Stability of the GraA Antitoxin Depends on Growth Phase, ATP Level, and Global Regulator MexT

Abstract

Bacterial type II toxin-antitoxin systems consist of a potentially poisonous toxin and an antitoxin that inactivates the toxic protein by binding to it. Most of the toxins regulate stress survival, but their activation depends on the stability of the antitoxin that has to be degraded in order for the toxin to be able to attack its cellular targets. The degradation of antitoxins is usually rapid and carried out by ATP-dependent protease Lon or Clp, which is activated under stress conditions. The graTA system of Pseudomonas putida encodes the toxin GraT, which can affect the growth rate and stress tolerance of bacteria but is under most conditions inactivated by the unusually stable antitoxin GraA. Here, we aimed to describe the stability features of the antitoxin GraA by analyzing its degradation rate in total cell lysates of P. putida. We show that the degradation rate of GraA depends on the growth phase of bacteria being fastest in the transition from exponential to stationary phase. In accordance with this, higher ATP levels were shown to stabilize GraA. Differently from other antitoxins, the main cellular proteases Lon and Clp are not involved in GraA stability. Instead, GraA seems to be degraded through a unique pathway involving an endoprotease that cleaves the antitoxin into two unequal parts. We also identified the global transcriptional regulator MexT as a factor for destabilization of GraA, which indicates that the degradation of GraA may be induced by conditions similar to those that activate MexT. IMPORTANCEToxin-antitoxin (TA) modules are widespread in bacterial chromosomes and have important roles in stress tolerance. As activation of a type II toxin is triggered by proteolytic degradation of the antitoxin, knowledge about the regulation of the antitoxin stability is critical for understanding the activation of a particular TA module. Here, we report on the unusual degradation pathway of the antitoxin GraA of the recently characterized GraTA system. While GraA is uncommonly stable in the exponential and late-stationary phases, its degradation increases in the transition phase. The degradation pathway of GraA involves neither Lon nor Clp, which usually targets antitoxins, but rather an unknown endoprotease and the global regulator MexT, suggesting a new type of regulation of antitoxin stability.

Antitoxin HigA inhibits virulence gene mvfR expression in Pseudomonas aeruginosa

“…Lon proteases other than ClpXP play an important role in degrading HigA in PAO1. However, the degradation of GraA in P. putida KT2440 involves neither Lon nor Clp protease but rather an unknown endoprotease and the global regulator MexT (Tamman et al, ). In addition, homologues of mvfR are absent in the nonpathogenic strain P. putida KT2440, suggesting that the function of GraTA and HigB/HigA is divergent in different Pseudomonas hosts.…”

Toxin-Antitoxin Modules Are Pliable Switches Activated by Multiple Protease Pathways

“…While no adaptors have been identified for the Lon protease, there is no a priori reason to expect there are none [ 142 ]. A newly identified TA module, named GraTA, is degraded by a unique yet unidentified ATP-independent protease, and this module has also recently been demonstrated to target ribosome biogenesis in a unique mechanism [ 154 , 155 ]. This was the first report of antitoxin degradation independent of Lon or ClpP.…”

The toxin GraT inhibits ribosome biogenesis

“…This suggests that the GraT‐caused stress protection is costly and requires strict control by the antitoxin GraA. Indeed, GraA is an efficient antidote and suppresses GraT on multiple levels, first acting as an autorepressor of the graTA operon and also sequestering the toxin by binding it into an inactive complex (Tamman et al ., ).…”