DNA from low-biodiversity fracture water collected at 2.8-kilometer depth in a South African gold mine was sequenced and assembled into a single, complete genome. This bacterium, Candidatus Desulforudis audaxviator , composes >99.9% of the microorganisms inhabiting the fluid phase of this particular fracture. Its genome indicates a motile, sporulating, sulfate-reducing, chemoautotrophic thermophile that can fix its own nitrogen and carbon by using machinery shared with archaea. Candidatus Desulforudis audaxviator is capable of an independent life-style well suited to long-term isolation from the photosphere deep within Earth's crust and offers an example of a natural ecosystem that appears to have its biological component entirely encoded within a single genome.

We present a genome-scale metabolic model for the archaeal methanogen Methanosarcina barkeri. We characterize the metabolic network and compare it to reconstructions from the prokaryotic, eukaryotic and archaeal domains. Using the model in conjunction with constraint-based methods, we simulate the metabolic fluxes and resulting phenotypes induced by different environmental and genetic conditions. This represents the first large-scale simulation of either a methanogen or an archaeal species. Model predictions are validated by comparison to experimental growth measurements and phenotypes of M. barkeri on different substrates. The predicted growth phenotypes for wild type and mutants of the methanogenic pathway have a high level of agreement with experimental findings. We further examine the efficiency of the energy-conserving reactions in the methanogenic pathway, specifically the Ech hydrogenase reaction, and determine a stoichiometry for the nitrogenase reaction. This work demonstrates that a reconstructed metabolic network can serve as an analysis platform to predict cellular phenotypes, characterize methanogenic growth, improve the genome annotation and further uncover the metabolic characteristics of methanogenesis.

We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCRamplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 g of total RNA, representing approximately 7.5 ؋ 10 6

Sediments from a high-level nuclear waste plume were collected as part of investigations to evaluate the potential fate and migration of contaminants in the subsurface. The plume originated from a leak that occurred in 1962 from a waste tank consisting of high concentrations of alkali, nitrate, aluminate, Cr(VI), 137 Cs, and 99 Tc. Investigations were initiated to determine the distribution of viable microorganisms in the vadose sediment samples, probe the phylogeny of cultivated and uncultivated members, and evaluate the ability of the cultivated organisms to survive acute doses of ionizing radiation. The populations of viable aerobic heterotrophic bacteria were generally low, from below detection to ϳ10 4 CFU g ؊1 , but viable microorganisms were recovered from 11 of 16 samples, including several of the most radioactive ones (e.g., >10 Ci of 137 Cs/g). The isolates from the contaminated sediments and clone libraries from sediment DNA extracts were dominated by members related to known gram-positive bacteria. Gram-positive bacteria most closely related to Arthrobacter species were the most common isolates among all samples, but other phyla high in G؉C content were also represented, including Rhodococcus and Nocardia. Two isolates from the second-most radioactive sample (>20 Ci of 137 Cs g ؊1 ) were closely related to Deinococcus radiodurans and were able to survive acute doses of ionizing radiation approaching 20 kGy. Many of the gram-positive isolates were resistant to lower levels of gamma radiation. These results demonstrate that gram-positive bacteria, predominantly from phyla high in G؉C content, are indigenous to Hanford vadose sediments and that some are effective at surviving the extreme physical and chemical stress associated with radioactive waste.As a result of World War II and the subsequent Cold War, a large nuclear complex was developed in the United States, including large land tracts in Nevada, Idaho, and Washington state. Over a 40-year period, approximately 104 metric tons of plutonium was extracted from irradiated uranium at various sites within this complex. The result of the fuel chemical reprocessing at the Hanford Site, near Richland, Washington, and the Savannah River Site, near Aiken, South Carolina, was an accumulation of approximately 90 million gallons of highlevel radioactive waste (HLW

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Total DNA from sediment samples was isolated by a direct lysis technique. Purified DNA was used as template either undiluted or diluted 1:10 prior to polymerase chain reaction (PCR) amplification of 16S rRNA genes. Full-length inserts were analysed for restriction fragment length polymorphisms (RFLP) with the enzyme Cfo1, and the resulting distribution and abundance of RFLP patterns compared between the undiluted and diluted PCR reactions. Results indicate that for low PCR template concentrations, in the range from a few picograms to tens of picograms DNA, proportional representation of specific RFLP types was not reproducible upon template dilution, confirming that PCR amplification of 16S rDNA cannot be used directly to infer microbial abundance. In particular, only 15-24% of the RFLP types recovered from a sample were present in both the undiluted and diluted extracts. We propose that very low template concentrations in the PCR generate random fluctuations in priming efficiency, which led to the contrast in RFLP types observed in the libraries from the undiluted and diluted extracts.

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Nitrilotriacetate (NTA), a synthetic chelating agent, has been used for various radionuclide processing and decontamination procedures. NTA has been codisposed with radionuclides and heavy metals to soils and subsurface sediments (4, 27, 32), where it can greatly increase the mobility of these metals in the environment by forming soluble complexes with them. Microbial degradation of NTA may ultimately aid in immobilizing these radionuclides. Several NTA-degrading bacteria have been isolated (2,7,13,18,23,42). An NTA monooxygenase (NTA-Mo) of Chelatobacter heintzii ATCC 29600 has been identified (16) (47). NTA oxidation is proposed to be catalyzed by a heterodimer of components A (cA) and B (cB), but it is unclear how these two components interact with each other or what the function of each component is (44). In order to provide additional information on the function of cA and cB and to facilitate understanding natural attenuation or engineered bioremediation of NTA in the environment through the use of specific PCR primers or gene probes, we cloned, sequenced, and analyzed a gene cluster involved in NTA degradation. On the basis of DNA sequence and activity analysis, the two components were shown to be two separate enzymes, an monooxygenase that oxidized NTA at the expense of reduced flavin mononucleotide (FMNH 2 ) and O 2 and an NADH:flavin mononucleotide (FMN) oxidoreductase that uses NADH to reduce FMN to FMNH 2 .(A preliminary account of this work was presented at the 1995 American Society for Microbiology General Meeting [46], and the DNA sequence and gene organization have been published in a master's degree thesis [46]). MATERIALS AND METHODSBacterial strains and plasmids. The plasmids used or constructed in this study are listed in Table 1. C. heintzii ATCC 29600 was obtained from the American Type Culture Collection (Rockville, Md.) and was cultured with using a mineral salt medium with NTA as the sole carbon source (42). Escherichia coli HB101 was used as the host for pRK311, strain DH5␣ was used as the host for pBS, and strain JM105 was used as the host for pTrc99A. E. coli strains were routinely grown at 37ЊC in Luria-Bertani (LB) medium or on LB agar (37). Tetracycline and ampicillin (Sigma, St. Louis, Mo.) were used at 25 and 100 g/ml, respectively.Gene cloning. The two components of NTA-Mo were purified according to the method of Uetz et al. (44), subjected to discontinuous sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (24), and then electroblotted onto a polyvinylidene difluoride membrane (28, 31) for N-terminal amino acid sequence analysis on an ABI 470 protein sequencer at the Department of Biochemistry and Biophysics, Washington State University.Oligonucleotides were 5Ј end labeled with 32 P by polynucleotide kinase (37). C. heintzii ATCC 29600 genomic DNA was isolated by a combination of largescale CsCl gradient preparation and hexadecyltrimethyl ammonium bromide and phenol extraction (3, 37). The PolarPlex Chemiluminescent Kit of Millipore (Bedford, Mass.) was used to ...