Pseudomonas aeruginosa is a ubiquitous and opportunistic bacterium that inhibits the growth of different microorganisms, including Gram-positive bacteria and fungi such as Candida spp. and Aspergillus fumigatus. In this study, we investigated the interaction between P. aeruginosa and Cryptococcus spp. We found that P. aeruginosa PA14 and, to a lesser extent, PAO1 significantly inhibited the growth of Cryptococcus spp. The inhibition of growth was observed on solid medium by the visualization of a zone of inhibition of yeast growth and in liquid culture by viable cell counting. Interestingly, such inhibition was only observed when P. aeruginosa and Cryptococcus were co-cultured. Minimal inhibition was observed when cell–cell contact was prevented using a separation membrane, suggesting that cell contact is required for inhibition. Using mutant strains of Pseudomonas quinoline signaling, we showed that P. aeruginosa inhibited the growth of Cryptococcus spp. by producing antifungal molecules pyocyanin, a redox-active phenazine, and 2-heptyl-3,4-dihydroxyquinoline (PQS), an extracellular quorum-sensing signal. Because both P. aeruginosa and Cryptococcus neoformans are commonly found in lung infections of immunocompromised patients, this study may have important implication for the interaction of these microbes in both an ecological and a clinical point of view.
Chitinase and chitin-oligosaccaride can be used in multiple field, so it is important to develop a high-yield chitinase producing strain. Here, a recombinant Pichia pastoris with 4 copies of ChiA gene from Bacillus licheniformis and co-expression of molecular chaperon HAC1 was constructed. The amount of recombinant ChiA in the supernatant of high-cell-density fermentation reaches a maximum of 12.7 mg/mL, which is 24-fold higher than that reported in the previous study. The recombinant ChiA can hydrolyze 30% collodidal chitin with 74% conversion ratio, and GlcNAc is the most abundant hydrolysis product, followed by N, N′-diacetylchitobiose. Combined with BsNagZ, the hydrolysate of ChiA can be further transformed into GlcNAc with 88% conversion ratio. Additionally, the hydrolysate of ChiA can obviously accelerate the germination growth of rice and wheat, increasing the seedling height and root length by at least 1.6 folds within 10 days.
Pseudomonas aeruginosa (Pa) is a versatile opportunistic pathogen. Quorum sensing (QS) is essential for Pa pathogenicity by activating virulence factor production. Among the three known QS systems in Pa, Pseudomonas Quinolone Signal (PQS) is an alkyquinolone, one of >50 quinolone compounds produced that differ in alkyl chain length and desaturation. The condensing enzyme PqsD catalyzes the branching point of quinolone synthesis, utilizing either malonyl‐CoA or 3‐oxo‐fatty acids of various chain lengths. We showed that PqsD exhibited higher activity using fatty acid substrates with 10, 12 or 14 carbons than those with 6 or 8 carbons, suggesting the fatty acid chain length is critical in positioning the carboxylic group for efficient catalysis. In addition, PqsD showed preference for malonyl‐CoA over 3‐oxo‐decanoic acid in vitro. Alanine mutants of the active site triad (C112, H257, and N287) were assayed for PqsD activity. All 3 residues were essential for condensation, whereas N287 was not required in the formation of anthraniloyl‐PqsD intermediate. Inhibitors of PqsD were identified by virtual screen and the IC50s of the top‐scoring hit compounds were determined. Our results suggest that regulation of the substrate supply in vivo may be important in determining the composition of quinolone compounds, which in turn affect the virulence factor production in Pa. (Supported by NIH P20 RR017677 and UL1 RR029882)
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