Expression and Functional Analysis of the Argonaute Protein of Thermus thermophilus (TtAgo) in E. coli BL21(DE3)

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. colifabI 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.

Prokaryotic Argonaute Protein from Natronobacterium gregoryi Requires RNAs To Activate for DNA Interference In Vivo

“…Thus, expression of NgAgo in BL21(DE3) leads to the linearization of the pET28a-based expression plasmid in such a way that it cannot be recircularized and therefore is unable to replicate within cells. These data are also entirely consistent with our previous result that induction of pAgos (NgAgo, TtAgo, and RsAgo) expression induced the elimination of their expression plasmids ( 25 ).…”

“…Consistent with the observations obtained in Fig. 1 and our previous report ( 25 ), NgAgo induction with IPTG led to the elimination of its own expression plasmid, as no visible colony growth was observed on the LB+Kan+IPTG plates ( Fig. 2A , middle).…”

“…In our previous study ( 25 ), we showed that plasmid-based expression of either NgAgo or TtAgo (designated pET28a-NgAgo and pET28a-TtAgo) in E. coli BL21(DE3) resulted in rapid loss of their cognate expression vectors but had no adverse effect on the host cell genome. We hypothesized that this occurred via targeted endonuclease activity by NgAgo and TtAgo on their expression plasmids.…”

“…6D ). We generated and tested two mutants of NgAgo: (i) the double mutant NgAgoDM (D663A, D738A), which targets the DEDX active center of the NgAgo protein, constructed based on structural similarity with TtAgo and determined to be catalytically important in previous in vitro nuclease activity studies ( 6 ), and (ii) NgAgo (R40C), a single point mutation constructed and verified using the same method described in our previous in vivo loss-of-function mutation screen ( 25 ). Although NgAgoDM showed a decrease in protection compared with wild-type NgAgo, reducing PFU production by 1 order of magnitude, it still retained significant activity compared with control BL21(DE3) cells.…”

Detection of E. coli Bacteria in Milk by an Acoustic Wave Aptasensor with an Anti-Fouling Coating

“…The bacteria pellet was then resuspended in 1 mL of PBS, after which the solution's optical density at 600 nm was measured by the UV-Vis spectrometer UV-1600PC (VWR International, Mississauga, Canada). The CFU was calculated from the optical density according to the following papers [31][32][33]. For samples in PBS, the resuspension was diluted to the desired concentration.…”