- MICROBIAL DIVERSITY IN COASTAL MARSH ECOSYSTEMS—A high throughput 16S rRNA sequencing/analysis pipeline was developed to characterize spatial and temporal variability in composition and diversity of bacterioplankton and sediment bacteria. Approximately 100-200 species are present in bacterioplankton 16S rRNA gene libraries of Sapelo Island water at any given time (Table 2). Statistical comparisons indicate little spatial variability but significant temporal variability in the bacterioplankton community composition. More than 700 species are present in sediment core libraries from various depths, and over 3000 in the composite library. The diversity of sediment communities is not well represented in 16S rRNA databases. Like the water column, temporal variability is high in sediment bacterial communities.
- AN ENVIRONMENTAL SEQUENCE DATABASE—A model relational database was created for 16S rRNA sequences that also stores ecological metadata (e.g. study site, collection date, macro- and, microenvironment, taxonomic assignment, tidal stage, temperature, methodology; Sheldon et al. 2002). The database serves not only as a sequence repository but also an independent research tool for mining sequences with particular environmental characteristics (habitat, season, salinity zone, depth interval) or taxonomic affiliations.
- AUTOMATED TAXONOMIC ANALYSIS—An automated taxonomic assignment program ("RDPquery") was developed that queries the Ribosomal Database Project (RDP) site and returns a taxonomic assignment for 16S rRNA sequences based on sequence similarity. Any 16S rRNA sequence collection can be analyzed or re-analyzed automatically to keep information current, and the RDPquery results can be incorporated directly into database records. The program has now been converted to a stand-alone Java application for general distribution.
- QUANTITATIVE CLONE LIBRARY ANALYSIS - A method for statistical comparisons was developed of the composition of two or more rRNA gene libraries (Singleton et al. 2001, 2004). Along with the downloadable LIBSHUFF program, an archived web version (WebLIBSHUFF) is available.
- ENVIRONMENTAL TRANSCRIPTOMICS (SIMO AND MLMO COLLABORATION)—A collaborative effort with the Mono Lake Microbial Observatory (MLMO) produced a protocol to directly retrieve mRNA sequences from natural bacterioplankton communities (Poretsky et al., in press). Transcript sequences are indications of what the organisms are doing right now. Transcript sequences retrieved from salt marsh communities have generated new hypotheses about nutrient utilization and provided novel functional sequences for environmental gene expression studies.
- MOLECULAR ANALYSIS OF FLOW CYTOMETRIC SORTED POPULATIONS—Flow cytometric sorting coupled to 16S rRNA analysis is used in SIMO to physically isolate assemblages of bacterioplankon based on their ecological roles. From enrichments or unperturbed natural communities, high-activity cells are separated from low-activity cells based on increases in nucleic acid content or incorporation of the thymidine analog BrdU. DNA is extracted from sorted populations and used in 16S rRNA clone library construction (Mou et al. 2004). This approach provides information on the taxonomic composition of functional assemblages as well as physically separates cells from the bulk community for subsequent molecular analyses.
Copyright on all images, and material by Mary Ann Moran, 2006.