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The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri.

Miyashiro T, Oehlert D, Ray VA, Visick KL, Ruby EG - Microbiologyopen (2014)

Bottom Line: We found that overexpression of sypK, which encodes a putative oligosaccharide translocase, is sufficient to activate qrr1, and, in addition, this effect appears to depend on the kinase activity of the sensor LuxQ.Finally, we found that induction of the syp locus by overexpression of sypG was sufficient to activate qrr1 levels.Together, our results show how conditions that promote biofilm formation impact the quorum-signaling network in V. fischeri, and further highlight the integrated nature of the regulatory circuits involved in complex bacterial behaviors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Eberly College of Science, The Pennsylvania State University, University Park, Pennsylvania, 16802; Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, 53706.

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Mutants with a transposon insertion in the sypIJKL operon have enhanced qrr1 expression. (A) Levels of qrr1 expression in WT (ES114), ΔluxO (TIM306), sypI::Tn5 (DRO5F11), sypJ::Tn5 (DRO1B3), and sypI::Tn5 [NT] (DRO222) harboring the reporter plasmid pTM268. The nonfluorescent strain ES114 harboring pVSV105 was used to calculate cellular levels of GFP and mCherry. Graphical and error bars represent the averages and standard deviations of triplicate biological replicates, respectively. One-way ANOVA with Tukey's multiple comparisons test show significance (P-value <0.01) between columns, except for the comparison labeled not significant (n.s.). Experiment was performed three times, with similar results. (B) Transposon insertion sites of mutants examined in (A). Genes disrupted by a transposon are highlighted in gray. Arrows above transposon insertions indicate direction of erm gene transcription. (C) Luminescence levels of WT (ES114), Δlux (EVS102), sypI::Tn5 [NT] (DRO222), Δqrr1 (TIM305), ΔluxO (TIM306), and ΔlitR (TIM358) in response to 120 nmol/L 3-oxo-C6. Graphical and error bars represent the averages and standard deviations of triplicate biological replicates, respectively. One-way ANOVA with Tukey's multiple comparisons test on log-transformed data show significance (P-value <0.01) between columns, except for the comparison labeled not significant (n.s.). Experiment was performed three times, with similar results.
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fig02: Mutants with a transposon insertion in the sypIJKL operon have enhanced qrr1 expression. (A) Levels of qrr1 expression in WT (ES114), ΔluxO (TIM306), sypI::Tn5 (DRO5F11), sypJ::Tn5 (DRO1B3), and sypI::Tn5 [NT] (DRO222) harboring the reporter plasmid pTM268. The nonfluorescent strain ES114 harboring pVSV105 was used to calculate cellular levels of GFP and mCherry. Graphical and error bars represent the averages and standard deviations of triplicate biological replicates, respectively. One-way ANOVA with Tukey's multiple comparisons test show significance (P-value <0.01) between columns, except for the comparison labeled not significant (n.s.). Experiment was performed three times, with similar results. (B) Transposon insertion sites of mutants examined in (A). Genes disrupted by a transposon are highlighted in gray. Arrows above transposon insertions indicate direction of erm gene transcription. (C) Luminescence levels of WT (ES114), Δlux (EVS102), sypI::Tn5 [NT] (DRO222), Δqrr1 (TIM305), ΔluxO (TIM306), and ΔlitR (TIM358) in response to 120 nmol/L 3-oxo-C6. Graphical and error bars represent the averages and standard deviations of triplicate biological replicates, respectively. One-way ANOVA with Tukey's multiple comparisons test on log-transformed data show significance (P-value <0.01) between columns, except for the comparison labeled not significant (n.s.). Experiment was performed three times, with similar results.

Mentions: We screened over 100,000 colonies and isolated ˜100 clones with elevated levels of GFP. In this study, we report our characterization of two mutants isolated from this genetic screen. Both mutants displayed qrr1 expression levels that were approximately threefold higher than wild-type cells (Fig. 2A). Sequencing of the transposon insertion site in each mutant revealed an insertion in either VF_A1028 (sypI) or VF_A1029 (sypJ) (Fig. 2B). To determine whether the phenotype of elevated qrr1 expression was linked to the transposon, we reintroduced the sypI transposon insertion into ES114 by transformation. The resulting strain, DRO222 (designated sypI::Tn5 [NT]), showed qrr1 expression levels comparable to the original transposon insertion mutant (sypI::Tn5), indicating that the transposon insertion in sypI is linked to elevated qrr1 expression (Fig. 2A). Hereafter, the sypI::Tn5 [NT] mutant is termed the sypI mutant.


The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri.

Miyashiro T, Oehlert D, Ray VA, Visick KL, Ruby EG - Microbiologyopen (2014)

Mutants with a transposon insertion in the sypIJKL operon have enhanced qrr1 expression. (A) Levels of qrr1 expression in WT (ES114), ΔluxO (TIM306), sypI::Tn5 (DRO5F11), sypJ::Tn5 (DRO1B3), and sypI::Tn5 [NT] (DRO222) harboring the reporter plasmid pTM268. The nonfluorescent strain ES114 harboring pVSV105 was used to calculate cellular levels of GFP and mCherry. Graphical and error bars represent the averages and standard deviations of triplicate biological replicates, respectively. One-way ANOVA with Tukey's multiple comparisons test show significance (P-value <0.01) between columns, except for the comparison labeled not significant (n.s.). Experiment was performed three times, with similar results. (B) Transposon insertion sites of mutants examined in (A). Genes disrupted by a transposon are highlighted in gray. Arrows above transposon insertions indicate direction of erm gene transcription. (C) Luminescence levels of WT (ES114), Δlux (EVS102), sypI::Tn5 [NT] (DRO222), Δqrr1 (TIM305), ΔluxO (TIM306), and ΔlitR (TIM358) in response to 120 nmol/L 3-oxo-C6. Graphical and error bars represent the averages and standard deviations of triplicate biological replicates, respectively. One-way ANOVA with Tukey's multiple comparisons test on log-transformed data show significance (P-value <0.01) between columns, except for the comparison labeled not significant (n.s.). Experiment was performed three times, with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig02: Mutants with a transposon insertion in the sypIJKL operon have enhanced qrr1 expression. (A) Levels of qrr1 expression in WT (ES114), ΔluxO (TIM306), sypI::Tn5 (DRO5F11), sypJ::Tn5 (DRO1B3), and sypI::Tn5 [NT] (DRO222) harboring the reporter plasmid pTM268. The nonfluorescent strain ES114 harboring pVSV105 was used to calculate cellular levels of GFP and mCherry. Graphical and error bars represent the averages and standard deviations of triplicate biological replicates, respectively. One-way ANOVA with Tukey's multiple comparisons test show significance (P-value <0.01) between columns, except for the comparison labeled not significant (n.s.). Experiment was performed three times, with similar results. (B) Transposon insertion sites of mutants examined in (A). Genes disrupted by a transposon are highlighted in gray. Arrows above transposon insertions indicate direction of erm gene transcription. (C) Luminescence levels of WT (ES114), Δlux (EVS102), sypI::Tn5 [NT] (DRO222), Δqrr1 (TIM305), ΔluxO (TIM306), and ΔlitR (TIM358) in response to 120 nmol/L 3-oxo-C6. Graphical and error bars represent the averages and standard deviations of triplicate biological replicates, respectively. One-way ANOVA with Tukey's multiple comparisons test on log-transformed data show significance (P-value <0.01) between columns, except for the comparison labeled not significant (n.s.). Experiment was performed three times, with similar results.
Mentions: We screened over 100,000 colonies and isolated ˜100 clones with elevated levels of GFP. In this study, we report our characterization of two mutants isolated from this genetic screen. Both mutants displayed qrr1 expression levels that were approximately threefold higher than wild-type cells (Fig. 2A). Sequencing of the transposon insertion site in each mutant revealed an insertion in either VF_A1028 (sypI) or VF_A1029 (sypJ) (Fig. 2B). To determine whether the phenotype of elevated qrr1 expression was linked to the transposon, we reintroduced the sypI transposon insertion into ES114 by transformation. The resulting strain, DRO222 (designated sypI::Tn5 [NT]), showed qrr1 expression levels comparable to the original transposon insertion mutant (sypI::Tn5), indicating that the transposon insertion in sypI is linked to elevated qrr1 expression (Fig. 2A). Hereafter, the sypI::Tn5 [NT] mutant is termed the sypI mutant.

Bottom Line: We found that overexpression of sypK, which encodes a putative oligosaccharide translocase, is sufficient to activate qrr1, and, in addition, this effect appears to depend on the kinase activity of the sensor LuxQ.Finally, we found that induction of the syp locus by overexpression of sypG was sufficient to activate qrr1 levels.Together, our results show how conditions that promote biofilm formation impact the quorum-signaling network in V. fischeri, and further highlight the integrated nature of the regulatory circuits involved in complex bacterial behaviors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Eberly College of Science, The Pennsylvania State University, University Park, Pennsylvania, 16802; Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, 53706.

Show MeSH
Related in: MedlinePlus