Quorum Quenching in a Novel Acinetobacter sp. XN-10 Bacterial Strain against Pectobacterium carotovorum subsp. carotovorum

Abstract

Quorum sensing (QS) is a cell density-dependent mechanism that regulates the expression of specific genes in microbial cells. Quorum quenching (QQ) is a promising strategy for attenuating pathogenicity by interfering with the QS system of pathogens. N-Acyl-homoserine lactones (AHLs) act as signaling molecules in many Gram-negative bacterial pathogens and have received wide attention. In this study, a novel, efficient AHL-degrading bacterium, Acinetobacter sp. strain XN-10, was isolated from agricultural contaminated soil and evaluated for its degradation efficiency and potential use against QS-mediated pathogens. Strain XN-10 could effectively degrade N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), N-hexanoyl-L-homoserine lactone (C6HSL), N-(3-oxododecanoyl)-L-homoserine lactone (3OC12HSL), and N-(3-oxooctanoyl)-L-homoserine lactone (3OC8HSL), which all belong to the AHL family. Analysis of AHL metabolic products by gas chromatography-mass spectrometry (GC-MS) led to the identification of N-cyclohexyl-propanamide, and pentanoic acid, 4-methyl, methyl ester as the main intermediate metabolites, revealing that AHL could be degraded by hydrolysis and dehydroxylation. All intermediates were transitory and faded away without any non-cleavable metabolites at the end of the experiment. Furthermore, strain XN-10 significantly attenuated the pathogenicity of Pectobacterium carotovorum subsp. carotovorum (Pcc) to suppress tissue maceration in carrots, potatoes, and Chinese cabbage. Taken together, our results shed light on the QQ mechanism of a novel AHL-degrading bacterial isolate, and they provide useful information which show potential for biocontrol of infectious diseases caused by AHL-dependent bacterial pathogens.

Keywords: Acinetobacter sp. XN-10; N-acyl homoserine lactones; Pectobacterium carotovorum subsp. carotovorum; biocontrol; quorum quenching; quorum sensing.

Figure 1

Degradation activity of N-acyl-homoserine lactone (AHL) by strain XN-10. The degradation activity of AHL by strain XN-10 was tested using various concentrations of N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) (i.e., 5, 10, 15, 20, 25, and 30 μmol·L⁻¹) (B–G) compared to a blank control (A) only containing 30 μmol·L⁻¹ of OHHL. The residual content of OHHL in the sample was determined according to the distance of the report strain on the agar strip from the top to blue. The columns containing the various concentrations of OHHL did not turn blue, indicating that the OHHL was completely degraded by strain XN-10.

Figure 2

Phylogenetic analysis based on the 16S rRNA sequence of strain XN-10 and other representative Acinetobacter strains. The phylogenetic tree was constructed with the neighbor-joining (NJ) method. Numbers in parentheses represent the sequence accession numbers in GenBank. Numbers at the nodes indicate bootstrap values from the neighborhood-joining analysis of 1000 resampled datasets. Bars represent sequence divergence.

Figure 3

Antibiotic sensitivity of strain XN-10. This figure shows the results of the sensitivity of strain XN-10 to various concentrations of each antibiotic. The green in the figure indicates that the strain has strong resistance to this concentration of antibiotics, and the red indicates that the strain is susceptible to this concentration of antibiotics.

Figure 4

The relationship between N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) degradation and growth of strain XN-10. Symbols: OHHL concentration; and cell growth. Values represent the mean of three repeats. Each experiment was conducted with three replicates. Bars indicate standard deviation of the mean.

Figure 5

The degradation abilities of strain XN-10 to different AHL signal molecules. OHHL: N-(3-oxohexanoyl)-L-homoserine lactone; C6HSL: N-hexanoyl-L-homoserine lactone; 3OC12HSL:N-(3-oxododecanoyl)-L-homoserine lactone; 3OC8HSL: N-(3-oxooctanoyl)-L-homoserine lactone.

Figure 6

Biocontrol test of strain XN-10 against Pectobacterium carotovorum subsp. carotovorum (Pcc) on potato slices under laboratory conditions. (a) Attenuated maceration of Pcc strain Z3-3 by strain XN-10 on potatoes. A: inoculation of Z3-3 alone; B: co-inoculation of Z3-3 and Escherichia coli DH5α; C: co-inoculation of Z3-3 and Bacillus thuringiensis subsp. israelensis B23; and D: co-inoculation of Z3-3 and strain XN-10. (b) Tissue maceration rates in each treatment.

Figure 7

Biocontrol test of strain XN-10 against Pectobacterium carotovorum subsp. carotovorum (Pcc) on carrot slices under laboratory conditions. (a) Attenuated maceration of Pcc strain Z3-3 by strain XN-10 on carrots. A: inoculation of Z3-3 alone; B: co-inoculation of Z3-3 and Escherichia coli DH5α; C: co-inoculation of Z3-3 and Bacillus thuringiensis subsp. israelensis B23; and D: co-inoculation of Z3-3 and strain XN-10. (b) Tissue maceration rates in each treatment.

Figure 8

Biocontrol test of strain XN-10 against Pectobacterium carotovorum subsp. carotovorum (Pcc) on Chinese cabbage under laboratory conditions. (a) Attenuated maceration of Pcc strain Z3-3 by strain XN-10 on Chinese cabbage. A: inoculation of Z3-3 alone; B: co-inoculation of Z3-3 and Escherichia coli DH5α; C: co-inoculation of Z3-3 and Bacillus thuringiensis subsp. israelensis B23; and D: co-inoculation of Z3-3 and strain XN-10. (b) Tissue maceration rates in each treatment.

Figure 9

Proposed N-acyl-homoserine lactone (AHL) degradation pathway in strain XN-10.