Transcription of AAT•ATT triplet repeats in Escherichia coli is silenced by H-NS and IS1E transposition

Abstract

Background: The trinucleotide repeats AAT•ATT are simple DNA sequences that potentially form different types of non-B DNA secondary structures and cause genomic instabilities in vivo.

Methodology and principal findings: The molecular mechanism underlying the maintenance of a 24-triplet AAT•ATT repeat was examined in E. coli by cloning the repeats into the EcoRI site in plasmid pUC18 and into the attB site on the E. coli genome. Either the AAT or the ATT strand acted as lagging strand template in a replication fork. Propagations of the repeats in either orientation on plasmids did not affect colony morphology when triplet repeat transcription using the lacZ promoter was repressed either by supplementing LacI(Q)in trans or by adding glucose into the medium. In contrast, transparent colonies were formed by inducing transcription of the repeats, suggesting that transcription of AAT•ATT repeats was toxic to cell growth. Meanwhile, significant IS1E transposition events were observed both into the triplet repeats region proximal to the promoter side, the promoter region of the lacZ gene, and into the AAT•ATT region itself. Transposition reversed the transparent colony phenotype back into healthy, convex colonies. In contrast, transcription of an 8-triplet AAT•ATT repeat in either orientation on plasmids did not produce significant changes in cell morphology and did not promote IS1E transposition events. We further found that a role of IS1E transposition into plasmids was to inhibit transcription through the repeats, which was influenced by the presence of the H-NS protein, but not of its paralogue StpA.

Conclusions and significance: Our findings thus suggest that the longer AAT•ATT triplet repeats in E. coli become vulnerable after transcription. H-NS and its facilitated IS1E transposition can silence long triplet repeats transcription and preserve cell growth and survival.

Figure 1. Organizations of AAT•ATT repeats in both plasmids and chromosomal attB site and the papillation assays when in plasmid.

A) Organization of the AAT•ATT repeats in the lacZ gene in pUC18 plasmid and in the attB site of the genome; B) Papillation assays when the AAT•ATT repeats were subcloned in plasmids. (1) JM83 (pAAT₂₄); (2) JM83Δhns (pAAT₂₄); (3) JM83 (pATT₂₄); (4) JM83Δhns (pATT₂₄), which behaves similarly when a lacIQ plasmid was coexpressed, or 5% glucose was added in the LB plate (For clarity the data were not shown); (5) JM83 ΔstpA::cat (pAAT₂₄); (6) JM83ΔstpA::cat(pAAT₂₄) with 5% glucose; (7) JM83ΔstpA::cat(pATT₂₄); (8) JM83ΔstpA::cat (pATT₂₄) with 5% glucose; (9) W3110ΔstpA::cat (pAAT₂₄); (10) W3110ΔstpA::cat(pAAT₂₄) with 5% glucose; (11) W3110ΔstpA::cat(pATT₂₄); (12) W3110ΔstpA::cat(pATT₂₄) with 5% glucose; (13) AB1157 recF (pAAT₂₄); (14) AB1157 recF (pAAT₂₄) with 5% glucose; (15) AB1157 recF (pATT₂₄); and (16) AB1157 recF (pATT₂₄) with 5% glucose.

Figure 2. Characterization of the IS1E insertion positions.

A) a. Transposition of IS1E elements into plasmids, plasmid isolated from the healthy colonies, and b. Restriction digestion of the plasmid DNA with transposons. Lanes 1, molecular weight (pBR322/BstN1); Lane 2 and 3, pAAT₂₄ and pATT₂₄ without IS1E transposition, digested by PstI; Lane 4, pAAT₂₄, with IS1E transposition, digested by PstI; Lane 5, pAAT ₂₄ with IS1E transposition, digested by EcoR1;Lane 6 and 7, pAAT₂₄ with IS1E transposition, digested by PstI and EcoRI respectively; Lane 8 and 9 pAAT₂₄ with IS1E transposition, digested by using PvuII. B) Mapping the IS1E inserting elements in the promoter and the AAT repeats region by DNA sequencing, the positions of the mostly recognized sites were shown.

Figure 3. IS1E transposition into AAT•ATT repeats.

A) Plasmids of AAT orientation propagated in LB medium were recovered and digested by Pst I (lane 1), EcoR I (lane 2); and propagated in LB medium in the presence of IPTG, and were digested by PstI (lane 3), and EcoRI (lane 4); Plasmids of ATT orientation propagated in LB medium with or without IPTG, digested by Pst I (lane 5), EcoR I (lane 6); Pst I (lane 7 with IPTG induction) and EcoR I (lane 8 with IPTG induction). M1 and M2 are DNA molecular weights; B) Schematic illustration of the directions of IS1E transposition into the AAT•ATT repeats in light of the transcription of either AAT or ATT orientation.

Figure 4. The α–complementation assay for the role of IS1E insertions.

a) α-complementation by an AAT•ATT repeats free pAAT24IS1E-A plasmid, and b) α-complementation by pUC18 plasmid (For detail see text).

Figure 5. Effects of hns gene on the growth of AAT•ATT repeats-carrying strains in chromosome.

Significant filamentous cells were observed in JM83Δhns -AAT and JM83Δhns -ATT,but cannot be seen with JM83 -AAT and JM83 -ATT, nor be seen with JM83(pAAT₂₄), JM83(pATT₂₄), JM83Δhns (pAAT₂₄), JM83Δhns(pATT₂₄) and JM83(pUC18), JM83Δhns (pUC18) (data not shown).

Figure 6. A model illustrating the response of E.coli cell to the transcription of AAT•ATT repeats in plasmid and genome.

RNA transcription by using lacZ promoter opened the double stranded AAT•ATT repeats that could promote the repeats to form DNA secondary structures that may recruit histone-like proteins, H-NS, to the repeats DNA, causing depletion of this protein in the chromosome, which further resulting in DNA instability and/or cell death. Repression by H-NS deactivated the transcription while also facilitating IS1E transposion, the repeats transcription is therefore further silenced by the transposition, over time, the repeats might be able to be converted into mixed repeats by IS1E and eventually form “Junk DNA”.