Identification of novel autoinducer-2 receptors in Clostridia reveals plasticity in the binding site of the LsrB receptor family

Abstract

Edited by Chris Whitfield Autoinducer-2 (AI-2) is unique among quorum-sensing signaling molecules, as it is produced and recognized by a wide variety of bacteria and thus facilitates interspecies communication. To date, two classes of AI-2 receptors have been identified: the LuxP-type, present in the Vibrionales, and the LsrB-type, found in a number of phylogenetically distinct bacterial families. Recently, AI-2 was shown to affect the colonization levels of a variety of bacteria in the microbiome of the mouse gut, including members of the genus Clostridium, but no AI-2 receptor had been identified in this genus. Here, we identify a noncanonical, functional LsrB-type receptor in Clostridium saccharobutylicum. This novel LsrB-like receptor is the first one reported with variations in the binding-site amino acid residues that interact with AI-2. The crystal structure of the C. saccharobutylicum receptor determined at 1.35 Å resolution revealed that it binds the same form of AI-2 as the other known LsrB-type receptors, and isothermal titration calorimetry (ITC) assays showed that binding of AI-2 occurs at a submicromolar concentration. Using phylogenetic analysis, we inferred that the newly identified noncanonical LsrB receptor shares a common ancestor with known LsrB receptors and that noncanonical receptors are present in bacteria from different phyla. This led us to identify putative AI-2 receptors in bacterial species in which no receptors were known, as in bacteria belonging to the Spirochaetes and Actinobacteria phyla. Thus, this work represents a significant step toward understanding how AI-2-mediated quorum sensing influences bacterial interactions in complex biological niches.

Sensing of autoinducer-2 by functionally distinct receptors in prokaryotes

“…We further determined the binding affinity between AI-2 and these two receptors by isothermal titration calorimetry (ITC) and found that it has a disassociation constant ( K d ) of 0.14 ± 0.02 μM and 0.12 ± 0.01 μM for PctA-LBD and TlpQ-LBD, respectively (Fig. 2b, c ), which are comparable to the K d values of AI-2 for such established receptors as LuxP (0.16 μM) 30 and LsrB (0.19–0.81 μM) 10 . Under the same experimental conditions, low-affinity binding of AI-2 to PctB-LBD (181 ± 17 μM) and PctC-LBD (99 ± 13 μM) was detected (Supplementary Fig.…”

“…1b–e ) led us to speculate that these two LBDs may interact with AI-2. We first determined the ability of signal released from recombinant proteins purified from an E. coli strain capable of AI-2 production 10 to stimulate light production in the Vibrio harveyi strain MM32 lacking luxN and luxS 7 . Upon denaturing by heat treatment, purified LBDs of PctA and TlpQ from the luxS + E. coli strain released ligands capable of inducing light production in strain MM32 at levels comparable to those by LsrB, an established AI-2-binding protein 10 , whereas these two LBDs purified from an E. coli strain lacking luxS did not release ligands that detectably induced bioluminescence in strain MM32 (Fig.…”

“…The two forms of AI-2 ( S -THMF-borate and R -THMF) can interconvert and addition of borate is known to shift the equilibrium of AI-2 molecules toward the S -THMF-borate form 7 , 10 . We thus investigated the role of boron in the binding of AI-2 to PctA-LBD and TlpQ-LBD.…”

An Efficient Synthesis of Optically Active [4-13C] Labelled Quorum Sensing Signal Autoinducer-2

“…It has been shown that many species of Gram-positive and Gram-negative bacteria can detect different forms of this molecule and thus all these forms are known collectively as AI-2. Specifically, many Vibrio have the LuxP-type of AI-2 receptor which recognises borated isomer 6 [ 8 ], whereas members belonging to phylogenetically distinct families such as the human pathogens Salmonella typhimurium and Bacillus anthracis , as well as different Clostridium and the plant symbiont Sinorhizobium meliloti having the LsrB-type of receptors, recognise the non-borated diastereoisomer 5 [ 9 , 10 ]. Bacteria that have the LsrB-type of receptor internalise this signal via an ABC transporter.…”

“…Once inside the cell the non-borated form of AI-2 is phosphorylated and in bacteria such as Escherichia coli the phosphorylated compound is cleaved by a thiolase (LsrF) into metabolites which are channelled into glycolysis and the citric acid cycle, as means to terminate quorum sensing [ 11 ]. This thiolase is not present in all bacteria that have the LsrB receptor, indicating that AI-2 metabolism might not be as conserved as the receptors [ 10 ].…”