Quorum signaling via AI-2 communicates the "Metabolic Burden" associated with heterologous protein production in Escherichia coli.

Recent reports have shown that bacterial cell-cell communication or quorum sensing is quite prevalent in pathogenic Escherichia coli, especially at high cell density; however, the role of quorum sensing in nonpathogenic E. coli is less clear and, in particular, there is no information regarding the role of quorum sensing in overexpression of plasmid-encoded genes. In this work, it was found that the activity of a quorum signaling molecule, autoinducer-2 (AI-2), decreased significantly following induction of several plasmid-encoded genes in both low and high-cell-density cultures of E. coli. Furthermore, we show that AI-2 signaling level was linearly related to the accumulation level of each protein product and that, in general, the highest rates of recombinant protein accumulation resulted in the greatest attenuation of AI-2 signaling. Importantly, our findings demonstrate for the first time that recombinant E. coli communicate the stress or burden of overexpressing heterologous genes through the quorum-based AI-2 signaling pathway.     

Stationary-phase quorum-sensing signals affect autoinducer-2 and gene expression in Escherichia coli

Quorum sensing via autoinducer-2 (AI-2) has been identified in different strains, including those from Escherichia, Vibrio, Streptococcus, and Bacillus species, and previous studies have suggested the existence of additional quorum-sensing signals working in the stationary phase of Escherichia coli cultures. To investigate the presence and global effect of these possible quorum-sensing signals other than AI-2, DNA microarrays were used to study the effect of stationary-phase signals on the gene expression of early exponential-phase cells of the AI-2-deficient strain E. coli DH5alpha. For statistically significant differential gene expression (P < 0.05), 14 genes were induced by supernatants from a stationary culture and 6 genes were repressed, suggesting the involvement of indole (induction of tnaA and tnaL) and phosphate (repression of phoA, phoB, and phoU). To study the stability of the signals, the stationary-phase supernatant was autoclaved and was used to study its effect on E. coli gene expression. Three genes were induced by autoclaved stationary-phase supernatant, and 34 genes were repressed. In total, three genes (ompC, ptsA, and btuB) were induced and five genes (nupC, phoB, phoU, argT, and ompF) were repressed by both fresh and autoclaved stationary-phase supernatants. Furthermore, supernatant from E. coli DH5alpha stationary culture was found to repress E. coli K-12 AI-2 concentrations by 4.8-fold +/- 0.4-fold, suggesting that an additional quorum-sensing system in E. coli exists and that gene expression is controlled as a network with different signals working at different growth stages.     

Engineering Escherichia coli for enhanced sensitivity to the autoinducer-2 quorum sensing signal

The autoinducer-2 (AI-2) quorum sensing system is involved in a range of population-based bacterial behaviors and has been engineered for cell–cell communication in synthetic biology systems. Investigation into the cellular mechanisms of AI-2 processing has determined that overexpression of uptake genes increases AI-2 uptake rate, and genomic deletions of degradation genes lowers the AI-2 level required for activation of reporter genes. Here, we combine these two strategies to engineer an Escherichia coli strain with enhanced ability to detect and respond to AI-2. In an E. coli strain that does not produce AI-2, we monitored AI-2 uptake and reporter protein expression in a strain that overproduced the AI-2 uptake or phosphorylation units LsrACDB or LsrK, a strain with the deletion of AI-2 degradation units LsrF and LsrG, and an “enhanced” strain with both overproduction of AI-2 uptake and deletion of AI-2 degradation elements. By adding up to 40 μM AI-2 to growing cell cultures, we determine that this “enhanced” AI-2 sensitive strain both uptakes AI-2 more rapidly and responds with increased reporter protein expression than the others. This work expands the toolbox for manipulating AI-2 quorum sensing processes both in native environments and for synthetic biology applications.     

Interspecific Quorum Sensing Mediates the Resuscitation of Viable but Nonculturable Vibrios

Entry and exit from dormancy are essential survival mechanisms utilized by microorganisms to cope with harsh environments. Many bacteria, including the opportunistic human pathogen Vibrio vulnificus, enter a form of dormancy known as the viable but nonculturable (VBNC) state. VBNC cells can resuscitate when suitable conditions arise, yet the molecular mechanisms facilitating resuscitation in most bacteria are not well understood. We discovered that bacterial cell-free supernatants (CFS) can awaken preexisting dormant vibrio populations within oysters and seawater, while CFS from a quorum sensing mutant was unable to produce the same resuscitative effect. Furthermore, the quorum sensing autoinducer AI-2 could induce resuscitation of VBNC V. vulnificus in vitro, and VBNC cells of a mutant unable to produce AI-2 were unable to resuscitate unless the cultures were supplemented with exogenous AI-2. The quorum sensing inhibitor cinnamaldehyde delayed the resuscitation of wild-type VBNC cells, confirming the importance of quorum sensing in resuscitation. By monitoring AI-2 production by VBNC cultures over time, we found quorum sensing signaling to be critical for the natural resuscitation process. This study provides new insights into the molecular mechanisms stimulating VBNC cell exit from dormancy, which has significant implications for microbial ecology and public health.     

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.     

Cell-to-cell signalling in Escherichia coli and Salmonella enterica

Cell-to-cell signalling in prokaryotes that leads to co-ordinated behaviour has been termed quorum sensing. This type of signalling can have profound impacts on microbial community structure and host-microbe interactions. The Gram-negative quorum-sensing systems were first discovered and extensively characterized in the marine Vibrios. Some components of the Vibrio systems are present in the classical genetic model organisms Escherichia coli and Salmonella enterica. Both organisms encode a signal receptor of the LuxR family, SdiA, but not a corresponding signal-generating enzyme. Instead, SdiA of Salmonella detects and responds to signals generated only by other microbial species. Conversely, E. coli and Salmonella encode the signal-generating component of a second system (a LuxS homologue that generates AI-2), but the sensory apparatus for AI-2 differs substantially from the Vibrio system. The only genes currently known to be regulated by AI-2 in Salmonella encode an active uptake and modification system for AI-2. Therefore, it is not yet clear whether Salmonella uses AI-2 as a signal molecule or whether AI-2 has some other function. In E. coli, the functions of both SdiA and AI-2 are unclear due to pleiotropy. Genetic strategies to identify novel signalling systems have been performed with E. coli and Providencia stuartii. Several putative signalling systems have been identified, one that uses indole as a signal and another that releases what appears to be a peptide. The latter system has homologues in E. coli and Salmonella, as well as other bacteria, plants and animals. In fact, the protease components from Providencia and Drosophila are functionally interchangeable.     

Bacterial autoinduction: looking outside the cell for new metabolic engineering targets

Recent evidence has demonstrated that cell-to-cell signaling is a fundamental activity carried out by numerous microorganisms. A number of specialized processes are reported to be regulated by density-dependent signaling molecules including antibiotic production, bioluminescence, biofilm formation, genetic competence, sporulation, swarming motility and virulence. However, a more centralized role for quorum sensing is emerging where quorum signaling pathways overlap with stress and starvation circuits to regulate cellular adaptation to changing environmental conditions. The interplay of these phenomena is especially critical in the expression of recombinant proteins where elicitation of stress responses can dramatically impact cellular productivity.     

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.     

The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule

Many bacteria control gene expression in response to cell population density, and this phenomenon is called quorum sensing. In Gram-negative bacteria, quorum sensing typically involves the production, release and detection of acylated homoserine lactone signalling molecules called autoinducers. Vibrio harveyi, a Gram-negative bioluminescent marine bacterium, regulates light production in response to two distinct autoinducers (AI-1 and AI-2). AI-1 is a homoserine lactone. The structure of AI-2 is not known. We have suggested previously that V. harveyi uses AI-1 for intraspecies communication and AI-2 for interspecies communication. Consistent with this idea, we have shown that many species of Gram-negative and Gram-positive bacteria produce AI-2 and, in every case, production of AI-2 is dependent on the function encoded by the luxS gene. We show here that LuxS is the AI-2 synthase and that AI-2 is produced from S-adenosylmethionine in three enzymatic steps. The substrate for LuxS is S-ribosylhomocysteine, which is cleaved to form two products, one of which is homocysteine, and the other is AI-2. In this report, we also provide evidence that the biosynthetic pathway and biochemical intermediates in AI-2 biosynthesis are identical in Escherichia coli, Salmonella typhimurium, V. harveyi, Vibrio cholerae and Enterococcus faecalis. This result suggests that, unlike quorum sensing via the family of related homoserine lactone autoinducers, AI-2 is a unique, 'universal' signal that could be used by a variety of bacteria for communication among and between species.     

LuxS-Dependent AI-2 Regulates Versatile Functions in Enterococcus faecalis V583

Bacteria utilize a quorum sensing (QS) system to coordinate gene expression by monitoring the concentration of molecules known as autoinducers (AI). In the present study, we confirmed the presence of a LuxS/AI-2 dependent QS system in vancomycin-resistant Enterococcus faecalis V583. Then, the cellular targets controlled by AI-2 were identified by comparative proteomics analysis in order to elucidate the possible role of AI-2 in E. faecalis. Results demonstrated 15 proteins that are differentially expressed upon the addition of AI-2, including proteins involved in metabolism, translation, energy production and/or conversion, and cell wall biogenesis. Glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase associated with carbohydrate metabolism and energy production were up-regulated upon inducing by AI-2. In addition, externally added AI-2 could down-regulate acetyl-coenzyme A carboxylase and ornithine carbamoyltransferase, two key enzyme involved in metabolism. All these data suggest that AI-2 signaling may play a role in the regulation of a number of important metabolic properties of E. faecali. We further investigated the role of AI-2 in biofilm formation by E. faecalis, showing the addition of AI-2 to E. faecalis V583 cultures resulted in increased biofilm formation. Our results provide important clues to the role of a LuxS/AI-2 dependent QS system in vancomycin-resistant E. faecalis.     

Quorum sensing and production of autoinducer-2 in Campylobacter spp., Escherichia coli O157:H7, and Salmonella enterica serovar Typhimurium in foods

Autoinducer molecules are utilized by gram-negative and gram-positive bacteria to regulate density-dependent gene expression by a mechanism known as quorum sensing. PCR and DNA sequencing results showed that Campylobacter jejuni and Campylobacter coli possessed luxS, which is responsible for autoinducer-2 (AI-2) production. Using a Vibrio harveyi luminescence assay, the production of AI-2 was observed in milk, chicken broth, and brucella broth by C. coli, C. jejuni, Salmonella enterica serovar Typhimurium, and Escherichia coli O157:H7 under different conditions.