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Pentachlorophenol-4-monooxygenase is an aromatic flavoprotein monooxygenase which hydroxylates pentachlorophenol and a wide range of polyhalogenated phenols at their para position. The PCP-degrading Sphingomonas species UG30 was recently shown to mineralize p-nitrophenol. In this study, the UG30 pcpB gene encoding the pentachlorophenol-4-monooxygenase gene was cloned for use to study its potential role in p-nitrophenol degradation. The UG30 pcpB gene consists of 1614 bp with a predicted translational product of 538 amino acids and a molecular mass of 59,933 Da. The primary sequence of pentachlorophenol-4-monooxygenase contained a highly conserved FAD binding site at its N-terminus associated with a beta alpha beta fold. UG30 has been shown previously to convert p-nitrophenol to 4-nitrocatechol. We observed that pentachlorophenol-4-monooxygenase catalyzed the hydroxylation of 4-nitrocatechol to 1,2,4-benzenetriol. About 31.2% of the nitro substituent of 4-nitrocatechol (initial concentration of 200 microM) was cleaved to yield nitrite over 2 h, indicating that the enzyme may be involved in the second step of p-nitrophenol degradation. The enzyme also hydroxylated p-nitrophenol at the para position, but only to a very slight extent. Our results confirm that pentachlorophenol-4-monooxygenase is not the primary enzyme in the initial step of p-nitrophenol metabolism by UG30.

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An in-well sediment incubator (ISI) was developed to investigate the stability and dynamics of sediment-associated microbial communities to prevailing subsurface oxidizing or reducing conditions. Herein we describe the use of these devices at the Old Rifle Uranium Mill Tailings Remedial Action (UMTRA) site. During a seven-month period in which oxidized Rifle Aquifer background sediment (RABS) were deployed in previously biostimulated wells under ironreducing conditions, cell densities of known iron-reducing bacteria, including Geobacteraceae, increased significantly, showing the microbial community response to local subsurface conditions. Phospholipid fatty acid (PLFA) profiles of RABS following in situ deployment were strikingly similar to those of adjacent sediment cores, suggesting ISI results could be extrapolated to the native material of the test plots. Results for ISI deployment with laboratory-reduced sediments showed only slight changes in community composition and pointed toward the ability of the ISI to monitor microbial community stability and response to subsurface conditions. INTRODUCTIONEnhanced bioremediation, whether for petroleum hydrocarbons, chlorinated solvents, or radionuclides, frequently involves subsurface injection of an amendment (electron acceptor or donor) to stimulate microbial activity. Following injection, temporal groundwater monitoring programs are commonly instituted to track contaminant concentrations, geochemical parameters, and changes in microbial community composition. Although groundwater monitoring can provide valuable insight into biological processes, important microbial populations, and contaminant transformations may be strongly associated with the subsurface matrix (Bekins et al., 1999;Hazen et al., 1991;Thomas et al., 1998) as opposed to the planktonic communities. Collection of sediment cores allows interrogation of sediment-associated transformations. However, repeated drilling events are expensive and not always practical, especially in deeper aquifer systems. To combat this problem, a number of researchers have employed in situ microcosms in which native sediment or a sediment surrogate is deployed and recovered from existing monitoring wells (Bennett et al., 2000;Hendricks et al., 2005;Reardon et al., 2004). The in situ microcosm approach, however, is potentially limited by the extent to which the results relate to the native sediment and the aquifer as a whole (Bennett et al., 2000). Therefore, the cornerstone of the in-well sediment incubator approach is that analysis of recovered microcosms provides interpretable results consistent with sediment core samples. In aerobic aquifers typical of many Department of Energy (DOE) legacy waste sites, uranium is present in the oxidized U(VI) form, which is more soluble and, thus, more mobile (Anderson et al., 2003;Istok et al., 2004;Wall & Krumholz, 2006). A wide variety of dissimilatory metal-reducing bacteria, most notably of the genera Geobacter and Shewanella, have been shown to enzymatically reduce U(VI) to less ...

To search for extraterrestrial life surrogate extreme environments on Earth have been chosen for investigation. An example of a surrogate site is the Canadian subpermafrost. Investigations into microbial communities occurred by access fracture borehole water in the Lupin gold mine, and drill rock cores and drilling waters in the High Lake region of Nunavut, Canada. Membrane lipid analyses uses GC/MS and HPLC/ES/MS/MS to provide estimates of biomass, phospholipid (PLFA) and respiratory quinone composition, and compositional changes related to membrane stress caused by nutritional limitations or exposure to toxic conditions. Lupin fracture borehole waters were collected from 800 to 1200 meters, while the High Lake rock cores were collected from 335 to 535 meters. Biomass estimates based on PLFA ranged from 0.25 to 22 pmol L -1 for the Lupin waters. High Lake drill waters had biomass that ranged from below detection limits (bdl) to 595 pmol/ml, while rock core samples had biomass estimates ranging from bdl to 32 pmol g -1 . PLFA profiles revealed the presence of both Gram +/-bacteria and sulfatereducing bacteria. Specific PLFA ratios indicate that the bacterial communities were physiologically stressed. Menaquinones were the most abundant but varied in the dominant isoprene units between the two sites. Ubiquinone to menaquinone ratio indicated that these samples have been anoxic for a long time. Methods to detect life signatures at surrogate sites on Earth will be critical for assessing extraterrestrial life. Currently, the membrane lipid analyses provide additional information not easily provided by other molecular techniques.