Involvement of bacterial quorum-sensing signals in spoilage of bean sprouts

Abstract

Bacterial communication signals, acylated homoserine lactones (AHLs), were extracted from samples of commercial bean sprouts undergoing soft-rot spoilage. Bean sprouts produced in the laboratory did not undergo soft-rot spoilage and did not contain AHLs or AHL-producing bacteria, although the bacterial population reached levels similar to those in the commercial sprouts, 10(8) to 10(9) CFU/g. AHL-producing bacteria (Enterobacteriaceae and pseudomonads) were isolated from commercial sprouts, and strains that were both proteolytic and pectinolytic were capable of causing soft-rot spoilage in bean sprouts. Thin-layer chromatography and liquid chromatography-high-resolution mass spectrometry revealed the presence of N-3-oxo-hexanoyl-l-homoserine lactone in spoiled bean sprouts and in extracts from pure cultures of bacteria. During normal spoilage, the pH of the sprouts increased due to proteolytic activity, and the higher pH probably facilitated the activity of pectate lyase. The AHL synthetase gene (I gene) from a spoilage Pectobacterium was cloned, sequenced, and inactivated in the parent strain. The predicted amino acid sequence showed 97% homology to HslI and CarI in Erwinia carotovora. Spoilage of laboratory bean sprouts inoculated with the AHL-negative mutant was delayed compared to sprouts inoculated with the wild type, and the AHL-negative mutant did not cause the pH to rise. Compared to the wild-type strain, the AHL-negative mutant had significantly reduced protease and pectinase activities and was negative in an iron chelation (siderophore) assay. This is the first study demonstrating AHL regulation of iron chelation in Enterobacteriaceae. The present study clearly demonstrates that the bacterial spoilage of some food products is influenced by quorum-sensing-regulated phenotypes, and understanding these processes may be useful in the development of novel food preservation additives that specifically block the quorum-sensing systems.

FIG. 1.

Appearance of bean sprouts, where the soaking water has been inoculated with different bacterial strains to determine their spoilage potentials. (A) Control (uninoculated); (B) bean sprouts inoculated with the nonspoiling strain C1JM; (C) bean sprouts inoculated with the spoiling strain Pectobacterium sp. strain A2JM; and (D) bean sprouts inoculated with the AHL-deficient A2JM luxI mutant.

FIG. 2.

Siderophore-indicative plates spotted with Pectobacterium sp. strain A2JM, the A2JM luxI mutant (I⁻), and the A2JM luxR mutant (R⁻). The bottom plate was complemented with 1 μM 3-oxo-C₆-HSL.

FIG. 3.

pH (A) and bacterial counts (B) of bean sprouts during sprouting. Sprouts inoculated with Pectobacterium sp. strain A2JM (♦), the A2JM luxI mutant (○), the A2JM luxI mutant complemented with 3-oxo-C₆-HSL (•), and the control (★) are shown. The values are the means and standard deviations of duplicate determinations of the production of bean sprouts.

FIG. 4.

Pectinase activity in the soaking water from the bean sprouts inoculated with Pectobacterium sp. strain A2JM (♦), the A2JM luxI mutant (○), the A2JM luxI mutant complemented with 3-oxo-C₆-HSL (•), the A2JM luxR mutant (▵), the A2JM luxR mutant complemented with 3-oxo-C₆-HSL (▴), and the control (★). The values are the means and standard deviations of triplicate determinations.

FIG. 5.

Protease activity in the soaking water from the bean sprouts inoculated with Pectobacterium sp. strain A2JM (♦), the A2JM luxI mutant (○), the A2JM luxI mutant complemented with 3-oxo-C₆-HSL (•), the A2JM luxR mutant (▵), the A2JM luxR mutant complemented with 3-oxo-C₆-HSL (▴), and the control (★). The values are the means and standard deviations of triplicate determinations.