Pseudomonas aeruginosa is an important opportunistic pathogen in cystic fibrosis patients and immunocompromised individuals, and the toxin–antitoxin (TA) system is involved in bacterial virulence and phage resistance. However, the roles of TA systems in P. aeruginosa are relatively less studied and no phage Cro-like regulators were identified as TA components. Here, we identified and characterized a chromosome-encoded prophage Cro-like antitoxin (CrlA) in the clinical isolate P. aeruginosa WK172. CrlA neutralized the toxicity of the toxin CrlA (CrlT) which cleaves mRNA, and they formed a type II TA system. Specifically, crlA and crlT are co-transcribed and their protein products interact with each other directly. The autorepression of CrlA is abolished by CrlT through the formation of the CrlTA complex. Furthermore, crlTA is induced in the stationary phase, and crlA is expressed at higher levels than crlT. The excess CrlA inhibits the infection of lytic Pseudomonas phages. CrlA is widely distributed among Pseudomonas and in other bacterial strains and may provide antiphage activities.
Regulatory systems that maintain prophage quiescence integrate phage and host gene expression with environmental conditions1,2. In the opportunistic bacterial pathogen Pseudomonas aeruginosa, Pf filamentous bacteriophages play critical roles in biofilm formation and virulence3-5, but mechanisms governing Pf prophage activation in biofilms are largely unknown. Here, we report a new type of prophage regulatory module in a widely-distributed P. aeruginosa lineage that not only controls virion production of co-resident Pf prophages, but also mediates defense against diverse lytic phages. By comparing two lineages of the prototype P. aeruginosa strain PAO1 that harbor different Pf prophages, we identified a prophage-encoded kinase-kinase-phosphatase (KKP) system that controls Pf production in biofilms. KKP components exhibit dynamic stoichiometry, where high kinase levels in planktonic conditions maintain phosphorylation of the host H-NS protein MvaU, repressing prophage activation. During biofilm formation, phosphatase expression is heightened, leading to MvaU dephosphorylation and alleviating repression of prophage gene expression. KKP clusters are present in hundreds of diverse temperate prophages and other mobile elements across Gram-negative bacteria. Characterization of KKP modules from different species revealed that, in addition to regulating Pf phage lysogeny, KKP functions as a tripartite toxin-antitoxin system that mediates host defense from predatory lytic phages. KKP represents a new phosphorylation-based mechanism for prophage regulation and for phage defense. The dual function of this module raises the question of whether other newly described phage defense systems6-9 also regulate intrinsic prophage biology in diverse hosts.
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