Genomic Recoding Broadly Obstructs the Propagation of Horizontally Transferred Genetic Elements

Abstract

Horizontally transferred genetic elements such as viruses and conjugative plasmids move DNA between organisms, increasing genetic diversity but destabilizing engineered biological systems. Here, we used a genomically recoded Escherichia coli strain lacking UAG stop codons and the recognition protein release factor 1 to study how an alternative genetic code influences horizontally transferred genetic element propagation. The alternative genetic code conferred resistance to multiple viruses (λ, M13, P1, MS2) at titers up to 10(11) PFU/ml and impaired conjugative plasmids (F and RK2) up to 10(5)-fold. By recoding UAG codons to UAA in viruses and plasmids, we restored viral infectivity and conjugative function. Propagating viruses on a mixed community of cells with standard and alternative genetic codes reduced viral titer, and over time viruses adapted to the alternative genetic code. This work demonstrates that altering the genetic code broadly obstructs the propagation of horizontally transferred genetic elements and supports the use of genomic recoding as a strategy to stabilize engineered biological systems.

Figure 1. The alternate genetic code obstructs viral infection

(A) Schematic depicting viral infection of cells with standard genetic codes or alternate genetic codes with no assigned meaning for the UAG codon. (B) Relative titers of viruses on strains +UAG+RF1, ΔUAG+RF1, and ΔUAGΔRF1. (C) Mutation analysis of 94 λ plaques isolated after recoding using MAGE. Colors represent number of mutations and the bar pattern represents proportion of mutants with UAG-to-UAA recoding in egrN or lgrQ. (D) Relative titers of λ phages with varying recoded loci (x-axis). (E) Relative titers of wild-type and recoded M13 phages infected on hosts with wild-type or partially recoded (Fpr) pF. For all relative titers, data are mean with standard deviation, n=3. “0” indicates zero plaque forming units (PFU)/mL. P-values are as follows: * is P ≤ 0.05, ** is P ≤ 0.01, *** is P ≤ 0.001, and **** is P ≤ 0.0001.

Figure 2. The alternate genetic code obstructs conjugation

Conjugation efficiency from donors with standard and alternate genetic codes (x-axis) to recipients with standard and alternate genetic codes (bars) for wild-type and recoded (A) pF and (B) pRK2 conjugative plasmids. “0” indicates transfer efficiency was below limit of detection of 1%. Data are mean with standard deviation, n=3. P-values are as follows: * is P ≤ 0.05, ** is P ≤ 0.01, *** is P ≤ 0.001, and **** is P ≤ 0.0001. (C) Mutation analysis of 96 pF variants isolated after recoding using MAGE and conjugation from +UAG+RF1 and ΔUAGΔRF1. (D) Mutation analysis of 96 pRK2 variants isolated after recoding using MAGE and conjugation to +UAG+RF1 or ΔUAGΔRF1. For mutation analysis, colors represent number of mutations and pattern represents mutants with UAG-to-UAA recoding in indicated genes.

Figure 3. Recoded organisms reduce viral population fitness in microbial communities and select for viral mutations that eliminate UAG codon use

(A) Schematic of microbial community assays. Phages are infected on a co-culture containing varying ratios of ΔUAG+RF1 and ΔUAGΔRF1, extracted the next day, and propagated on a co-culture with the same cell ratio. Viral populations of λ were quantified by infection on ΔUAG+RF1, and ability of phage MS2 to infect ΔUAGΔRF1 was assayed by plating on ΔUAGΔRF1 containing pFpr. (B) Titers of phage λ viral populations propagated on microbial communities containing cells with standard and alternate genetic codes. Lines are mean of 3 biological replicates for each population. (C) Location of mutations eliminating UAG codon usage in the MS2 genome (Calendar, 2006; Fiers et al., 1976). (D) Relative titers of wild-type and recoded MS2 (MS2rec2) phages infected on ΔUAGΔRF1 with pF or pFpr, which is required for phage infection. Data are mean with standard deviation, n=3. P-values are as follows: * is P ≤ 0.05, ** is P ≤ 0.01, *** is P ≤ 0.001, and **** is P ≤ 0.0001.