Phage display uncovers a sequence motif that drives polypeptide binding to a conserved regulatory exosite of O-GlcNAc transferase.

The modification of nucleocytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an important regulator of cell physiology. O-GlcNAc is installed on over a thousand proteins by just one enzyme, O-GlcNAc transferase (OGT). How OGT is regulated is therefore a topic of interest. To gain insight into these questions, we used OGT to perform phage display selection from an unbiased library of ~109 peptides of 15 amino acids in length. Following rounds of selection and deep mutational panning, we identified a high-fidelity peptide consensus sequence, [Y/F]-x-P-x-Y-x-[I/M/F], that drives peptide binding to OGT. Peptides containing this sequence bind to OGT in the high nanomolar to low micromolar range and inhibit OGT in a noncompetitive manner with low micromolar potencies. X-ray structural analyses of OGT in complex with a peptide containing this motif surprisingly revealed binding to an exosite proximal to the active site of OGT. This structure defines the detailed molecular basis driving peptide binding and explains the need for specific residues within the sequence motif. Analysis of the human proteome revealed this motif within 52 nuclear and cytoplasmic proteins. Collectively, these data suggest a mode of regulation of OGT by which polypeptides can bind to this exosite to cause allosteric inhibition of OGT through steric occlusion of its active site. We expect that these insights will drive improved understanding of the regulation of OGT within cells and enable the development of new chemical tools to exert fine control over OGT activity.

Genetically encoded multivalent liquid glycan array displayed on M13 bacteriophage.

The central dogma of biology does not allow for the study of glycans using DNA sequencing. We report a liquid glycan array (LiGA) platform comprising a library of DNA 'barcoded' M13 virions that display 30-1,500 copies of glycans per phage. A LiGA is synthesized by acylation of the phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido-modified glycans. Pulldown of the LiGA with lectins followed by deep sequencing of the barcodes in the bound phage decodes the optimal structure and density of the recognized glycans. The LiGA is target agnostic and can measure the glycan-binding profile of lectins, such as CD22, on cells in vitro and immune cells in a live mouse. From a mixture of multivalent glycan probes, LiGAs identify the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and in vivo.

Genetically Encoded Fragment-Based Discovery from Phage-Displayed Macrocyclic Libraries with Genetically Encoded Unnatural Pharmacophores.

Genetically encoded macrocyclic peptide libraries with unnatural pharmacophores are valuable sources for the discovery of ligands for many targets of interest. Traditionally, generation of such libraries employs "early stage" incorporation of unnatural building blocks into the chemically or translationally produced macrocycles. Here, we describe a divergent late-stage approach to such libraries starting from readily available starting material: genetically encoded libraries of peptides. A diketone linchpin 1,5-dichloropentane-2,4-dione converts peptide libraries displayed on phage to 1,3-diketone bearing macrocyclic peptides (DKMP): shelf-stable precursors for Knorr pyrazole synthesis. Ligation of diverse hydrazine derivatives onto DKMP libraries displayed on phage that carries silent DNA-barcodes yields macrocyclic libraries in which the amino acid sequence and the pharmacophore are encoded by DNA. Selection of this library against carbonic anhydrase enriched macrocycles with benzenesulfonamide pharmacophore and nanomolar Kd. The methodology described in this manuscript can graft diverse pharmacophores into many existing genetically encoded phage libraries and significantly increase the value of such libraries in molecular discoveries.

Influence of labeling on the glycan affinities and specificities of glycan-binding proteins. A case study involving a C-terminal fragment of human galectin-3.

Glycan interactions with glycan-binding proteins (GBPs) play essential roles in a wide variety of cellular processes. Currently, the glycan specificities of GBPs are most often inferred from binding data generated using glycan arrays, wherein the GBP is incubated with oligosaccharides immobilized on a glass surface. Detection of glycan-GBP binding is typically fluorescence-based, involving the labeling of the GBP with a fluorophore or with biotin, which binds to fluorophore-labeled streptavidin, or using a fluorophore-labeled antibody that recognizes the GBP. While it is known that covalent labeling of a GBP may influence its binding properties, these effects have not been well studied and are usually overlooked when analyzing glycan array data. In the present study, electrospray ionization mass spectrometry (ESI-MS) was used to quantitatively evaluate the impact of GBP labeling on oligosaccharide affinities and specificities. The influence of three common labeling approaches, biotinylation, labeling with a fluorescent dye and introducing an iodination reagent, on the affinities of a series of human milk and blood group oligosaccharides for a C-terminal fragment of human galectin-3 was evaluated. In all cases labeling resulted in a measurable decrease in oligosaccharide affinity, by as much as 90%, and the magnitude of the change was sensitive to the nature of the ligand. These findings demonstrate that GBP labeling may affect both the absolute and relative affinities and, thereby, obscure the true glycan binding properties. These results also serve to illustrate the utility of the direct ESI-MS assay for quantitatively evaluating the effects of protein labeling on ligand binding.

Freeze-Float Selection of Ice Nucleators.

In this manuscript, we developed a screening system that employs the difference in density between liquid water and ice (0.9998 g/cm3 vs 0.9168 g/cm3 at 0 °C) to identify ice-nucleating agents (INAs) that are encapsulated into droplets of water suspended in silicone oil of intermediate density (0.939 g/cm3). Droplets of liquid water stably reside at the interface of the silicone oil and perfluoro oil (1.6658 g/cm3); freezing causes the aqueous droplets to float to the top of the silicone oil layer. We demonstrated the feasibility of this screening system by using droplets that contained well-defined ice-nucleator Snomax. The droplets with and without Snomax froze at different temperatures and separated into two groups in our system. We employed the screening system to test samples that have different ice-nucleating activities. Starting from known ice-nucleating active bacteria Pseudomonas syringae, we confirmed that droplets that contain an increasing amount of ice-nucleating bacteria per droplet exhibit a dose-dependent increase in ice nucleation. When droplets containing different amounts of P. syringae were separated using a freeze-float setup, we observed that the droplets that floated at higher temperature contained more ice-nucleating active bacteria. The outlined system, thus, permits simple power-free separation of droplets that contain effective INA from those that contain weak or no INA. Such a setup can be used as a starting point for the development of high-throughput approaches for the discovery of new INAs.

Light-responsive bicyclic peptides.

In this paper, we describe a method for the synthesis of light-responsive (LR) bicyclic macrocycles from linear peptides composed of 20 natural amino acids. Small molecules, peptide macrocycles, and protein conjugates that reversibly turn their function on and off in response to visible light enabled the fields of photopharmacology and optochemical genetics. Bioactive LR molecules could be produced by grafting azobenzene or other LR-structures onto molecules with known biological functions (e.g., alpha-helical peptides). It is also possible to discover such LR ligands de novo by selecting compounds with a desired function-such as binding to a target-from a library of LR-compounds or a genetically-encoded (GE) library of LR-macrocycles. The bicyclic topology of ligands offers added value such as improved binding and stability when compared to monocyclic peptides, but approaches for the design of bicyclic light-responsive architectures are limited. To address this need, we developed a tridentate C2-symmetric hydroxyl amine and di-chlorobenzene containing azobenzene (HADCAz) LR-linker with two orthogonally reactive functionalities (chlorobenzyl and hydroxylamine) to convert a linear unprotected peptide into a bicyclic peptide in a one-pot, two-step reaction. This linker reversibly isomerizes from the trans to cis form upon irradiation with blue light (365 nm). The resulting bicyclic peptide contains two loops of amino acids, one of which is constrained with an azobenzene moiety that can change the conformation in response to visible light. A scalable synthetic route to the HADCAz linker allowed us to demonstrate its application in multiple synthetic bicyclic peptides with loops that contain 2-5 amino acids.

Genetically-encoded fragment-based discovery (GE-FBD) of glycopeptide ligands with differential selectivity for antibodies related to mycobacterial infections.

Accurate identification of tuberculosis (TB), caused by Mycobacterium tuberculosis, is important for global disease management. Point-of-care serological tests may improve TB diagnosis; however, specificities of available serodiagnostics are sub-optimal. We employed genetically encoded fragment-based discovery (GE-FBD) to select ligands for antibodies directed against the mycobacterial cell wall component lipoarabinomannan (LAM), a potent antigen. GE-FBD employed a phage displayed library of 108 heptapeptides, chemically modified with an arabinofuranosyl hexasaccharide fragment of LAM (Ara6), and the anti-LAM antibody CS-35 as a bait. The selection gave rise to glycopeptides with an enhanced affinity and selectivity for CS-35 but not for 906.4321 antibody, both of which bind to Ara6 with a comparable affinity. Multivalent assays incorporating the discovered ligands Ara6-ANSSFAP, Ara6-DAHATLR and Ara6-TTYVVNP exhibited up to 19-fold discrimination between CS-35 and 906.4321. The use of the Ara6 antigen alone failed to distinguish these antibodies. Thus, GE-FBD gives rise to ligands that differentiate monoclonal antibodies with enhanced specificity. This technology could facilitate the development of effective point-of-care serological tests for mycobacterial and other infections.

Genetically-encoded fragment-based discovery of glycopeptide ligands for DC-SIGN.

We have employed genetically-encoded fragment-based discovery to identify novel glycopeptides with affinity for the dendritic cell receptor DC-SIGN. Starting from libraries of 108 mannose-conjugated peptides, we identified glycopeptides that exhibited up to a 650-fold increase in multivalent binding affinity for DC-SIGN, which is also preserved in cells. Monovalently, our most potent glycopeptides have a similar potency to a Man3 oligosaccharide, representing a 15-fold increase in activity compared to mannose. These compounds represent the first examples of glycopeptide ligands that target the CRD of DC-SIGN. The natural framework of glycopeptide conjugates and the simplicity of orthogonal conjugation to make these glycopeptides anticipates a promising future for development of DC-SIGN-targeting moieties.

Synergic "Click" Boronate/Thiosemicarbazone System for Fast and Irreversible Bioorthogonal Conjugation in Live Cells.

Fast, high-yielding, and selective bioorthogonal "click" reactions employing nontoxic reagents are in high demand for their great utility in the conjugation of biomolecules in live cells. Although a number of click reactions were developed for this purpose, many are associated with drawbacks and limitations that justify the development of alternative systems for both single- or dual-labeling applications. Recent reports have highlighted the potential of boronic ester formation as a bioorthogonal click reaction between abiotic boronic acids and diols. Boronic ester formation is a fast dehydrative process; however it is intrinsically reversible in aqueous medium. We designed and optimized a synergic system based on two bifunctional reagents, a thiosemicarbazide-functionalized nopoldiol and an ortho-acetyl arylboronic acid. Both reagents were shown to be chemically stable and nontoxic to HEK293T cells at concentrations as high as 50 µM. The resulting boronate/thiosemicarbazone adduct is a medium-sized ring that forms rapidly and irreversibly without any catalyst at low µM concentrations, in neutral buffer, with a rate constant of 9 M-1 s-1 as measured by NMR spectroscopy. Control experiments in the presence of competing boronic acids showed no crossover side-products and confirmed the stability and lack of reversibility of the boronate/thiosemicarbazone conjugates. Formation of the conjugates is not affected by the presence of biological diols such as fructose, glucose, and catechol, and the thiosemicarbazide-functionalized nopoldiol is inert to aldehyde electrophiles of the sort found on protein-bound glyoxylyl units. The suitability of this system in the cell-surface labeling of live cells was demonstrated using a SNAP-tag approach to install the boronic acid reagent onto the extracellular domain of the Beta-2 adrenergic receptor in HEK293T cells, followed by incubation with the optimal thiosemicarbazide-functionalized nopoldiol reagent labeled with fluorescein dye. Successful visualization by fluorescence microscopy was possible with a reagent concentration as low as 10 µM, thus confirming the potential of this system in biological applications.

A novel strategy to enhance angiogenesis in vivo using the small VEGF-binding peptide PR1P.

Therapeutic angiogenesis is an experimental frontier in vascular biology that seeks to deliver angiogenic growth factors to ischemic or injured tissues to promote targeted formation of new blood vessels as an alternative approach to surgical revascularization procedures. Vascular endothelial growth factor (VEGF) is a potent angiogenic signal protein that is locally upregulated at sites of tissue injury. However, therapies aimed at increasing VEGF levels experimentally by injecting VEGF gene or protein failed to improve outcomes in human trials in part due to its short half-life and systemic toxicity. We recently designed a novel 12-amino acid peptide (PR1P) whose sequence was derived from an extracellular VEGF-binding domain of the pro-angiogenic glycoprotein prominin-1. In this study, we characterized the molecular binding properties of this novel potential therapeutic for targeted angiogenesis and provided the foundation for its use as an angiogenic molecule that can potentiate endogenous VEGF. We showed that PR1P bound VEGF directly and enhanced VEGF binding to endothelial cells and to VEGF receptors VEGFR2 and neuropilin-1. PR1P increased angiogenesis in the murine corneal micropocket assay when combined with VEGF, but had no activity without added VEGF. In addition, PR1P also enhanced angiogenesis in murine choroidal neovascularization and wound-healing models and augmented reperfusion in a murine hind-limb ischemia model. Together our data suggest that PR1P enhanced angiogenesis by potentiating the activity of endogenous VEGF. In so doing, this novel therapy takes advantage of endogenous VEGF gradients generated in injured tissues and may improve the efficacy of and avoid systemic toxicity seen with previous VEGF therapies.

Tandem Wittig/Diels-Alder diversification of genetically encoded peptide libraries.

In this paper, we developed a tandem of two carbon-carbon bond-forming reactions to chemically diversify the libraries of peptides displayed on a bacteriophage. The Wittig reaction of a biotin-ester from a stabilized phosphorane ylide with model peptides containing N-terminal glyoxal exhibits reaction rates of 0.07 to 5 M-1 s-1 in water at pH 6.5-8. The log(k) scaled linearly with pH from pH 6 to 8; above pH 9 the reaction is accompanied by the hydrolysis of the ester functionality. Capture of the phage displaying the biotinylated product by streptavidin beads confirmed the rate of this reaction in a library of 108 peptides (k = 0.23 M-1 s-1 at pH = 6.5) and also confirmed the regioselectivity of this modification. Olefins introduced into the Wittig reaction can act as Michael acceptors: addition of glutathione, cysteamine, and DYKDDDDKC ("FLAG-Cys") peptide occurs with k = 0.12-4.1 M-1 s-1 at pH 7.8. Analogous reactions with the DYKDDDDKC peptide take place on phage-displayed peptides modified via the Wittig reaction. This reaction is manifested as a progressive emergence of a FLAG-epitope on the phage and detected by the capture of this phage using an anti-FLAG antibody. Olefins introduced into the Wittig reaction also act as dienophiles in the Diels-Alder reaction with cyclopentadiene. The conversion of the dienophile to norbornene-like adducts on the phage was observed by monitoring the disappearance of the thiol-reactive olefin on the phage. This report broadens the reaction scope of genetically-encoded peptide libraries displayed on the phage, expanding the structural diversity of these platforms and increasing their potential to be used in screening against important protein targets. The possibility of monitoring tandem reactions by the use of different labels illustrates the feasibility of obtaining highly functionalized peptides with chemical motifs impossible to achieve using conventional translational machinery.

Silent Encoding of Chemical Post-Translational Modifications in Phage-Displayed Libraries.

In vitro selection of chemically modified peptide libraries presented on phage, while a powerful technology, is limited to one chemical post-translational modification (cPTM) per library. We use unique combinations of redundant codons to encode cPTMs with "silent barcodes" to trace multiple modifications within a mixed modified library. As a proof of concept, we produced phage-displayed peptide libraries Ser-[X]4-Gly-Gly-Gly, with Gly and Ser encoded using unique combinations of codons (TCA-[X]4-GGAGGAGGA, AGT-[X]4-GGTGGTGGT, etc., where [X]4 denotes a random NNK library). After separate chemical modification and pooling, mixed-modified libraries can be panned and deep-sequenced to identify the enriched peptide sequence and the accompanying cPTM simultaneously. We panned libraries bearing combinations of modifications (sulfonamide, biotin, mannose) against matched targets (carbonic anhydrase, streptavidin, concanavalin A) to identify desired ligands. Synthesis and validation of sequences identified by deep sequencing revealed that specific cPTMs are significantly enriched in panning against the specific targets. Panning on carbonic anhydrase yielded a potent ligand, sulfonamide-WIVP, with Kd = 6.7 ± 2.1 nM, a 20-fold improvement compared with the control ligand sulfonamide-GGGG. Silent encoding of multiple cPTMs can be readily incorporated into other in vitro display technologies such as bacteriophage T7 or mRNA display.

Allene Functionalized Azobenzene Linker Enables Rapid and Light-Responsive Peptide Macrocyclization.

In this manuscript, we describe the efficient synthesis of a bis(allenamide) functionalized water-soluble azobenzene reagent (BSBDA) and its application as a new tool for the rapid generation of visible light-responsive macrocyclic peptides and peptide libraries displayed on the surface of bacteriophage. The allenamide functionality promotes cysteine ligation in model peptides and those displayed on phage with rates 2-3 orders of magnitude faster than the established alkyl halide containing azobenzenes.

Phage-displayed macrocyclic glycopeptide libraries.

In this report, we describe an efficient way to generate libraries of macrocyclic glycopeptides in one step by reacting phage-displayed libraries of peptides with dichloro-oxime derivatives. We showed that the reactions do not interfere with the ability of phage to replicate in bacteria. The reactions are site-selective for phage-displayed peptides and they do not modify any other proteins of phage. The technology described in this report will be instrumental for genetic selection of macrocyclic glycopeptides for diverse glycan-binding proteins.

Heat-enhanced peptide synthesis on Teflon-patterned paper.

In this report, we describe the methodology for 96 parallel organic syntheses of peptides on Teflon-patterned paper assisted by heating with an infra-red lamp. SPOT synthesis is an important technology for production of peptide arrays on a paper-based support for rapid identification of peptide ligands, epitope mapping, and identification of bio-conjugation reactions. The major drawback of the SPOT synthesis methodology published to-date is suboptimal reaction conversion due to mass transport limitations in the unmixed reaction spot. The technology developed in this report overcomes these problems by changing the environment of the reaction from static to dynamic (flow-through), and further accelerating the reaction by selective heating of the reaction support in contact with activated amino acids. Patterning paper with Teflon allows for droplets of organic solvents to be confined in a zone on the paper array and flow through the paper at a well-defined rate and provide a convenient, power-free setup for flow-through solid-phase synthesis and efficient assembly of peptide arrays. We employed an infra-red (IR) lamp to locally heat the cellulosic support during the flow-through delivery of the reagents to each zone of the paper-based array. We demonstrate that IR-heating in solid phase peptide synthesis shortened the reaction time necessary for amide bond formation down to 3 minutes; in some couplings of alpha amino acids, conversion rates increased up to fifteen folds. The IR-heating improved the assembly of difficult sequences, such as homo-oligomers of all 20 natural amino acids.

Prospective identification of parasitic sequences in phage display screens.

Phage display empowered the development of proteins with new function and ligands for clinically relevant targets. In this report, we use next-generation sequencing to analyze phage-displayed libraries and uncover a strong bias induced by amplification preferences of phage in bacteria. This bias favors fast-growing sequences that collectively constitute <0.01% of the available diversity. Specifically, a library of 10(9) random 7-mer peptides (Ph.D.-7) includes a few thousand sequences that grow quickly (the 'parasites'), which are the sequences that are typically identified in phage display screens published to date. A similar collapse was observed in other libraries. Using Illumina and Ion Torrent sequencing and multiple biological replicates of amplification of Ph.D.-7 library, we identified a focused population of 770 'parasites'. In all, 197 sequences from this population have been identified in literature reports that used Ph.D.-7 library. Many of these enriched sequences have confirmed function (e.g. target binding capacity). The bias in the literature, thus, can be viewed as a selection with two different selection pressures: (i) target-binding selection, and (ii) amplification-induced selection. Enrichment of parasitic sequences could be minimized if amplification bias is removed. Here, we demonstrate that emulsion amplification in libraries of ∼ 10(6) diverse clones prevents the biased selection of parasitic clones.

Genetically encoded fragment-based discovery of glycopeptide ligands for carbohydrate-binding proteins.

We describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based discovery (GE-FBD) uses selection of phage-displayed glycopeptides to dock a glycan fragment at the CRD and guide selection of synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10(8) glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40-50-fold enhancement in affinity over methyl α-d-mannopyranoside (MeMan). Lectin array suggested specificity: Man-WYD derivative bound only to 3 out of 17 proteins­ConA, LcH, and PSA­that bind to Man. An X-ray structure of ConA:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking, but their extra-CRD binding modes are significantly different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.

Rapid, hydrolytically stable modification of aldehyde-terminated proteins and phage libraries.

We describe the rapid reaction of 2-amino benzamidoxime (ABAO) derivatives with aldehydes in water. The ABAO combines an aniline moiety for iminium-based activation of the aldehyde and a nucleophilic group (Nu:) ortho to the amine for intramolecular ring closure. The reaction between ABAO and aldehydes is kinetically similar to oxime formations performed under stoichiometric aniline catalysis. We characterized the reaction by both NMR and UV spectroscopy and determined that the rate-determining step of the process is formation of a Schiff base, which is followed by rapid intramolecular ring closure. The relationship between apparent rate constant and pH suggests that a protonated benzamidoxime acts as an internal general acid in Schiff-base formation. The reaction is accelerated by substituents in the aromatic ring that increase the basicity of the aromatic amine. The rate of up to 40 M(-1) s(-1) between an electron-rich aldehyde and 5-methoxy-ABAO (PMA), which was observed at pH 4.5, places this reaction among the fastest known bio-orthogonal reactions. Reaction between M13 phage-displayed library of peptides terminated with an aldehyde moiety and 1 mM biotin-ABAO derivative reaches completion in 1 h at pH 4.5. Finally, the product of reaction, dihydroquinazoline derivative, shows fluorescence at 490 nm suggesting a possibility of developing fluorogenic aldehyde-reactive probes based on ABAO framework.

Rapid enumeration of phage in monodisperse emulsions.

Phage-based detection assays have been developed for the detection of viable bacteria for applications in clinical diagnosis, monitoring of water quality, and food safety. The majority of these assays deliver a positive readout in the form of newly generated progeny phages by the bacterial host of interest. Progeny phages are often visualized as plaques, or holes, in a lawn of bacteria on an agar-filled Petri dish; however, this rate-limiting step requires up to 12 h of incubation time. We have previously described an amplification of bacteriophages M13 inside droplets of media suspended in perfluorinated oil; a single phage M13 in a droplet yields 10(7) copies in 3-4 h. Here, we describe that encapsulation of reporter phages, both lytic T4-LacZ and nonlytic M13, in monodisperse droplets can also be used for rapid enumeration of phage. Compartmentalization in droplets accelerated the development of the signal from the reporter enzyme; counting of "positive" droplets yields accurate enumeration of phage particles ranging from 10(2) to 10(6) pfu/mL. For enumeration of T4-LacZ phage, the fluorescent signal appeared in as little as 90 min. Unlike bulk assays, quantification in emulsion is robust and insensitive to fluctuations in environmental conditions (e.g., temperature). Power-free emulsification using gravity-driven flow in the absence of syringe pumps and portable fluorescence imaging solutions makes this technology promising for use at the point of care in low-resource environments. This droplet-based phage enumeration method could accelerate and simplify point-of-care detection of the pathogens for which reporter bacteriophages have been developed.

Towards rapid on-site phage-mediated detection of generic Escherichia coli in water using luminescent and visual readout.

Wild-type T4 bacteriophage and recombinant reporter lac Z T4 bacteriophage carrying the ß-galactosidase gene were used for detection of generic Escherichia coli by monitoring the release of ß-galactosidase upon phage-mediated cell lysis. The reaction was performed on a paper-based portable culture device to limit the diffusion of reagents and, hence, increase the sensitivity of the assay, and to avoid handling large sample volumes, making the assay suitable for on-site analysis. Chromogenic (chlorophenol red-ß-D-galactopyranoside, CPRG) and bioluminescent (6-O-ß-galactopyranosyl-luciferin, Beta-Glo(®)) ß-galactosidase substrates were tested in the assay. Water samples were first filtered through 0.45-µm pore size filters to concentrate bacteria. The filters were then placed into the paper-based device containing nutrient medium and incubated at 37 °C for 4 h. Bacteriophage with the respective indicator substrate was added to the device, and signal (color, luminescence) development was recorded with a digital camera, luminometer, or luminescence imaging device. It was demonstrated that as low as 40 or <10 colony-forming units (cfu) ml(-1) of E. coli can be detected visually within 8 h when wild-type T4 bacteriophage or recombinant lacZ T4 bacteriophage were used in the assay, respectively. Application of the bioluminescent ß-galactosidase substrate allowed reliable detection of <10 cfu ml(-1) within 5.5 h. The specificity of the assay was demonstrated using a panel of microorganisms including Aeromonas hydrophila, Enterobacter cloacae, E. coli, and Salmonella Typhimurium.