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A protocol that couples poly(A) polymerase with a multifunctional reverse transcriptase (RT) for targeted cloning of environmental prokaryotic cDNA is described. The reverse transcriptase PCR (RT-PCR) cDNA synthesis kit demonstrates the feasibility of directly accessing prokaryote RNA (rRNA and/or mRNA) in environmental samples, but the results also show potentially important problems.

Multiple soil samples were obtained from a nonradioactive model low-level-waste pit at the Idaho National Laboratory to understand the effect of the cellulosic waste products on the soil microbial community. The obtained results suggested that the presence of the cellulosic material significantly influenced the bacterial community structure in a stratified soil system.

Novel arsenite [As(III)] oxidase structural genes (aoxAB) are cloned from Hydrogenobaculum bacteria isolated from an acidic geothermal spring. Reverse transcriptase PCR has shown expression throughout the outflow channel and the aoxB cDNA clones have displayed distribution patterns relative to the physicochemical gradients in the spring.

The relative importance of [H.sub.2] and [H.sub.2]S as energy sources for supporting chemolothotrophic metabolism in an acidic geothermal spring in Yellowstone National Park is assessed. The studies have shown that phylogeny might not be reliable for predicting physiology and that [H.sub.2]S can dominate over [H.sub.2] as an energy source in terms of availability, apparent in situ consumption rates and growth-supporting energy.

As(III) oxidation in Dragon Spring is reexamined in order to assess the potential for As(III) chemolithotrophs to inhabit the geothermal feature. The findings have shown that [H.sub.2]S is a potent inhibitor of As(III) oxidation and will affect the opportunities and population distribution of As(III) chemolithotrophs.

An arsenite-oxidizing Hydrogenobaculum strain is isolated from a geothermal spring that was previously shown to contain microbial populations engaged in arsenite oxidation. The isolate is sensitive to both arsenite and arsenate and behaves as an obligate chemolithoautograph that uses H2 as its sole energy source and has an optimum temperature of 55 to 60 degrees and an optimum pH of 3.0.

Geothermal waters contain numerous potential electron donors capable of supporting chemolithotrophy-based primary production. Thermodynamic predictions of energy yields for specific electron donor and acceptor pairs in such systems are available, although direct assessments of these predictions are rare. This study assessed the relative importance of dissolved H2 and H2S as energy sources for the support of chemolithotrophic metabolism in an acidic geothermal spring in Yellowstone National Park. H2S and H2 concentration gradients were observed in the outflow channel, and vertical H2S and °2 gradients were evident within the microbial mat. H2S levels and microbial consumption rates were approximately three orders of magnitude greater than those of H2. Hydrogenobaculum-like organisms dominated the bacterial component of the microbial community, and isolates representing three distinct 16S rRNA gene phylotypes (phylotype = 100% identity) were isolated and characterized. Within a phylotype, °2 requirements varied, as did energy source utilization: some isolates could grow only with H2S, some only with H2, while others could utilize either as an energy source. These metabolic phenotypes were consistent with in situ geochemical conditions measured using aqueous chemical analysis and in-field measurements made by using gas chromatography and microelectrodes. Pure-culture experiments with an isolate that could utilize H2S and H2 and that represented the dominant phylotype (70% of the PCR clones) showed that H2S and H2 were used simultaneously, without evidence of induction or catabolite repression, and at relative rate differences comparable to those measured in ex situ field assays. Under in situ-relevant concentrations, growth of this isolate with H2S was better than that with H2. The major conclusions drawn from this study are that phylogeny may not necessarily be reliable for predicting physiology and that H2S can dominate over H2 as an energy source in terms of availability, apparent in situ consumption rates, and growth-supporting energy. [ABSTRACT FROM AUTHOR]