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DspI, a putative enoyl-coenzyme A (CoA) hydratase/isomerase, was proposed to be involved in the synthesis of cis-2-decenoic acid (CDA), a quorum sensing (QS) signal molecule in the pathogen Pseudomonas aeruginosa (P. aeruginosa). The present study provided a structural basis for the dehydration reaction mechanism of DspI during CDA synthesis. Structural analysis reveals that Glu126, Glu146, Cys127, Cys131 and Cys154 are important for its enzymatic function. Moreover, we show that the deletion of dspI results in a remarkable decreased in the pyoverdine production, flagella-dependent swarming motility, and biofilm dispersion as well as attenuated virulence in P. aeruginosa PA14. This study thus unravels the mechanism of DspI in diffusible signal factor (DSF) CDA biosynthesis, providing vital information for developing inhibitors that interfere with DSF associated pathogenicity in P. aeruginosa.Pseudomona aeruginosa (P. aeruginosa), as a common nosocomial gram-negative pathogenic bacterium, can inhabit a wide variety of ecological niches and infect diverse hosts. Owing to its intrinsic multi-drug resistance, P. aeruginosa is one of the leading causes of healthcare-associated infections, calling for effective treatments or agents with novel anti-infective mechanisms. In response to cell density or confinement to niches, P. aeruginosa adopts various signal molecules to mediate virulence factors biosynthesis and/or biofilm formation. Therefore, inhibiting these signaling pathways represents attractive strategies for developing novel therapeutics against P. aeruginosa infection 1-4 .In many pathogenic bacteria, quorum-sensing (QS) signaling is an important regulatory switch contributing to bacteria virulence and persistence 5 . By producing and releasing hormone-like chemical signal molecules involved in bacterial QS system, bacteria can communicate intercellularly to regulate a variety of physiological activities, such as motility, virulence, antibiotic production and biofilm dispersion. In the past few years, the diffusible signal factor (DSF) family has been disclosed as a new type of QS system signal that is common in gram-negative bacterial pathogens 6,7 . The first identified DSF family molecule cis-11-methyl-2-dodecenoic acid, was found in the plant pathogen Xanthomonas campestris pv. campestris (X.cc) 8 , which regulates many biological functions including cell growth, biofilm dispersion and virulence 9 . Cis-2-decenoic acid (CDA) is a new member of the DSF family that was found in P. aeruginosa and functions as an auto-inducer for biofilm dispersion 10,11 . Additionally, as an inter-kingdom signaling molecule, CDA also regulates the biofilm formation and dispersion in a number of other pathogens [12][13][14][15] .So far, multiple DSF family molecules have been detected in various pathogens 7 . A particular group of particular enoyl-coenzyme A (CoA) hydratase/isomerases includes RpfF from X.cc, which appears to be the key enzyme in DSF biosynthesis 7,[16][17][18][19][20][21] . In P. aeruginosa, the gene PA0745 ...

Ionizing irradiation kills pathogens by destroying nucleic acids without protein structure destruction. However, how pathogens respond to irradiation stress has not yet been fully elucidated. Here, we observed that Pseudomonas aeruginosa PAO1 could release nucleic acids into the extracellular environment under X-ray irradiation. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), X-ray irradiation was observed to induce outer membrane vesicle (OMV) formation in P. aeruginosa PAO1. The size distribution of the OMVs of the irradiated PAO1 was similar to that of the OMVs of the non-irradiated PAO1 according to nanoparticle tracking analysis (NTA). The pyocin-related proteins are involved in OMV production in P. aeruginosa PAO1 under X-ray irradiation conditions, and that this is regulated by the key SOS gene recA. The OMV production was significantly impaired in the irradiated PAO1 Δlys mutant, suggesting that Lys endolysin is associated with OMV production in P. aeruginosa PAO1 upon irradiation stress. Meanwhile, no significant difference in OMV production was observed between PAO1 lacking the pqsR, lasR, or rhlR genes and the parent strain, demonstrating that the irradiation-induced OMV biosynthesis of P. aeruginosa was independent of the Pseudomonas quinolone signal (PQS).

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