Agrobacterium bioassay strain for ultrasensitive detection of N-acylhomoserine lactone-type quorum-sensing molecules: detection of autoinducers in Mesorhizobium huakuii - PubMed (original) (raw)

Agrobacterium bioassay strain for ultrasensitive detection of N-acylhomoserine lactone-type quorum-sensing molecules: detection of autoinducers in Mesorhizobium huakuii

Jun Zhu et al. Appl Environ Microbiol. 2003 Nov.

Abstract

An ultrasensitive bioassay system for the detection of N-acylhomoserine lactones (AHLs) was constructed in Agrobacterium tumefaciens by using the T7 expression system to overproduce the AHL receptor TraR. This strain detected many diverse AHLs, some at extremely low concentrations. We used this strain to detect for the first time AHLs made by Mesorhizobium huakuii, which symbiotically fixes nitrogen in association with the legume Astragalus sinicus, a source of green manure throughout eastern Asia.

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Figures

FIG. 1.

TraR expression in a T7 system in A. tumefaciens. (A) TraR protein abundance in P_tetR_-traR and PT7-traR strains. Strains WCF47(pCF218)(pCF372) and KYC55(pJZ372)(pJZ384)(pJZ410) were cultured overnight in the presence of 100 nM OOHL, diluted serially in fourfold increments, size fractionated by SDS-PAGE, and immunodetected with rabbit polyclonal antiserum. (B) Pulse-labeling of TraR. Strain KYC55(pJZ372)(pJZ384)(pJZ410) was cultured to mid-logarithmic phase in AT medium in the absence or presence of 100 nM OOHL at 28°C, incubated with rifampin for 30 min, and then pulse-labeled with [35S]methionine for 5 min. The cultures were then chased with nonradiolabeled methionine and terminated by freezing at −80°C at various intervals as indicated. Cleared lysates were size fractionated by SDS-PAGE, and gels were analyzed with a Storm B840 PhosphorImager (Molecular Dynamics).

FIG. 2.

3-Oxooctanoyl-HSL dose-response curves of A. tumefaciens strains KYC55 (pJZ372)(pJZ384)(pJZ410) (squares) and WCF47(pCF218)(pCF372) (diamonds). Synthetic 3-oxooctanoyl-HSL was diluted as indicated and added to 2 ml of AT medium supplemented with approximately 107 bacterial cells, incubated with aeration for 12 h, and assayed for β-galactosidase specific activity.

FIG. 3.

PT7-traR sensitively detects acyl-HSLs in M. huakuii spent culture supernatant. A 100-ml volume of supernatant from stationary-phase M. huakuii 93 growing in TY medium was extracted with ethyl acetate, dried, and resuspended in 1 ml of distilled H2O. The indicated volumes were added to 1 ml of AT medium containing approximately 107 cells of either strain KYC55(pJZ372)(pJZ384)(pJZ410) (squares) or WCF47(pCF218)(pCF372) (diamonds). The cultures were incubated with aeration for 12 h and assayed for β-galactosidase specific activity.

FIG. 4.

TLC of autoinducers synthesized by M. huakuii. Lanes: 1, standard mixture of 3-oxo derivatives with acyl chain lengths of C6 (2.5 pmol), C8 (0.25 pmol), and C12 (0.5 nmol); 2, 3-hydroxyl derivatives with acyl chain lengths of C6 (20 pmol), C8 (20 pmol), and C10 (20 pmol); 3, HSLs with fully reduced acyl chain lengths of C6 (100 pmol), C8 (30 pmol), and C10 (40 pmol); 4, activities of autoinducers obtained from 1 ml of M. huakuii growing in TY medium to stationary phase; 5, activities of autoinducers obtained from 1 μl of A. tumefaciens R10(pCF218). TLC plates were chromatographed as described previously and overlaid with 100 ml of agar containing AT medium, X-Gal (5-bromo-4-chloro-3-indolyl-β-

-galactopyranoside), and approximately 107 bacteria per ml [strains KYC55(pJZ372)(pJZ384)(pJZ410) (left panel) and WCF47(pCF218)(pCF372) (right panel)]. TLC plates were incubated overnight at 28°C and examined for X-Gal hydrolysis.

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Agrobacterium bioassay strain for ultrasensitive detection of N-acylhomoserine lactone-type quorum-sensing molecules: detection of autoinducers in Mesorhizobium huakuii - PubMed (original) (raw)