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Bacteria with antagonistic activity inhibit the growth of other bacteria through different mechanisms, including the production of antibiotics. As a result, these microorganisms are a prolific source of such compounds. However, searching for antibiotic-producing strains requires high-throughput techniques due to the vast diversity of microorganisms. Here, we screened and isolated bacteria with antagonistic activity against Escherichia coli expressing the green fluorescent protein (E. coli-GFP). We used microfluidics to co-encapsulate and co-culture single cells from different strains within picoliter gel beads and analyzed them using fluorescence-activated cell sorting (FACS). To test the methodology, we used three bacterial isolates obtained from Mexican maize, which exhibit high, moderate, or no antagonistic activity against E. coli-GFP, as determined previously using agar plate assays. Single cells from each strain were separately co-incubated into gel beads with E. coli-GFP. We monitored the development of the maize bacteria microcolonies and tracked the growth or inhibition of E. coli-GFP using bright-field and fluorescent microscopy. We correlated these images with distinctive light scatter and fluorescence signatures of each incubated bead type using FACS. This analysis enabled us to sort gel beads filled with an antagonistic strain, starting from a mixture of the three different types of maize bacteria and E. coli-GFP. Likewise, culturing the FACS-sorted beads on agar plates confirmed the isolation and recovery of the two antagonistic strains. In addition, enrichment assays demonstrated the methodology's effectiveness in isolating rare antibiotic-producer strains (0.01% abundance) present in a mixture of microorganisms. These results show that associating light side scatter and fluorescent flow cytometry signals with microscopy images provides valuable controls to establish successful high-throughput methods for sorting beads in which microbial interaction assays are performed. [ABSTRACT FROM AUTHOR]

Water-in-oil microdroplets in microfluidics are well-defined individual picoliter reaction compartments and, as such, have great potential for quantitative high-throughput biological screening. This, however, depends upon contents of the droplets not leaking out into the oil phase. To assess the mechanism of possible leaking, the retention of various fluorescein derivatives from droplets formed in mineral oil and stored for hours in a reservoir on chip was studied. Leaking into the oil phase was observed and was shown to be dependent on the nature of the compounds and on the concentration of the silicone-based polymeric surfactant Abil EM 90 used. In experiments in which droplets filled with fluorescein were mixed with droplets filled with only buffer, the rate of efflux from filled droplets to empty droplets was dependent on the number of neighboring droplets of different composition. Buffer droplets with five fluorescein-containing neighbors took up the fluorophore 4.5 times faster than buffer droplets without fluorescein neighbors. The addition of bovine serum albumin (BSA) substantially reduced leaking. A formulation with 5% BSA reduces leaking of the fluorophore from 45% to 3%. Inclusion of BSA enabled experiments to be carded out over periods up to 18 h, without substantial leaking (

We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitoring the fluorescence at several time points within the device and exhibits kinetic behavior similar to that observed in bulk solution. Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements.

Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. [beta]-Phosphoglucomutase catalyses the isomerization of [beta]-glucose-1-phosphate to [beta]-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using [sup.19]F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which Mg[F.sup.-.sub.3] mimics the transferring P[O.sup.- .sub.3] moiety. Here we present a detailed characterization of the metal ion-fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of [beta]-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography. enzyme mechanism | fluoride inhibition | NMR structure | phosphoryl transfer | isosteric isoelectronic | transition state analogue

A microfluidic device capable of storing picoliter droplets containing single bacteria at constant volumes is prepared in PDMS. The gene expression and enzymatic activity is simultaneously and continuously monitored by measuring the fluorescence intensity of both the mRFP1 (red fluorescent protein) inside the cells and a fluorescent product formed as a result of the enzymatic activity outside the cells.

Steady- and presteady-state kinetic analysis is used for examining the isomerization of [beta]-glucose-1-phosphate ([beta]G1P) to [beta]-glucose-6-phosphate (G6P) catalyzed by [beta]-phosphoglucomutase ([beta]PGM). The studies have shown that the preference for transition-state analogue (TSA) formation when fluoride is present and the hydrolysis of substrates when it is not, has ruled out the formation of a stable pentavalent phosphorane intermediate in the active site of [beta]PGM.

The catalytic promiscuity of Pseudomonas aeruginosa arylsulfatase (PAS) is explored. The magnitudes and relative similarity of the kinetic parameters have shown that a functional switch from sulfatase to phosphatase activities is feasible, either by gene duplication or by direct evolution by an intermediate enzyme with dual specificity.

Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. β-Phosphoglucomutase catalyses the isomerization of β-glucose-1-phosphate to β-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using 19F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which MgF3- mimics the transferring PO3- moiety. Here we present a detailed characterization of the metal ion-fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of β-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography. [ABSTRACT FROM AUTHOR]

High catalytic proficiencies observed for the native and promiscuous reaction of the Pseudomonas aeruginosa arylsulfatase (PAS; the picture shows transition states of the two substrates with corresponding binding constants K(tx)) suggest that the trade-off between high activity and tight specificity can be substantially relaxed.