Biological laser printing of genetically modified Escherichia coli for biosensor applications

One of the primary requirements of cell- or tissue-based sensors is the placement of cells and cellular material at or near the sensing elements of the device. The ability to achieve precise, reproducible and rapid placement of cells is the focus of this study. We have developed a technique, biological laser printing or BioLP, which satisfies these requirements and has advantages over current technologies. BioLP is capable of rapidly depositing patterns of active biomolecules and living cells onto a variety of material surfaces. Unlike ink jet or manual spotting techniques, this process delivers small volume (nl to fl) aliquots of biomaterials without the use of an orifice, thus eliminating potential clogging issues and enabling diverse classes of biomaterials to be deposited. This report describes the use of this laser-based printing method to transfer genetically-modified bacteria capable of responding to various chemical stressors onto agar-coated slides and into microtiter plates. The BioLP technology enables smaller spot sizes, increased resolution, and improved reproducibility compared to related technologies.     

Nonobese diabetic mouse congenic analysis reveals chromosome 11 locus contributing to diabetes susceptibility, macrophage STAT5 dysfunction, and granulocyte-macrophage colony-stimulating factor overproduction

Unstimulated monocytes of at-risk/type 1 diabetic humans and macrophages of the NOD mouse have markedly elevated autocrine GM-CSF production and persistent STAT5 phosphorylation. We analyzed the relationship between GM-CSF production and persistent STAT5 phosphorylation in NOD macrophages using reciprocal congenic mouse strains containing either diabetes-susceptible NOD (B6.NODC11), or diabetes-resistant C57L (NOD.LC11) loci on chromosome 11. These intervals contain the gene for GM-CSF (Csf2; 53.8 Mb) and those for STAT3, STAT5A, and STAT5B (Stat3, Stat5a, and Stat5b; 100.4-100.6 Mb). High GM-CSF production and persistent STAT5 phosphorylation in unactivated NOD macrophages can be linked to a region (44.9-55.7 Mb) containing the Csf2 gene, but not the Stat3/5a/5b genes. This locus, provisionally called Idd4.3, is upstream of the previously described Idd4.1 and Idd4.2 loci. Idd4.3 encodes an abundance of cytokine genes that use STAT5 in their macrophage activation signaling and contributes approximately 50% of the NOD.LC11 resistance to diabetes.     

Properties and conditions of formation of the fibronectin-collagen complex in a culture of human embryo fibroblasts

In our previous study the macromolecular complexes mostly consisting of fibronectin and procollagen were isolated from human fibroblast culture media using immobilized antibodies against fibronectin. At present an attempt was made to elucidate at what stage the formation of the fibronectin-collagen complex occurs--either in the course of incubation of the immobilized antibodies with labelled proteins secreted by fibroblasts, or in the extracellular space while labelling fibroblasts, or intracellularly. The results obtained show that the fibronectin-collagen complex: 1) pre-exists even before incubation with the immobilized antibodies and 2) it is of intracellular origin. Thus, the considerable amount of the fibroblast-secreted fibronectin (no less than 20%) is released from the cell not in the free form but in the complex with procollagen. It was suggested that the fibronectin-collagen complex presents a stage in the formation of the insoluble extracellular matrix.     

Iron- and 4-hydroxy-2-alkylquinoline-containing periplasmic inclusion bodies of Pseudomonas aeruginosa: a chemical analysis

Dark aggregated particles were seen on pellets of iron-rich, mid-logarithmic phase Pseudomonas aeruginosa. Transmission electron microscopy of these cells showed inclusion bodies in periplasmic vacuoles. Aggregated particles isolated from the spent medium of these cells contained iron as indicated by atomic absorption spectroscopy and by electron paramagnetic resonance spectroscopy that revealed Fe(3+). Scanning electron microscopy/energy dispersive X-ray analysis of whole cells revealed the presence of iron-containing particles beneath the surface of the cell, indicating that the isolated aggregates were the intracellular inclusion bodies. Collectively, mass spectroscopy and nuclear magnetic resonance spectroscopy of the isolated inclusion bodies revealed the presence of 3,4-dihydroxy-2-heptylquinoline which is the Pseudomonas quinolone signaling compound (PQS) and an iron chelator; 4-hydroxy-2-heptylquinoline (pseudan VII), which is an iron chelator, antibacterial compound and precursor of PQS; 4-hydroxy-2-nonylquinoline (pseudan IX) which is an iron chelator and antibacterial compound; 4-hydroxy-2-methylquinoline (pseudan I), and 4-hydroxy-2-nonylquinoline N-oxide.     

Improved detection of antibiotic compounds by bacterial reporter strains achieved by manipulations of membrane permeability and efflux capacity

The occurrence of pharmaceuticals, including antibacterial compounds, in the environment has been acknowledged as an emerging and troubling issue in environmental safety; their usage is constantly on the rise, and their effects on the environment are only partially understood. Such compounds can accumulate, contaminate the ecosystem, and contribute to the spreading of antibiotic resistance among bacteria, hindering human health. Bioluminescent Escherichia coli reporter strains, engineered to detect antibiotic compounds by fusing the promoter of the global regulator soxS to the Photorhabdus luminescens luxCDABE cassette, were further modified by altering their membrane permeability and efflux capabilities. This was accomplished by introducing several mutations in the efflux system (ΔemrE, ΔacrB, and ΔtolC) and by overexpressing OmpF, a porin located in the outer membrane that allows passive diffusion of molecules. Combinations of these alterations had a cumulative effect in lowering the detection threshold of several antibiotics, in some of the cases to concentrations reported from pharmaceutical-polluted environments.     

A bacterial reporter panel for the detection and classification of antibiotic substances

The ever‐growing use of pharmaceutical compounds, including antibacterial substances, poses a substantial pollution load on the environment. Such compounds can compromise water quality, contaminate soils, livestock and crops, enhance resistance of microorganisms to antibiotic substances, and hamper human health. We report the construction of a novel panel of genetically engineered Escherichia coli reporter strains for the detection and classification of antibiotic substances. Each of these strains harbours a plasmid that carries a fusion of a selected gene promoter to bioluminescence (luxCDABE) reporter genes and an alternative tryptophan auxotrophy‐based non‐antibiotic selection system. The bioreporter panel was tested for sensitivity and responsiveness to diverse antibiotic substances by monitoring bioluminescence as a function of time and of antibiotic concentrations. All of the tested antibiotics were detected by the panel, which displayed different response patterns for each substance. These unique responses were analysed by several algorithms that enabled clustering the compounds according to their functional properties, and allowed the classification of unknown antibiotic substances with a high degree of accuracy and confidence.     

Modeling and measurement of a whole-cell bioluminescent biosensor based on a single photon avalanche diode

Whole-cell biosensors are potential candidates for on-line and in situ environmental monitoring. In this work we present a new design of a whole-cell bioluminescence biosensor for water toxicity detection, based on genetically engineered Escherichia coli bacteria, carrying a recA::luxCDABE promoter-reporter fusion. Sensitive optical detection is achieved using a single photon avalanche photodiode (SPAD) working in the Geiger mode. The present work describes a simple mathematical model for the kinetic process of the bioluminescence based SOS toxin response of E. coli bacteria. We find that initially the bioluminescence signal depends on the time square and we show that the spectral intensity of the bioluminescence signal is inverse proportional to the frequency. We get excellent agreement between the theoretical model and the measured light signal. Furthermore, we present experimental results of the bioluminescent signal measurement using a SPAD and a photomultiplier, and demonstrate improvement of the measurement by applying a matched digital filter. Low intensity bioluminescence signals were measured after the whole-cell sensors were exposed to various toxicant concentrations (5, 15 and 20ppm).     

Antioxidant defense system in tissues of semiaquatic mammals

Activity of antioxidant enzymes (superoxide dismutase and catalase) and low molecular weight antioxidants (tocopherol and retinol) was studied in tissues of 8 semiaquatic mammalian species. In animals from different taxa, the enhancement of antioxidant defense can be achieved either by increased enzymatic activity of tissues or elevated concentration of low molecular weight antioxidants. The level of enzymatic and nonenzymatic (low molecular weight) antioxidants, determined in tissues and organs of diving mammals, is likely to be considered as an integral complex, which has evolved to ensure the greatest efficiency of metabolic systems during adaptation to species-specific habitats.     

Na-Dithionite Promotes Photosynthetic Sulfide Utilization by the Cyanobacterium Oscillatoria limnetica

The light- and sulfide-dependent induction process leading to photosystem I-mediated sulfide utilization by Oscillatoria limnetica, for either H₂ evolution or CO₂ photoassimilation, was studied. The identical dependence on pH of the lag length, the inhibition of leucine incorporation and final H₂S concentration imply that the latter exerts a deleterious effect on nonadapted cells.

Na-dithionite (Na₂S₂O₄), Na-sulfite (Na₂SO₃), or ethanol cannot serve as photosynthetic electron donors. However, when these compounds were added to the sulfide-containing system, the need for induction was eliminated. At pH 6.9, in the presence of 3.5 millimolar sulfide, these substances (at concentrations of 10 millimolar, 5 millimolar, or 0.4 molar, respectively) completely abolished the delay preceding sulfide-dependent H₂ evolution. It is suggested that all three compounds expose a site capable of directly accepting sulfide electrons.

Only dithionite could adapt the cells to sulfide utilization on its own. Sulfite or ethanol acted only in the presence of sulfide. It is implied that this specific activity of dithionite is related to its characteristic low redox potential.

Sulfide-dependent H₂ evolution was insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but was inhibited by the plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, in the presence as well as in the absence of dithionite. In both cases, therefore, the plastoquinone was implied in the electron transport from sulfide.