ParE toxin encoded by the broad‐host‐range plasmid RK2 is an inhibitor of Escherichia coli gyrase

Abstract

Summary Broad‐host‐range plasmid RK2 encodes a post‐segregational killing system, parDE, which contributes to the stable maintenance of this plasmid in Escherichia coli and many distantly related bacteria. The ParE protein is a toxin that inhibits cell growth, causes cell filamentation and eventually cell death. The ParD protein is a specific ParE antitoxin. In this work, the in vitro activities of these two proteins were examined. The ParE protein was found to inhibit DNA synthesis using an E. coli oriC supercoiled template and a replication‐proficient E. coli extract. Moreover, ParE inhibited the early stages of both chromosomal and plasmid DNA replication, as measured by the DnaB helicase‐ and gyrase‐dependent formation of FI*, a highly unwound form of supercoiled DNA. The presence of ParD prevented these inhibitory activities of ParE. We also observed that the addition of ParE to supercoiled DNA plus gyrase alone resulted in the formation of a cleavable gyrase–DNA complex that was converted to a linear DNA form upon addition of sodium dodecyl sulphate (SDS). Adding ParD before or after the addition of ParE prevented the formation of this cleavable complex. These results demonstrate that the target of ParE toxin activity in vitro is E. coli gyrase.

MazF ribonucleases promote Mycobacterium tuberculosis drug tolerance and virulence in guinea pigs

“…The toxin components of the TA modules exert their effects in different ways by targeting essential cellular functions such as DNA replication, protein synthesis, cell division, peptidoglycan biosynthesis and ribosome assembly; however, RNA cleavage is the most prevalent mode of action [10][11][12][13][14][15][16] . These ribonucleases cleave RNA in either a ribosome-dependent (for example, RelE, HigB and YoeB) or ribosome-independent manner (for example, MazF, ToxN and VapC) 17,18 .…”

The ParE2–PaaA2 toxin–antitoxin complex fromEscherichia coliO157 forms a heterodocecamer in solution and in the crystal

“…This toxin-neutralizing segment binds to or even complements the fold of the toxin and inhibits its biochemical activity (Kamada et al, 2003;Kamada & Hanaoka, 2005;Garcia-Pino et al, 2008;Li et al, 2009;De Jonge et al, 2009;Brown et al, 2009). This activity depends on the family of toxin considered and may include poisoning DNA gyrase (Bernard & Couturier, 1992;Jiang et al, 2002), cleaving RNA in a ribosome-dependent or independent fashion (Zhang et al, 2003;Zhang & Inouye, 2011), direct ribosome inhibition (Liu et al, 2008), chemical modification of ribosomes (Vesper et al, 2011), modifying initiator tRNA or a number of other activities that directly alter the basic physiology of the cell (Yamamoto et al, 2009;Tan et al, 2010;Mutschler et al, 2011).…”

“…The parDE family of TA modules encodes a toxin (ParE) that poisons gyrase and thus interferes with transcription and replication (Jiang et al, 2002). While not as well characterized in terms of mechanism and structure as the CcdB proteins De Jonge et al, 2009), recent data indicate that the binding site on gyrase for the two types of toxins is different (Yuan et al, 2010).…”

Engineered Phagemids for Nonlytic, Targeted Antibacterial Therapies

“…This enables production of both targeted and broad-spectrum antibacterial treatments, depending upon bacteriophage selection. While some toxins tested here had little effect on the target E. coli strain, the selected toxins have a broad-range activity across many bacterial species [18][19][20][21][22]25 . Additionally, since our choices for antibacterial peptides are broad spectrum [11][12] , this system should provide a plug-and-play therapeutic that can be readily modified to suit its target and will therefore function in many target bacteria.…”

“…The last toxin, ParE, acts by halting the F1* formation from both chromosomal and plasmid DNA replication origins by inhibiting bacterial gyrase, causing filamentation and cell death 22 .…”