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. 2018 May 9:8:155.
doi: 10.3389/fcimb.2018.00155. eCollection 2018.

Isolation and Characterization of N-acyl Homoserine Lactone-Producing Bacteria From Cattle Rumen and Swine Intestines

Yang Yang  1   2   3 , Mingxu Zhou  2   3 , Philip R Hardwidge  4 , Hengmi Cui  1 , Guoqiang Zhu  2   3
Affiliations

Isolation and Characterization of N-acyl Homoserine Lactone-Producing Bacteria From Cattle Rumen and Swine Intestines

Yang Yang et al. Front Cell Infect Microbiol. .

Abstract

Quorum sensing systems regulate gene expression in response to bacterial population density. Acyl-homoserine lactones are a class of quorum sensing molecules found in cattle rumen that are thought to regulate the gene expression of enterohemorrhagic Escherichia coli and thus help this pathogen survive in animal gastrointestinal tracts. However, the specific bacteria that produce these signaling molecules in bovine and porcine gastrointestinal tracts are unknown. Here we developed methods to concentrate gastrointestinal fluids and screen the bacteria that produce acyl-homoserine lactones. We isolated a Pseudomonas aeruginosa strain YZ1 from cattle rumen, and an Aeromonas hydrophila strain YZ2 from pig intestine. Mass spectrometry analysis of culture supernatants indicated at least three specific classes of acyl-homoserine lactones produced by YZ1, and a C4-acyl-homoserine lactone produced by YZ2. Transformation of E. coli with P. aeruginosa or A. hydrophila luxI homologs,which can produce short- or long-chain acyl-homoserine lactones conferred upon E. coli the ability to synthesize acyl-homoserine lactones and affected gene expression, motility, and acid tolerance of E. coli. This is the first study reporting the isolation and characterization of acyl-homoserine lactone synthase-positive bacteria from cattle rumen and swine intestines.

Keywords: E. coli; acyl-homoserine lactone; cattle rumen; pig intestine; quorum sensing.

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Figures

Figure 1
Figure 1
Isolation of the AHL-producing bacteria YZ1 and YZ2. (A,B) JZA1-based β-galactosidase assays of AHL production. β-galactosidase activity is plotted as a function of the culture supernatant added to JZA1 cultures. Strains (YZ1, A; YZ2, B) were grown for different times before the addition of supernatants, as indicated. Asterisks indicate significantly different β-galactosidase activity as compared with activity induced by DH5α supernatant. (C,D) Phylogenetic analyses of YZ1 and YZ2. The bar represents evolutionary distance as number of changes per nucleotide position, determined by measuring the lengths of the horizontal lines connecting the corresponding species. GenBank accession numbers are indicated. (E,F) Biosensor strain C. violaceum CV026-based cross-streaking assays for AI-1 activity (purple color). P. aeruginosa strains YZ1 (E) A. hydrophila YZ2 (F) the positive control Y. enterocolitica GIM1.266, and the negative control DH5α were struck across the biosensor strain as indicated.
Figure 2
Figure 2
Bioluminescence assays of AHL production. (A) Bioluminescence (relative light units) of reporter strain E. coli pSB1142 after the addition of the indicated culture supernatants from YZ1. (B) Bioluminescence (relative light units) of reporter strain E. coli pSB401 after the addition of the indicated culture supernatants from YZ1. (C) Bioluminescence (relative light units) of reporter strain E. coli pSB1142 after the addition of the indicated culture supernatants from YZ2. (D) Bioluminescence (relative light units) of reporter strain E. coli pSB401 after the addition of the indicated culture supernatants from YZ2. Asterisks indicate significantly different reporter strain bioluminescence as compared with activity induced by DH5α supernatant.
Figure 3
Figure 3
Mass spectrometry analysis of AHLs in YZ1 and YZ2 extracts. (A) Identification of C4-HSL produced by YZ1. (B) Identification of C8-HSL produced by YZ1. (C) Identification of 3-oxo-C12-HSL produced by YZ1. (D) Identification of C4-HSL produced by YZ2.
Figure 4
Figure 4
Cross-streaking assays for AI-1 activity from E. coli 8624 transformed with lasI or rhlI and E. coli 107/86 transformed with ahyI. (A) Biosensor strain C. violaceum CV026-based cross-streaking assay. The indicated E. coli 8624 recombinant strains were struck across the CV026 biosensor strain to detect short-chain AHLs (purple color). (B) Biosensor strain JZA1 assay. The indicated E. coli 8624 recombinant strains were struck across the JZA1 biosensor strain to detect long-chain AHLs (blue color). (C) CV026-based cross-streaking assay. The indicated E. coli 107/86 recombinant strain was struck across the CV026 biosensor strain to detect short-chain AHLs.
Figure 5
Figure 5
Phenotypic analysis of E. coli 8624 and 107/86 recombinant strains. (A) Relative fliC, sdiA, and gadA transcript abundance (as compared with E. coli 8624 transformed with pBR322) are plotted as a function of E. coli transformation with luxI homologs. Asterisks indicate significantly different gene expression, as compared with E. coli 8624 transformed with pBR322. (B) Motility diameters were measured after 12 h growth of the indicated strains on 0.3% swim agar plates. Asterisks indicate significantly different motility halos, as compared with E. coli 8624 transformed with pBR322. (C) Acid tolerance was quantified by enumerating bacteria survival as a function of time after growth seeded in acidified LB (pH 2.5) supplemented with 1.0 mM glutamate. Asterisks indicate significantly different survival, as compared with E. coli 8624 transformed with pBR322. (D) Relative fliC, sdiA, and gadA transcript abundance (as compared with E. coli 107/86 transformed with pBR322) are plotted as a function of E. coli transformation with ahyI from YZ2. Asterisks indicate significantly different gene expression, as compared with E. coli 107/86 transformed with pBR322. (E) Motility diameters were measured after 12 h growth of the indicated strains on 0.3% swim agar plates. Asterisks indicate significantly different motility halos, as compared with E. coli 107/86 transformed with pBR322. (F) Acid tolerance was quantified by enumerating bacteria survival as a function of time after growth seeded in acidified LB (pH 2.5) supplemented with 1.0 mM glutamate. Asterisks indicate significantly different survival, as compared with E. coli 107/86 transformed with pBR322.
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