Small subunit (16S) rRNA gene surveys generating near full-length 16S rRNA clones offer a unique opportunity for in-depth phylogenetic analysis to highlight the breadth of diversity within various major bacterial phyla encountered in soil. This study offers a detailed phylogenetic analysis of the Proteobacteria-affiliated clones identified from 13 001 nearly full-length 16S rRNA gene clones derived from Oklahoma tall-grass prairie soil. Proteobacteria was the most abundant phylum in the community, and comprised 25% of the total clones. The most abundant and diverse class within the Proteobacteria was Alphaproteobacteria, followed by the Delta-, Beta-and Gammaproteobacteria. Members of the Epsilon-and Zetaproteobacteria were not detected in the dataset. Our analysis identified 15 novel order-level and 48 novel family-level Proteobacteria lineages. In addition, we show that the majority of Proteobacteria clones in the dataset belong to orders and families containing no described cultivated representatives (50% and 65%, respectively). An examination of the ecological distribution of the six most abundant Proteobacteria lineages in this dataset with no characterized pure culture representatives provided important information regarding their global distribution and environmental preferences. This level of novel phylogenetic diversity indicates that our understanding of the functions of soil microorganisms, even those belonging to phyla with numerous and diverse well-characterized cultured representatives such as the Proteobacteria, remains far from adequate.

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Immobilization of uranium in groundwater can be achieved through microbial reduction of U(VI) to U(IV) upon electron donor addition. Microbial community structure was analyzed in ethanol-biostimulated and control sediments from a high-nitrate (>130 mM), low-pH, uranium-contaminated site in Oak Ridge, TN. Analysis of small subunit (SSU) rRNA gene clone libraries and polar lipid fatty acids from sediments revealed that biostimulation resulted in a general decrease in bacterial diversity. Specifically, biostimulation resulted in an increase in the proportion of Betaproteobacteria (10% of total clones in the control sediment versus 50 and 79% in biostimulated sediments) and a decrease in the proportion of Gammaproteobacteria and Acidobacteria. Clone libraries derived from dissimilatory nitrite reductase genes (nirK and nirS) were also dominated by clones related to Betaproteobacteria (98% and 85% of total nirK and nirS clones, respectively). Within the nirK libraries, one clone sequence made up 59 and 76% of sequences from biostimulated sediments but only made up 10% of the control nirK library. Phylogenetic analysis of SSU rRNA and nirK gene sequences from denitrifying pure cultures isolated from the site indicate that all belong to a Castellaniella species; nearly identical sequences also constituted the majority of biostimulated SSU rRNA and nirK clone libraries. Thus, by combining culture-independent with culture-dependent techniques, we were able to link SSU rRNA clone library information with nirK sequence data and conclude that a potentially novel Castellaniella species is important for in situ nitrate removal at this site.

Soil bacterial communities typically exhibit a distribution pattern in which most bacterial species are present in low abundance. Due to the relatively small size of most culture-independent sequencing surveys, a detailed phylogenetic analysis of rare members of the community is lacking. To gain access to the rarely sampled soil biosphere, we analyzed a data set of 13,001 near-full-length 16S rRNA gene clones derived from an undisturbed tall grass prairie soil in central Oklahoma. Rare members of the soil bacterial community (empirically defined at two different abundance cutoffs) represented 18.1 to 37.1% of the total number of clones in the data set and were, on average, less similar to their closest relatives in public databases when compared to more abundant members of the community. Detailed phylogenetic analyses indicated that members of the soil rare biosphere either belonged to novel bacterial lineages (members of five novel bacterial phyla identified in the data set, as well as members of multiple novel lineages within previously described phyla or candidate phyla), to lineages that are prevalent in other environments but rarely encountered in soil, or were close relatives to more abundant taxa in the data set. While a fraction of the rare community was closely related to more abundant taxonomic groups in the data set, a significant portion of the rare biosphere represented evolutionarily distinct lineages at various taxonomic cutoffs. We reason that these novelty and uniqueness patterns provide clues regarding the origins and potential ecological roles of members of the soil's rare biosphere.

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