The role of the cell envelope in the solvent tolerance mechanisms of Pseudomonas putida was investigated. The responses of a solvent-tolerant strain, P. putida Idaho, and a solvent-sensitive strain, P. putida MW1200, were examined in terms of phospholipid content and composition and of phospholipid biosynthetic rate following exposure to a nonmetabolizable solvent, o-xylene. Following o-xylene exposure, P. putida MW1200 exhibited a decrease in total phospholipid content. In contrast, P. putida Idaho demonstrated an increase in phospholipid content 1 to 6 h after exposure. Analysis of phospholipid biosynthesis showed P. putida Idaho to have a higher basal rate of phospholipid synthesis than MW1200. This rate increased significantly following exposure to xylene. Both strains showed little significant turnover of phospholipid in the absence of xylene. In the presence of xylene, both strains showed increased phospholipid turnover. The rate of turnover was significantly greater in P. putida Idaho than in P. putida MW1200. These results suggest that P. putida Idaho has a greater ability than the solvent-sensitive strain MW1200 to repair damaged membranes through efficient turnover and increased phospholipid biosynthesis.

Solvent-tolerant and-sensitive Pseudomonas putida strains were studied to determine their cell envelope changes following exposure to o-xylene. Both strains produced trans-unsaturated fatty acids. The tolerant strain showed an increase in total fatty acids, an increase in saturated fatty acids, and modified lipopolysaccharide. It is suggested that these envelope modifications aid in survival at high concentrations of organic solvents.

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The microbial community diversity and composition of meromictic Soap Lake were studied using culturedependent and culture-independent approaches. The water column and sediments were sampled monthly for a year. Denaturing gradient gel electrophoresis of bacterial and archaeal 16S rRNA genes showed an increase in diversity with depth for both groups. Late-summer samples harbored the highest prokaryotic diversity, and the bacteria exhibited less seasonal variability than the archaea. Most-probable-number assays targeting anaerobic microbial guilds were performed to compare summer and fall samples. In both seasons, the anoxic samples appeared to be dominated by lactate-oxidizing sulfate-reducing prokaryotes. High numbers of lactate-and acetate-oxidizing ironreducing bacteria, as well as fermentative microorganisms, were also found, whereas the numbers of methanogens were low or methanogens were undetectable. The bacterial community composition of summer and fall samples was also assessed by constructing 16S rRNA gene clone libraries. A total of 508 sequences represented an estimated >1,100 unique operational taxonomic units, most of which were from the monimolimnion, and the summer samples were more diverse than the fall samples (Chao1 ‫؍‬ 530 and Chao1 ‫؍‬ 295, respectively). For both seasons, the mixolimnion sequences were dominated by Gammaproteobacteria, and the chemocline and monimolimnion libraries were dominated by members of the low-G؉C-content group, followed by the Cytophaga-Flexibacter-Bacteroides (CFB) group; the mixolimnion sediments contained sequences related to uncultured members of the Chloroflexi and the CFB group. Community overlap and phylogenetic analyses, however, not only demonstrated that there was a high degree of spatial turnover but also suggested that there was a degree of temporal variability due to differences in the members and structures of the communities.

The potential for aerobic methyl tert-butyl ether (MTBE) degradation was investigated with microcosms containing aquifer sediment and groundwater from four MTBE-contaminated sites characterized by oxygenlimited in situ conditions. MTBE depletion was observed for sediments from two sites (e.g., 4.5 mg/liter degraded in 15 days after a 4-day lag period), whereas no consumption of MTBE was observed for sediments from the other sites after 75 days. For sediments in which MTBE was consumed, 43 to 54% of added [U-14 C]MTBE was mineralized to 14 CO 2 . Molecular phylogenetic analyses of these sediments indicated the enrichment of species closely related to a known MTBE-degrading bacterium, strain PM1. At only one site, the presence of water-soluble gasoline components significantly inhibited MTBE degradation and led to a more pronounced accumulation of the metabolite tert-butyl alcohol. Overall, these results suggest that the effects of oxygen and water-soluble gasoline components on in situ MTBE degradation will vary from site to site and that phylogenetic analysis may be a promising predictor of MTBE biodegradation potential.The magnitude and remediation cost of methyl tert-butyl ether (MTBE) contamination in drinking water have rapidly become a national concern. It has been estimated that 250,000 of the approximately 385,000 confirmed leaking underground storage tank (LUST) releases in the United States involve MTBE (15). In California, at least 10,000 LUST sites are estimated to be contaminated with MTBE (13). Several states, including California, have set primary maximum concentration levels for MTBE at or below 20 g/liter, and at an even lower level of 12 g/liter for tert-butyl alcohol (TBA), an MTBE metabolite. The U.S. Environmental Protection Agency has listed MTBE as a possible human carcinogen, whereas TBA is a known animal carcinogen (7). MTBE appears to be more mobile and less biodegradable than BTEX compounds (benzene, toluene, ethylbenzene, and xylenes), and consequently, MTBE plumes have extended over kilometer-scale distances, as is the case at Port Hueneme, Calif., and East Patchogue, N.Y.Previous microcosm studies reported little or no biodegradation of MTBE under a variety of aerobic (11,14) and anaerobic (18,23,26) conditions. More recent microcosm (3) and column (6) studies suggest that limited intrinsic biodegradation of MTBE may occur. One research group observed MTBE mineralization activity in stream-bed sediments from both contaminated and pristine sites under aerobic conditions (4,5). Mixed cultures capable of MTBE degradation have been isolated from activated sludge (10,20). Pure bacterial cultures capable of MTBE metabolism have been reported (12,17,22), including strain PM1, which uses MTBE as a sole carbon source and electron donor (12), and propane-oxidizing strains that cometabolize MTBE (22). In microcosm and field experiments, Salanitro et al. (21) showed that oxygenation in combination with bioaugmentation with an MTBE-degrading consortium resulted in more rapid MTBE degradation, alt...