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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 swimming motility of peanut-nodulating strains.Swimming motility (expressed as halo diameter; cm) of peanut-nodulating strains in reduced 1/10 TY medium with 0.3% agar, 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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f3-sensors-12-02851: Effect of AHLs on swimming motility of peanut-nodulating strains.Swimming motility (expressed as halo diameter; cm) of peanut-nodulating strains in reduced 1/10 TY medium with 0.3% agar, 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: In general, the motility of 62B, P8A, and P5 was increased as a result of exposure to various types and concentrations of AHLs (Figure 3). In the case of 62B and P8A, a low autoinducer concentration (5 μM) resulted in a significant increase in motility, and this effect was maintained or increased in the presence of higher autoinducer concentrations (10 and 20 μM) (Figure 3(A,B,C)). P5 also responded positively to the presence of autoinducers, but to a lesser degree than 62B or P8A. We concluded that, regardless of the type and concentration of autoinducer, these rhizobial strains show increased motility in the presence of exogenous signaling molecules. Interestingly, USDA 4438, a reference strain that did not induce blue haloes in biosensor strains, displayed a more complex swimming behavior when various exogenous AHLs were added. Low concentrations of 3OC10 AHL (5 or 10 μM) significantly decreased the motility of USDA 4438, whereas the maximal autoinducer concentration tested (20 μM) significantly increased its swimming ability in comparison to controls without addition of exogenous AHLs (Figure 3(A)). The addition to USDA 4438 of 5 or 10 μM 3OC12 AHL caused a slight but significant increase in motility (Figure 3(B)), and the addition of 10 μM 3OC14 AHL also caused a slight increase (Figure 3(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 swimming motility of peanut-nodulating strains.Swimming motility (expressed as halo diameter; cm) of peanut-nodulating strains in reduced 1/10 TY medium with 0.3% agar, 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

f3-sensors-12-02851: Effect of AHLs on swimming motility of peanut-nodulating strains.Swimming motility (expressed as halo diameter; cm) of peanut-nodulating strains in reduced 1/10 TY medium with 0.3% agar, 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: In general, the motility of 62B, P8A, and P5 was increased as a result of exposure to various types and concentrations of AHLs (Figure 3). In the case of 62B and P8A, a low autoinducer concentration (5 μM) resulted in a significant increase in motility, and this effect was maintained or increased in the presence of higher autoinducer concentrations (10 and 20 μM) (Figure 3(A,B,C)). P5 also responded positively to the presence of autoinducers, but to a lesser degree than 62B or P8A. We concluded that, regardless of the type and concentration of autoinducer, these rhizobial strains show increased motility in the presence of exogenous signaling molecules. Interestingly, USDA 4438, a reference strain that did not induce blue haloes in biosensor strains, displayed a more complex swimming behavior when various exogenous AHLs were added. Low concentrations of 3OC10 AHL (5 or 10 μM) significantly decreased the motility of USDA 4438, whereas the maximal autoinducer concentration tested (20 μM) significantly increased its swimming ability in comparison to controls without addition of exogenous AHLs (Figure 3(A)). The addition to USDA 4438 of 5 or 10 μM 3OC12 AHL caused a slight but significant increase in motility (Figure 3(B)), and the addition of 10 μM 3OC14 AHL also caused a slight increase (Figure 3(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