Improving Salmonella vector with rec mutation to stabilize the DNA cargoes

Abstract

Background: Salmonella has been employed to deliver therapeutic molecules against cancer and infectious diseases. As the carrier for target gene(s), the cargo plasmid should be stable in the bacterial vector. Plasmid recombination has been reduced in E. coli by mutating several genes including the recA, recE, recF and recJ. However, to our knowledge, there have been no published studies of the effect of these or any other genes that play a role in plasmid recombination in Salmonella enterica.

Results: The effect of recA, recF and recJ deletions on DNA recombination was examined in three serotypes of Salmonella enterica. We found that (1) intraplasmid recombination between direct duplications was RecF-independent in Typhimurium and Paratyphi A, but could be significantly reduced in Typhi by a ΔrecA or ΔrecF mutation; (2) in all three Salmonella serotypes, both ΔrecA and ΔrecF mutations reduced intraplasmid recombination when a 1041 bp intervening sequence was present between the duplications; (3) ΔrecA and ΔrecF mutations resulted in lower frequencies of interplasmid recombination in Typhimurium and Paratyphi A, but not in Typhi; (4) in some cases, a ΔrecJ mutation could reduce plasmid recombination but was less effective than ΔrecA and ΔrecF mutations. We also examined chromosome-related recombination. The frequencies of intrachromosomal recombination and plasmid integration into the chromosome were 2 and 3 logs lower than plasmid recombination frequencies in Rec+ strains. A ΔrecA mutation reduced both intrachromosomal recombination and plasmid integration frequencies.

Conclusions: The ΔrecA and ΔrecF mutations can reduce plasmid recombination frequencies in Salmonella enterica, but the effect can vary between serovars. This information will be useful for developing Salmonella delivery vectors able to stably maintain plasmid cargoes for vaccine development and gene therapy.

Figure 1

Illustration of plasmids carrying intact or truncated tetA genes. Plasmids are not drawn to scale. (A) Plasmid pACYC184 carries an intact tetA gene (1191 bp), which is the source of all truncated tetA genes used in this study. Plasmid pYA4463 carries two copies of truncated tetA genes, truncated at the 5' or 3' ends as indicated, which results in a 466-bp direct tandem duplication (shown as open arrows). Plasmid pYA4590 has two similar copies of truncated tetA genes, resulting in 602 bp of repetitive sequence (shown as open arrows) separated by 1041-bp kan cassette. (B) Plasmid pYA4464 has a 3'tet truncated gene. Plasmid pYA4465 has a 5'tet truncated gene. There are 751 bp of common sequences (shown as open arrows) between the two truncated tetA genes. (C) Plasmid pYA4463 dimer is the intermolecular recombination product of two pYA4463 molecules. Plasmid pYA4590 dimer is the intermolecular recombination product of two pYA4590 molecules. Plasmid pYA4464-pYA4465 is the intermolecular recombination product of pYA4464 and pYA4465.

Figure 2

Strategies for measuring DNA recombination. (A) Truncated tetA genes. Two truncated tetA genes were derived from an intact tetA gene and its promoter (P). 5'tet, includes the tetA promoter and the 5' portion of tetA gene. 3'tet, consists of the 3' portion of the tetA gene. The overlapping region (between 5'tet and 3'tet) varies from 466 to 789 bp depending on the system. Homologous recombination can occur between the two truncated tetA genes at the overlapping region, leading to the formation of a functional tetA gene. (B) Intermolecular recombination. Each DNA molecule carries either 5'tet or 3'tet. A single crossover between the two molecules occurs at the regions of homology, and leads to a functional tetA gene. (C) Intramolecular recombination. The two truncated tetA genes were placed on one molecule in the same orientation. A single crossover between the regions of homology leads to a functional tetA gene.

Figure 3

Verification of plasmid recombination product by agarose gel separation. (A) Plasmid DNA was isolated from Tc^R clones derived from χ3761(pYA4463) and digested by XbaI and SalI. (B) Plasmid DNA was isolated from Tc^R clones of χ3761(pYA4590) and digested by KpnI and EcoRI. (C) Plasmid DNA was isolated from Tc^R or Tc^S clones of χ3761(pYA4464, pYA4465). The purified plasmids were digested with NcoI and BglII.

Figure 4

UV sensitivity of S. Typhimurium rec mutants. Log phase cultures of S. Typhimurium were diluted and spread on LB agar. Multiple dilutions were exposed to 254 nm UV in a dark room at each designated dose. Then the plates were wrapped with aluminum foil and placed at 37°C overnight. Surviving fractions were calculated and shown except ΔrecA strains χ9833 and χ9833(pYA5001), for which no survivors were recovered at any UV dose. wt: χ3761; ΔrecF: χ9070; ΔrecJ: χ9072; ΔrecA(RecA⁺): χ9833(pYA5002); ΔrecF(vector): χ9070(pYA5001); ΔrecF(Typhimurium RecF⁺): χ9070(pYA5005); ΔrecF(Typhi RecF⁺): χ9070(pYA5006). Survival of Rec⁺ strains [χ3761, χ9833(pYA5002), χ9070(pYA5005) and χ9070(pYA5006)] was significantly greater than survival of the Rec^- strains [χ9070, χ9072 and χ9070(pYA5001)] at the UV doses indicated (P ≤ 0.002; *).