Identification of Functional LsrB-Like Autoinducer-2 Receptors

Abstract

Although a variety of bacterial species have been reported to use the interspecies communication signal autoinducer-2 (AI-2) to regulate multiple behaviors, the molecular mechanisms of AI-2 recognition and signal transduction remain poorly understood. To date, two types of AI-2 receptors have been identified: LuxP, present in Vibrio spp., and LsrB, first identified in Salmonella enterica serovar Typhimurium. In S. Typhimurium, LsrB is the ligand binding protein of a transport system that enables the internalization of AI-2. Here, using both sequence analysis and structure prediction, we establish a set of criteria for identifying functional AI-2 receptors. We test our predictions experimentally, assaying key species for their abilities to import AI-2 in vivo, and test their LsrB orthologs for AI-2 binding in vitro. Using these experimental approaches, we were able to identify AI-2 receptors in organisms belonging to phylogenetically distinct families such as the Enterobacteriaceae, Rhizobiaceae, and Bacillaceae. Phylogenetic analysis of LsrB orthologs indicates that this pattern could result from one single origin of the functional LsrB gene in a gammaproteobacterium, suggesting possible posterior independent events of lateral gene transfer to the Alphaproteobacteria and Firmicutes. Finally, we used mutagenesis to show that two AI-2-interacting residues are essential for the AI-2 binding ability. These two residues are conserved in the binding sites of all the functional AI-2 binding proteins but not in the non-AI-2-binding orthologs. Together, these results strongly support our ability to identify functional LsrBtype AI-2 receptors, an important step in investigations of this interspecies signal.Autoinducer-2 (AI-2) is a small molecule produced and secreted by a large number of bacterial species belonging to very widespread branches within the kingdom Bacteria (15, 46, 64). AI-2 or its synthase, LuxS, has been implicated in the regulation of many bacterial behaviors including biofilm formation, virulence, competence, and the production of secondary metabolites like antibiotics (17,60,64). While in some cases, AI-2 is clearly acting through a canonical quorum-sensing mechanism (61), in others a role in central metabolism has been proposed (62). One of the obstacles to an understanding of the function of AI-2 in any given species is a lack of knowledge of the molecular mechanisms of AI-2 recognition, signal transduction, and/or processing.Undoubtedly, one of the major difficulties in identifying AI-2 receptors is the complexity of the chemistry of this signal molecule. The product of the reaction catalyzed by LuxS is 4,5-dihydroxy-2,3-pentadione (DPD), which, in solution, spontaneously rearranges into a variety of chemically distinct forms collectively called AI-2 (31, 46). We have shown that these forms are in equilibrium and can thus interconvert and that the availability of the different forms of AI-2 is highly dependent on the chemistry of the environment (31). Additionally, different organisms recognize dist...

Processing the Interspecies Quorum-sensing Signal Autoinducer-2 (AI-2)

“…Although the conservation of catalytic residues among the proteins from this family is low, this is not the case among the previously identified LsrG orthologs (12). In this group there was 100% conservation of 7 residues in and around the active site (which include the residues Glu-54 and His-65 and His-70) out of a total of only 9 completely conserved residues (in addition to the start Met).…”

“…The conservation of other active site residues, including those shown here to be essential for function, supports the prediction that the function of LsrG in the regulation of the Lsr system is conserved in the range of species with LsrG homologues. The lsr operon and lsrG homologues are present in bacteria including important human pathogens such as S. typhimurium, B. anthracis, and Y. pestis (12) and thus can impact communications in multispecies bacterial communities that include pathogens such as these.…”

“…Sequence Comparison with LsrG from Other Organisms-In earlier work (12) we used bioinformatics analysis to identify bacterial species that have a functional lsr operon. Using the sequences for LsrG from these species, we created a multiple sequence alignment via ClustalW2 (29).…”

“…We have previously shown that certain bacteria can employ this strategy by processing AI-2 and thus quenching interspecies signaling (11). These bacteria include most members from the Enterobacteriaceae family (like the commensal Escherichia coli K12 and the pathogens E. coli O157 and Salmonella typhimurium) but also more distantly related bacteria such as the plant symbiont Sinorhizobium meliloti and the pathogen Bacillus anthracis (12).…”

Modification of quorum sensing in bacterial cultures

“…This high-affinity substratebinding periplasmic protein also interacts with the ABC transport system, called Lsr (36). The LsrB family is typically present in enteric bacteria and the members of the Rhizobiaceae and Bacillaceae and have high affinity for the R-THMF isomer of DPD (35,37).…”