TheActinobacillus actinomycetemcomitansRibose Binding Protein RbsB Interacts with Cognate and Heterologous Autoinducer 2 Signals

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Abstract
Autoinducer 2 (AI-2) produced by the oral pathogenActinobacillus actinomycetemcomitansinfluences growth of the organism under iron limitation and regulates the expression of iron uptake genes. However, the cellular components that mediate the response ofA. actinomycetemcomitansto AI-2 have not been fully characterized. Analysis of the complete genome sequence ofA. actinomycetemcomitans(www.oralgen.lanl.gov) indicated that the RbsB protein was related to LuxP, the AI-2 receptor ofVibrio harveyi. To determine if RbsB interacts with AI-2, the bioluminescence of the reporter strainV. harveyiBB170 (sensor 1−, sensor 2+) was determined after stimulation with partially purified AI-2 fromA. actinomycetemcomitansor conditioned medium fromV. harveyicultures in the presence and absence of purified six-His-tagged RbsB. RbsB efficiently inhibitedV. harveyibioluminescence induced by bothA. actinomycetemcomitansAI-2 andV. harveyiAI-2 in a dose-dependent manner, suggesting that RbsB competes with LuxP for AI-2. Fifty percent inhibition occurred with approximately 0.3 nM RbsB forA. actinomycetemcomitansAI-2 and 15 nM RbsB forV. harveyiAI-2. RbsB-mediated inhibition ofV. harveyibioluminescence was reversed by the addition of 50 mM ribose, suggesting thatA. actinomycetemcomitansAI-2 and ribose bind at the same site of RbsB. The RbsB/AI-2 complex was thermostable sinceA. actinomycetemcomitansAI-2 could not be recovered by heating. This was not due to heat inactivation ofA. actinomycetemcomitansAI-2 since signal activity was unaffected by heating in the absence of RbsB. Furthermore, an isogenicA. actinomycetemcomitansmutant that was unable to expressrbsBwas deficient in depletingA. actinomycetemcomitansAI-2 from solution relative to the wild-type organism. Inactivation ofrbsBalso influenced the ability of the organism to grow under iron-limiting conditions. The mutant strain attained a cell density of approximately 30% that of the wild-type organism under iron limitation. In addition, real-time PCR showed that the expression ofafuABC, encoding a major ferric ion transporter, was reduced by approximately eightfold in therbsBmutant. This phenotype was similar to that of a LuxS-deficient mutant ofA. actinomycetemcomitansthat is unable to produce AI-2. Together, our results suggest that RbsB may play a role in the response ofA. actinomycetemcomitansto AI-2.