Metagenomic analysis of two enhanced biological phosphorus removal (EBPR) sludge communities.

Enhanced biological phosphorus removal (EBPR) is one of the best-studied microbially mediated industrial processes because of its ecological and economic relevance. Despite this, it is not well understood at the metabolic level. Here we present a metagenomic analysis of two lab-scale EBPR sludges dominated by the uncultured bacterium, "Candidatus Accumulibacter phosphatis." The analysis sheds light on several controversies in EBPR metabolic models and provides hypotheses explaining the dominance of A. phosphatis in this habitat, its lifestyle outside EBPR and probable cultivation requirements. Comparison of the same species from different EBPR sludges highlights recent evolutionary dynamics in the A. phosphatis genome that could be linked to mechanisms for environmental adaptation. In spite of an apparent lack of phylogenetic overlap in the flanking communities of the two sludges studied, common functional themes were found, at least one of them complementary to the inferred metabolism of the dominant organism. The present study provides a much needed blueprint for a systems-level understanding of EBPR and illustrates that metagenomics enables detailed, often novel, insights into even well-studied biological systems.

Biome-level biogeography of streambed microbiota.

A field study was conducted to determine the microbial community structures of streambed sediments across diverse geographic and climatic areas. Sediment samples were collected from three adjacent headwater forest streams within three biomes, eastern deciduous (Pennsylvania), southeastern coniferous (New Jersey), and tropical evergreen (Guanacaste, Costa Rica), to assess whether there is biome control of stream microbial community structure. Bacterial abundance, microbial biomass, and bacterial and microbial community structures were determined using classical, biochemical, and molecular methods. Microbial biomass, determined using phospholipid phosphate, was significantly greater in the southeastern coniferous biome, likely due to the smaller grain size, higher organic content, and lower levels of physical disturbance of these sediments. Microbial community structure was determined using phospholipid fatty acid (PLFA) profiles and bacterial community structure from terminal restriction fragment length polymorphism and edited (microeukaryotic PLFAs removed) PLFA profiles. Principal component analysis (PCA) was used to investigate patterns in total microbial community structure. The first principal component separated streams based on the importance of phototrophic microeukaryotes within the community, while the second separated southeastern coniferous streams from all others based on increased abundance of fungal PLFAs. PCA also indicated that within- and among-stream variations were small for tropical evergreen streams and large for southeastern coniferous streams. A similar analysis of bacterial community structure indicated that streams within biomes had similar community structures, while each biome possessed a unique streambed community, indicating strong within-biome control of stream bacterial community structure.

Limitations of the widely used GAM42a and BET42a probes targeting bacteria in the Gammaproteobacteria radiation.

The 23S rRNA-targeted probes GAM42a and BET42a provided equivocal results with the uncultured gammaproteobacterium 'Candidatus Competibacter phosphatis' where some cells bound GAM42a and other cells bound BET42a in fluorescence in situ hybridization (FISH) experiments. Probes GAM42a and BET42a span positions 1027-1043 in the 23S rRNA and differ from each other by one nucleotide at position 1033. Clone libraries were prepared from PCR products spanning the 16S rRNA genes, intergenic spacer region and 23S rRNA genes from two mixed cultures enriched in 'Candidatus C. phosphatis'. With individual clone inserts, the 16S rDNA portion was used to confirm the source organism as 'Candidatus C. phosphatis' and the 23S rDNA portion was used to determine the sequence of the GAM42a/BET42a probe target region. Of the 19 clones sequenced, 8 had the GAM42a probe target (T at position 1033) and 11 had G at position 1033, the only mismatch with GAM42a. However, none of the clones had the BET42a probe target (A at 1033). Non-canonical base-pairing between the 23S rRNA of 'Candidatus C. phosphatis' with G at position 1033 and GAM42a (G-A) or BET42a (G-T) is likely to explain the probing anomalies. A probe (GAM42_C1033) was optimized for use in FISH, targeting cells with G at position 1033, and was found to highlight not only some 'Candidatus C. phosphatis' cells, but also other bacteria. This demonstrates that there are bacteria in addition to 'Candidatus C. phosphatis' with the GAM42_C1033 probe target and not the BET42a or GAM42a probe target.