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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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Related in: MedlinePlus

Detection of AHLs with long acyl chain by thin layer chromatography.The TLC plate was overlaid with the biosensor A. tumefaciens NTL4 (pZLR4). Lane 1, long chain 3-oxo-standards. Lane 2, 10 μL ethyl acetate extract from supernatant of liquid culture of strain 62B.
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f2-sensors-12-02851: Detection of AHLs with long acyl chain by thin layer chromatography.The TLC plate was overlaid with the biosensor A. tumefaciens NTL4 (pZLR4). Lane 1, long chain 3-oxo-standards. Lane 2, 10 μL ethyl acetate extract from supernatant of liquid culture of strain 62B.

Mentions: Based on the results of the β-galactosidase assay, we selected strains having moderate (62B) and high (P8A) activity for further characterization of the autoinducers by TLC and LC-MS/MS analysis. Using bacterial extracts of strains 62B and P8A, we detected the presence of four different signaling molecules corresponding to standards of C6 AHL and 3OC10, 3OC12, and 3OC14 AHLs. Figure 2 shows the TLC patterns of AHLs produced by strain 62B detected by the biosensor A. tumefaciens NTL4 (pZLR4). Extracts of strains 62B and P8A (not shown) typically produced four spots detected by NTL4 (pZLR4).


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)

Detection of AHLs with long acyl chain by thin layer chromatography.The TLC plate was overlaid with the biosensor A. tumefaciens NTL4 (pZLR4). Lane 1, long chain 3-oxo-standards. Lane 2, 10 μL ethyl acetate extract from supernatant of liquid culture of strain 62B.
© Copyright Policy
Related In: Results  -  Collection

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

f2-sensors-12-02851: Detection of AHLs with long acyl chain by thin layer chromatography.The TLC plate was overlaid with the biosensor A. tumefaciens NTL4 (pZLR4). Lane 1, long chain 3-oxo-standards. Lane 2, 10 μL ethyl acetate extract from supernatant of liquid culture of strain 62B.
Mentions: Based on the results of the β-galactosidase assay, we selected strains having moderate (62B) and high (P8A) activity for further characterization of the autoinducers by TLC and LC-MS/MS analysis. Using bacterial extracts of strains 62B and P8A, we detected the presence of four different signaling molecules corresponding to standards of C6 AHL and 3OC10, 3OC12, and 3OC14 AHLs. Figure 2 shows the TLC patterns of AHLs produced by strain 62B detected by the biosensor A. tumefaciens NTL4 (pZLR4). Extracts of strains 62B and P8A (not shown) typically produced four spots detected by NTL4 (pZLR4).

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