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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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Overexpression of sypK activates qrr1 expression. Levels of qrr1 expression in the reporter strain TIM303 harboring vector (pTM214), Ptrc-sypK (pTM367), or Ptrc-sypL (pTM368) grown ±100 μmol/L IPTG. The wild-type strain ES114 harboring pTM214 was used as the GFP-negative control for quantifying GFP levels. Graphical and error bars represent the averages and standard deviations of triplicate biological replicates, respectively. Comparisons with significance based on two-way ANOVA with Tukey's multiple comparisons are shown with *(P-value <0.01). Comparisons between pTM367-harboring strains with strains harboring either pTM214 or pTM368 are significant (P-value <0.001). Experiment was performed three times, with similar results.
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fig04: Overexpression of sypK activates qrr1 expression. Levels of qrr1 expression in the reporter strain TIM303 harboring vector (pTM214), Ptrc-sypK (pTM367), or Ptrc-sypL (pTM368) grown ±100 μmol/L IPTG. The wild-type strain ES114 harboring pTM214 was used as the GFP-negative control for quantifying GFP levels. Graphical and error bars represent the averages and standard deviations of triplicate biological replicates, respectively. Comparisons with significance based on two-way ANOVA with Tukey's multiple comparisons are shown with *(P-value <0.01). Comparisons between pTM367-harboring strains with strains harboring either pTM214 or pTM368 are significant (P-value <0.001). Experiment was performed three times, with similar results.

Mentions: To determine whether increased expression of sypK or sypL leads to qrr1 expression, we separately cloned sypK and sypL on a plasmid downstream of the IPTG-inducible trc promoter. For experiments involving overexpression vectors, we monitored qrr1 expression using a GFP transcriptional reporter integrated into the chromosome at the Tn7 insertion site. We found that basal expression of sypK from the trc promoter was sufficient to increase qrr1 levels at least threefold higher than wild-type levels (Fig. 4). Addition of IPTG to the growth medium resulted in a slight but significant increase in qrr1 expression. In contrast, we did not detect any change in qrr1 expression when using the IPTG-inducible sypL construct. These results indicate that overexpression of sypK is sufficient to activate qrr1 expression.


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)

Overexpression of sypK activates qrr1 expression. Levels of qrr1 expression in the reporter strain TIM303 harboring vector (pTM214), Ptrc-sypK (pTM367), or Ptrc-sypL (pTM368) grown ±100 μmol/L IPTG. The wild-type strain ES114 harboring pTM214 was used as the GFP-negative control for quantifying GFP levels. Graphical and error bars represent the averages and standard deviations of triplicate biological replicates, respectively. Comparisons with significance based on two-way ANOVA with Tukey's multiple comparisons are shown with *(P-value <0.01). Comparisons between pTM367-harboring strains with strains harboring either pTM214 or pTM368 are significant (P-value <0.001). Experiment was performed three times, with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263508&req=5

fig04: Overexpression of sypK activates qrr1 expression. Levels of qrr1 expression in the reporter strain TIM303 harboring vector (pTM214), Ptrc-sypK (pTM367), or Ptrc-sypL (pTM368) grown ±100 μmol/L IPTG. The wild-type strain ES114 harboring pTM214 was used as the GFP-negative control for quantifying GFP levels. Graphical and error bars represent the averages and standard deviations of triplicate biological replicates, respectively. Comparisons with significance based on two-way ANOVA with Tukey's multiple comparisons are shown with *(P-value <0.01). Comparisons between pTM367-harboring strains with strains harboring either pTM214 or pTM368 are significant (P-value <0.001). Experiment was performed three times, with similar results.
Mentions: To determine whether increased expression of sypK or sypL leads to qrr1 expression, we separately cloned sypK and sypL on a plasmid downstream of the IPTG-inducible trc promoter. For experiments involving overexpression vectors, we monitored qrr1 expression using a GFP transcriptional reporter integrated into the chromosome at the Tn7 insertion site. We found that basal expression of sypK from the trc promoter was sufficient to increase qrr1 levels at least threefold higher than wild-type levels (Fig. 4). Addition of IPTG to the growth medium resulted in a slight but significant increase in qrr1 expression. In contrast, we did not detect any change in qrr1 expression when using the IPTG-inducible sypL construct. These results indicate that overexpression of sypK is sufficient to activate qrr1 expression.

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