N 2 fixation by diazotrophic bacteria associated with the roots of the smooth cordgrass, Spartina alterniflora, is an important source of new nitrogen in many salt marsh ecosystems. However, the diversity and phylogenetic affiliations of these rhizosphere diazotrophs are unknown. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified nifH sequence segments was used in previous studies to examine the stability and dynamics of the Spartina rhizosphere diazotroph assemblages in the North Inlet salt marsh, near Georgetown, S.C. In this study, plugs were taken from gel bands from representative DGGE gels, the nifH amplimers were recovered and cloned, and their sequences were determined. A total of 59 sequences were recovered, and the amino acid sequences predicted from them were aligned with sequences from known and unknown diazotrophs in order to determine the types of organisms present in the Spartina rhizosphere. We recovered numerous sequences from diazotrophs in the ␥ subdivision of the division Proteobacteria (␥-Proteobacteria) and from various anaerobic diazotrophs. Diazotrophs in the ␣-Proteobacteria were poorly represented. None of the Spartina rhizosphere DGGE band sequences were identical to any known or previously recovered environmental nifH sequences. The Spartina rhizosphere diazotroph assemblage is very diverse and apparently consists mainly of unknown organisms.

Despite the ubiquity of ammonium in geothermal environments and the thermodynamic favorability of aerobic ammonia oxidation, thermophilic ammonia-oxidizing microorganisms belonging to the crenarchaeota kingdom have only recently been described. In this study, we analyzed microbial mats and surface sediments from 21 hot spring samples (pH 3.4 to 9.0; temperature, 41 to 86°C) from the United States, China, and Russia and obtained 846 putative archaeal ammonia monooxygenase large-subunit (amoA) gene and transcript sequences, representing a total of 41 amoA operational taxonomic units (OTUs) at 2% identity. The amoA gene sequences were highly diverse, yet they clustered within two major clades of archaeal amoA sequences known from water columns, sediments, and soils: clusters A and B. Eighty-four percent (711/846) of the sequences belonged to cluster A, which is typically found in water columns and sediments, whereas 16% (135/846) belonged to cluster B, which is typically found in soils and sediments. Although a few amoA OTUs were present in several geothermal regions, most were specific to a single region. In addition, cluster A amoA genes formed geographic groups, while cluster B sequences did not group geographically. With the exception of only one hot spring, principal-component analysis and UPGMA (unweighted-pair group method using average linkages) based on the UniFrac metric derived from cluster A grouped the springs by location, regardless of temperature or bulk water pH, suggesting that geography may play a role in structuring communities of putative ammoniaoxidizing archaea (AOA). The amoA genes were distinct from those of low-temperature environments; in particular, pair-wise comparisons between hot spring amoA genes and those from sympatric soils showed less than 85% sequence identity, underscoring the distinctness of hot spring archaeal communities from those of the surrounding soil system. Reverse transcription-PCR showed that amoA genes were transcribed in situ in one spring and the transcripts were closely related to the amoA genes amplified from the same spring. Our study demonstrates the global occurrence of putative archaeal amoA genes in a wide variety of terrestrial hot springs and suggests that geography may play an important role in selecting different assemblages of AOA.

Pseudomonas putida strain AJ and Ochrobactrum strain TD were isolated from hazardous waste sites based on their ability to use vinyl chloride (VC) as the sole source of carbon and energy under aerobic conditions. Strains AJ and TD also use ethene and ethylene oxide as growth substrates. Strain AJ contained a linear megaplasmid (approximately 260 kb) when grown on VC or ethene, but it contained no circular plasmids. While strain AJ was growing on ethylene oxide, it was observed to contain a 100-kb linear plasmid, and its ability to use VC as a substrate was retained. The linear plasmids in strain AJ were cured, and the ability of strain AJ to consume VC, ethene, and ethylene oxide was lost following growth on a rich substrate (LuriaBertani broth) through at least three transfers. Strain TD contained three linear plasmids, ranging in size from approximately 90 kb to 320 kb, when growing on VC or ethene. As with strain AJ, the linear plasmids in strain TD were cured following growth on Luria-Bertani broth and its ability to consume VC and ethene was lost. Further analysis of these linear plasmids may help reveal the pathway for VC biodegradation in strains AJ and TD and explain why this process occurs at many but not all sites where groundwater is contaminated with chloroethenes. Metabolism of VC and ethene by strains AJ and TD is initiated by an alkene monooxygenase. Their yields during growth on VC (0.15 to 0.20 mg of total suspended solids per mg of VC) are similar to the yields reported for other isolates (i.e., Mycobacterium sp., Nocardioides sp., and Pseudomonas sp.).Millions of tons of vinyl chloride (VC) are produced each year, primarily for the manufacture of polyvinyl chloride (25). However, the occurrence of VC in groundwater is typically not a consequence of direct releases to the environment. VC contamination of groundwater results mainly from the transformation of other chlorinated aliphatic compounds, including the reductive dechlorination of polychlorinated ethenes and the dehydrohalogenation of 1,2-dichloroethane (41). It has recently been demonstrated that VC is also formed naturally in soils, presumptively during oxidative reactions involving humic substances, chloride ions, and an oxidant (23). This process may have started as long as 400 million years ago (23), so it seems reasonable to expect that biodegradation processes also developed long ago.Reduction of VC to ethene is typically the rate-limiting step in the overall reduction of chlorinated ethenes, which can lead to the accumulation of VC in groundwater (13, 31). The comparatively low rate of VC reduction may be related to this reaction being cometabolic in some strains of Dehalococcoides, although other strains have recently been shown to be capable of respiring with VC (9, 19). Oxidative acetogenesis of VC has also been documented for anaerobic sediments (2), although the extent of this process at most locations is not yet known.In locations where anaerobic groundwater transitions to aerobic conditions, VC that migrates from the anaero...

DNA was extracted from dry standing dead Spartina alterniflora stalks as well as dry Spartina wrack from the North Inlet (South Carolina) and Sapelo Island (Georgia) salt marshes. Partial nifH sequences were PCR amplified, the products were separated by denaturing gradient gel electrophoresis (DGGE), and the prominent DGGE bands were sequenced. Most sequences (109 of 121) clustered with those from ␣-Proteobacteria, and 4 were very similar (>99%) to that of Azospirillum brasilense. Seven sequences clustered with those from known ␥-Proteobacteria and five with those from known anaerobic diazotrophs. The diazotroph assemblages associated with dead Spartina biomass in these two salt marshes were very similar, and relatively few major lineages were represented.

The detection and identification of microorganisms in natural communities is a great challenge to biologists. Microarray-based genomic technology provides a promising high-throughput alternative to traditional microbial characterization. A novel prototype microarray containing whole genomic DNA, termed community genome array (CGA), was constructed and evaluated. Microarray hybridizations at 55 degrees C using 50% formamide permitted the examined bacteria to be distinguished at the species level, while strain-level differentiation was obtained at hybridization temperatures of 65 or 75 degrees C. The detection limit was estimated to be approximately 0.2 ng with genomic DNA from a single pure culture using a reduced hybridization volume (3 microL). Using mixtures of known amounts of DNA or a known number of cells from 14 or 16 different species, respectively, about 5 ng of genomic DNA or 2.5 x 10(5) cells were detected under the hybridization conditions used. In addition, strong linear relationships were observed between hybridization signal intensity and target DNA concentrations for pure cultures, a mixture of DNA templates, and a population of mixed cells (r2 = 0.95-0.98, P < 0.01). Finally, the prototype CGA revealed differences in microbial community composition in soil, river, and marine sediments. The results suggest that CGA hybridization has potential as a specific, sensitive, and quantitative tool for detection and identification of microorganisms in environmental samples.