Potential of a Quorum Quenching Bacteria Isolate Ochrobactrum intermedium D-2 Against Soft Rot Pathogen Pectobacterium carotovorum subsp. carotovorum

Abstract

Quorum quenching (QQ) is a promising strategy for preventing and controlling quorum sensing (QS)-mediated bacterial infections. It interferes with QS by the inhibition of signal synthesis, the detection of enzyme-catalyzed degradation, and the modification of signals. N-Acyl homoserine lactones (AHLs) represent a family of widely conserved QS signals involved in the regulation of virulence factor production in many Gram-negative bacterial pathogens. In this study, AHL-degrading bacterial strains were isolated, and the most efficient one was evaluated for its potential against QS-mediated pathogens. Results showed that an AHL-degrading bacteria Ochrobactrum intermedium D-2 effectively attenuated maceration produced by the pathogen Pectobacterium carotovorum subsp. carotovorum (Pcc) on radish and potato slices. Strain D-2 exhibited a superior AHL degradation activity and efficiently degraded various AHLs, including N-hexanoyl-L-homoserine lactone (C6HSL), N-(3-oxohexanoyl)-L-homoserine lactone (3OC6HSL), N-(3-oxooctanoyl)-L-homoserine lactone (3OC8HSL), and N-(3-oxododecanoyl)-L-homoserine lactone (3OC12HSL). Analysis of the degradation products of AHL by gas chromatography-mass spectrometry led to the identification of N-cyclohexyl-propanamide and propanamide as the main intermediate products, suggesting that AHL was degraded by hydrolysis. Annotation and analysis of the whole genome sequence of strain D-2 revealed the presence of an AHL-lactonase, termed AidF. Moreover, the application of strain D-2 was able to substantially reduce the disease severity caused by Pcc on host plants. These results reveal the biochemical basis of a highly efficient AHL-degrading bacterial isolate and present the potential to attenuate Pcc virulence through QQ.

Keywords: N-acyl homoserine lactone; Ochrobactrum intermedium; degradation; quorum quenching; quorum sensing.

FIGURE 1

Phylogenetic tree based on 16S rDNA sequences of strain D-2 and representative Ochrobactrum species. Numbers in parentheses represent Genbank accession number. Numbers at the nodes indicate bootstrap values. Bar represents sequence divergence.

FIGURE 2

Degradation of 3OC6HSL during the growth of strain D-2. Black square, 3OC6HSL degradation; Black triangle, growth of strain D-2; Black rotundity, 3OC6HSL control.

FIGURE 3

Maceration attenuating test of strain D-2 against soft rot disease on radish slices. Bacillus thuringiensis subsp. israelensis B23 and Escherichia coli DH5α served as positive and negative controls. (A) Panel A, Z3-3 alone on radish slices; Panel B, Z3-3 + E. coli DH5α; Panel C, Z3-3 + B23; Panel D, Z3-3 + agricultural streptomycin; Panel E, Z3-3 + D-2. (B) Numbers of maceration area in each treatment.

FIGURE 4

Maceration attenuating test of strain D-2 against soft rot disease on potato slices. Bacillus thuringiensis subsp. israelensis B23 and Escherichia coli DH5α served as positive and negative controls. (A) Panel A, Z3-3 alone on potato slices; Panel B, Z3-3 + E. coli DH5α; Panel C, Z3-3 + B23; Panel D, Z3-3 + D-2. (B) Numbers of maceration area in each treatment.

FIGURE 5

Mass spectra of AHL degradation products by strain D-2. (A) AHL; (B) Propanamide; (C) N-cyclohexyl-propanamide.

FIGURE 6

Proposed AHL degradation pathway in strain D-2.

FIGURE 7

The circular genome diagram of Ochrobactrum intermedium D-2. Genomic analysis revealing the circular replicons in the genome of strain D-2. From the outside to the inside of the chromosome, the circles are as follows: circle 1, genomic location; circle 2, GC content; circle 3 and 4, location of encoding genes on the positive (red) and negative (green) strand; circles 5 and 6, locations of ncRNA on the positive (blue) and negative (purple) strand; circles 7, genomic long fragment repeats.