Sinorhizobium meliloti, a bacterium lacking the autoinducer-2 (AI-2) synthase, responds to AI-2 supplied by other bacteria

Many bacterial species respond to the quorum-sensing signal autoinducer-2 (AI-2) by regulating different niche-specific genes. Here, we show that Sinorhizobium meliloti, a plant symbiont lacking the gene for the AI-2 synthase, while not capable of producing AI-2 can nonetheless respond to AI-2 produced by other species. We demonstrate that S. meliloti has a periplasmic binding protein that binds AI-2. The crystal structure of this protein (here named SmlsrB) with its ligand reveals that it binds (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (R-THMF), the identical AI-2 isomer recognized by LsrB of Salmonella typhimurium. The gene encoding SmlsrB is in an operon with orthologues of the lsr genes required for AI-2 internalization in enteric bacteria. Accordingly, S. meliloti internalizes exogenous AI-2, and mutants in this operon are defective in AI-2 internalization. S. meliloti does not gain a metabolic benefit from internalizing AI-2, suggesting that AI-2 functions as a signal in S. meliloti. Furthermore, S. meliloti can completely eliminate the AI-2 secreted by Erwinia carotovora, a plant pathogen shown to use AI-2 to regulate virulence. Our findings suggest that S. meliloti is capable of 'eavesdropping' on the AI-2 signalling of other species and interfering with AI-2-regulated behaviours such as virulence.     

Processing the interspecies quorum-sensing signal autoinducer-2 (AI-2): characterization of phospho-(S)-4,5-dihydroxy-2,3-pentanedione isomerization by LsrG protein

The molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD) is produced by many different species of bacteria and is the precursor of the signal molecule autoinducer-2 (AI-2). AI-2 mediates interspecies communication and facilitates regulation of bacterial behaviors such as biofilm formation and virulence. A variety of bacterial species have the ability to sequester and process the AI-2 present in their environment, thereby interfering with the cell-cell communication of other bacteria. This process involves the AI-2-regulated lsr operon, comprised of the Lsr transport system that facilitates uptake of the signal, a kinase that phosphorylates the signal to phospho-DPD (P-DPD), and enzymes (like LsrG) that are responsible for processing the phosphorylated signal. Because P-DPD is the intracellular inducer of the lsr operon, enzymes involved in P-DPD processing impact the levels of Lsr expression. Here we show that LsrG catalyzes isomerization of P-DPD into 3,4,4-trihydroxy-2-pentanone-5-phosphate. We present the crystal structure of LsrG, identify potential catalytic residues, and determine which of these residues affects P-DPD processing in vivo and in vitro. We also show that an lsrG deletion mutant accumulates at least 10 times more P-DPD than wild type cells. Consistent with this result, we find that the lsrG mutant has increased expression of the lsr operon and an altered profile of AI-2 accumulation and removal. Understanding of the biochemical mechanisms employed by bacteria to quench signaling of other species can be of great utility in the development of therapies to control bacterial behavior.     

luxS-dependent gene regulation in Escherichia coli K-12 revealed by genomic expression profiling

The bacterial quorum-sensing autoinducer 2 (AI-2) has received intense interest because the gene for its synthase, luxS, is common among a large number of bacterial species. We have identified luxS-controlled genes in Escherichia coli under two different growth conditions using DNA microarrays. Twenty-three genes were affected by luxS deletion in the presence of glucose, and 63 genes were influenced by luxS deletion in the absence of glucose. Minimal overlap among these gene sets suggests the role of luxS is condition dependent. Under the latter condition, the metE gene, the lsrACDBFG operon, and the flanking genes of the lsr operon (lsrR, lsrK, tam, and yneE) were among the most significantly induced genes by luxS. The E. coli lsr operon includes an additional gene, tam, encoding an S-adenosyl-l-methionine-dependent methyltransferase. Also, lsrR and lsrK belong to the same operon, lsrRK, which is positively regulated by the cyclic AMP receptor protein and negatively regulated by LsrR. lsrK is additionally transcribed by a promoter between lsrR and lsrK. Deletion of luxS was also shown to affect genes involved in methionine biosynthesis, methyl transfer reactions, iron uptake, and utilization of carbon. It was surprising, however, that so few genes were affected by luxS deletion in this E. coli K-12 strain under these conditions. Most of the highly induced genes are related to AI-2 production and transport. These data are consistent with the function of LuxS as an important metabolic enzyme but appear not to support the role of AI-2 as a true signal molecule for E. coli W3110 under the investigated conditions.     

AI-3 synthesis is not dependent on luxS in Escherichia coli

The quorum-sensing (QS) signal autoinducer-2 (AI-2) has been proposed to promote interspecies signaling in a broad range of bacterial species. AI-2 is spontaneously derived from 4,5-dihydroxy-2,3-pentanedione that, along with homocysteine, is produced by cleavage of S-adenosylhomocysteine (SAH) and S-ribosylhomocysteine by the Pfs and LuxS enzymes. Numerous phenotypes have been attributed to AI-2 QS signaling using luxS mutants. We have previously reported that the luxS mutation also affects the synthesis of the AI-3 autoinducer that activates enterohemorrhagic Escherichia coli virulence genes. Here we show that several species of bacteria synthesize AI-3, suggesting a possible role in interspecies bacterial communication. The luxS mutation leaves the cell with only one pathway, involving oxaloacetate and l-glutamate, for de novo synthesis of homocysteine. The exclusive use of this pathway for homocysteine production appears to alter metabolism in the luxS mutant, leading to decreased levels of AI-3. The addition of aspartate and expression of an aromatic amino acid transporter, as well as a tyrosine-specific transporter, restored AI-3-dependent phenotypes in an luxS mutant. The defect in AI-3 production, but not in AI-2 production, in the luxS mutant was restored by expressing the Pseudomonas aeruginosa S-adenosylhomocysteine hydrolase that synthesizes homocysteine directly from SAH. Furthermore, phenotype microarrays revealed that the luxS mutation caused numerous metabolic deficiencies, while AI-3 signaling had little effect on metabolism. This study examines how AI-3 production is affected by the luxS mutation and explores the roles of the LuxS/AI-2 system in metabolism and QS.     

Differential interaction of Aggregatibacter (Actinobacillus) actinomycetemcomitans LsrB and RbsB proteins with autoinducer 2

Our previous studies showed that the Aggregatibacter actinomycetemcomitans RbsB protein interacts with cognate and heterologous autoinducer 2 (AI-2) signals and suggested that the rbsDABCK operon encodes a transporter that may internalize AI-2 (D. James et al., Infect. Immun. 74:4021-4029, 2006.). However, A. actinomycetemcomitans also possesses genes related to the lsr operon of Salmonella enterica serovar Typhimurium which function to import AI-2. Here, we show that A. actinomycetemcomitans LsrB protein competitively inhibits the interaction of the Vibrio harveyi AI-2 receptor (LuxP) with AI-2 from either A. actinomycetemcomitans or V. harveyi. Interestingly, LsrB was a more potent inhibitor of LuxP interaction with AI-2 from V. harveyi whereas RbsB competed more effectively with LuxP for A. actinomycetemcomitans AI-2. Inactivation of lsrB in wild-type A. actinomycetemcomitans or in an isogenic RbsB-deficient strain reduced the rate by which intact bacteria depleted A. actinomycetemcomitans AI-2 from solution. Consistent with the results from the LuxP competition experiments, the LsrB-deficient strain depleted AI-2 to a lesser extent than the RbsB-deficient organism. Inactivation of both lsrB and rbsB virtually eliminated the ability of the organism to remove AI-2 from the extracellular environment. These results suggest that A. actinomycetemcomitans possesses two proteins that differentially interact with AI-2 and may function to inactivate or facilitate internalization of AI-2.     

Synthesis of autoinducer 2 by the lyme disease spirochete, Borrelia burgdorferi

Defining the metabolic capabilities and regulatory mechanisms controlling gene expression is a valuable step in understanding the pathogenic properties of infectious agents such as Borrelia burgdorferi. The present studies demonstrated that B. burgdorferi encodes functional Pfs and LuxS enzymes for the breakdown of toxic products of methylation reactions. Consistent with those observations, B. burgdorferi was shown to synthesize the end product 4,5-dihydroxy-2,3-pentanedione (DPD) during laboratory cultivation. DPD undergoes spontaneous rearrangements to produce a class of pheromones collectively named autoinducer 2 (AI-2). Addition of in vitro-synthesized DPD to cultured B. burgdorferi resulted in differential expression of a distinct subset of proteins, including the outer surface lipoprotein VlsE. Although many bacteria can utilize the other LuxS product, homocysteine, for regeneration of methionine, B. burgdorferi was found to lack such ability. It is hypothesized that B. burgdorferi produces LuxS for the express purpose of synthesizing DPD and utilizes a form of that molecule as an AI-2 pheromone to control gene expression.     

Let LuxS speak up in AI-2 signaling

Quorum sensing is a process of bacterial cell-cell communication that uses small diffusible molecules to coordinate diverse behaviors in response to population density. The only quorum-sensing system shared by Gram-positive and Gram-negative bacteria involves the production of autoinducer-2 (AI-2). The AI-2 synthase LuxS is widely distributed among the Bacteria, which suggests that AI-2 is a language for interspecies communication. However, LuxS is also an integral component of the activated methyl cycle in bacteria. LuxS-based quorum sensing has been intensively studied in the past decade, mostly in relation to the AI-2 molecule and the downstream effects of luxS knockouts; few studies have focused on the gene and protein activity itself. Ongoing attempts to dissect the metabolic and signaling roles of LuxS leave little doubt that unraveling the regulation of luxS expression and cellular LuxS activity is the key to understanding LuxS-based quorum sensing.     

群体感应信号分子AI-2研究进展

群体感应(QS)是细菌根据种群密度的变化调控基因表达,协调群体行为的机制。除具有种特异性的信号分子AI-1外,近年来发现一类新的信号分子AI-2在调控细菌基因表达中起重要作用。AI-2的结构和生物合成途径已被确定,其产生依赖于一种称为LuxS的蛋白。目前认为AI-2在细菌种间交流中起通用信号分子(universalsignal)的作用。了解细菌的QS调控过程以及种间细胞交流的新机制,有助于对细菌病害进行防治。     

Gene network homology in prokaryotes using a similarity search approach: queries of quorum sensing signal transduction

Bacterial cell-cell communication is mediated by small signaling molecules known as autoinducers. Importantly, autoinducer-2 (AI-2) is synthesized via the enzyme LuxS in over 80 species, some of which mediate their pathogenicity by recognizing and transducing this signal in a cell density dependent manner. AI-2 mediated phenotypes are not well understood however, as the means for signal transduction appears varied among species, while AI-2 synthesis processes appear conserved. Approaches to reveal the recognition pathways of AI-2 will shed light on pathogenicity as we believe recognition of the signal is likely as important, if not more, than the signal synthesis. LMNAST (Local Modular Network Alignment Similarity Tool) uses a local similarity search heuristic to study gene order, generating homology hits for the genomic arrangement of a query gene sequence. We develop and apply this tool for the E. coli lac and LuxS regulated (Lsr) systems. Lsr is of great interest as it mediates AI-2 uptake and processing. Both test searches generated results that were subsequently analyzed through a number of different lenses, each with its own level of granularity, from a binary phylogenetic representation down to trackback plots that preserve genomic organizational information. Through a survey of these results, we demonstrate the identification of orthologs, paralogs, hitchhiking genes, gene loss, gene rearrangement within an operon context, and also horizontal gene transfer (HGT). We found a variety of operon structures that are consistent with our hypothesis that the signal can be perceived and transduced by homologous protein complexes, while their regulation may be key to defining subsequent phenotypic behavior.     

AI-2 biosynthesis module in a magnetic nanofactory alters bacterial response via localized synthesis and delivery

Nanofactories are nano-dimensioned and comprised of modules serving various functions that alter the response of targeted cells when deployed by locally synthesizing and delivering cargo to the surfaces of the targeted cells. In its basic form, a nanofactory consists of a minimum of two functional modules: a cell capture module and a synthesis module. In this work, magnetic nanofactories that alter the response of targeted bacteria by the localized synthesis and delivery of the "universal" bacterial quorum sensing signal molecule autoinducer AI-2 are demonstrated. The magnetic nanofactories consist of a cell capture module (chitosan-mag nanoparticles) and an AI-2 biosynthesis module that contains both AI-2 biosynthetic enzymes Pfs and LuxS on a fusion protein (His-LuxS-Pfs-Tyr, HLPT) assembled together. HLPT is hypothesized to be more efficient than its constituent enzymes (used separately) at conversion of the substrate SAH to product AI-2 on account of the proximity of the two enzymes within the fusion protein. HLPT is demonstrated to be more active than the constituent enzymes, Pfs and LuxS, over a wide range of experimental conditions. The magnetic nanofactories (containing bound HLPT) are also demonstrated to be more active than free, unbound HLPT. They are also shown to elicit an increased response in targeted Escherichia coli cells, due to the localized synthesis and delivery of AI-2, when compared to the response produced by the addition of AI-2 directly to the cells. Studies investigating the universality of AI-2 and unraveling AI-2 based quorum sensing in bacteria using magnetic nanofactories are envisioned. The prospects of using such multi-modular nanofactories in developing the next generation of antimicrobials based on intercepting and interrupting quorum sensing based signaling are discussed.     

嗜水气单胞菌群体感应信号分子AI-2的细胞外生物 合成及活性检测

【目的】对嗜水气单胞菌群体感应信号分子AI-2进行细胞外生物合成及活性检测。【方法】对LuxS、MtnN-1、MtnN-2蛋白进行氨基酸序列分析、表达及纯化。以S-腺苷同型半胱氨酸(SAH)为底物,利用纯化的LuxS分别与MtnN-1及MtnN-2蛋白共同作用合成AI-2,并利用哈维氏弧菌报告菌株BB170检测AI-2活性。【结果】嗜水气单胞菌培养液上清中AI-2活性在8 h达到空白对照的16.96倍。氨基酸序列分析表明,嗜水气单胞菌与水生病原菌哈维式弧菌和迟钝爱德华氏LuxS一致性达到76%以上,MtnN-1与MtnN-2氨基酸序列一致性为26.37%,其中MtnN-2与哈维氏弧菌和迟钝爱德华氏菌Pfs一致性达到53%以上。成功表达及纯化了LuxS、MtnN-1和MtnN-2蛋白,细胞外LuxS和MtnN-1共同作用合成的AI-2活性是空白对照的45.04倍,LuxS和MtnN-2共同作用合成的AI-2活性是空白对照的63.62倍。【结论】嗜水气单胞菌能够合成信号分子AI-2。MtnN-1和MtnN-2氨基酸序列尽管存在较大差异,但两者均能与LuxS共同催化AI-2的细胞外生物合成。     

Biological activity and identification of a peptide inhibitor of LuxS from Streptococcus suis serotype 2

The virulence of bacterial communities may be regulated by mechanisms involving the synthesis of the quorum-sensing signal autoinducer 2 (AI-2), which allows both intra- and interspecies communication. AI-2 is produced in bacteria that express the gene luxS . In the present study, expressed and purified LuxS from Streptococcus suis serotype 2 (SS2) was used to catalyze the substrate S -ribosylhomocysteine in a reaction that leads to the production of AI-2. The biological activity of the in vitro synthesized AI-2 was demonstrated in a Vibrio harveyi strain BB170 bioassay; real-time PCR results showed that biosynthesis of AI-2 can increase the virulence of SS2. Phage-encoded peptides that specifically interact with the LuxS enzyme were selected following three rounds of phage display. One such peptide inhibitor (TNRHNPHHLHHV) of LuxS was shown to partially inhibit the activity of the enzyme. Furthermore, 14 peptides containing the consensus sequence HSIR showed high affinity with LuxS. The selected and characterized specific inhibitor as well as the high-affinity ligands may facilitate the identification of new vaccination targets, opening up new approaches to the development of therapeutic drugs.     

Three parallel quorum-sensing systems regulate gene expression in Vibrio harveyi

In a process called quorum sensing, bacteria communicate using extracellular signal molecules termed autoinducers. Two parallel quorum-sensing systems have been identified in the marine bacterium Vibrio harveyi. System 1 consists of the LuxM-dependent autoinducer HAI-1 and the HAI-1 sensor, LuxN. System 2 consists of the LuxS-dependent autoinducer AI-2 and the AI-2 detector, LuxPQ. The related bacterium, Vibrio cholerae, a human pathogen, possesses System 2 (LuxS, AI-2, and LuxPQ) but does not have obvious homologues of V. harveyi System 1. Rather, System 1 of V. cholerae is made up of the CqsA-dependent autoinducer CAI-1 and a sensor called CqsS. Using a V. cholerae CAI-1 reporter strain we show that many other marine bacteria, including V. harveyi, produce CAI-1 activity. Genetic analysis of V. harveyi reveals cqsA and cqsS, and phenotypic analysis of V. harveyi cqsA and cqsS mutants shows that these functions comprise a third V. harveyi quorum-sensing system that acts in parallel to Systems 1 and 2. Together these communication systems act as a three-way coincidence detector in the regulation of a variety of genes, including those responsible for bioluminescence, type III secretion, and metalloprotease production.