Transmissible spongiform encephalopathies (TSEs, prion diseases) are a class of fatal neurodegenerative diseases affecting a variety of mammalian species including humans. A misfolded form of the prion protein (PrPTSE) is the major, if not sole, component of the infectious agent. Prions are highly resistant to degradation and to many disinfection procedures suggesting that, if prions enter wastewater treatment systems through sewers and/or septic systems (e.g., slaughterhouses, necropsy laboratories, rural meat processors, private game dressing) or through leachate from landfills that have received TSE-contaminated material, prions could survive conventional wastewater treatment. Here, we report the results of experiments examining the partitioning and persistence of PrPTSE during simulated wastewater treatment processes including activated and mesophilic anaerobic sludge digestion. Incubation with activated sludge did not result in significant PrPTSE degradation. PrPTSE and prion infectivity partitioned strongly to activated sludge solids and are expected to enter biosolids treatment processes. A large fraction of PrPTSE survived simulated mesophilic anaerobic sludge digestion. The small reduction in recoverable PrPTSE after 10-d anaerobic sludge digestion appeared attributable to a combination of declining extractability with time and microbial degradation. Our results suggest that if prions were to enter municipal wastewater treatment systems, most of the agent would partition to activated sludge solids, survive mesophilic anaerobic digestion, and be present in treated biosolids.

RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, quantitation of viral RNAs can predict disease progression and therapeutic efficacy. Likewise, gene expression analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmacological agents. As the focus of the Human Genome Project moves toward gene expression analysis, the field will require a flexible RNA analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs (refs 3,4). We have applied the principles of invasive cleavage and engineered an improved 5'-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)-based signal amplification method for detecting RNA in both total RNA and cell lysate samples. This detection format, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional enzymatic signal enhancement. In this report, we describe the assay and present data demonstrating its capabilities for sensitive (<100 copies per reaction), specific (discrimination of 95% homologous sequences, 1 in > or =20,000), and quantitative (1.2-fold changes in RNA levels) detection of unamplified RNA in both single- and biplex-reaction formats.

Escherichia coli was engineered for the production of even-and odd-chain fatty acids (FAs) by fermentation. Co-production of thiolase, hydroxybutyryl-CoA dehydrogenase, crotonase and trans-enoyl-CoA reductase from a synthetic operon allowed the production of butyrate, hexanoate and octanoate. Elimination of native fermentation pathways by genetic deletion (DldhA, DadhE, DackA, Dpta, DfrdC) helped eliminate undesired by-products and increase product yields. Initial butyrate production rates were high (0.7 g l "1 h "1 ) but quickly levelled off and further study suggested this was due to product toxicity and/or acidification of the growth medium. Results also showed that endogenous thioesterases significantly influenced product formation. In particular, deletion of the yciA thioesterase gene substantially increased hexanoate production while decreasing the production of butyrate. E. coli was also engineered to co-produce enzymes for even-chain FA production (described above) together with a coenzyme B 12 -dependent pathway for the production of propionyl-CoA, which allowed the production of odd-chain FAs (pentanoate and heptanoate). The B 12 -dependent pathway used here has the potential to allow the production of odd-chain FAs from a single growth substrate (glucose) in a more energy-efficient manner than the prior methods.

Species-specific sequences were identified within the V4 variable region of 16S rRNA of two bacterial species capable of aromatic hydrocarbon metabolism, Pseudomonas putida F1 andBurkholderia sp. strain JS150, and a third, Bacillus subtilis ATCC 7003, that can function as a secondary degrader. Fluorescent in situ hybridization (FISH) with species-specific oligonucleotides was used for direct counting of these species throughout a phenol biodegradation experiment in batch culture. Traditional differential plate counting methods could not be used due to the similar metabolism and interactions of the primary degraders and difficulties in selecting secondary degraders in mixed culture. In contrast, the FISH method provided reliable quantitative results without interference from those factors.

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