Fig 1.
The AvcID system provides phage defense in liquid culture.
Growth curves for E. coli with active (pAvcID) or inactive (pAvcID*) after infection with T5 (A) or T7 (B) phage at varying multiplicities of infection (MOI). Data represents the mean ± SEM of three biological replicate cultures.
Fig 2.
Transcriptional shutoff leads to the degradation of avcI.
Shown are Northern blots of avcI RNA using a biotinylated probe complementary to avcI (top) and Western blots of AvcD-6xHis using anti-6xHis antibody (bottom) during rifampicin treatment (250 μg/mL) (A), spectinomycin treatment (200 μg/mL) (B), T5 infection (C), and T7 infection (D) at a MOI of 5.
Fig 3.
AvcD is activated by T5 and T7 phages.
In vivo abundance of dCTP (A) and dCMP (B) in an E. coli host carrying pAvcID or pAvcID* with its native promoter before and after infection of T5 (MOI = 5) or T7 phage (MOI = 5). Nucleotides were measured using UPLC-MS/MS and normalized to total protein. Data represents the mean ± SEM of three biological replicate cultures, Two-way ANOVA with Dunnett’s post-hoc test, and ns indicates not significant.
Fig 4.
AvcID reduces the functionality of T5 but not T7 phage.
Survival of E. coli encoding the indicated AvcID systems as measured by CFU after infection with T5 (MOI 0.1) (A) or T7 (MOI 0.01) (D). PFU quantification over time in cultures of pAvcID or pAvcID* containing cells infected with T5 (B) or T7 (E). Relative T5 (C) or T7 (F) genome abundance comparing E. coli expressing pAvcID or inactive AvcID* over time. Percent viable phage after infecting cells containing AvcID with T5 or T7 phages (G). Data represents the mean ± SEM of three biological replicate cultures.
Fig 5.
TEM of AvcID-induced Phage Defense.
Transmission Electron Microscopy (TEM) of T5 (A, B) or T7 (C) from E. coli host carrying pAvcID (A, C) or pAvcID* (B). Samples were negative stained with 1% (w/v) uranyl acetate. Scale bar 100 nm. All samples were analyzed in three biological replicates with similar results.
Fig 6.
Ung and AvcID do not function together to provide phage protection.
(A) Growth curves for E. coli MG1655 or Δung mutant with pAvcID or pAvcID* after infection with T5 phage at varying multiplicities of infection (MOI). Data represents the mean ± SEM of three biological replicate cultures. (B) Measurement of phage titer on WT E. coli MG1655 or Δung E. coli encoding either active or inactive AvcID system infected with T5 phage. Data represents the mean ± SEM of three biological replicate cultures. (C) Measurement of phage titer on WT E. coli MG1655 co-expressing Dut and either active or inactive AvcID system infected by T5 phage. Data represents the mean ± SEM of three biological replicate cultures.
Fig 7.
AvcID reduces the functionality of T7412 mutant phage.
Representative images of tenfold serial dilution plaque assays of T7WT (A), T7412 (B), or T7C74 (C) phages spotted on E. coli MG1655 expressing either active (top) or inactive (bottom) avcID system. Images are representative of three replicates. (D) CFU quantification of E. coli MG1655 over time in cultures indicated AvcID systems after infection with mutant T7412. (E) PFU quantification over time in cultures of indicated AvcID systems-containing cells infected with T7412. (F) Relative T7412 genome abundance comparing E. coli expressing pAvcID or inactive AvcID* over time. (G) Percent viable phage after infecting cells containing AvcID with T7412. Data represents the mean ± SEM of three biological replicate cultures. (H) Representative images of tenfold serial dilution plaque assays of T7WT (top) or T7412 (bottom) phages spotted on E. coli BL21 or BL21(D3E) expressing either active or inactive avcID system. Images are representative of three replicates.