Expression and recombination of the antigenic variation vlsE gene of the Lyme disease spirochete Borrelia burgdorferi were analyzed in the tick vector. To assess vlsE expression, Ixodes scapularis nymphs infected with the B. burgdorferi strain B31 were fed on mice for 48 or 96 h or to repletion and then crushed and acetone fixed either immediately thereafter (ticks collected at the two earlier time points) or 4 days after repletion. Unfed nymphs also were examined. At all of the time points investigated, spirochetes were able to bind a rabbit antibody raised against the conserved invariable region 6 of VlsE, as assessed by indirect immunofluorescence, but not preimmune serum from the same rabbit. This same antibody also bound to B31 spirochetes cultivated in vitro. Intensity of fluorescence appeared highest in cultured spirochetes, followed by spirochetes present in unfed ticks. Only a dim fluorescent signal was observed on spirochetes at the 48 and 96 h time points and at day 4 postrepletion. Expression of vlsE in vitro was affected by a rise in pH from 7.0 to 8.0 at 34°C. Hence, vlsE expression appears to be sensitive to environmental cues of the type found in the B. burgdorferi natural history. To assess vlsE recombination, nymphs were capillary fed the B. burgdorferi B31 clonal isolate 5A3. Ticks thus infected were either left to rest for 4 weeks (Group I) or fed to repletion on a mouse (Group II). The contents of each tick from both groups were cultured and 10 B. burgdorferi clones from the spirochetal isolate of each tick were obtained. The vlsE cassettes from several of these clones were amplified by PCR and sequenced. Regardless of whether the isolate was derived from Group I or Group II ticks, no changes were observed in the vlsE sequence. In contrast, vlsE cassettes amplified from B. burgdorferi clones derived from a mouse that was infected with B31-5A3 capillary-fed nymphs showed considerable recombination. It follows that vlsE recombination does not occur in the tick vector.

Cyclic nitramine explosives are synthesized globally mainly as military munitions, and their use has resulted in environmental contamination. Several biodegradation pathways have been proposed, and these are based mainly on end-product characterization because many of the metabolic intermediates are hypothetical and unstable in water. Biodegradation mechanisms for cyclic nitramines include (a) formation of a nitramine free radical and loss of nitro functional groups, (b) reduction of nitro functional groups, (c) direct enzymatic cleavage, (d) alpha-hydroxylation, or (e) hydride ion transfer. Pathway intermediates spontaneously decompose in water producing nitrite, nitrous oxide, formaldehyde, or formic acid as common end-products. In vitro enzyme and functional gene expression studies have implicated a limited number of enzymes/genes involved in cyclic nitramine catabolism. Advances in molecular biology methods such as high-throughput DNA sequencing, microarray analysis, and nucleic acid sample preparation are providing access to biochemical and genetic information on cultivable and uncultivable microorganisms. This information can provide the knowledge base for rational engineering of bioremediation strategies, biosensor development, environmental monitoring, and green biosynthesis of explosives. This paper reviews recent developments on the biodegradation of cyclic nitramines and the potential of genomics to identify novel functional genes of explosive metabolism.

Xenobiotics such as explosives and pesticides released into the environment can have lethal and sublethal impacts on soil organisms such as earthworms with potential subsequent impacts at highertrophic levels. To better understand the molecular toxicological mechanisms of 2,4,6-trinitrotoluene (TNT), a commonly used explosive, in Eisenia fetida, earthworms were exposed to a gradient of TNT-spiked soils for 28 days and impacts on gene expression were examined using a 4032 cDNA microarray. Reproduction was increased at low doses of TNT, whereas high doses of TNT reduced juvenile production. On the basis of reproduction responses to TNT, four treatments, that is, control, 2, 10.6, and 38.7 mg/kg, were selected for gene expression studies in a balanced interwoven loop design microarray experiment in which the expression of 311 transcripts was significantly affected. Reverse-transcription quantitative polymerase chain reaction (RT-QPCR) data on 68 selected differentially and nondifferentially expressed transcripts showed a significant correlation with microarray results. The expression of genes involved in multiple biological processes was altered, including muscle contraction, neuronal signaling and growth, ubiquitinylation, fibrinolysis and coagulation, iron and calcium homeostasis, oxygen transport, and immunity. Chitinase activity assays confirmed down-regulation of chitinase genes as indicated by array and RT-QPCR data. An acute toxicity test provided evidence that dermal contact with TNT can cause bleeding, inflammation, and constriction, which may be explained by gene expression results. Sublethal doses of TNT affected the nervous system, caused blood disorders similar to methemoglobinemia, and weakened immunity in E. fetida.