Walther R. Ellis

Reagentless detection of microorganisms by intrinsic fluorescence

Quick and accurate detection of microbial contamination is accomplished by a unique combination of leading-edge technologies described in this and the accompanying paper. In this contribution, a hand-held prototype instrument is described which is capable of statistically sampling the environment for microbial contamination and determining cell viability. The technology is sensitive enough to detect very low levels ( approximately 20 cells/cm(2) or cm(3)) of microbes in seconds.

Taxonomic identification of microorganisms by capture and intrinsic fluorescence detection.

Quick and accurate detection of microbial contamination is accomplished by a unique combination of leading edge technologies described in this and the accompanying article. Microbe capture chips, used with a prototype fluorescence detector, are capable of statistically sampling the environment for pathogens (including spores), identifying the specific pathogens/exotoxins, and determining cell viability where appropriate.

Conversion in the peptides coating cadmium:sulfide crystallites in Candida glabrata

Cultures of Candida glabrata treated with CdCl2 form intracellular Cd(II) complexes that evolve with the time of culturing. Initially, glutathione (gamma ECG) appears to be the major buffering component. One type of Cd(II)-glutathione complex exists as a cadmium:sulfide (CdS) crystallite coated with glutathione. A time dependent change in the coating of the CdS particles occurs with a decrease in the (gamma ECG) content and a corresponding increase in the abundance of (gamma EC)nG peptides with (gamma EC)2G becoming the predominant peptide. The des-Gly variant (gamma EC)2 appears in significant concentration only in late cultures. The evolution in isopeptide coating appears to be dependent on the sulfide content of the CdS particles. Cellular conditions that enhance the generation of sulfide ions facilitate the conversion from gamma ECG to (gamma EC)2G.

Magnetic circular dichroism studies of exogenous ligand and substrate binding to the non-heme ferrous active site in phthalate dioxygenase

BACKGROUND Mononuclear non-heme iron centers are found in the active sites of a variety of enzymes that require molecular oxygen for catalysis. The mononuclear non-heme iron is believed to be the active site for catalysis, and is presumed to bind and activate molecular oxygen. The mechanism of this reaction is not understood. Phthalate dioxygenase is one such enzyme. Because it also contains a second iron site, the Rieske site, it is difficult to obtain information on the structure of the active site. We therefore used magnetic circular dichroism (MCD) spectroscopy to probe the mononuclear, non-heme Fe2+ site in this biodegradative enzyme. RESULTS The MCD spectrum of the resting enzyme shows features indicative of one six-coordinate Fe2+ site; substrate binding converts the site to two different five-coordinate species, opening up a coordination position for O2 binding. MCD spectra of the corresponding apoenzyme have been subtracted to account for temperature-independent contributions from the Rieske site. Azide binds both to the resting enzyme to produce a new six-coordinate species, showing that one of the ferrous ligands is exchangeable, and also to the enzyme-substrate complex to form a ternary species. The low azide binding constant for the substrate-enzyme species relative to the resting enzyme indicates steric interaction and close proximity between exogenous ligand and the substrate. CONCLUSIONS We have been able to provide some detailed structural insight into exogenous ligand and substrate binding to the non-heme Fe2+ site, even in the presence of the enzymes [2Fe-2S] Rieske center. Further mechanistic studies are now required to maximize the molecular-level detail available from these spectroscopic studies.

Affinity Capture Mass Spectrometry of Biomarker Proteins Using Peptide Ligands from Biopanning

Affinity capture mass spectrometry was used to isolate and ionize protein A from Staphylococcus aureus from both a commercial source and cell culture lysate using matrix assisted laser desorption/ionization (MALDI) mass spectrometry. Two surfaces are compared: gold surfaces with immunoglobulin G covalently immobilized and silica surfaces with a covalently bound small peptide discovered via biopanning. A detection limit of 2.22 bacterial cells/mL of culture fluid was determined for the immobilized peptide surfaces. This study emphasizes the ability to use peptide ligands to effectively capture a biomarker protein out of a complex mixture. This demonstrates the potential to use biopanning to generate capture ligands for a large variety of target proteins and subsequently detect the captured protein using MALDI mass spectrometry.

Separation of protein inclusion bodies from Escherichia coli lysates using sedimentation field‐flow fractionation

Sedimentation field-flow fractionation has been used to separate my- ohemerythrin inclusion bodies from components of growth media, soluble proteins, and unlysed cells that are present in Escherichia coli cell lysates. Collected . fractions were concentrated and then analyzed by sodium dodecyl sulfate SDS polyacrylamide gel electrophoresis to confirm the presence of myohemerythrin inclusion bodies and to determine their position in the elution sequence. The fractograms of samples prepared using two different cell lysing methods were compared. Q 1997 John Wiley & Sons, Inc. JM icro Sep9: 233)239, 1997

Functional Role of Leucine-103 in Myohemerythrin†

Hemerythrins (Hrs) and myohemerythrins (Mhrs) are nonheme iron proteins that function as O2 carriers in four marine invertebrate phyla. Available amino acid sequences and X-ray structures indicate that a conserved leucine, residue 103 in the Themiste zostericola Mhr sequence, occupies a site distal to the Fe-O-Fe center. The side-chain methyl groups of the analogous leucine in Themiste dyscrita oxyHr are in van der Waals contact with bound O2 in the X-ray crystal structure, and this residue may therefore play a role in stabilizing bound dioxygen with respect to autoxidation. In order to test this hypothesis, the gene for T. zostericola Mhr was synthesized and expressed in Escherichia coli. Two mutant Mhrs, L103V and L103N, were also prepared. Optical spectra and kinetics data for these three proteins are presented. Importantly, neither mutant forms a stable oxy adduct; instead, rapid autoxidation results in formation of the corresponding met forms. In addition, the L103N Mhr displays unusually rapid reduction kinetics, suggesting that the amide functionality of Asn-103 destabilizes most bound ligands and additionally promotes rapid semi-metR <==> semi-metO isomerization.

Is what you eat and drink safe? Detection and identification of microbial contamination in foods and water

Current technologies for assessing the microbial load in food and water require cellular outgrowth to increase the cell count to a detectable level. This step therefore makes real-time assessment of microbial loads impossible. To circumvent this problem, we have developed a high-sensitivity, multiwavelength fluorescence detector and signal- collection and processing software. This detection system enables one to distinguish between abiotic matter, sporulated bacteria, vegetative bacteria, dead bacteria, and other biomaterials containing aromatic amino acids. Our detection limit is in the 10-100 cell/cm/sup 2/ range (or per cm/sup 3/ if bulk aqueous samples are used), and there is no sample contact. To supplement this detection system, we have developed a variety of ligands, including hemin derivatives, peptides, glycoconjugates, and siderophores, for cell capture to circumvent problems associated with antibodies. These materials are tethered to disposable surfaces (glass, plastics) in arrays for use in capturing biomaterials (e.g., live bacterial cells, dead cells, spores, and toxins); identification is based on which sector of an array it binds to. The detection system can serve as a reader for these coated materials, and variants have been tested for use in scanning food surfaces and food wash water.

Real-time In-situ detection of microbes

Currently, no methods exist for the real-time detection and quantification of microbes in the environment or for the detection and identification of pathogenic organisms in clinical specimens. We have developed technologies which overcome these limitations and provide detection limits as low as a ten microbial cells per cm2 on abiotic surfaces, and per mL in fluids. The detection and quantification of microbes [total microbial load] is based on the intrinsic fluorescence of microbial metabolites and protein cofactors, and provides an estimate of the total microbial load as well as the relative distribution of live cells, dead cells, and endospores. Unlike existing methods, no additional reagents or sample contact is needed. This technology has been applied to the in-situ measurements of two sub-glacial microbial communities at sites in the Svalbard Archipelago, Norway, and to the efficacy of disinfection of contact lenses. In the rapid spread of a life-threatening infection, early diagnosis is of great importance. In such situations, pathogen counts will be very low, which also presents a significant challenge to diagnostic methods. We have developed a point of care disposable diagnostic based on the en masse capture of blood-borne microbes from 1 mL of fresh whole blood with surface-tethered, small molecule ligands. Quantification is based on the intrinsic fluorescence of captured cells.

Molecular engineering of sensors for biological materials

Molecular engineering methods have been used to develop sensors for the capture of DNA, viruses, and bacterial cells, spores, and toxins. The capture technology exploits the molecular basis of pathogenesis and capture events are detected using the intrinsic fluorescence of the captured microbial components. Capabilities include statistically sampling the environment in minutes and sensitivity of /spl sim/100 cells/cm/sup 3/.