No effect of femtosecond laser pulses on M13, E. coli, DNA, or protein.

Data showing what appears to be nonthermal inactivation of M13 bacteriophage (M13), Tobacco mosaic virus, Escherichia coli (E. coli), and Jurkatt T-cells following exposure to 80-fs pulses of laser radiation have been published. Interest in the mechanism led to attempts to reproduce the results for M13 and E. coli. Bacteriophage plaque-forming and bacteria colony-forming assays showed no inactivation of the microorganisms; therefore, model systems were used to see what, if any, damage might be occurring to biologically important molecules. Purified plasmid DNA (pUC19) and bovine serum albumin were exposed to and analyzed by agarose gel electrophoresis (AGE) and polyacrylamide gel electrophoresis (PAGE), respectively, and no effect was found. DNA and coat proteins extracted from laser-exposed M13 and analyzed by AGE or PAGE found no effect. Raman scattering by M13 in phosphate buffered saline was measured to determine if there was any physical interaction between M13 and femtosecond laser pulses, and none was found. Positive controls for the endpoints measured produced the expected results with the relevant assays. Using the published methods, we were unable to reproduce the inactivation results or to show any interaction between ultrashort laser pulses and buffer/water, DNA, protein, M13 bacteriophage, or E. coli.

Fluorescent proteins as biosensors by quenching resonance energy transfer from endogenous tryptophan: detection of nitroaromatic explosives.

Ensuring domestic safety from terrorist attack is a daunting challenge because of the wide array of chemical agents that must be screened. A panel of purified fluorescent protein isoforms (FPs) was screened for the ability to detect various explosives, explosive simulants, and toxic agents. In addition to their commonly used visible excitation wavelengths, essentially all FPs can be excited by UV light at 280 nm. Ultraviolet illumination excites electrons in endogenous tryptophan (W) residues, which then relax by Förster Resonance Energy Transfer (FRET) to the chromophore of the FP, and thus the FPs emit with their typical visible spectra. Taking advantage of the fact that tryptophan excitation can be quenched by numerous agents, including nitroaromatics like TNT and nitramines like RDX, it is demonstrated that quenching of visible fluorescence from UV illumination of FPs can be used as the basis for detecting these explosives and explosive degradation products. This work provides the foundation for production of an array of genetically-modified FPs for in vitro biosensors capable of rapid, simultaneous, sensitive and selective detection of a wide range of explosive or toxic agents.

Basis for the extraordinary genetic stability of anthrax.

Over 500 isolates of anthrax bacillus from around the world represent one of the most genetically homogeneous microbes. There are three possibilities for this genetic stability: (1) anthrax has an extraordinarily high fidelity repair system, (2) genetic damage to anthrax is usually lethal, and/or (3) a highly demanding and selective process exists in its environment that is necessary for the completion of its life cycle. Using probes made from genes selected by growth of an Escherichia coli expression vector Bacillus anthracis library on hypertrophic high nitrate concentration medium, genes unique to B. anthracis were isolated. High nitration conditions generated stable chromosomal mutants that displayed altered morphology and life-cycle progression. Therefore, life-cycle progression connected to nitration, associated with host inflammatory response, selects for mutants that show life-cycle progression tightly coupled to progression of the inflammatory response to anthrax. Significant variation from this coupled progression leads to failure of anthrax to complete its life-cycle at the death of its host.