Abstract
The prokaryotic Argonaute proteins (pAgos) have been reported to cleave or interfere with DNA targets in a guide-dependent or independent manner. It is often difficult to characterize pAgos in vivo due to the extreme environments favored by their hosts. In the present study, we expressed functional Thermus thermophilus pAgo (TtAgo) in E. coli BL21 (DE3) cells at 37 °C. Initial attempts to express TtAgo in BL21(DE3) cells at 37 °C failed. This was not because of TtAgo mediated general toxicity to the host cells, but instead because of TtAgo-induced loss of its expression plasmid. We employed this discovery to establish a screening system for isolating loss-of-function mutants of TtAgo. The E. coli fabI gene was used to help select for full-length TtAgo loss of function mutants, as overexpression of fabI renders the cell to be resistant to the triclosan. We isolated and characterized eight mutations in TtAgo that abrogated function. The ability of TtAgo to induce loss of its expression vector in vivo at 37 °C is an unreported function that is mechanistically different from its reported in vitro activity. These results shed light on the mechanisms by which TtAgo functions as a defense against foreign DNA invasion.
Highlights
Accepted: 25 March 2021Argonaute proteins are present in all three domains of life and are the subject of much recent attention for their ability to bind a nucleic acid guide sequence and subsequently cleave single-stranded DNA or RNA targets in vitro [1,2,3,4,5], interfering with DNA integrity [6] and gene expression [7]
As shown by spot plating assays (Figure 1A), the transformed cells grew well on LB medium supplemented with kanamycin without isopropyl β-D-thiogalactopyranoside (IPTG) induction; after the addition of IPTG into the medium, no colonies formed on plates with kanamycin selection
By spot plating assays (Figure 6A), measuring plasmid yields (Figure 6B) and CFU assay (Figure 6C) in parallel with pET28a-Thermus thermophilus pAgo (TtAgo) vector, we found that a C-terminal modification of TtAgo did not abolish its activity in E. coli, with only a slight decrease in its activity when compared with pET28a-TtAgo in the CFU assay
Summary
Argonaute proteins are present in all three domains of life and are the subject of much recent attention for their ability to bind a nucleic acid guide sequence and subsequently cleave single-stranded DNA or RNA targets in vitro [1,2,3,4,5], interfering with DNA integrity [6] and gene expression [7]. They can enhance homologous sequence-directed recombination by interacting with RecA [6], and play roles in DNA replication in vivo [7]. By mining the expanding repertoire of microbial genome sequences, many pAgo genes have been reported, such as Thermus thermophilus (TtAgo) [4,12,21], archaeon Pyrococcus furiosus (PfAgo) [1,22], archaeal organism Methanocaldococcus jannaschii (MjAgo) [5,23,24], CRISPR-associated Marinitoga piezophila (MpAgo) [11,25,26], Clostridium butyricum (CbAgo)
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