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Detection of quorum-sensing-related molecules in Vibrio scophthalmi.

García-Aljaro C, Eberl L, Riedel K, Blanch AR - BMC Microbiol. (2008)

Bottom Line: This observation was further supported by the decrease in the presence of these signal molecules after cloning and expression of lactonase AiiA from Bacillus cereus in the V. scophthalmi strains.One of the signal molecules was identified as N-(3-hydroxy dodecanoyl)-L-homoserine lactone.Analysis of the translated sequence revealed that the sequenced luxS gene carried the conserved domain, which is common to luxS sequences found in other species, and which is essential for LuxS enzymatic activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, University of Barcelona, Barcelona, Spain. cristina.garcia@cnm.es

ABSTRACT

Background: Cell-to-cell communication (also referred to as quorum sensing) based on N-acyl-homoserine lactones (AHLs) is a widespread response to environmental change in Gram-negative bacteria. AHLs seem to be highly variable, both in terms of the acyl chain length and in the chemical structure of the radicals. Another quorum sensing pathway, the autoinducer-2-based system, is present both in Gram-positive and Gram-negative bacteria. In this study the presence of signal molecules belonging to both quorum sensing signalling pathways was analysed in the marine symbiotic species Vibrio scophthalmi.

Results: Three AHL-like signal molecules were detected in V. scophthalmi supernatants with the Agrobacterium tumefaciens sensor assay. This observation was further supported by the decrease in the presence of these signal molecules after cloning and expression of lactonase AiiA from Bacillus cereus in the V. scophthalmi strains. One of the signal molecules was identified as N-(3-hydroxy dodecanoyl)-L-homoserine lactone. V. scophthalmi was also shown to carry a functional LuxS synthase. The coding sequence for a luxS-like gene was obtained showing a maximum similarity of 78% with Vibrio vulnificus. Analysis of the translated sequence revealed that the sequenced luxS gene carried the conserved domain, which is common to luxS sequences found in other species, and which is essential for LuxS enzymatic activity.

Conclusion: The data are consistent with the presence of quorum-sensing signal molecules from both AHL- and autoinducer 2-based quorum sensing systems in V. scophthalmi, which are homologous to others previously described in various Vibrio species. How this bacterium interacts with other bacteria and eukaryotic cells to compete ecologically with other intestinal bacteria present in the fish Scophthalmus maximus warrants further investigation.

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Chromatograph obtained from the lower AHL signalling spot from the TLC. A peak of 300.2414 was obtained with a Mariner TOF MS, whose exact mass was later confirmed as m/z 300.2167 after HPLC and mass spectrometry with electrospray ionisation analysis. This molecule matched the empirical formula C16H29NO4 (with an error of 0.39 ppm) for M and hypothetically corresponds to a molecular structure of a hydroxylated-C12HSL. A peak of 282.2256 was also observed which could represent loss of OH group due to fragmentation of the molecule, and a 599.4826 peak, an aggregation of two molecules. Other peaks were originated from components of the medium used for growing the bacterium.
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Figure 2: Chromatograph obtained from the lower AHL signalling spot from the TLC. A peak of 300.2414 was obtained with a Mariner TOF MS, whose exact mass was later confirmed as m/z 300.2167 after HPLC and mass spectrometry with electrospray ionisation analysis. This molecule matched the empirical formula C16H29NO4 (with an error of 0.39 ppm) for M and hypothetically corresponds to a molecular structure of a hydroxylated-C12HSL. A peak of 282.2256 was also observed which could represent loss of OH group due to fragmentation of the molecule, and a 599.4826 peak, an aggregation of two molecules. Other peaks were originated from components of the medium used for growing the bacterium.

Mentions: Detailed analyses of the AHL-like molecules detected were carried out exclusively with the strain V. scophthalmi A102, since both strains presented the same TLC pattern. Two different culture media, mTSB and mPW, were evaluated for growing V. scophthalmi A102 in order to carry out the extraction of AHLs. Due to the high background observed with the mTSB medium, we decided to use the mPW culture medium. Only that extract corresponding to the lower Rf spot resulted in a structure composition similar to an AHL, which hypothetically corresponded to an N-(3-hydroxy dodecanoyl)-L-homoserine lactone (N-3-hydroxy-C12-HSL). This molecule matched the empirical formula C16H29NO4 + H+ with mass (m/z) 300.2167 and an error of 0.39 ppm (see Fig. 2). As far as we know, this constitutes the first description of an AHL in V. scophthalmi; moreover, it has proven different from those produced by other Vibrio species. It was not possible to identify any recognizable AHL-like structures in the other signalling spot extracts, even after carrying out four independent extraction experiments.


Detection of quorum-sensing-related molecules in Vibrio scophthalmi.

García-Aljaro C, Eberl L, Riedel K, Blanch AR - BMC Microbiol. (2008)

Chromatograph obtained from the lower AHL signalling spot from the TLC. A peak of 300.2414 was obtained with a Mariner TOF MS, whose exact mass was later confirmed as m/z 300.2167 after HPLC and mass spectrometry with electrospray ionisation analysis. This molecule matched the empirical formula C16H29NO4 (with an error of 0.39 ppm) for M and hypothetically corresponds to a molecular structure of a hydroxylated-C12HSL. A peak of 282.2256 was also observed which could represent loss of OH group due to fragmentation of the molecule, and a 599.4826 peak, an aggregation of two molecules. Other peaks were originated from components of the medium used for growing the bacterium.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2527009&req=5

Figure 2: Chromatograph obtained from the lower AHL signalling spot from the TLC. A peak of 300.2414 was obtained with a Mariner TOF MS, whose exact mass was later confirmed as m/z 300.2167 after HPLC and mass spectrometry with electrospray ionisation analysis. This molecule matched the empirical formula C16H29NO4 (with an error of 0.39 ppm) for M and hypothetically corresponds to a molecular structure of a hydroxylated-C12HSL. A peak of 282.2256 was also observed which could represent loss of OH group due to fragmentation of the molecule, and a 599.4826 peak, an aggregation of two molecules. Other peaks were originated from components of the medium used for growing the bacterium.
Mentions: Detailed analyses of the AHL-like molecules detected were carried out exclusively with the strain V. scophthalmi A102, since both strains presented the same TLC pattern. Two different culture media, mTSB and mPW, were evaluated for growing V. scophthalmi A102 in order to carry out the extraction of AHLs. Due to the high background observed with the mTSB medium, we decided to use the mPW culture medium. Only that extract corresponding to the lower Rf spot resulted in a structure composition similar to an AHL, which hypothetically corresponded to an N-(3-hydroxy dodecanoyl)-L-homoserine lactone (N-3-hydroxy-C12-HSL). This molecule matched the empirical formula C16H29NO4 + H+ with mass (m/z) 300.2167 and an error of 0.39 ppm (see Fig. 2). As far as we know, this constitutes the first description of an AHL in V. scophthalmi; moreover, it has proven different from those produced by other Vibrio species. It was not possible to identify any recognizable AHL-like structures in the other signalling spot extracts, even after carrying out four independent extraction experiments.

Bottom Line: This observation was further supported by the decrease in the presence of these signal molecules after cloning and expression of lactonase AiiA from Bacillus cereus in the V. scophthalmi strains.One of the signal molecules was identified as N-(3-hydroxy dodecanoyl)-L-homoserine lactone.Analysis of the translated sequence revealed that the sequenced luxS gene carried the conserved domain, which is common to luxS sequences found in other species, and which is essential for LuxS enzymatic activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, University of Barcelona, Barcelona, Spain. cristina.garcia@cnm.es

ABSTRACT

Background: Cell-to-cell communication (also referred to as quorum sensing) based on N-acyl-homoserine lactones (AHLs) is a widespread response to environmental change in Gram-negative bacteria. AHLs seem to be highly variable, both in terms of the acyl chain length and in the chemical structure of the radicals. Another quorum sensing pathway, the autoinducer-2-based system, is present both in Gram-positive and Gram-negative bacteria. In this study the presence of signal molecules belonging to both quorum sensing signalling pathways was analysed in the marine symbiotic species Vibrio scophthalmi.

Results: Three AHL-like signal molecules were detected in V. scophthalmi supernatants with the Agrobacterium tumefaciens sensor assay. This observation was further supported by the decrease in the presence of these signal molecules after cloning and expression of lactonase AiiA from Bacillus cereus in the V. scophthalmi strains. One of the signal molecules was identified as N-(3-hydroxy dodecanoyl)-L-homoserine lactone. V. scophthalmi was also shown to carry a functional LuxS synthase. The coding sequence for a luxS-like gene was obtained showing a maximum similarity of 78% with Vibrio vulnificus. Analysis of the translated sequence revealed that the sequenced luxS gene carried the conserved domain, which is common to luxS sequences found in other species, and which is essential for LuxS enzymatic activity.

Conclusion: The data are consistent with the presence of quorum-sensing signal molecules from both AHL- and autoinducer 2-based quorum sensing systems in V. scophthalmi, which are homologous to others previously described in various Vibrio species. How this bacterium interacts with other bacteria and eukaryotic cells to compete ecologically with other intestinal bacteria present in the fish Scophthalmus maximus warrants further investigation.

Show MeSH
Related in: MedlinePlus