Recombination and Selection Can Remove blaTEM Alleles from Bacterial Populations

The overall abundance of blaTEM alleles in bacterial populations has caused multiple blaTEM alleles to coexist in single strains of bacteria (1, 4-7, 9-11, 15, 16), which has created the opportunity for recombination to occur among blaTEM alleles. The occurrence of recombination between two blaTEM alleles on a single plasmid strongly resembles gene conversion (14) and will cause the fragment between the recombination sites to be removed if the alleles are oriented in the same direction (3). This will typically result in the removal of one blaTEM allele from the plasmid while the remaining allele will usually be a chimera (Fig. ​(Fig.1).1). The potential for the removal of one blaTEM allele from a plasmid creates a situation in which closely related alleles may compete directly for retention in bacterial plasmids. While the blaTEM allele that is left on a plasmid following recombination is random, subsequent selection is not. Therefore, the combined effects of recombination and selection could result in the presence of a single allele on each copy of the plasmid and the removal of one of the alleles entirely from the population. FIG. 1. Schematic model of the recombination occurring in plasmid containing two regions of high homology. The dashed line marks the region containing two blaTEM genes (TEM-X and TEM-Y) where recombination takes place (A). As a result, two circular DNA molecules ... To experimentally investigate whether allelic competition occurs in an environment with potential for both recombination and selection, we cloned the two blaTEM alleles blaTEM-1 and blaTEM-12 into the pAC3 vector (2) in the same orientation (Fig. ​(Fig.1A),1A), validated the construction by sequencing, and expressed them from wild-type E. coli K-12. We passaged these bacteria through L broth containing either 32 μg/ml ampicillin or 4 μg/ml ceftazidime. Every 24 h, the saturated cultures were diluted 100 times in fresh broth, and the remaining bacteria were used to isolate plasmid for quantitative PCR (qPCR) analysis. Each 25-μl qPCR reaction mixture contained 106 to 107copies (60 pg of DNA) of the plasmid, 1× Brilliant master mixture (Stratagene, La Jolla, CA), 200 nmol of each molecular beacon probe (Biosearch Technologies), and 900 nmol of each primer (Table ​(Table1).1). To measure the ratio between the origin of replication (Ori) and TEM alleles, recombination markers, and TET amplicons (Fig. ​(Fig.1),1), real-time PCR with TaqMan probes was performed. Each 25-μl qPCR reaction mixture contained 106 to 107copies of the plasmid DNA, 1× Brilliant multiplex master mixture (Stratagene, La Jolla, CA), 200 nmol of each TaqMan probe (Biosearch Technologies), and appropriate concentrations of each primer (Table ​(Table1).1). The specificity of all primers and probes for the appropriate sequence was verified. Real-time PCR experiments were performed on a Stratagene Mx3000 multiplex qPCR system, with the quantitative PCR setting. The cycling conditions were either 1 cycle of 10 min at 95°C, followed by 40 cycles of 30 s at 95°C, 45 s at 55°C, and 30 s at 72°C, or 1 cycle of 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 45 s at 58°C, for the assay using molecular beacon and TaqMan probes, respectively. The fluorescence signal was collected at the end of each annealing step, using appropriate filters. TABLE 1. DNA oligonucleotides used for qPCR assays Based on results from previous competition experiments (13) and enzymatic studies (8, 12), we predicted that for bacteria passaged through ampicillin, blaTEM-12 would be eliminated from the plasmid and population and that for bacteria passaged through ceftazidime, blaTEM-1 would be eliminated from the plasmid and population. When Escherichia coli K-12 carrying the pAC3/TEM-1_TEM-12 construct was cultured in the presence of 4 μg/ml ceftazidime, the complete removal of one allele from each copy of the plasmid present in the population usually occurred after six passages (Fig. ​(Fig.2A).2A). To determine whether the same allele remained on each copy of the plasmid or if both alleles were still present in the population, we performed single-nucleotide polymorphism (SNP)-specific qPCR as previously described (13) using the probes that distinguish between the wild type and the strain carrying the mutated blaTEM alleles (Fig. ​(Fig.2B).2B). We found that blaTEM-12 had swept completely through the bacterial population at the same rate with which recombination had removed one allele. We verified that result by sequencing and also found that the allele present after the experiment was identical in sequence to the allele originally inserted into the construct. To ensure that the blaTEM-12 allele was retained in the population as a result of recombination rather than the occurrence of a novel point mutation, creating new blaTEM-12 alleles, we performed qPCR using a set of probes to detect the silent mutation G238* in the blaTEM-12 allele which was introduced prior to cloning. We followed the frequency of that silent mutation through the population over the course of the experiments and found that its frequency corresponded exactly with the frequency of the R164S substitution (Fig. ​(Fig.2C).2C). These results show that blaTEM-12 did replace blaTEM-1 in the population via recombination. FIG. 2. Recombination of the pAC3TEM1-TEM12 plasmid in wild-type E. coli K-12 cultured in 4 μg/ml ceftazidime for 9 days (data are shown for a representative culture). On day 0, four independent cultures were inoculated in Luria broth supplemented with ... When we passaged E. coli K-12 carrying the pAC3/TEM-1_TEM-12 vector through ampicillin, we saw a result similar to that described above, except that the recombination removed one allele in fewer replicates than in ceftazidime (75% instead of 100%). As expected for the ampicillin passage, blaTEM-1 rather than blaTEM-12 went to fixation in the population (Fig. 3A and B). FIG. 3. Recombination of the pAC3TEM1-TEM12 plasmid in wild-type E. coli K-12 cultured in 32 μg/ml ampicillin. For experimental details, see the legend to Fig. ​Fig.2.2. (A) qPCR analysis of isolated plasmids using TaqMan probes. (B) SNP analysis ... To create controls for both of these sets of experiments, we passaged DH5α containing pAC3/TEM-1_TEM-12 through ceftazidime and ampicillin. We used DH5α because it contains the recA1 mutation, which inhibits the occurrence of recombination. For the DH5α experiments, we never observed the occurrence of recombination. This shows that the potential for recombination is necessary for the affect we observed (Fig. ​(Fig.44). FIG. 4. Recombination of the pAC3TEM1-TEM12 plasmid in DH5α cultured in 4 μg/ml ceftazidime. For experimental details see the legend to Fig. ​Fig.2.2. qPCR analysis of isolated plasmids using TaqMan probes. As an additional control, we also passaged K-12 carrying the pAC3/TEM-1_TEM-12 plasmid through broth containing tetracycline. Over the course of the experiment, we did not observe the occurrence of recombination in this control strain. These results show that the combined effects of recombination and selection can eliminate the cooccurrence of multiple alleles from a single plasmid, even when the alleles confer different resistance phenotypes. Natural plasmids are often larger and have a lower copy number than the constructs we introduced, which may reduce the frequency of recombination among similar alleles. Additionally, selection may be strong enough to maintain the multiple plasmidic alleles of a resistance gene if bacteria are exposed to multiple antimicrobials. However, these results indicate that competition may occur among plasmidic alleles and that resistance phenotypes may be eliminated from bacterial populations by the dynamics resulting from that competition.