We report an approach for spatially selective assembly of an enzyme onto selected patterns of microfabricated chips. Our approach is based on electrodeposition of the aminopolysaccharide chitosan onto selected electrode patterns and covalent conjugation of a target enzyme to chitosan upon biochemical activation of a genetically fused "pro-tag." We report assembly of S-adenosylhomocysteine nucleosidase (Pfs) fused with a C-terminal pentatyrosine pro-tag. Pfs is a member of the bacterial autoinducer-2 biosynthesis pathway, catalyzing the irreversible cleavage of S-adenosylhomocysteine. The assembled Pfs retains its catalytic activity and structure, as demonstrated by retained antibody recognition. Assembly is controlled by the electrode area, resulting in reproducible rates of catalytic conversion for a given area, and thus allowing for area-based manipulation of catalysis and small molecule biosynthesis. Our approach enables optimization of small molecule biosynthesis in 1-step as well as multistep enzymatic reactions, including entire metabolic pathways, and we envision a wide variety of potential applications.

Cell-cell communication and coordinated population-based behavior among single cell organisms have gained considerable attention in the recent years. The ability to send, receive, and process information allows unicellular organisms to act as multicellular entities and increases their chances of survival in complex environments. Quorum sensing (QS), a density-dependent cell-signaling mechanism, is one way by which bacteria 'talk' to one another. QS is commonly associated with adverse health effects such as biofilm formation, bacteria pathogenicity, and virulence. But there exists great potential to harness QS circuitry and its properties for other applications, enabling even wider societal impact. Interesting avenues are envisioned for the detection of chemicals and pathogens, the navigation of interspecies communication, the synchronization and control of cell phenotype, and the creation of novel materials based on synthetic biology. In this review, we first highlight the recent discoveries of the molecular underpinnings of QS function, with emphasis on the formation of biofilms. We then discuss how researchers have used QS circuitry to their advantage to build synthetic networks, rewire native metabolic pathways, and engineer cells for a variety of applications.

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. Biotechnol. Bioeng.(c) 2008 Wiley Periodicals, Inc.

Multicellular bacterial communities (biofilms) abound in nature, and their successful formation and survival is likely to require cell-cell communication - including quorum sensing - to co-ordinate appropriate gene expression. The only mode of quorum sensing that is shared by both Gram-positive and Gram-negative bacteria involves the production of the signalling molecule autoinducer 2 by LuxS. A survey of the current literature reveals that luxS contributes to biofilm development in some bacteria. However, inconsistencies prevent biofilm development being attributed to the production of AI2 in all cases.

We report a biofunctionalization strategy for the assembly of catalytically active enzymes within a completely packaged bioMEMS device, through the programmed generation of electrical signals at spatially and temporally defined sites. The enzyme of a bacterial metabolic pathway, S-adenosylhomocysteine nucleosidase (Pfs), is genetically fused with a pentatyrosine "pro-tag" at its C-terminus. Signal responsive assembly is based on covalent conjugation of Pfs to the aminopolysaccharide, chitosan, upon biochemical activation of the pro-tag, followed by electrodeposition of the enzyme-chitosan conjugate onto readily addressable sites in microfluidic channels. Compared to traditional physical entrapment and surface immobilization approaches in microfluidic environments, our signal-guided electrochemical assembly is unique in that the enzymes are assembled under mild aqueous conditions with spatial and temporal programmability and orientational control. Significantly, the chitosan-mediated enzyme assembly can be reversed, making the bioMEMS reusable for repeated assembly and catalytic activity. Additionally, the assembled enzymes retain catalytic activity over multiple days, demonstrating enhanced enzyme stability. We envision that this assembly strategy can be applied to rebuild metabolic pathways in microfluidic environments for antimicrobial drug discovery.

Quorum sensing (QS) is an important determinant of bacterial phenotype. Many cell functions are regulated by intricate and multimodal QS signal transduction processes. The LuxS/AI-2 QS system is highly conserved among Eubacteria and AI-2 is reported as a 'universal' signal molecule. To understand the hierarchical organization of AI-2 circuitry, a comprehensive approach incorporating stochastic simulations was developed. We investigated the synthesis, uptake, and regulation of AI-2, developed testable hypotheses, and made several discoveries:(1) the mRNA transcript and protein levels of AI-2 synthases, Pfs and LuxS, do not contribute to the dramatically increased level of AI-2 found when cells are grown in the presence of glucose;(2) a concomitant increase in metabolic flux through this synthesis pathway in the presence of glucose only partially accounts for this difference. We predict that 'high-flux' alternative pathways or additional biological steps are involved in AI-2 synthesis; and (3) experimental results validate this hypothesis. This work demonstrates the utility of linking cell physiology with systems-based stochastic models that can be assembled de novo with partial knowledge of biochemical pathways.

We report the assembly of seven different antibodies (and two antigens) into functional supramolecular structures that are specifically designed to facilitate integration into devices using entirely biologically based bottom-up fabrication. This is enabled by the creation of an engineered IgG-binding domain (HG3T) with an N-terminal hexahistidine tag that facilitates purification and a C-terminal enzyme-activatable pentatyrosine "pro-tag" that facilitates covalent coupling to the pH stimuli-responsive polysaccharide, chitosan. Because we confer pH-stimuli responsiveness to the IgG-binding domain, it can be electrodeposited or otherwise assembled into many configurations. Importantly, we demonstrate the loading of both HG3T and antibodies can be achieved in a linear fashion so that quantitative assessment of antibodies and antigens is feasible. Our demonstration formats include: conventional multiwell plates, micropatterned electrodes, and fiber networks. We believe biologically based fabrication (i.e., biofabrication) provides bottom-up hierarchical assembly of a variety of nanoscale components for applications that range from point-of-care diagnostics to smart fabrics. Biotechnol. Bioeng.(c) 2008 Wiley Periodicals, Inc.

Bacterial autoinducer 2 (AI-2) is proposed to be an interspecies mediator of cell-cell communication that enables cells to operate at the multicellular level. Many environmental stimuli have been shown to affect the extracellular AI-2 levels, carbon sources being among the most important. In this report, we show that both AI-2 synthesis and uptake in Escherichia coli are subject to catabolite repression through the cyclic AMP (cAMP)-CRP complex, which directly stimulates transcription of the lsr (for "luxS regulated") operon and indirectly represses luxS expression. Specifically, cAMP-CRP is shown to bind to a CRP binding site located in the upstream region of the lsr promoter and works with the LsrR repressor to regulate AI-2 uptake. The functions of the lsr operon and its regulators, LsrR and LsrK, previously reported in Salmonella enterica serovar Typhimurium, are confirmed here for E. coli. The elucidation of cAMP-CRP involvement in E. coli autoinduction impacts many areas, including the growth of E. coli in fermentation processes.

Bacterial Quorum Sensing (QS) is a cell-cell communication process, mediated by signaling molecules, that alters various phenotypes including pathogenicity. Methods to interrupt these communication networks are being pursued as next generation antimicrobials. We present a technique for interrupting communication among bacteria that exploits their native and highly specific machinery for processing the signaling molecules themselves. Specifically, our approach is to bring native intracellular signal processing mechanisms to the extracellular surroundings and "quench" crosstalk among a variety of strains. In this study, the QS system based on the interspecies signaling molecule, autoinducer-2 (AI-2), is targeted because of its prevalence among prokaryotes (it functions in over 80 bacterial species). We demonstrate that the Escherichia coli AI-2 kinase, LsrK, can phosphorylate AI-2 in vitro, and when LsrK treated AI-2 is added ex vivo to E. coli populations, the native QS response is significantly reduced. Further, LsrK-mediated degradation of AI-2 attenuates the QS response among Salmonella typhimurium and Vibrio harveyi even though the AI-2 signal transduction mechanisms and the phenotypic responses are species-specific. Analogous results are obtained from a synthetic ecosystem where three species of bacteria (enteric and marine) are co-cultured. Finally, the addition of LsrK and ATP to growing co-cultures of E. coli and S. typhimurium, exhibits significantly reduced native "cross-talk" that ordinarily exists among and between species in an ecosystem. We believe this nature-inspired enzymatic approach for quenching QS systems will spawn new methods for controlling cell phenotype and potentially open new avenues for controlling bacterial pathogenicity.

The regulatory network for the uptake of E. coli autoinducer, AI-2, is comprised of a transporter complex, LsrABCD, its repressor, LsrR and cognate signal kinase, LsrK. This network is an integral part of the AI-2 quorum sensing (QS) system. Because LsrR and LsrK directly regulate AI-2 uptake, we hypothesized they might play a wider role in regulating other QS-related cellular functions. In this study, we characterized physiological changes due to the genomic deletion of lsrR and lsrK. We discovered that many genes were co-regulated by lsrK and lsrR but in a distinctly different manner than the lsr operon (where LsrR serves as a repressor that is derepressed by the binding of phospho-AI-2 to the LsrR protein). An extended model for AI-2 signaling is proposed that is consistent with all current data on AI-2, LuxS, and the LuxS-regulon. Additionally, we found that both the quantity and architecture of biofilms were regulated by this distinct mechanism, as lsrK and lsrR knockouts behaved identically. Similar biofilm architectures probably resulted from the concerted response of a set of genes including flu and wza, the expression of which are influenced by lsrRK. We also found for the first time that the generation of several small RNAs (including DsrA, which was previously linked to QS systems in V. harveyi) was affected by LsrR. Our results suggest that AI-2 is indeed a quorum sensing signal in E. coli, especially when it acts through transcriptional regulator, LsrR.

The cross-species bacterial communication signal autoinducer 2 (AI-2), produced by the purified enzymes Pfs (nucleosidase) and LuxS (terminal synthase) from S-adenosylhomocysteine, directly increased Escherichia coli biofilm mass 30-fold. Continuous-flow cells coupled with confocal microscopy corroborated these results by showing the addition of AI-2 significantly increased both biofilm mass and thickness and reduced the interstitial space between microcolonies. As expected, the addition of AI-2 to cells which lack the ability to transport AI-2 (lsr null mutant) failed to stimulate biofilm formation. Since the addition of AI-2 increased cell motility through enhanced transcription of five motility genes, we propose that AI-2 stimulates biofilm formation and alters its architecture by stimulating flagellar motion and motility. It was also found that the uncharacterized protein B3022 regulates this AI-2-mediated motility and biofilm phenotype through the two-component motility regulatory system QseBC. Deletion of b3022 abolished motility, which was restored by expressing b3022 in trans. Deletion of b3022 also decreased biofilm formation significantly, relative to the wild-type strain in three media (46 to 74%) in 96-well plates, as well as decreased biomass (8-fold) and substratum coverage (19-fold) in continuous-flow cells with minimal medium (growth rate not altered and biofilm restored by expressing b3022 in trans). Deleting b3022 changed the wild-type biofilm architecture from a thick (54-mum) complex structure to one that contained only a few microcolonies. B3022 positively regulates expression of qseBC, flhD, fliA, and motA, since deleting b3022 decreased their transcription by 61-, 25-, 2.4-, and 18-fold, respectively. Transcriptome analysis also revealed that B3022 induces crl (26-fold) and flhCD (8- to 27-fold). Adding AI-2 (6.4 muM) increased biofilm formation of wild-type K-12 MG1655 but not that of the isogenic b3022, qseBC, fliA, and motA mutants. Adding AI-2 also increased motA transcription for the wild-type strain but did not stimulate motA transcription for the b3022 and qseB mutants. Together, these results indicate AI-2 induces biofilm formation in E. coli through B3022, which then regulates QseBC and motility; hence, b3022 has been renamed the motility quorum-sensing regulator gene (the mqsR gene).

We describe a "biolithographic" technique in which the unique properties of biopolymeric materials and the selective catalytic activities of enzymes are exploited for patterning surfaces under simple and bio-friendly conditions. We begin by coating a reactive film of the polysaccharide chitosan onto an inorganic surface (glass or silicon wafer). Chitosan's pH-responsive solubility facilitates film deposition, while the nucleophilic properties of this polysaccharide allow simple chemistries or biochemistries to be used to covalently attach species to the film. The thermally responsive protein gelatin is then cast on top of the chitosan film, and the gelatin gel serves as a sacrificial "thermoresist". Pattern transfer is accomplished by applying a heated stamp to melt specific regions of the gelatin thermoresist and selectively expose the underlying chitosan. Finally, molecules are conjugated to the exposed chitosan sublayer and the sacrificial gelatin layer is removed (either by treating with warm water or protease). To demonstrate the concept, we patterned a reactive dye (NHS-fluorescein), a model 20-base oligonucleotide (using standard glutaraldehyde coupling chemistries), and a model green fluorescent protein (using tyrosinase-initiated conjugation). Because gelatin can be applied and removed under mild conditions, sequential thermo-biolithographic steps can be performed without destroying previously patterned biomacromolecules. These studies represent the first step toward exploiting nature's exquisite specificity for lithographic patterning.

Biological nanofactories, which are engineered to contain modules that can target, sense and synthesize molecules, can trigger communication between different bacterial populations. These communications influence biofilm formation, virulence, bioluminescence and many other bacterial functions in a process called quorum sensing. Here, we show the assembly of a nanofactory that can trigger a bacterial quorum sensing response in the absence of native quorum molecules. The nanofactory comprises an antibody (for targeting) and a fusion protein that produces quorum molecules when bound to the targeted bacterium. Our nanofactory selectively targets the appropriate bacteria and triggers a quorum sensing response when added to two populations of bacteria. The nanofactories also trigger communication between two bacterial populations that are otherwise non-communicating. We envision the use of these nanofactories in generating new antimicrobial treatments that target the communication networks of bacteria rather than their viability.

Quorum sensing (QS) enables an individual bacterium's metabolic state to be communicated to and ultimately control the phenotype of an emerging population. Harnessing the hierarchical nature of this signal transduction process may enable the exploitation of individual cell characteristics to direct or "program" entire populations of cells. We re-engineered the native QS regulon so that individual cell signals (autoinducers) are used to guide high level expression of recombinant proteins in E. coli populations. Specifically, the autoinducer-2 (AI-2) QS signal initiates and guides the overexpression of green fluorescent protein (GFP), chloramphenicol acetyl transferase (CAT) and >-galactosidase (LacZ). The new process requires no supervision or input (e.g., sampling for optical density measurement, inducer addition, or medium exchange) and represents a low-cost, high-yield platform for recombinant protein production. Moreover, rewiring a native signal transduction circuit exemplifies an emerging class of metabolic engineering approaches that target regulatory functions.

We report the in situ generation of pH gradients in microfluidic devices for biofabrication of freestanding, semi-permeable chitosan membranes. The pH-stimuli-responsive polysaccharide chitosan was enlisted to form a freestanding hydrophilic membrane structure in microfluidic networks where pH gradients are generated at the converging interface between a slightly acidic chitosan solution and a slightly basic buffer solution. A simple and effective pumping strategy was devised to realize a stable flow interface thereby generating a stable, well-controlled and localized pH gradient. Chitosan molecules were deprotonated at the flow interface, causing gelation and solidification of a freestanding chitosan membrane from a nucleation point at the junction of two converging flow streams to an anchoring point where the two flow streams diverge to two output channels. The fabricated chitosan membranes were about 30-60 microm thick and uniform throughout the flow interface inside the microchannels. A T-shaped membrane formed by sequentially fabricating orthogonal membranes demonstrates flexibility of the assembly process. The membranes are permeable to aqueous solutions and are removed by mildly acidic solutions. Permeability tests suggested that the membrane pore size was a few nanometres, i.e., the size range of antibodies. Building on the widely reported use of chitosan as a soft interconnect for biological components and microfabricated devices and the broad applications of membrane functionalities in microsystems, we believe that the facile, rapid biofabrication of freestanding chitosan membranes can be applied to many biochemical, bioanalytical, biosensing applications and cellular studies.

Biological nanofactories, which are engineered to contain modules that can target, sense and synthesize molecules, can trigger communication between different bacterial populations. These communications influence biofilm formation, virulence, bioluminescence and many other bacterial functions in a process called quorum sensing. Here, we show the assembly of a nanofactory that can trigger a bacterial quorum sensing response in the absence of native quorum molecules. The nanofactory comprises an antibody (for targeting) and a fusion protein that produces quorum molecules when bound to the targeted bacterium. Our nanofactory selectively targets the appropriate bacteria and triggers a quorum sensing response when added to two populations of bacteria. The nanofactories also trigger communication between two bacterial populations that are otherwise non-communicating. We envision the use of these nanofactories in generating new antimicrobial treatments that target the communication networks of bacteria rather than their viability.

Bacterial Quorum Sensing (QS) is a cell-cell communication process, mediated by signaling molecules, that alters various phenotypes including pathogenicity. Methods to interrupt these communication networks are being pursued as next generation antimicrobials. We present a technique for interrupting communication among bacteria that exploits their native and highly specific machinery for processing the signaling molecules themselves. Specifically, our approach is to bring native intracellular signal processing mechanisms to the extracellular surroundings and "quench" crosstalk among a variety of strains. In this study, the QS system based on the interspecies signaling molecule, autoinducer-2 (AI-2), is targeted because of its prevalence among prokaryotes (it functions in over 80 bacterial species). We demonstrate that the Escherichia coli AI-2 kinase, LsrK, can phosphorylate AI-2 in vitro, and when LsrK treated AI-2 is added ex vivo to E. coli populations, the native QS response is significantly reduced. Further, LsrK-mediated degradation of AI-2 attenuates the QS response among Salmonella typhimurium and Vibrio harveyi even though the AI-2 signal transduction mechanisms and the phenotypic responses are species-specific. Analogous results are obtained from a synthetic ecosystem where three species of bacteria (enteric and marine) are co-cultured. Finally, the addition of LsrK and ATP to growing co-cultures of E. coli and S. typhimurium, exhibits significantly reduced native "cross-talk" that ordinarily exists among and between species in an ecosystem. We believe this nature-inspired enzymatic approach for quenching QS systems will spawn new methods for controlling cell phenotype and potentially open new avenues for controlling bacterial pathogenicity.

Halogenated furanones, a group of natural products initially isolated from marine red algae, are known to inhibit bacterial biofilm formation, swarming, and quorum sensing. However, their molecular targets and the precise mode of action remain elusive. Herein, we show that a naturally occurring brominated furanone covalently modifies and inactivates LuxS (S-ribosylhomocysteine lyase, EC 4.4.1.21), the enzyme which produces autoinducer-2 (AI-2).

Analysis of different architectures of quorum sensing networks has been the center of attention in recent times. The approach employs mathematical models to uncover the factors behind the dynamics. Quorum sensing networks mostly display autoregulation such as Pseudomonas aeruginosa and Vibrio cholerae. However, Es cherichia coli autoinducer 2 (AI-2) synthesis does not display autoinduction (i.e. autoregulation). This and other features have raised questions about the actual function of AI-2 inside the cell. In this paper we propose a model for lsr operon regulation which explains or at least is consistent with AI-2 uptake in E. coli. The model was employed to determine the main factors that control the concentration of the signal and the uptake activation. We investigated deterministic and stochastic variants of the network model and we found no states that could lead to the typical bistability in quorum sensing systems. However, stochastic simulations predict a transient bifurcation (positively regulated by AI-2 synthesis) that could provide some advantage in adapting to new environments. LsrR inactivation was found to play a crucial role in the uptake activation compared to AI-2 synthesis, lsr transcription and AI-2 excretion. Our hypothesis is that positive regulation of the level of expression is the main factor in understanding the function of the lsr operon. This is in contrast to the conventionally held belief that the main factor is the onset of activation.

We report a versatile approach for the immobilization of unmodified monosaccharides onto iron oxide nanoparticles. Covalent coupling of the carbohydrate onto iron oxide nanoparticle surfaces was accomplished by the CH insertion reaction of photochemically activated phosphate-functionalized perfluorophenylazides (PFPAs), and the resulting glyconanoparticles were characterized by IR, TGA, and TEM. The surface-bound d-mannose showed the recognition ability toward Concanavalin A and Escherichia coli strain ORN178 that possesses mannose-specific receptor sites. Owing to the simplicity and versatility of the technique, together with the magnetic property of iron oxide nanoparticles, the methodology developed in this study serves as a general approach for the preparation of magnetic glyconanoparticles to be used in clinical diagnosis, sensing, and decontamination.

Chemotaxis is the migration of cells in gradients of chemoeffector molecules. Although multiple, competing gradients must often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to gradients of multiple signals. In this work, we developed a microfluidic device for generating precise and stable gradients of signaling molecules. We used the device to investigate the effects of individual and combined chemoeffector gradients on Escherichia coli chemotaxis. Laminar flow-based diffusive mixing was used to generate gradients, and the chemotactic responses of cells expressing green fluorescent protein (GFP) were determined using fluorescence microscopy. Quantification of the migration profiles indicated that E. coli was attracted to the quorum-sensing molecule autoinducer-2 (AI-2) but repelled from the stationary-phase signal indole. Cells also migrated towards higher concentrations of isatin (2-3-indole-dione), an oxidized derivative of indole. Attraction to AI-2 overcame repulsion by indole in equal, competing gradients. Our data suggest that concentration-dependent interactions between signals may be an important determinant of chemotaxis leading to bacterial colonization.

LuxS catalyses the synthesis of the quorum sensing signalling molecule autoinducer-2. We show that in Salmonella Typhimurium, deletion of the luxS gene polarises flagellar phase variation towards the more immunogenic phase-1 flagellin. This phenotype is complementable by luxS in trans but independent of quorum sensing signals.

Two-component sensor kinase signaling systems are widespread in bacteria, but gaining mechanistic insight into how kinase activity is controlled by ligand binding has proved challenging. Here, we discuss this problem in the context of our structural and functional studies of bacterial quorum sensing receptors. Specifically, this chapter focuses on the transmembrane sensor kinase complex LuxPQ, which serves as the receptor for the "universal" quorum sensing signal molecule autoinducer-2 (AI-2). Methods are presented for the overproduction, purification, crystallization, and functional characterization of LuxPQ's ligand-binding (periplasmic) domain.

Small-molecule agonists and antagonists of bacterial quorum sensing can enhance our understanding of this form of cell-cell communication. A recent effort has discovered effective modulators of the autoinducer-1 circuit for bacterial quorum sensing by the synthesis and evaluation of a small library of aryl-substituted acyl-homoserine lactone analogues. This series highlights the sensitivity to structure of the contrasting responses of agonism and antagonism of the natural signal and identifies an analogue that provokes the same response as the natural signal but at 10-fold lower concentration, a "superagonist".

Many bacteria use a molecule known as autoinducer-2 for interspecies communication, a form of quorum sensing. Enteric bacteria secrete this molecule and later import and degrade it. A new study explores the molecular mechanism behind this curious signal-destroying process.