PrrT/A, a Pseudomonas aeruginosa Bacterial Encoded Toxin-Antitoxin System Involved in Prophage Regulation and Biofilm Formation.

Toxin-antitoxin (TA) systems are genetic modules that consist of a stable protein-toxin and an unstable antitoxin that neutralizes the toxic effect. In type II TA systems, the antitoxin is a protein that inhibits the toxin by direct binding. Type II TA systems, whose roles and functions are under intensive study, are highly distributed among bacterial chromosomes. Here, we identified and characterized a novel type II TA system PrrT/A encoded in the chromosome of the clinical isolate 39016 of the opportunistic pathogen Pseudomonas aeruginosa. We have shown that the PrrT/A system exhibits classical type II TA characteristics and novel regulatory properties. Following deletion of the prrA antitoxin, we discovered that the system is involved in a range of processes including (i) biofilm and motility, (ii) reduced prophage induction and bacteriophage production, and (iii) increased fitness for aminoglycosides. Taken together, these results highlight the importance of this toxin-antitoxin system to key physiological traits in P. aeruginosa. IMPORTANCE The functions attributed to bacterial TA systems are controversial and remain largely unknown. Our study suggests new insights into the potential functions of bacterial TA systems. We reveal that a chromosome-encoded TA system can regulate biofilm and motility, antibiotic resistance, prophage gene expression, and phage production. The latter presents a thus far unreported function of bacterial TA systems. In addition, with the emergence of antimicrobial-resistant bacteria, especially with the rising of P. aeruginosa resistant strains, the investigation of TA systems is critical as it may account for potential new targets against the resistant strains.

An Efficient, Counter-Selection-Based Method for Prophage Curing in Pseudomonas aeruginosa Strains.

Prophages are bacteriophages in the lysogenic state, where the viral genome is inserted within the bacterial chromosome. They contribute to strain genetic variability and can influence bacterial phenotypes. Prophages are highly abundant among the strains of the opportunistic pathogen Pseudomonas aeruginosa and were shown to confer specific traits that can promote strain pathogenicity. The main difficulty of studying those regions is the lack of a simple prophage-curing method for P. aeruginosa strains. In this study, we developed a novel, targeted-curing approach for prophages in P. aeruginosa. In the first step, we tagged the prophage for curing with an ampicillin resistance cassette (ampR) and further used this strain for the sacB counter-selection marker's temporal insertion into the prophage region. The sucrose counter-selection resulted in different variants when the prophage-cured mutant is the sole variant that lost the ampR cassette. Next, we validated the targeted-curing with local PCR amplification and Whole Genome Sequencing. The application of the strategy resulted in high efficiency both for curing the Pf4 prophage of the laboratory wild-type (WT) strain PAO1 and for PR2 prophage from the clinical, hard to genetically manipulate, 39016 strain. We believe this method can support the research and growing interest in prophage biology in P. aeruginosa as well as additional Gram-negative bacteria.

Characterization of PfiT/PfiA toxin-antitoxin system of Pseudomonas aeruginosa that affects cell elongation and prophage induction.

Toxin-antitoxin (TA) systems are small genetic modules usually consisting of two elements-a toxin and an antitoxin. The abundance of TA systems among various bacterial strains may indicate an important evolutionary role. Pseudomonas aeruginosa, which can be found in a variety of niches in nature, is an opportunistic pathogen for various hosts. While P. aeruginosa strains are very versatile and diverse, only a few TA systems were characterized in this species. Here, we describe a newly characterized TA system in P. aeruginosa that is encoded within the filamentous Pf4 prophage. This system, named PfiT/PfiA, is a homologue of the ParE/YefM TA system. It is a type II TA system, in which the antitoxin is a protein that binds the toxic protein and eliminates the toxic effect. PfiT/PfiA carries several typical type II characteristics. Specifically, it constitutes two small genes expressed in a single operon, PfiT inhibits growth and PfiA eliminates this effect, PfiA binds PfiT, and PfiT expression results in elongated cells. Finally, we assigned a novel function to this TA system, where an imbalance between PfiT and PfiA, favouring the toxin, resulted in cell elongation and an increase in virion production.

SawR a new regulator controlling pyomelanin synthesis in Pseudomonas aeruginosa.

Surface Acoustic Waves (SAW) were previously shown to inhibit biofilm formation, increase bacterial susceptibility to antibiotic treatment and alter the transcription pattern of Pseudomonas aeruginosa. Here we characterize one gene, sawR (PA3133), that is highly overexpressed when P. aeruginosa is exposed to SAW. SawR is a putative transcription factor belonging to the TetR regulator family. When overexpressed sawR causes numerous phenotypes, including the accumulation of a brown pigment which we identified as pyomelanin. In this study we describe how sawR regulates pyomelanin synthesis. We show that sawR down-regulates the expression levels of hmgA and this causes the accumulation of homogentisic acid which in turn undergoes oxidation and polymerization to pyomelanin. Using bioinformatics, we were able to identify a specific amino acid, arginine 23, which is found within the sawR DNA binding domain and is crucial for its regulatory activity. Our results indicate that sawR does not affect any other genes in the phenylalanine/tyrosine metabolic pathway and its repressive ability on hmgA is not mediated by the hmgA repressor PA2010 (i.e. hmgR). Taken together, our results shed light on the regulatory cascade controlling pyomelanin synthesis and uncover yet another unknown regulator involved in its regulation.