Preparation of CJ236 Competent Cells

E. coli strain CJ236 was plated on LB-agar plates with 15 µg/mL chloramphenicol (Cm), 125 µg/mL deoxythymidine (dThd) and grown at 30°C. Although it is usual to proceed with competent cell preparation from a single colony (especially if using a new cell stock validated by the manufacturer), we chose to first confirm the desired strain phenotype, an optional step. A colony with the proper temperature-sensitive dut-1 phenotype was identified by replica plating on M9 minimal media agar [12] supplemented with and without 125 µg/mL dThd and incubated at 30°C and 42°C for ∼40 hours. A colony was selected which displayed the desired phenotype of stunted growth at 42°C, which was improved with dThd (Personal correspondence with B. Weiss). This colony was used to prepare chemically competent cells [13]. To prevent genetic drift and reversal of the dut-1 ung-1 phenotype it is best to propagate CJ236 at ≤30°C in dThd supplemented media. These conditions reduce uracil incorporation in DNA (an unfavorable mutagenic event leading to reversions of this phenotype) since uracil incorporation is unnecessary when propagating the strain. However, during preparation of uracil-containing ssDNA or dsDNA template, the strain should be grown at 37°C without dThd for increased uracil incorporation.

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Figure 2. Completeness and frequency of codon substitutions observed in 454 sequencing of the comprehensive codon mutagenesis library of TEM-1.

(a) Number of the 63 possible codon substitutions observed and (b) frequency of codon substitutions observed as a function of position in the gene. For each of the 287 codons of TEM-1 the frequency of each of the 63 possible codon substitutions is shown, except for the 3% of the 18,081 codon substitutions that were not observed. The frequency is based on 454 sequencing in which 738,615 codon substitutions were observed in 787,488 reads. The frequency is normalized to the frequency that would occur if all substitutions were evenly distributed among the 18,081 possible substitutions (i.e. frequency = 1.0 means that the substitution was observed 738,615/18,081 = 41 times). The number of codon substitutions observed resulting from sequencing errors is small (∼4% of the 738,615 codon substitutions observed).

https://doi.org/10.1371/journal.pone.0052031.g002

Preparation of Uracil-containing ssDNA Template

pSkunk3-BLA is a 4.4 kB phagemid derived from pDIM-C8-BLA [14] in which the coding sequence of the Cm resistance gene was replaced with the streptomycin/spectinomycin (Sm/Spec) resistance gene. This phagemid was used to transform CJ236 cells which were then plated on LB-agar with 50 µg/mL Spec, 15 µg/mL Cm, and 125 µg/mL dThd and incubated overnight at 30°C. A single colony was used to inoculate 10 ml of LB supplemented with Cm, Spec, and dThd as above, which was incubated with shaking at 30°C overnight. The cell density of the culture was determined from the OD_600nm using the correlation 2×10⁸ CFU/mL-OD_600nm. In a 20 ml test tube, 2 ml of TBG media [15] with 50 µg/mL Spec was inoculated with 2×10⁷ CFU from the overnight culture and 1×10⁸ pfu R408 helper phage for an MOI of 5. This culture was incubated for 6 hours at 37°C with shaking at 300 rpm. The culture was then centrifuged for 5 minutes at 16,100×g to pellet the cells, and the phage-containing supernatant recovered. Then 300 µL of 2.5 M NaCl/20% PEG was added to the supernatant and the mixture was incubated at 4°C for 1 hour to precipitate the phage. The phage was pelleted by centrifugation at 20,817×g for 10 minutes at 4°C. The liquid supernatant was discarded and the phage pellet resuspended in 150 µL PBS. The Qiagen QIAprep Spin M13 kit (#27704) was then used to purify ssDNA from the phage as per the manufacturer’s directions. The absorbance at 260 nm of the ssDNA sample was measured using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific) and converted to a concentration using the relation 1.0 A_260nm = 33 ng/µL.

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Table 2. Potential adaptive amino acid substitutions in TEM-1 identified from genetic selections for tazobactam resistance codon substitutions.

https://doi.org/10.1371/journal.pone.0052031.t002

Site-directed PFunkel Mutagenesis using a ssDNA Template

All steps were performed in a pre-programmed Eppendorf Mastercycler personal thermocycler. A mutagenic oligo (5′-gacaccacgatgcatgcagcaatggc) encoding a c542a mutation in the bla gene was phosphorylated in a 50 µL reaction containing 1X T4 PNK buffer, 1 mM ATP, 5 mM DTT, 3.0 uM oligo and 10 units T4 PNK. The reaction was incubated at 37°C for 1 hour, and the enzyme inactivated at 65°C for 20 minutes.

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Figure 3. Tazobactam resistance of selected alleles.

The increase in ampicillin or piperacillin resistance is reported as the fold increase (over TEM-1) in the minimum inhibitory concentration (MIC) of the antibiotic in the presence of 6 µg/ml tazobactam. MIC assays performed in √2-fold increments of antibiotic concentration. Median MIC values of three replicates were used. Data for all replicates is in Tables S6 and S7.

https://doi.org/10.1371/journal.pone.0052031.g003

The PFunkel reaction was prepared in a 0.5 mL eppendorf tube containing 1X PfuTurbo Cx hotstart DNA polymerase buffer, 10 mM DTT, 0.5 mM NAD+, 0.2 mM dNTPs, 1 µL of the kinase reaction, 1 µg (0.75 pmol) of dU-ssDNA template, 2.5 units PfuTurbo Cx hotstart DNA polymerase, and 200 cohesive end units Taq ligase in a total volume of 100 µL. The free Mg²⁺ concentration should be maintained between 0.5–2.5 mM since low concentration reduces polymerase fidelity while high concentration leads to nonspecific annealing of oligos [16]. The volume of kinase reaction added should therefore be minimized to maintain Mg²⁺ concentration in the mutagenesis reaction close to the 2 mM Mg²⁺ provided in the 1X polymerase buffer. The following denaturation/annealing/extension/ligation steps were performed: 95°C for 3 min, 55°C for 90 sec, 68°C for 15 min and 45°C for 15 min. Then 3.8 pmol of oligo P320 (5′-P-gcagaaattcgaaagcaaattcgac) was added and one more cycle of 95°C for 30 sec, 55°C for 45 sec, 68°C for 10 min and 45°C for 15 min was performed. Then 10 units of UDG and 30 units of ExoIII were added and incubated at 37°C for 1 hr followed by an inactivation step at 70°C for 20 min.

Five µL of the unpurified reaction was used to directly transform 100 µL of DH5α chemically competent cells [13]. The entire transformation was plated on an LB-agar plate with 50 µg/mL Spec and incubated overnight at 37°C. To obtain more transformants, the remaining DNA was purified using the Zymo DNA Clean & Concentrator kit according to the manufacturer’s instructions and eluted in 15 µL of 1X EB. One uL was electroporated into 50 µL DH5α electrocompetent cells and then incubated with SOC recovery media for 1 hr at 37°C with shaking at 250 rpm. The transformation was plated on LB-agar with 50 µg/mL Spec and incubated overnight at 37°C.

For the experiments of Table S2, the reaction was scaled down to 200 ng template and 20 µl volume.

Multi-site PFunkel Mutagenesis using a ssDNA Template

Four oligos were designed to introduce NNN random bases at codon positions 42, 104, 182, and 238 in the bla gene with respective sequences: 5′-gatcagttgggtnnncgagtgggttac, 5′-gaatgacttggttnnntactcaccagtcac, 5′-cgtgacaccacgnnncctgcagcaatg, 5′-aaatctggagccnnngagcgtgggtct. These oligos were combined in equimolar amounts and phosphorylated in a 50 µL reaction containing 1X T4 PNK buffer, 1 mM ATP, 5 mM DTT, 6.0 µM total oligo and 10 units T4 PNK. The reaction was incubated at 37°C for 1 hour, and the enzyme inactivated at 65°C for 20 minutes.

The annealing reaction was prepared in a 0.5 mL eppendorf tube containing 1X PfuTurbo Cx hotstart DNA polymerase buffer, 2 uL of kinase reaction, and 1 ug of pSkunk3-bla ssDNA template in a total volume of 77 uL. The annealing was performed by heating to 95°C for 3 min, then 55°C for 10 min, and holding at 55°C.

Meanwhile, in a separate PCR tube, 1X PfuTurbo Cx hotstart DNA polymerase buffer and 2.75 units of PfuTurbo Cx hotstart DNA polymerase were combined in a total volume of 5.5 µL. The hotstart polymerase was heat activated by heating to 95°C for 3 min.

After the annealing step, 10 mM DTT, 0.5 mM NAD+, 0.2 mM dNTPs, 5 µL of the activated polymerase solution, and 200 cohesive end units Taq ligase was added bringing the total volume to 100 µL. The reaction was mixed by slowly and gently pipetting up and down. Extension and ligation of the mutant strand was performed at 65°C for 15 min and 45°C for 15 min. A total of 3.8 pmol of oligo P320 was added and one more cycle of 95°C for 30 sec, 55°C for 45 sec, 65°C for 10 min and 45°C for 15 min was performed. Five units of UDG and 2 units of ExoIII were added and the mixture was incubated at 37°C for 30 min followed by an inactivation step at 70°C for 20 min. The DNA was then purified using the Zymo DNA Clean & Concentrator kit according to the manufacturer’s instructions and eluted in 15 µL of DI water. This volume was vacuum concentrated down to 1–2 µL, electroporated into 50 µL DH5α electrocompetent cells and then incubated with SOC recovery media for 1 hr at 37°C with shaking at 250 rpm. The entire volume was then plated on a Nalgene Bioassay dish (D4803; 245 mm×245 mm×25 mm) containing LB-agar with 50 µg/mL Spec and incubated overnight at 37°C.

For the experiments of Table S2, the reaction was scaled down to 200 ng template and 20 µl volume.

Comprehensive Codon Mutagenesis by PFunkel using a ssDNA Template

All steps were performed in a pre-programmed Eppendorf Mastercycler personal thermocycler. Equimolar amounts of 287 different mutagenic oligos were combined in a single tube at a total oligo concentration of 100 µM. The oligos were phosphorylated in a 50 µL reaction containing 1X T4 PNK buffer, 1 mM ATP, 5 mM DTT, 0.038 µM oligos and 10 units T4 PNK. The reaction was incubated at 37°C for 1 hour, and the enzyme inactivated at 65°C for 20 minutes.

The PFunkel reaction was prepared in a 0.5 mL eppendorf tube containing 1X PfuTurbo Cx hotstart DNA polymerase buffer, 10 mM DTT, 0.5 mM NAD+, 0.2 mM dNTPs, 1 µL of the kinase reaction, 1 µg (0.75 pmol) of dU-ssDNA template, 2.5 units PfuTurbo Cx hotstart DNA polymerase, and 200 cohesive end units Taq ligase in a total volume of 100 µL. The following denaturation/annealing/extension steps were performed: 95°C for 2 min, 15 cycles of 95°C for 30 sec, 55°C for 45 sec, and 68°C for 6.5 min. At the 95°C step of cycles 6 and 11, 1 µL of the kinase reaction was added and mixed in by stirring with the pipette tip. The reaction was then incubated at 45°C for 15 min for ligation to occur. Then 3.8 pmol of oligo P320 (5∶1 molar ratio oligo to template) was added and one more cycle of 95°C for 30 sec, 55°C for 45 sec, and 68°C for 10 min was carried out. The reaction was again incubated at 45°C for 15 min. Then 10 units of UDG and 30 units of ExoIII were added and incubated at 37°C for 1 hr followed by an inactivation step at 70°C for 20 min. The DNA was then purified using the Zymo DNA Clean & Concentrator kit according to the manufacturer’s instructions and eluted in 15 µL of DI water. This volume was then vacuum concentrated down to 1–2 µL. For CCM-1 the DNA was electroporated into 50 µL DH5α electrocompetent cells and then incubated with SOC recovery media for 1 hr at 37°C with shaking at 250 rpm. The entire volume was then plated on a Nalgene Bioassay dish (D4803; 245 mm×245 mm×25 mm) containing LB-agar with 50 µg/mL Spec and incubated overnight at 37°C. For CCM-2, the DNA was used to transform NEB 5-alpha F’I^q competent cells as per the manufacturer’s instructions, and then plated on a Nalgene Bioassay dish containing LB-agar with 50 µg/mL Spec, 15 µg/mL tetracycline, and 2 w/v% glucose.

454 GS FLX High-throughput Sequencing

Transformants were recovered from agar plates with LB broth, and plasmid DNA recovered using the Qiagen QIAprep Spin Miniprep kit (27106). The plasmid DNA was linearized by restriction endonuclease digestion with NdeI. PCR amplicons of each of the three bla libraries were created using Titanium Lib-A fusion primers that included a 10-base MID barcode. Each 25 µL PCR reaction had 1–2 ng linearized template DNA, 0.4 µM each primer, 200 µM each dNTP, 1X HF Phusion buffer, and 2 units Phusion high-fidelity polymerase. Cycler conditions were 98°C for 30 sec, 30 cycles of 98°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec, and then 72°C for 5 min. PCR products were visualized on an ethidium bromide 1% agarose gel, and then gel purified using the QIAquick Gel Extraction Kit (28706). Amplicons were furthered purified using the Agencourt AMPure XP PCR Purification kit (A63880), to remove short DNA fragments, primers, and primer dimers. DNA concentration was determined using the Quant-iT Picogreen dsDNA Assay kit (P7589). Amplicons from each sub-library were diluted to 1E9 molecules/µL in 1X TE, equal volumes pooled together and then further diluted to 1E7 molecules/µL in DI water. 454 sequencing was performed by Tufts University Core Facility on a Roche 454 GS FLX+ instrument. The sequencing data was then analyzed using the Galaxy open web-based platform [17]–[19] and custom Matlab scripts.

Identification of Adaptive Codon Substitutions for Tazobactam Resistance in TEM-1

Library CCM-2 was plated at a density of about 500 CFU/cm² (non-selective conditions) on LB-agar plates supplemented with 50 µg/ml Spec, 300 µM IPTG, 100 µg/ml ampicillin and 4 or 6 µg/ml tazobactam. Plates were incubated at 37°C for 17 hours. The tazobactam concentration chosen was 1.3 or 2-fold higher than the concentration at which cells bearing wildtype TEM-1 could grow effectively. Large colonies on the plates were chosen at random for sequencing.

Selected single base mutations were re-introduced into TEM-1 by site-directed PFunkel mutagenesis on the 20 µl volume scale. The MIC for ampicillin and piperacillin of the mutants was assessed with and without 6 µg/ml tazobactam by spotting 10⁴ CFU on Mueller-Hinton agar plates containing 50 µg/ml Spec, 300 µM IPTG, and √2-fold increments of either ampicillin or piperacillin. Plates were incubated at 37°C for 12 hrs.

Preparation of Uracil-containing dsDNA Template for Phage-less PFunkel

A 10 mL LB culture of CJ236 cells with the pSkunk3-bla plasmid was incubated overnight at 37°C with shaking at 250 rpm. Plasmid dU-dsDNA was then isolated using the Qiagen QIAprep Spin Miniprep kit (27106) and the concentration quantified using a Nanodrop ND-1000 spectrophotometer.

Site-directed PFunkel Mutagenesis using a Plasmid dsDNA Template

All steps were performed in a pre-programmed Eppendorf Mastercycler personal thermocycler. A mutagenic oligo (5′-gacaccacgatgcatgcagcaatggc) encoding a c542a mutation in TEM-1 was phosphorylated in a 50 µL reaction containing 1X T4 PNK buffer, 1 mM ATP, 5 mM DTT, 1.5 µM oligo and 10 units T4 PNK. The reaction was incubated at 37°C for 1 hour and the enzyme inactivated at 65°C for 20 minutes.

The PFunkel reaction was prepared in a 0.5 mL tube containing 1X PfuTurbo Cx hotstart DNA polymerase buffer, 10 mM DTT, 0.5 mM NAD+, 0.2 mM dNTPs, 1 µL of the kinase reaction, 1 µg (0.38 pmol) of dU-dsDNA template, 2.5 units PfuTurbo Cx hotstart DNA polymerase, and 200 cohesive end units Taq ligase in a total volume of 100 µL. The free Mg²⁺ concentration should be maintained between 0.5–2.5 mM since low concentration reduces polymerase fidelity while high concentration leads to nonspecific annealing of oligos [16]. The volume of kinase reaction added should therefore be minimized to maintain Mg²⁺ concentration in the mutagenesis reaction close to the 2 mM Mg²⁺ provided in the 1X polymerase buffer. The following denaturation/annealing/extension/ligation steps were performed: 95°C for 3 min, 55°C for 90 sec, 68°C for 15 min and 45°C for 15 min. Next, 10 units of UDG and 30 units of Exo III were added and the reaction was incubated at 37°C for 1 hr followed by an inactivation step at 70°C for 20 min. A total of 3.8 pmol of oligo P320 (5′-P-gcagaaattcgaaagcaaattcgac) was added and one more cycle of 95°C for 30 sec, 55°C for 45 sec, 68°C for 10 min and 45°C for 15 min was performed. The DNA was purified using the Zymo DNA Clean & Concentrator kit according to the manufacturer’s instructions and eluted in 15 µL of DI water. This solution was vacuum concentrated down to 1–2 µL, electroporated into 50 µL DH5α electrocompetent cells, which were incubated with SOC recovery media for 1 hr at 37°C with shaking at 250 rpm. The transformation was plated on LB-agar with 50 µg/mL Spec and incubated overnight at 37°C.

Multi-site PFunkel Mutagenesis using a Plasmid dsDNA Template

Four oligos were designed to introduce the four mutations A42G, E104K, M182T, and G238S in the bla gene with respective sequences: 5′-gatcagttgggtgga cgagtgggttac, 5′-ctcagaatgacttggttaagtactcaccagtcacag, 5′-gtgacaccacgacgcctgcagcaatggcaacaac, 5′-gctgataaatctggagccagtgagcgtgggtctcg. These oligos were combined in equimolar amounts and phosphorylated in a 50 µL reaction containing 1X T4 PNK buffer, 1 mM ATP, 5 mM DTT, 3 µM total oligo and 10 units T4 PNK. The reaction was incubated at 37°C for 1 hour and the enzyme inactivated at 65°C for 20 minutes.

The annealing reaction was prepared in a 0.5 mL eppendorf tube containing 1X PfuTurbo Cx hotstart DNA polymerase buffer, 2 µL of kinase reaction, and 1 µg of dU-dsDNA template in a total volume of 77 µL. The annealing was performed by heating to 95°C for 3 min, then 55°C for 10 min, and holding at 55°C.

Meanwhile, in a separate PCR tube, 1X PfuTurbo Cx hotstart DNA polymerase buffer and 2.75 units of PfuTurbo Cx hotstart DNA polymerase polymerase were combined in a total volume of 5.5 µL. The hotstart polymerase was heat activated by heating to 95°C for 3 min.

After the annealing step, 10 mM DTT, 0.5 mM NAD+, 0.2 mM dNTPs, 5 µL of the activated polymerase solution, and 200 cohesive end units Taq ligase was added bringing the total volume to 100 µL. The reaction was mixed by slowly and gently pipetting up and down. Extension and ligation of the mutant strand was performed at 65°C for 15 min and 45°C for 15 min. Five units of UDG and 2 units of ExoIII were added and the mixture was incubated at 37°C for 1 hr followed by an inactivation step at 70°C for 20 min. A total of 3.8 pmol of oligo P320 was added and one more cycle of 95°C for 30 sec, 55°C for 45 sec, 68°C for 10 min and 45°C for 15 min was performed. The DNA was purified using the Zymo DNA Clean & Concentrator kit according to the manufacturer’s instructions and eluted in 15 µL of DI water. This solution was vacuum concentrated down to 1–2 µL, electroporated into 50 µL DH5α electrocompetent cells and then incubated with SOC recovery media for 1 hr at 37°C with shaking at 250 rpm. The transformation was plated on LB-agar with 50 µg/mL Spec and incubated overnight at 37°C.

Limitations of Kunkel Mutagenesis

We sought to minimize the occurrence of wild-type sequences in Kunkel mutagenesis, which has been reported to be as high as 30–50% [11]. We postulated that wild-type sequences arise for several reasons. First, the low operating temperature of the second strand synthesis step allows any contaminating short DNA fragments in the single stranded DNA prep, termed “junk” DNA, to prime the single stranded DNA. Such synthesis can either create a wild-type double stranded product or poison a mutation-bearing product by creating reaction side-products that possess a nick or a displaced strand [20]. Additionally, at lower temperatures, the mutagenic oligos are more prone to anneal non-specifically to the template. Such reaction side-products that are not in the cccDNA form are prone to degradation by cellular nucleases, removing the mutation. The presence of junk DNA is apparent from DNA gels of reaction products in which no mutagenic oligonucleotides were added, yet higher molecular weight products are produced [21]. Another postulated reason for the high occurrence of wild-type sequences in Kunkel mutagenesis is the repair of the mutation by mismatch-repair machinery or repair of the uracil-containing template strand in the cell after transformation.

Overview of PFunkel Mutagenesis using a ssDNA Template

The in vitro reaction steps of PFunkel mutagenesis (Figure 1, Table S1) are designed to eliminate products other than the desired mutated cccDNA plasmid molecules, resulting in high mutational efficiencies. Other than the initial kinase reaction to phosphorylate the mutagenic oligonucleotides, all reaction steps are conveniently performed in the same tube with no DNA purification required except as an optional final step to improve transformation efficiency. PFunkel is conveniently performed in a thermocycler.

Uracil-containing ssDNA is produced by propagating phagemid DNA containing the DNA to be mutated in an E. coli dut-1 ung-1 host, then infecting the culture with M13 helper phage and harvesting ssDNA from the resulting phage particles. E. coli dut-1 ung-1 strains express a heat-sensitive dUTPase that has 5% of wildtype activity at 25°C but <1% at 37°C, and are deficient in uracil DNA glycosylase activity [22]. The result of these mutations is the accumulation of high levels of intracellular dUTP that becomes incorporated in DNA in place of dTTP during DNA synthesis and is not removed due to the lack of UDG activity. ssDNA preparation takes only a day and requires no special laboratory equipment or highly specialized training [21].

To minimize second strand synthesis originating from junk DNA annealing to the uracil-containing ssDNA template, we shifted the operating temperature from the 25–37°C to 55–95°C. This operating temperature required a high-fidelity thermostable polymerase capable of using a uracil-containing template. Additionally, a polymerase lacking strand displacement activity would be advantageous for creating multiple mutations simultaneously at different sites in a gene. The only commercially available polymerase that met these criteria was PfuTurbo Cx hotstart DNA polymerase (Agilent), a variant of Pfu polymerase with the V93Q mutation [23]. This mutation inactivates the uracil-binding pocket of the enzyme that would normally cause it to stall at uracil bases. At ≤68°C PfuTurbo Cx hotstart DNA polymerase does not strand-displace but still maintains significant polymerase activity [24]. Taq ligase was chosen due to its effectiveness in ligating DNA nicks, robust activity from 45°C to 65°C, and ability to withstand many rounds of temperature cycling.

In order to create the dsDNA product with the designed mutation on both strands, an excess of a ‘reverse’ oligonucleotide that anneals outside of the gene on the newly created mutant strand is added, such that it primes synthesis of a new complementary strand that encodes the desired mutations and displaces the uracil-containing template. Treatment with UDG acts to excise the uracil bases from the original template strand leaving apyrimidinic (AP) sites. Treatment with exonuclease III (ExoIII), which has both AP-site endonuclease and 3′->5′ exonuclease activity [25], acts to create nicks at the AP sites and then digests the template strand at the nicks and from any 3′ end in the context of dsDNA.

Site-directed PFunkel Mutagenesis

A mutagenic oligonucleotide encoding a c542a (P183H in the protein) mutation in the TEM-1 ß-lactamase (TEM-1) gene was first 5′ phosphorylated in a kinase reaction. The phosphorylated oligo was then combined with the ssDNA uracil-containing template in molar ratio of 4∶1 together with the polymerase and ligase. The incubation temperatures were cycled to perform a denaturing, annealing, extension, and ligation step to complete the mutated second strand and seal the nick. A second primer that annealed to the new strand outside the gene was added to the reaction, and the denaturing, annealing, extension, and ligation steps were repeated. Exo III and UDG were then added to the reaction to remove the template and undesired side-products. All steps for this procedure took about 3 hrs to complete.

A transformation of 5 µL of the unpurified reaction with 100 µL of chemically competent cells yielded over 1000 transformants, illustrating that DNA purification is not necessary. The remaining DNA was purified using a spin column and 1/15^th of the product was electroporated into electrocompetent DH5α E. coli yielding 533,000 transformants. Sequencing of the TEM-1 gene from 23 colonies showed that all 23 (100%) contained the c542a mutation encoded by the oligo. No undesired mutations were observed. We further substantiated the high mutational efficiency of our method using eleven different oligos encoding either a 1 or 2 base substitution at different locations of the gene (Table S2).

Multi-site PFunkel Mutagenesis

Existing methods for site-directed mutagenesis at multiple distal sites simultaneously either have complex and multi-step procedures or have not been demonstrated to be efficient enough for library construction [5], [26]. PFunkel was designed in part to allow efficient construction of libraries in which site-saturation mutagenesis (or any user-defined mutational composition) can be performed at multiple sites simultaneously in a single reaction.

For simultaneous introduction of mutations at multiple distant sites in a gene, the basic PFunkel protocol is modified to increase the frequency of multiple mutations. The polymerase is added after the annealing of the mutagenic oligos. The rationale for the delayed addition of polymerase is to prevent a bias for mutations that result from oligos that anneal efficiently. DNA synthesis from such early annealing oligos might proceed to regions of the gene where other oligos are intended to anneal before the oligos for those locations have a chance to anneal, thus decreasing the frequency of multiple mutations in the resulting transformants. We also reduce the extension temperature to 65°C, to better ensure that PfuTurbo Cx hotstart DNA polymerase does not strand displace. Strand displacement of a strand created from one mutagenic oligo by a strand being synthesized starting from a second mutagenic oligo would reduce the frequency of multiple mutations. We do not know whether the shift of the extension temperature from 68°C to 65°C provides any benefit as we have not performed any direct comparison.

To demonstrate multiple-site mutagenesis using PFunkel, we synthesized four mutagenic oligos designed to create site-saturation libraries of four codons in different regions of the TEM-1 gene simultaneously. The oligos encoding NNN at codon positions A42, E104, M182, and G238 were combined with the ssDNA template such that each oligo was present in an oligo to template molar ratio of 4∶1. Electroporation of the entire reaction product after spin column purification yielded 5.8 million transformants. Sequencing of the TEM-1 gene of 10 colonies showed that 7 variants had mutations at all 4 designated codon positions, 2 had mutations at 3 positions, and 1 had mutations at 2 positions (Table S3). Twenty-nine of the 35 codon substitutions were unique and no undesired mutations were observed. We further substantiated multi-site PFunkel by constructing 11 different double, triple, or quadruple mutants at 73% efficiency (Table S2). The error rate for single or multi-site PFunkel mutagenesis was ∼5×10⁻⁵, higher than expected based on the error rate of PfuTurbo Cx in a PCR reaction (Text S1).

Comprehensive Codon Mutagenesis

We next used PFunkel to create a library designed to encompass all possible single codon substitutions in the TEM-1 gene (287 codons x 63 possible codon substitutions at each codon = 18,081 desired mutants). We did not desire library members with more than one codon substituted. Such a library is the equivalent of performing site-saturation mutagenesis at all positions in the gene simultaneously. The advantage of PFunkel is that one does not have to perform 287 separate mutagenesis reactions or 287 separate gene syntheses to create this library. The library would also be much closer to a true random mutagenesis library than one created by error prone PCR, which is biased towards certain base substitutions made by the polymerase and certain amino acid substitutions accomplished by single base mutations.

The 287 degenerate mutagenic oligos (one for each of the 287 codons to be mutated) were designed in silico using a Matlab script (Figure S1). The oligos were purchased in desalted 96-well format using machine-mixed degenerate bases and pooled. To minimize the occurrence of multiple mutations, the total oligo to ssDNA template ratio was kept low (1∶20), which makes two oligos annealing to the same ssDNA template unlikely. To increase the yield and efficiency of the reaction, we implemented a cycling reaction of denaturing, annealing, and extension to allow multiple chances for each oligo to productively anneal. Fifteen cycles were performed with additional oligos spiked in at the 6^th and 11^th cycle. Additional cycles and oligo additions can be performed if larger libraries are desired. Our scheme is analogous to that of a discontinuous fed batch reactor – a reaction strategy to minimize undesirable side products that occur with a high concentration of one of the reactants [27].

Although in principle the library could be created in a single tube, we divided the library into thirds corresponding to each 1/3 of the gene in order to facilitate characterization of the library by 454-GS-FLX Titanium sequencing, which has a read length of ∼400 bp for the sequencing of amplicons from pools of DNA. Transformation of the entire reaction product yielded ∼500,000 transformants for each library. Sequencing of 30 members of each library indicated that the libraries mostly consisted of single codon substitutions (87%) with the remainder being wildtype (13%) (Table 1). No clones with multiple mutations were observed. Two of the sequences contained a single mutation outside the region subjected to mutagenesis, which we attribute to polymerase error. We postulate that additional rounds of cycling and mutagenic oligo addition would lower the fraction of wild-type sequences closer to the theoretical minimum of 1.6% (i.e. 1/64 of the NNN containing oligos encode the wildtype codon). These three libraries collectively were named CCM-1.

We extensively analyzed CCM-1 using 454-GS-FLX Titanium sequencing. Our analysis indicates that 96–97% of the 18,081 desired codon substitutions are present in the library, and ≥97% of library members with a codon substitution contain only one codon substitution (Table 1, Text S1). The frequency of codon substitutions observed as a function of gene position shows that a few positions were hotspots for substitutions and that the frequency of codon substitutions has a broad distribution (Figure 2). Presumably the occurrence of hotspots reflects the suitably of the respective oligos for this mutagenesis technique. Codon substitutions with a single bp change were observed at about twice the expected frequency, and this comes at the expense of fewer codon substitutions with three bp changes (Table 1). A portion of the bias towards single base substitutions is likely due to polymerase errors during library construction, polymerase errors during the PCR-based amplicon preparation for sequencing, and 454 sequencing errors, all of which would be primarily single base substitutions. The remainder of the bias may reflect the increased mismatch between the mutagenic oligo and the template for codon substitutions with three mutated bases. Still, although codon substitutions with three bp changes may be somewhat disfavored, we observed 95% of the 7749 designed 3-bp change codon substitutions in the 454 sequencing results (Table 1). Since 454 sequencing errors with three bp changes in a codon are likely very rare, we believe most if not all of these 3-base substitutions are present in the library.

Identification of Adaptive Codon Substitutions in TEM-1 that Confer Increased Tazobactam Resistance with a Single Amino Acid Substitution

An extensive knowledge of the possible molecular determinants of bacterial resistance to b-lactam antibiotics and ß-lactamase inhibitors would inform the development and implementation of new antibiotics and inhibitors. We identified adaptive codon substitutions in TEM-1 conferring increased resistance to the ß-lactamase inhibitor tazobactam, which is used clinically in combination with the extended spectrum b-lactam antibiotic piperacillin in the drug Tazocin/Zosyn. We identified these adaptive mutations from library CCM-2– a second comprehensive codon mutagenesis library we constructed that lacked the oligonucleotide-derived bias for G’s in the substituted codon observed in CCM-1 (see Text S1, Figure S2). We subjected CCM-2 to a selection for an increase in resistance to tazobactam. Under the selective conditions, wildtype survived at a frequency of about 10⁻³. Sequencing of 279 colonies revealed 120 unique non-wildtype sequences. Since any particular amino acid substitution is relatively rare in the library, we used the criteria that an amino acid substitution had to be observed twice for us to categorize it as potentially adaptive in nature.

The set of these potentially adaptive substitutions (Table 2) overlapped one (M69L) but not other mutations previously found in alleles that increase tazobactam resistance, most notably R244S and N276D [30]. In addition, we identified 18 new, potentially adaptive amino acid substitutions, the most prevalent of which were 8 different amino acid substitutions at Y105 and the S235T mutation. The Y105 S/D/N and S235T mutations can occur with a single base change and are the most likely to appear naturally. We introduced these four mutations by single base substitution into TEM-1 and compared the ampicillin, piperacillin, and tazobactam resistance of these alleles to previously known tazobactam resistance alleles (Figure 3). We find that all four provide higher resistance to ampicillin in the presence of tazobactam than R244S and N276D, suggesting that our selection was too strong to identify R244S and N276D. The Y105N, Y105S, and S235T alleles show significant inhibitor resistance for both ampicillin and piperacillin hydrolysis – at or above that of the M69L allele, which is the most resistant allele observed to date for the piperacillin/tazobactam combination [30]. We predict that Y105N, Y105S, and S235T have the potential to emerge in the clinic. Their non-emergence to date, and the fact that they were not identified in previous selections for tazobactam resistance performed on error prone PCR libraries [31] may reflect the fact that the required base substitutions are not as common as the base substitutions for previously identified tazobactam resistance mutations. We speculate that we readily identified these mutations because PFunkel provides a less biased and much more comprehensive library of mutations than error prone PCR.

PFunkel Mutagenesis using a dsDNA Template

The methods described above require that the gene targeted for mutation is in a phagemid (a plasmid containing the f1 phage origin) and require the production of phage particles from which the dU-ssDNA template is isolated. Although preparation of such a template is straightforward, we sought to expand the method to be applicable to any plasmid and to simplify the protocol by eliminating the need for phage entirely. Phage-less PFunkel (Figure S3, Table S5) achieves this by utilizing a dU-dsDNA plasmid template. After the mutation-containing second strand synthesis, UDG and ExoIII are added to degrade both strands of the dU-dsDNA template. The newly-synthesized, circular ssDNA is then converted to dsDNA using the reverse oligonucleotide. Like PFunkel using a ssDNA template, the reaction can be performed in a single tube using a thermocycler.

We tested PFunkel using a dsDNA template for site-directed mutagenesis and multiple-site mutagenesis. For site-directed mutagenesis (using the c542a mutagenic primer), we obtained 708,000 transformants, and 10 of 10 randomly selected colonies had the desired mutation and no undesired mutations. For multiple-site mutagenesis, we attempted to create four specific mutations at distant sites in the gene. We obtained 445,000 transformants. Four of 10 colonies had all four mutations, the remainder either were wildtype (5 colonies) or had less than 4 mutations (1 colony). We speculate that the apparent lower efficiency of multi-site mutagenesis using a dsDNA template may result from the difficulty in getting all four primers to simultaneously anneal to a dsDNA template (as opposed to a ssDNA template) or difficulty in degrading the dsDNA template. Although we have not tested comprehensive codon mutagenesis using a dsDNA template, we believe it is feasible.