 21 bases, were created with a calculated G/C percentage of 38.10 and 47.62 for the left primer (nptIIpr1) and right primer (nptIIpr2), respectively. Melting temperature of the primer was calculated and found to be 58.56° and 59.47°C for nptIIpr1 and nptIIpr2, respectively. The self- complementarity score of the primers (taken as a measure of its tendency to anneal to itself or form secondary structure) was found to be 4.00 and 3.00 for nptIIpr1 and nptIIpr2, respectively. While the 3' self-complementarity of the primers (taken as a measure of its tendency to form a primer- dimer with itself) was found to be 2.00 for both primers. Primer sequences are shown in Table 4.
Table 4. Oligonucleotides used in this study.
Sequence ( 5ꞌ-3ꞌ) Restriction site present nptIIpr1: tgattgaacaagatggattgc BccI, AgsI nptIIpr2: gaactcgtcaagaaggcgata Genetics and Molecular Research 10 (3): 1445-1454 (2011) E. coli nptII gene and a recombinant strain construction Figure 1. nptII sequence of Tn5 transposon of Escherichia coli K12 (ATCC 10798).
Genetics and Molecular Research 10 (3): 1445-1454 (2011) Cloning of the nptII gene into the pGEM®-T Easy vector
The extracted plasmid DNA of E. coli, together with the degenerate primers, were used in PCR experiments, for amplifying the nptII gene.
A fragment of 795 bp corresponding to the nptII gene was amplified. The pGEM®-T Easy vector was selected to be used in the first step because it is convenient for cloning PCR products, as it is an open vector with a single 3ꞌ terminal thymidine (T) that overhangs at the insertion site to both ends. The presence of these single 3ꞌ-T overhangs at the insertion site, greatly improves the efficiency of ligation of a PCR product into the plasmids by provid- ing a compatible overhang for PCR products generated by certain thermostable polymerases (Mezei and Storts, 1994; Robles and Doers, 1994). It often adds a single deoxyadenosine in a template-independent fashion, to the 3ꞌ-ends of the amplified fragments (Clark, 1988; Newton and Graham, 1994).
The PCR fragment extracted from the gel was cloned in the pGEM®-T Easy vector, resulting in a pNPT plasmid (Figure 2). Figure 2. pNPT.
Many studies reported successful cloning of many bacterial aminoglycoside antibiotic genes, such as in E. coli (Clerget et al., 1982), Campylobacter jejuni (Tenover and Elvrum, 1988), Bacillus circulans (Sarwar and Akhtar, 1990), Pseudomonas aeruginosa (Schwocho et al., 1995), Citrobacter freundii (Wu et al., 1997), Streptomyces tenebrurius (Skeggs et al., 1987), and Campylobacter coli (Wang and Taylor, 1990). Genetics and Molecular Research 10 (3): 1445-1454 (2011) E. coli nptII gene and a recombinant strain construction Cloning of the nptII gene into the pKK388-1 plasmid
The second step was to clone the nptII gene extracted from the pNPT plasmid into pKK388-1. pKK388-1 is an E. coli expression vector with E. coli hybrid trc promoter and Apr (Brosius, 1988). The E. coli hybrid trc promoter was previously shown to promote efficient expression of genes in both E. coli (Brosius, 1988; El Shafey et al., 2009), and Corynebacte- rium glutamicum (Delaunay et al., 1999).
For this purpose, the pNPT plasmid was digested with EcoRI. This site was selected because it is absent in the PCR fragment and is represented in the pGEMT-Easy vector sur- rounding the insert; yet, at the same time, it is unique in the pKK388-1 plasmid and is found inside its polylinker. The digested pNPT plasmid was then electrophoresed to separate the digested fragment away from the rest of the plasmid. The fragment of the expected size was purified and extracted from the gel. On the other hand, pKK388-1 was open by EcoRI digestion. The extracted PCR fragment was then cloned in the open pKK388-1 plasmid, resulting in pNPTR (Figure 3). Figure 3. pNPTR.
Expression of recombinant aminoglycoside antibiotic genes in E. coli cells has been reported by many authors (e.g., Tenover and Elvrum, 1988; Sarwar and Akhtar, 1990; Wang and Taylor, 1990).
Transformation of pSH2 into DH5a
Plasmid pNPTR was dialyzed and transformed in the recA- and recA+ DH5a strains Genetics and Molecular Research 10 (3): 1445-1454 (2011) after its treatment to render it competent for receiving genetic materials, in an attempt to ex- press nptII gene in the two stains.
E. coli recA- DH5a harboring no plasmids were sensitive to ampicillin (Apr) and UV, while recA+ cells harboring pSH2 (Figure 4) were resistant to ampicillin, tetracycline and UV up to a dose 0.24 J/cm2. Cells grown on LB only (non-transformed DH5a), LB + ampicillin plates (recA+, nptII- DH5a::pSH2), and LB + neomycin (recA+, nptII+ DH5a::p) were picked up.
Figure 4. pSH2.
Transformed and non-transformed cells were tested for their sensitivity to the follow- ing antibiotics: ampicillin, kanamycin, chloramphenicol, tetracycline, neomycin, geneticin, paromomycin, and penicillin; in addition to their sensitivity to UV doses up to 0.24 J/cm2.
The results (Table 5) showed that nptII-transformed cells exhibited resistance to am- picillin, tetracycline, kanamycin, neomycin, geneticin, and paromomycin, while E. coli DH5a exhibited resistance to ampicillin and tetracycline only, and DH5a non-transformed cells did not exhibit resistance to any antibiotic tested.
Table 5. Sensitivity of transformed and non-transformed Escherichia coli DH5a cells to different antibiotics.
E. coli DH5a (recA-, nptII-) E. coli DH5a (recA+, nptII-) E. coli DH5a (recA+, nptII+) None = no antibiotic added; Amp = ampicillin; Tet = tetracycline; Kan = kanamycin; Neo = neomycin; Par = paromomycin; Gen = geneticin; Pen = penicillin.
Functionality of the recA gene was proved by relatively high resistance of DH5a transformed by (pSH2::recA) and (pNPTR::nptII) equally to UV up to a dose 0.24 J/cm2, Genetics and Molecular Research 10 (3): 1445-1454 (2011) E. coli nptII gene and a recombinant strain construction while non-transformed DH5a (ΔrecA) was sensitive to the tried range of UV (Figure 5).
Figure 5. Functionality of recA gene expressed as relative tolerance of transformed and non-transformed
Escherichia coli cells to UV radiation.
We concluded that the neomycin phosphotransferase gene of E. coli was cloned suc- cessfully and proved to confer kanamycin, neomycin, geneticin, and paromomycin resistance to recombination-positive DH5a, which in turn did not inhibit insertion of a new antibiotic resistance to the same cell already having ampicillin and tetracycline resistance genes, indi- cating the ability of the trc promoter to express two different genes carried by two different plasmids in the same cell.
It would also be interesting to follow up on this study with researchs concerned with the expression of more than two plasmids harboring different genes belonging to different families of antibiotic resistance genes cloned under the control of the trc promoter. The re- sulting recombinant strains would be of special interest for the emulation of the evolutionary mechanisms of resistance based on horizontal transfer of resistance genes. Such studies would also be interesting for the evaluation of new antibiotic generations destined for multidrug re- sistant strains that have evolved in last few years (Sandel et al., 2002; Lim et al., 2009; Jan et al., 2009; Johnson, 2010) as a result of the extensive use of different antibiotic drugs.
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