Data_Sheet_1_Ribosome inactivation by Escherichia coli GTPase RsgA inhibits T4 phage.pdf

IntroductionBacteria must combat phages, and myriad bacterial anti-phage systems have been discovered that reduce host metabolism, for example, by depleting energetic compounds like ATP and NAD+. Hence, these systems indirectly inhibit protein production. Surprisingly, direct reduction of ribosome activity has not been demonstrated to thwart phage.MethodsHere, by producing each of the 4,287 Escherichia coli proteins and selecting for anti-phage activity that leads to enhanced growth, we investigated the role of host proteins in phage inhibition.Results and discussionWe identified that E. coli GTPase RsgA inhibits lytic phage T4 by inactivating ribosomes.</p

Proteins whose production from pCA24N increased growth at low temperatures in the Δ<i>dinJ</i> Δ<i>Km</i>^<i>R</i> strain as identified by screening the complete ASKA overexpression library.

Proteins whose production from pCA24N increased growth at low temperatures in the ΔdinJ ΔKmR strain as identified by screening the complete ASKA overexpression library.</p

Mlc suppresses YafQ toxicity.

<p>Comparison of growth in LB medium for BW25113 <i>ΔdinJ ΔKm</i>^<i>R</i>/pCA24N (●) and BW25113 <i>ΔdinJ ΔKm</i>^<i>R</i>/pCA24N-<i>mlc</i> (♦) with 200 μM IPTG induction at 18°C (<b>A</b>) and 37°C (<b>B</b>). Data are averaged from three independent cultures, and one standard deviation is shown.</p

Gene inactivations that restore growth at 18°C.

<p>Gene inactivations that restore growth at 18°C.</p

Δ<i>dinJ</i> reduces metabolism only at 18°C.

<p>Comparison of metabolic activity via the Biolog assay for BW25113 wild-type (■), Δ<i>dinJ</i> Δ<i>Km</i>^<i>R</i> (●) and Δ<i>dinJ</i> Δ<i>yafQ</i> Δ<i>Km</i>^<i>R</i> (▲) at 18°C (<b>A</b>), 30°C (<b>B</b>), and 37°C (<b>C</b>). Data are the average of two independent cultures and one standard deviation is shown.</p

Δ<i>dinJ</i> reduces growth only at 18°C.

<p>Comparison of growth in LB medium for BW25113 wild-type (■), Δ<i>dinJ</i> Δ<i>Km</i>^<i>R</i> (●), Δ<i>dinJ</i> Δ<i>yafQ ΔKm</i>^<i>R</i> (▲), and Δ<i>yafQ</i> Δ<i>Km</i>^<i>R</i> (▼) at 18°C (<b>A</b>) and 37°C (<b>B</b>). Data are the average of three independent cultures and one standard deviation is shown.</p

Δ<i>mlc</i> reduces growth at 18°C and 37°C.

<p>Comparison of growth in LB medium for BW25113 wild-type (■) and BW25113 Δ<i>mlc</i> (●) at 18°C (<b>A</b>) and 37°C (<b>B</b>). Comparison of growth in LB medium for BW25113 Δ<i>mlc</i>/pCA24N (■) and BW25113 Δ<i>mlc</i>/pCA24N-<i>mlc</i> (●) with 0.5 mM IPTG induction at 18°C (<b>C</b>) and 37°C (<b>D</b>). Data are averaged from three independent cultures and one standard deviation is shown.</p

Toxin YafQ Reduces <i>Escherichia coli</i> Growth at Low Temperatures

<div><p>Toxin/antitoxin (TA) systems reduce metabolism under stress; for example, toxin YafQ of the YafQ/DinJ <i>Escherichia coli</i> TA system reduces growth by cleaving transcripts with in-frame 5’-AAA-G/A-3’ sites, and antitoxin DinJ is a global regulator that represses its locus as well as controls levels of the stationary sigma factor RpoS. Here we investigated the influence on cell growth at various temperatures and found that deletion of the antitoxin gene, <i>dinJ</i>, resulted in both reduced metabolism and slower growth at 18°C but not at 37°C. The reduction in growth could be complemented by producing DinJ from a plasmid. Using a transposon screen to reverse the effect of the absence of DinJ, two mutations were found that inactivated the toxin YafQ; hence, the toxin caused the slower growth only at low temperatures rather than DinJ acting as a global regulator. Corroborating this result, a clean deletion of <i>yafQ</i> in the Δ<i>dinJ</i> Δ<i>Km</i>^<i>R</i> strain restored both metabolism and growth at 18°C. In addition, production of YafQ was more toxic at 18°C compared to 37°C. Furthermore, by overproducing all the <i>E</i>. <i>coli</i> proteins, the global transcription repressor Mlc was found that counteracts YafQ toxicity only at 18°C. Therefore, YafQ is more effective at reducing metabolism at low temperatures, and Mlc is its putative target.</p></div

YafQ is more toxic at 18°C.

<p>Comparison of growth in LB medium for BW25113 Δ<i>dinJ</i> Δ<i>yafQ ΔKm</i>^<i>R</i>/pCA24N (■) and Δ<i>dinJ</i> Δ<i>yafQ</i> Δ<i>Km</i>^<i>R</i>/pCA24N-<i>yafQ</i> (●) at 37°C (<b>A</b>) and 18°C (<b>C</b>). Panels (<b>B</b>) and (<b>D</b>) expand data for the Δ<i>dinJ</i> Δ<i>yafQ</i> Δ<i>Km</i>^<i>R</i>/pCA24N-<i>yafQ</i> curves for (<b>A</b>) and (<b>C</b>), respectively. Data are averaged from three independent cultures, and one standard deviation is shown.</p

<i>E</i>. <i>coli</i> bacterial strains and plasmids used in this study.

<p><i>E</i>. <i>coli</i> bacterial strains and plasmids used in this study.</p