The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri.
Bottom Line: We isolated unique mutants with a transposon inserted into one of two genes within the syp locus, which is involved in biofilm formation.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.
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
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Mentions: Like many histidine kinases, LuxQ is bifunctional, with both kinase and phosphatase activities (Freeman and Bassler 1999a, 1999b). To determine which enzymatic activity of LuxQ is modulated by SypK, we used two LuxQ variants, A216P or H378A, that exhibit only kinase (K+/P−) or phosphatase (K−/P+) activities, respectively (Neiditch et al. 2006; Ray and Visick 2012). Consistent with the results shown in Figure 6, we found that qrr1 expression remained low in a ΔluxQ mutant, regardless of whether sypK was overexpressed (Fig. 7A). Introduction of luxQ into the ΔluxQ mutant in trans resulted in an increased expression of qrr1. The level of qrr1 expression in this strain was even higher than in wild-type cells (Fig. 6), presumably due to the overexpression of luxQ from the multicopy plasmid pVAR48. In V. cholerae, a similar activation of the analogous LuxU-LuxO phosphorelay by overexpression of the luxQ homologue has been observed (Shikuma et al. 2009). Similar to the results shown in Figure 6, the overexpression of sypK in V. fischeri harboring luxQ in trans also led to the increased qrr1 expression (Fig. 7A). In the presence of the K+/P− mutation luxQA216P, qrr1 was expressed, albeit at lower levels than in the presence of luxQ. This lower level of qrr1 expression may be attributed to the overall lower levels of the LuxQA216P variant relative to wild-type LuxQ (Fig. 7B). Overexpression of sypK in this background resulted in a high level of qrr1 expression that was comparable to the level induced by sypK in cells expressing wild-type LuxQ (Fig. 7A). Finally, the expression of qrr1 was low in cells harboring the K−/P+ variant of LuxQ regardless of sypK induction. As shown in Figure 7B, the LuxQH378A variant was produced at levels comparable to wild-type LuxQ. Taken together, these data suggest that the kinase activity of LuxQ is required for SypK-dependent regulation of qrr1 expression.
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.