Two dissimilar N-acyl-homoserine lactone acylases of Pseudomonas syringae influence colony and biofilm morphology

Abstract

Plant aerial surfaces comprise a complex habitat for microorganisms, and many plant-associated bacteria, such as the pathogen Pseudomonas syringae, exhibit density-dependent survival on leaves by utilizing quorum sensing (QS). QS is often mediated by diffusible signals called N-acyl-homoserine lactones (AHLs), and P. syringae utilizes N-3-oxo-hexanoyl-dl-homoserine lactone (3OC6HSL) to control traits influencing epiphytic fitness and virulence. The P. syringae pathovar syringae B728a genome sequence revealed two putative AHL acylases, termed HacA (Psyr_1971) and HacB (Psyr_4858), which are N-terminal nucleophile hydrolases that inactivate AHLs by cleaving their amide bonds. HacA is a secreted AHL acylase that degrades only long-chain (C > or = 8) AHLs, while HacB is not secreted and degrades all tested AHLs. Targeted disruptions of hacA, hacB, and hacA and hacB together do not alter endogenous 3OC6HSL levels under the tested conditions. Surprisingly, targeted disruptions of hacA alone and hacA and hacB together confer complementable phenotypes that are very similar to autoaggregative phenotypes seen in other species. While AHL acylases might enable P. syringae B728a to degrade signals of competing species and block expression of their QS-dependent traits, these enzymes also play fundamental roles in biofilm formation.

FIG. 1.

Western blot analyses of putative strain B728a AHL acylases expressed in E. coli and strain B728a. (A) Insoluble (i) and soluble (s) fractions collected from induced E. coli harboring plasmids pET30 (lanes 1 and 2), pET30-HacA (lanes 3 and 4), pET30-HacB (lanes 5 and 6), and pET30-Psyr_3871 (lanes 7 and 8). (B) Insoluble (i) and soluble (s) fractions collected from strain B728a harboring plasmids p519nGFP (lanes 1 and 2), p519-HacA (lanes 3 and 4), p519-HacB (lanes 5 and 6), and p519-Psyr_3871 (lanes 7 and 8).

FIG. 2.

Endogenous AHL (3OC6HSL) concentration (nM) in various strains grown in KB medium. Wild-type strain B728a (•), strain B728a harboring p519-HacA (▪), and strain B728a harboring p519-HacB (▴).

FIG. 3.

The enrichment and characterization of HacA and HacB in strain B728a. (A) SDS-PAGE analysis of soluble cell extracts (HacA, lane 2; HacB, lane 5), fractions after Ni-NTA affinity purification of soluble extracts (HacA, lane 3; HacB, lane 6), and fractions from media samples after nondenaturing Ni-NTA affinity purification (HacA, lane 4; HacB, lane 7). White arrows denote α subunits. Black arrows denote β subunits. Molecular masses of standards (M) are given in kilodaltons. (B) Schematic representations of HacA and HacB. Solid vertical lines indicate sites of autoproteolytic cleavage determined experimentally by amino acid sequencing. Dashed vertical lines indicate cleavage sites inferred from amino acid alignments with other characterized AHL acylases.

FIG. 4.

Colony morphologies of B728a strains on KB agar. Shown are the wild-type B728a (A), HacA⁻ (B), HacB⁻ (C), and HacA⁻B⁻ (D) strains.

FIG. 5.

Pellicles formed by various B728a strains in static cultures of KB medium. Shown are the wild-type B728a (A), B728a harboring p519-HacA (B), B728a harboring p519-HacB (C), HacA⁻ (D), HacB⁻ (E), and HacA⁻B⁻ (F) strains.

FIG. 6.

Complementation of the HacA⁻ and HacA⁻B⁻ phenotypes. Shown are HacA⁻ (A), HacA⁻ plus p519-HacA (B), HacA⁻ plus p519-HacB (C), HacA⁻ plus p519nGFP (D), HacA⁻B⁻ (E), HacA⁻B⁻ plus p519-HacA (F), HacA⁻B⁻ plus p519-HacB (G), and HacA⁻B⁻ plus p519nGFP (H).