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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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Effect of AHLs on cell aggregation of peanut-nodulating strains.Cell aggregation (%) of peanut-nodulating strains was calculated as described in “Experimental”. For the assay, TY medium was supplemented with various concentrations of 3OC10 AHL (A), 3OC12 AHL (B), and 3OC14 AHL (C). Values indicated by different letters are significantly different from each other according to Fisher’s LSD test (P < 0.05).
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f5-sensors-12-02851: Effect of AHLs on cell aggregation of peanut-nodulating strains.Cell aggregation (%) of peanut-nodulating strains was calculated as described in “Experimental”. For the assay, TY medium was supplemented with various concentrations of 3OC10 AHL (A), 3OC12 AHL (B), and 3OC14 AHL (C). Values indicated by different letters are significantly different from each other according to Fisher’s LSD test (P < 0.05).

Mentions: Under our experimental conditions, the peanut-nodulating Bradyrhizobium strains studied showed a low capacity for cell aggregation, with values ranging from ∼5–20%. In comparison to controls, the addition of exogenous AHLs caused decreased autoaggregative ability in all strains except P5, which showed a slight increase (Figure 5(A,B,C)). All concentration of 3OC10 AHL produced the greatest increases in autoaggregation of P5, with values ∼10-fold higher than those of controls (Figure 5(A)). 3OC12 (Figure 5(B)) and 3OC14 AHLs (Figure 5(C)) showed effects that were similar to but smaller than that of 3OC10 AHL. Autoaggregation of USDA 4438 was not affected by addition of 3OC10 AHL, but autoaggregation of 62B and P8A decreased in response to increasing concentrations of this autoinducer (Figure 5(A)). A negative dose-dependent effect by 3OC12 AHL was observed on autoaggregation of USDA 4438, and a negative dose-independent effect by this autoinducer was observed on autoaggregation of 62B and P8A (Figure 5(B)). Exposure to any concentration of 3OC14 AHL significantly reduced autoaggregation of USDA 4438 and 62B, and a high concentration (20 μM) of this autoinducer reduced autoaggregation of P8A (Figure 5(C)).


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)

Effect of AHLs on cell aggregation of peanut-nodulating strains.Cell aggregation (%) of peanut-nodulating strains was calculated as described in “Experimental”. For the assay, TY medium was supplemented with various concentrations of 3OC10 AHL (A), 3OC12 AHL (B), and 3OC14 AHL (C). Values indicated by different letters are significantly different from each other according to Fisher’s LSD test (P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

f5-sensors-12-02851: Effect of AHLs on cell aggregation of peanut-nodulating strains.Cell aggregation (%) of peanut-nodulating strains was calculated as described in “Experimental”. For the assay, TY medium was supplemented with various concentrations of 3OC10 AHL (A), 3OC12 AHL (B), and 3OC14 AHL (C). Values indicated by different letters are significantly different from each other according to Fisher’s LSD test (P < 0.05).
Mentions: Under our experimental conditions, the peanut-nodulating Bradyrhizobium strains studied showed a low capacity for cell aggregation, with values ranging from ∼5–20%. In comparison to controls, the addition of exogenous AHLs caused decreased autoaggregative ability in all strains except P5, which showed a slight increase (Figure 5(A,B,C)). All concentration of 3OC10 AHL produced the greatest increases in autoaggregation of P5, with values ∼10-fold higher than those of controls (Figure 5(A)). 3OC12 (Figure 5(B)) and 3OC14 AHLs (Figure 5(C)) showed effects that were similar to but smaller than that of 3OC10 AHL. Autoaggregation of USDA 4438 was not affected by addition of 3OC10 AHL, but autoaggregation of 62B and P8A decreased in response to increasing concentrations of this autoinducer (Figure 5(A)). A negative dose-dependent effect by 3OC12 AHL was observed on autoaggregation of USDA 4438, and a negative dose-independent effect by this autoinducer was observed on autoaggregation of 62B and P8A (Figure 5(B)). Exposure to any concentration of 3OC14 AHL significantly reduced autoaggregation of USDA 4438 and 62B, and a high concentration (20 μM) of this autoinducer reduced autoaggregation of P8A (Figure 5(C)).

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