The Gram-negative bacterium Pseudomonas aeruginosa is an important opportunistic pathogen in compromised individuals, such as patients with cystic fibrosis, severe burns or impaired immunity. In this work we aimed to screen novel essential genes of P. aeruginosa by shotgun antisense identification, a technique that was developed a decade ago for the Gram-positive bacterium Staphylococcus aureus and was under-used in Gram-negative bacteria for a considerable period of time. Following antisense screenings in the PAO1 strain of P. aeruginosa, we focused on a locus, PA2873, which was targeted by an antisense RNA construct that can impair cell growth. The PA2873 gene product was annotated as a hypothetical membrane protein endowed with a periplasmic region harbouring a structural domain belonging to the transglutaminase-like superfamily, a group of archaeal, bacterial and eukaryotic proteins homologous to animal transglutaminases. In this study, we show that the periplasmic portion of the PA2873 protein, which we named TgpA, does possess transglutaminase activity in vitro. This is the first report of transglutaminase activity in P. aeruginosa. In addition, we have provided strong evidences that TgpA plays a critical role in the viability of P. aeruginosa.