Dual role of a (p)ppGpp‐ and (p)ppApp‐degrading enzyme in biofilm formation and interbacterial antagonism

Abstract

The guanosine nucleotide‐based second messengers ppGpp and pppGpp (collectively: (p)ppGpp) enable adaptation of microorganisms to environmental changes and stress conditions. In contrast, the closely related adenosine nucleotides (p)ppApp are involved in type VI secretion system (T6SS)‐mediated killing during bacterial competition. Long RelA‐SpoT Homolog (RSH) enzymes regulate synthesis and degradation of (p)ppGpp (and potentially also (p)ppApp) through their synthetase and hydrolase domains, respectively. Small alarmone hydrolases (SAH) that consist of only a hydrolase domain are found in a variety of bacterial species, including the opportunistic human pathogen Pseudomonas aeruginosa. Here, we present the structure and mechanism of P. aeruginosa SAH showing that the enzyme promiscuously hydrolyses (p)ppGpp and (p)ppApp in a strictly manganese‐dependent manner. While being dispensable for P. aeruginosa growth or swimming, swarming, and twitching motilities, its enzymatic activity is required for biofilm formation. Moreover, (p)ppApp‐degradation by SAH provides protection against the T6SS (p)ppApp synthetase effector Tas1, suggesting that SAH enzymes can also serve as defense proteins during interbacterial competition.

Emerging and divergent roles of pyrophosphorylated nucleotides in bacterial physiology and pathogenesis

“…The toxicity of Tas1 can be alleviated by its cognate immunity protein, Tis1, which inhibits the toxin through direct binding. (p)ppApp-mediated toxicity can also be alleviated by the P. aeruginosa hydrolase, PaSAH (formerly PA0431) [113]. PaSAH may contribute to bacterial defense against Tas1-like effectors as the deletion of PaSAH from a Tas1-sensitive P. aeruginosa strain renders it hypersusceptible to T6SS-delivered Tas1 [113].…”

“…(p)ppApp-mediated toxicity can also be alleviated by the P. aeruginosa hydrolase, PaSAH (formerly PA0431) [113]. PaSAH may contribute to bacterial defense against Tas1-like effectors as the deletion of PaSAH from a Tas1-sensitive P. aeruginosa strain renders it hypersusceptible to T6SS-delivered Tas1 [113]. In addition, and in contrast to (p)ppGpp, (p)ppApp is inefficiently degraded by P. aeruginosa SpoT, which results in (p)ppApp accumulation and likely contributes to its function as an energy depleting toxin [12].…”

“…(p)ppApp-mediated toxicity can also be alleviated by the P . aeruginosa hydrolase, Pa SAH (formerly PA0431) [ 113 ]. Pa SAH may contribute to bacterial defense against Tas1-like effectors as the deletion of Pa SAH from a Tas1-sensitive P .…”

“…Pa SAH may contribute to bacterial defense against Tas1-like effectors as the deletion of Pa SAH from a Tas1-sensitive P . aeruginosa strain renders it hypersusceptible to T6SS-delivered Tas1 [ 113 ]. In addition, and in contrast to (p)ppGpp, (p)ppApp is inefficiently degraded by P .…”

Nonhydrolysable Analogues of (p)ppGpp and (p)ppApp Alarmone Nucleotides as Novel Molecular Tools

“…17,23 Second, representatives of several SAH subfamiliesincluding MESH1were shown to efficiently hydrolyze (p)ppApp. 17,20,24 Structural investigations of alarmone-bound RSH enzymes are complicated by, first, the rapid hydrolysis of the (p)ppGpp to GDP/GTP by HD-competent RSHs, second, the nonenzymatic degradation of the alarmone resulting in partially hydrolyzed ppG2′3′p. 14 While it is possible to use enzymes from thermophilic bacteriasuch as Thermus thermophilus Rel (Rel Tt ), an extremely inefficient (p)ppGpp-hydrolase at RT 16,25 studies of enzymes from mesophilic species remain challenging.…”