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Detection, characterization, and biological effect of quorum-sensing signaling molecules in peanut-nodulating bradyrhizobia.

Nievas F, Bogino P, Sorroche F, Giordano W - Sensors (Basel) (2012)

Bottom Line: The aims of this study were to identify and characterize QS signals produced by peanut-nodulating bradyrhizobial strains and to evaluate their effects on processes related to cell interaction.Strains displaying moderate to high levels of AHL-like inducer activity were subjected to chemical identification of signaling molecules by high-performance liquid chromatography coupled to mass spectrometry (LC-MS/MS).Our results clearly demonstrate the existence of cell communication mechanisms among bradyrhizobial strains symbiotic of peanut.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Río Cuarto, Córdoba, Argentina. fnievas@exa.unrc.edu.ar

ABSTRACT
Bacteria of the genus Bradyrhizobium are able to establish a symbiotic relationship with peanut (Arachis hypogaea) root cells and to fix atmospheric nitrogen by converting it to nitrogenous compounds. Quorum sensing (QS) is a cell-cell communication mechanism employed by a variety of bacterial species to coordinate behavior at a community level through regulation of gene expression. The QS process depends on bacterial production of various signaling molecules, among which the N-acylhomoserine lactones (AHLs) are most commonly used by Gram-negative bacteria. Some previous reports have shown the production of QS signaling molecules by various rhizobia, but little is known regarding mechanisms of communication among peanut-nodulating strains. The aims of this study were to identify and characterize QS signals produced by peanut-nodulating bradyrhizobial strains and to evaluate their effects on processes related to cell interaction. Detection of AHLs in 53 rhizobial strains was performed using the biosensor strains Agrobacterium tumefaciens NTL4 (pZLR4) and Chromobacterium violaceum CV026 for AHLs with long and short acyl chains, respectively. None of the strains screened were found to produce AHLs with short acyl chains, but 14 strains produced AHLs with long acyl chains. These 14 AHL-producing strains were further studied by quantification of β-galactosidase activity levels (AHL-like inducer activity) in NTL4 (pZLR4). Strains displaying moderate to high levels of AHL-like inducer activity were subjected to chemical identification of signaling molecules by high-performance liquid chromatography coupled to mass spectrometry (LC-MS/MS). For each AHL-producing strain, we found at least four different AHLs, corresponding to N-hexanoyl-DL-homoserine lactone (C(6)), N-(3-oxodecanoyl)-L-homoserine lactone (3OC(10)), N-(3-oxododecanoyl)-L-homoserine lactone (3OC(12)), and N-(3-oxotetradecanoyl)-L-homoserine lactone (3OC(14)). Biological roles of 3OC10, 3OC12, and 3OC14 AHLs were evaluated in both AHL-producing and -non-producing peanut-nodulating strains. Bacterial processes related to survival and nodulation, including motility, biofilm formation, and cell aggregation, were affected or modified by the exogenous addition of increasing concentrations of synthetic AHLs. Our results clearly demonstrate the existence of cell communication mechanisms among bradyrhizobial strains symbiotic of peanut. AHLs with long acyl chains appear to be signaling molecules regulating important QS physiological processes in these bacteria.

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Bioassay for detection of AHL-like molecules with long acyl chains using A. tumefaciens NTL4 (pZLR4) as biosensor strain.The plates show bioassay results obtained for control + (A. tumefaciens NTL4 pTiC58ΔaccR), control – (A. tumefaciens NTL4) (A), and peanut-nodulating strains (B, C, D). Strains USDA 4438 and C-145 were negative (B); 62B, 20AG, 15A, PC34 (C), 55AG, and PC3 were positive (D); P8A and P8B showed undefined results (D) for AHL production.
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f1-sensors-12-02851: Bioassay for detection of AHL-like molecules with long acyl chains using A. tumefaciens NTL4 (pZLR4) as biosensor strain.The plates show bioassay results obtained for control + (A. tumefaciens NTL4 pTiC58ΔaccR), control – (A. tumefaciens NTL4) (A), and peanut-nodulating strains (B, C, D). Strains USDA 4438 and C-145 were negative (B); 62B, 20AG, 15A, PC34 (C), 55AG, and PC3 were positive (D); P8A and P8B showed undefined results (D) for AHL production.

Mentions: AHL-like molecules in peanut-nodulating strains were detected using the biosensor strains A. tumefaciens NTL4 (pZLR4) and C. violaceum CV026. None of the tested strains showed positive results for the C. violaceum CV026 bioassay, indicating that the strains were unable to synthesize AHL-like molecules having short acyl chains. On the other hand, results of the A. tumefaciens NTL4 (pZLR4) bioassay revealed that some strains were able to produce AHL-like molecules with long acyl chains. A positive result consisted of the presence of a blue halo around a colony resulting from hydrolysis of X-Gal, as shown in Figure 1. Of the 53 strains analyzed, 14 (26%) were able to synthesize AHL-like molecules with long acyl chains (positive result), two (4%, indicated as +/−) showed an undefined result, and the remaining 37 (70%) showed a negative result for the production of AHL-like molecules by A. tumefaciens NTL4 (pZLR4) (Table A1).


Detection, characterization, and biological effect of quorum-sensing signaling molecules in peanut-nodulating bradyrhizobia.

Nievas F, Bogino P, Sorroche F, Giordano W - Sensors (Basel) (2012)

Bioassay for detection of AHL-like molecules with long acyl chains using A. tumefaciens NTL4 (pZLR4) as biosensor strain.The plates show bioassay results obtained for control + (A. tumefaciens NTL4 pTiC58ΔaccR), control – (A. tumefaciens NTL4) (A), and peanut-nodulating strains (B, C, D). Strains USDA 4438 and C-145 were negative (B); 62B, 20AG, 15A, PC34 (C), 55AG, and PC3 were positive (D); P8A and P8B showed undefined results (D) for AHL production.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376631&req=5

f1-sensors-12-02851: Bioassay for detection of AHL-like molecules with long acyl chains using A. tumefaciens NTL4 (pZLR4) as biosensor strain.The plates show bioassay results obtained for control + (A. tumefaciens NTL4 pTiC58ΔaccR), control – (A. tumefaciens NTL4) (A), and peanut-nodulating strains (B, C, D). Strains USDA 4438 and C-145 were negative (B); 62B, 20AG, 15A, PC34 (C), 55AG, and PC3 were positive (D); P8A and P8B showed undefined results (D) for AHL production.
Mentions: AHL-like molecules in peanut-nodulating strains were detected using the biosensor strains A. tumefaciens NTL4 (pZLR4) and C. violaceum CV026. None of the tested strains showed positive results for the C. violaceum CV026 bioassay, indicating that the strains were unable to synthesize AHL-like molecules having short acyl chains. On the other hand, results of the A. tumefaciens NTL4 (pZLR4) bioassay revealed that some strains were able to produce AHL-like molecules with long acyl chains. A positive result consisted of the presence of a blue halo around a colony resulting from hydrolysis of X-Gal, as shown in Figure 1. Of the 53 strains analyzed, 14 (26%) were able to synthesize AHL-like molecules with long acyl chains (positive result), two (4%, indicated as +/−) showed an undefined result, and the remaining 37 (70%) showed a negative result for the production of AHL-like molecules by A. tumefaciens NTL4 (pZLR4) (Table A1).

Bottom Line: The aims of this study were to identify and characterize QS signals produced by peanut-nodulating bradyrhizobial strains and to evaluate their effects on processes related to cell interaction.Strains displaying moderate to high levels of AHL-like inducer activity were subjected to chemical identification of signaling molecules by high-performance liquid chromatography coupled to mass spectrometry (LC-MS/MS).Our results clearly demonstrate the existence of cell communication mechanisms among bradyrhizobial strains symbiotic of peanut.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Río Cuarto, Córdoba, Argentina. fnievas@exa.unrc.edu.ar

ABSTRACT
Bacteria of the genus Bradyrhizobium are able to establish a symbiotic relationship with peanut (Arachis hypogaea) root cells and to fix atmospheric nitrogen by converting it to nitrogenous compounds. Quorum sensing (QS) is a cell-cell communication mechanism employed by a variety of bacterial species to coordinate behavior at a community level through regulation of gene expression. The QS process depends on bacterial production of various signaling molecules, among which the N-acylhomoserine lactones (AHLs) are most commonly used by Gram-negative bacteria. Some previous reports have shown the production of QS signaling molecules by various rhizobia, but little is known regarding mechanisms of communication among peanut-nodulating strains. The aims of this study were to identify and characterize QS signals produced by peanut-nodulating bradyrhizobial strains and to evaluate their effects on processes related to cell interaction. Detection of AHLs in 53 rhizobial strains was performed using the biosensor strains Agrobacterium tumefaciens NTL4 (pZLR4) and Chromobacterium violaceum CV026 for AHLs with long and short acyl chains, respectively. None of the strains screened were found to produce AHLs with short acyl chains, but 14 strains produced AHLs with long acyl chains. These 14 AHL-producing strains were further studied by quantification of β-galactosidase activity levels (AHL-like inducer activity) in NTL4 (pZLR4). Strains displaying moderate to high levels of AHL-like inducer activity were subjected to chemical identification of signaling molecules by high-performance liquid chromatography coupled to mass spectrometry (LC-MS/MS). For each AHL-producing strain, we found at least four different AHLs, corresponding to N-hexanoyl-DL-homoserine lactone (C(6)), N-(3-oxodecanoyl)-L-homoserine lactone (3OC(10)), N-(3-oxododecanoyl)-L-homoserine lactone (3OC(12)), and N-(3-oxotetradecanoyl)-L-homoserine lactone (3OC(14)). Biological roles of 3OC10, 3OC12, and 3OC14 AHLs were evaluated in both AHL-producing and -non-producing peanut-nodulating strains. Bacterial processes related to survival and nodulation, including motility, biofilm formation, and cell aggregation, were affected or modified by the exogenous addition of increasing concentrations of synthetic AHLs. Our results clearly demonstrate the existence of cell communication mechanisms among bradyrhizobial strains symbiotic of peanut. AHLs with long acyl chains appear to be signaling molecules regulating important QS physiological processes in these bacteria.

Show MeSH
Related in: MedlinePlus