1887

Abstract

The PAO1 genome has at least two genes, and encoding acylhomoserine lactone (AHL) acylases. Two additional genes, and , have been predicted to encode penicillin acylase proteins, but have not been characterized. Initial studies on a transposon insertion mutant suggested that the gene is not related to the AHL growth phenotype of . The close similarity (67 %) of to HacB, an AHL acylase of , prompted us to investigate whether the PA0305 protein might also function as an AHL acylase. The gene has been cloned and the protein (PA0305) has been overproduced, purified and subjected to functional characterization. Analysis of the purified protein showed that, like β-lactam acylases, PA0305 undergoes post-translational processing resulting in α- and β-subunits, with the catalytic serine as the first amino acid of the β-subunit, strongly suggesting that PA0305 is a member of the N-terminal nucleophile hydrolase superfamily. Using a biosensor assay, PA0305his was shown to degrade AHLs with acyl side chains ranging in length from 6 to 14 carbons. Kinetics studies using -octanoyl--homoserine lactone (C-HSL) and -(3-oxo-dodecanoyl)--homoserine lactone (3-oxo-C-HSL) as substrates showed that the enzyme has a robust activity towards these two AHLs, with apparent / values of 0.14×10 M s towards C-HSL and 7.8×10 Ms towards 3-oxo-C-HSL. Overexpression of the gene in showed significant reductions in both accumulation of 3-oxo-C-HSL and expression of virulence factors. A mutant strain with a deleted gene showed a slightly increased capacity to kill compared with the PAO1 wild-type strain and the PAO1 strain carrying a plasmid overexpressing . The harmful effects of the Δ strain on the animals were most visible at 5 days post-exposure and the mortality rate of the animals fed on the Δ strain was faster than for the animals fed on either the wild-type strain or the strain overexpressing In conclusion, the gene encodes an efficient acylase with activity towards long-chain homoserine lactones, including 3-oxo-C-HSL, the natural quorum sensing signal molecule in and we propose to name this acylase HacB.

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2011-07-01
2020-05-27
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