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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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Polar effects of transposon insertion on syp gene expression. Quantitative reverse-transcriptase PCR analysis of various genes in the sypI mutant (DRO222) relative to WT (ES114). Genes tested are VF_2254 (rpoD), VF_2177 (litR), VF_A1020 (sypA), VF_A1025 (sypF), VF_A1029 (sypJ), VF_A1030 (sypK), VF_A1031 (sypL), VF_A1032 (sypM), and VF_A1035 (sypP). Values are of quadruplicate biological replicates and normalized by wild-type levels. Error bars indicate ± 1 SD. Comparisons with significance based on unpaired t-tests are shown with *(P-value <0.01) and **(P-value <0.001), where P-values are adjusted using false discovery rate correction.
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fig03: Polar effects of transposon insertion on syp gene expression. Quantitative reverse-transcriptase PCR analysis of various genes in the sypI mutant (DRO222) relative to WT (ES114). Genes tested are VF_2254 (rpoD), VF_2177 (litR), VF_A1020 (sypA), VF_A1025 (sypF), VF_A1029 (sypJ), VF_A1030 (sypK), VF_A1031 (sypL), VF_A1032 (sypM), and VF_A1035 (sypP). Values are of quadruplicate biological replicates and normalized by wild-type levels. Error bars indicate ± 1 SD. Comparisons with significance based on unpaired t-tests are shown with *(P-value <0.01) and **(P-value <0.001), where P-values are adjusted using false discovery rate correction.

Mentions: To test this hypothesis, we examined the transcript levels of various genes in the sypI mutant. We found that litR levels were reduced 3.3-fold in the sypI mutant relative to wild-type cells (Fig. 3), consistent with the elevated expression of qrr1 in the mutant. We also found that the sypI mutant exhibited higher transcription levels of sypJ (4.7-fold), sypK (18.5-fold), and sypL (2.8-fold). Because our original screen also identified a sypJ mutant, we conclude that the elevated level of qrr1 expression in the sypI mutant is independent of sypJ. To determine whether the transposon insertion resulted in a general activation of the syp locus, we also examined several other syp genes. While the level of sypM, which is contained within the adjacent operon (Fig. 2B), was elevated 3.2-fold (Fig. 3), three other genes (sypA, sypF, and sypP) displayed wild-type levels of transcription in the sypI mutant. These results demonstrate that the transposon insertion leads to the increased expression of several downstream genes, but not of the entire locus.


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)

Polar effects of transposon insertion on syp gene expression. Quantitative reverse-transcriptase PCR analysis of various genes in the sypI mutant (DRO222) relative to WT (ES114). Genes tested are VF_2254 (rpoD), VF_2177 (litR), VF_A1020 (sypA), VF_A1025 (sypF), VF_A1029 (sypJ), VF_A1030 (sypK), VF_A1031 (sypL), VF_A1032 (sypM), and VF_A1035 (sypP). Values are of quadruplicate biological replicates and normalized by wild-type levels. Error bars indicate ± 1 SD. Comparisons with significance based on unpaired t-tests are shown with *(P-value <0.01) and **(P-value <0.001), where P-values are adjusted using false discovery rate correction.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: Polar effects of transposon insertion on syp gene expression. Quantitative reverse-transcriptase PCR analysis of various genes in the sypI mutant (DRO222) relative to WT (ES114). Genes tested are VF_2254 (rpoD), VF_2177 (litR), VF_A1020 (sypA), VF_A1025 (sypF), VF_A1029 (sypJ), VF_A1030 (sypK), VF_A1031 (sypL), VF_A1032 (sypM), and VF_A1035 (sypP). Values are of quadruplicate biological replicates and normalized by wild-type levels. Error bars indicate ± 1 SD. Comparisons with significance based on unpaired t-tests are shown with *(P-value <0.01) and **(P-value <0.001), where P-values are adjusted using false discovery rate correction.
Mentions: To test this hypothesis, we examined the transcript levels of various genes in the sypI mutant. We found that litR levels were reduced 3.3-fold in the sypI mutant relative to wild-type cells (Fig. 3), consistent with the elevated expression of qrr1 in the mutant. We also found that the sypI mutant exhibited higher transcription levels of sypJ (4.7-fold), sypK (18.5-fold), and sypL (2.8-fold). Because our original screen also identified a sypJ mutant, we conclude that the elevated level of qrr1 expression in the sypI mutant is independent of sypJ. To determine whether the transposon insertion resulted in a general activation of the syp locus, we also examined several other syp genes. While the level of sypM, which is contained within the adjacent operon (Fig. 2B), was elevated 3.2-fold (Fig. 3), three other genes (sypA, sypF, and sypP) displayed wild-type levels of transcription in the sypI mutant. These results demonstrate that the transposon insertion leads to the increased expression of several downstream genes, but not of the entire locus.

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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