The plant pathogen Pantoea ananatis produces N-acylhomoserine lactone and causes center rot disease of onion by quorum sensing

Abstract

A number of gram-negative bacteria have a quorum-sensing system and produce N-acyl-l-homoserine lactone (AHL) that they use them as a quorum-sensing signal molecule. Pantoea ananatis is reported as a common colonist of wheat heads at ripening and causes center rot of onion. In this study, we demonstrated that P. ananatis SK-1 produced two AHLs, N-hexanoyl-l-homoserine lactone (C6-HSL) and N-(3-oxohexanoyl)-l-homoserine lactone (3-oxo-C6-HSL). We cloned the AHL-synthase gene (eanI) and AHL-receptor gene (eanR) and revealed that the deduced amino acid sequence of EanI/EanR showed high identity to those of EsaI/EsaR from P. stewartii. EanR repressed the ean box sequence and the addition of AHLs resulted in derepression of ean box. Inactivation of the chromosomal eanI gene in SK-1 caused disruption of exopolysaccharide (EPS) biosynthesis, biofilm formation, and infection of onion leaves, which were recovered by adding exogenous 3-oxo-C6-HSL. These results demonstrated that the quorum-sensing system involved the biosynthesis of EPS, biofilm formation, and infection of onion leaves in P. ananatis SK-1.

FIG. 1.

Identification and characterization of AHLs produced by P. ananatis SK-1. (A) Cross-streaks of SK-1 and its mutants against C. violaceum CV026. Diffusible AHL production by SK-1 and SK-05R induced CV026 biosensor to produce a violacein. (B) TLC analysis of AHLs produced by strain SK-1. AHLs were visualized as pigments produced by CV026 biosensor and identified after comparison with the AHL standards C6-HSL and 3-oxo-C6-HSL.

FIG. 2.

Multiple alignments of EanI (A) and EanR (B). Gray and black shading indicates similar and identical amino acids, respectively. Sequence alignment was performed with CLUSTAL W (http://www.ddbj.nig.ac.jp/search/clustalw-j.html) and presented with Boxshade 3.21 (http://www.ch.embnet.org/software/BOX_form.html). The sequences used in the alignments with EanI were EsaI from P. stewartii (accession no. L32183) and CarI from E. carotovora (X74299). The sequences used in the alignments with EanR were EsaR from P. stewartii (L32184) and CarR from E. carotovora (AF041840).

FIG. 3.

(A) Nucleotide sequence analysis of the upstream region of eanR. Putative −35 and −10 promoter sequences, the ribosome binding site (RB), and the translation initiation site are shown in boldface type. The inverted repeat exhibiting similarity to lux box was underlined. (B) Nucleotide sequence comparison of consensus lux box sequences (13) and the upstream region of luxR homolog in P. ananatis SK-1, P. stewartii DC283, and S. marcescens SS-1. Conserved sequences are shown in boldface type.

FIG. 4.

Induction of the ean box-lacZ transcriptional fusion in E. coli DH5α by different AHL compounds. E. coli DH5α carrying pJN105ER and pQF50ER was grown in the LB medium with various synthetic AHLs. After 15 h of incubation, the β-galactosidase activity was measured. Arabinose was used for the induction of expression of EanR at a concentration of 0.4%. Ara⁻ indicates activity without arabinose and AHL. AHL⁻ indicates the result with 0.4% arabinose and without AHL. The results were reproduced in three repeated experiments, and error bars indicate standard deviations.

FIG. 5.

Production of EPS by wild-type and mutant strains of P. ananatis SK-1. Strains were streaked onto the TSB agar plates without (A) or with (B) 10 μM 3-oxo-C6-HSL. Mucoid and slimy colony morphology indicated EPS production after incubation at 30°C for 24 h.

FIG. 6.

Quantification of bacteria in biofilm formed on polypropylene plastic by wild-type and mutant strains of P. ananatis SK-1. Biofilms were allowed to form in a 96-well polypropylene microtiter dish, stained with crystal violet, and estimated by analysis at 570-nm absorbance. Six wells of each sample were used for measuring biofilm formation, and error bars indicate standard deviations.

FIG. 7.

Virulence assay of wild-type and mutant strains of P. ananatis SK-1. Bacterial strains were injected into onion leaves at two sites per leaf. 3-oxo-C6-HSL was spotted onto the leaves at 100 nmol per spot. Inoculated onion leaves were incubated at room temperature for 3 days and monitored for the development of the necrotic symptoms.