|Moran, Mary Ann||University of Georgia (UGA)||Principal Investigator|
|Kiene, Ronald P.||Dauphin Island Sea Lab (DISL)||Co-Principal Investigator|
|Whitman, William||University of Georgia (UGA)||Co-Principal Investigator|
|Rauch, Shannon||Woods Hole Oceanographic Institution (WHOI BCO-DMO)||BCO-DMO Data Manager|
Links are provided to published dimethylsulfoniopropionate (DMSP) demethylase protein, DmdA, sequence data from marine bacterioplankton that has been deposited in the NCBI GenBank Short Read Archive (SRA).
Experimental design, methods, and results are further described in:
Varaljay, V. A., E. C. Howard, S. Sun, M. A. Moran (2009). Deep Sequencing of a DMSP-Degrading Gene (dmdA) Using PCR Primer Pairs Designed from Marine Metagenomic Data. Applied and Environmental Microbiology, vol. 76, p. 609. doi: 10.1128/AEM.01258-09
See Varaljay et al. 2010 for detailed methods, which are paraphrased below.
"Surface water was collected between October 2000 and April 2005 at two sampling sites at the Sapelo Island Microbial Observatory (SIMO) in coastal Georgia: the Dean Creek site (a salt marsh tidal creek) and the Doboy Sound (a coastal ocean inlet). Approximately 20 liters of water was filtered sequentially through 8.0-um, 1.0-um, and 0.2-um pore size polycarbonate membrane filters, with two replicate samples obtained at each site. Cells captured on the 1.0-um (particle associated) and 0.2-um filter (free-living) were stored at -20 degrees C until DNA extraction using a PowerMax Soil DNA Isolation kit (MO BIO Laboratories, Inc.). 76 DNA extracts were used in the study, representing 38 samples of each size fraction. Samples were separately pooled by size fraction in equal amounts to produce composite free-living and particle-associated DNA samples.
Primer pairs giving single amplicons of the correct size from the composite SIMO DNA were chosen for analysis by sequencing. Amplicons suitable for 454 sequencing were prepared by modifying each primer pair with an adaptor sequence at the 5 end of the forward primer according to the method of Huber et al. Additional four-base key sequences in between the adaptor and primer sequence were used to distinguish inosine and degenerate primer sequences. PCRs were carried out in duplicate using 24 ng template DNA and then pooled before sequencing. Amplicons were cleaned using the AMPure purification method (Agencourt Bioscience Corp., Beverly, MA) according to the 454 Life Sciences protocol (Roche Diagnostics Corp., Branford, CT), with modifications to the volume of purified PCR products (30.0 l) and AMPure beads (50.4 l). Products were quantified spectrophotometrically and combined in equal concentrations in four separate pools based on primer and size fraction. Four-region 454 FLX LR70 sequencing was carried out at the University of South Carolina EnGenCore facility. Amplicon sequences were annotated by BLASTx analysis. This analysis was used to distinguish correct target sequences from closely related paralogous sequences and to classify amplicons by clade."
"To account for differences in the number of amplicons sequenced for each primer pair (ranging from 2,000 to 12,000 sequences), a resampling approach was used in which 1,000 sample populations of the same size were randomly drawn from the amplicon pools being compared. This approach was used to normalize the number of 90% dmdA clusters in comparisons between primer pairs and size fractions." (Varaljay et al. 2010)
BCO-DMO added the site coordinates, which were obtained from the Sapelo Island Microbial Observatory (SIMO) website.
|site_desc||Description of the general location where samples were collected.||text|
|taxon||Description of the taxon of study.||text|
|accession_number||Accession number and link to NCBI's Short Read Archive (SRA).||unitless|
|site||Name of the sampling site.||text|
|lat||Latitude of the sampling site. North = positive.||decimal degrees|
|lon||Longitude of the sampling site. East = positive.||decimal degrees|
shoreside Sapelo Island Microbial Observatory
Surface water was collected between October 2000 and April 2005 at two sampling sites at the Sapelo Island Microbial Observatory (SIMO) in coastal Georgia: the Dean Creek site (a salt marsh tidal creek) and the Doboy Sound (a coastal ocean inlet). Samples were collected for the project "En-Gen: A Functional Genomics Approach to Organic Sulfur Cycling in the Ocean". Dean Creek site: 81.2699°W, 31.3929°N Doboy Sound: 81.2915°W, 31.3862°N Both of these sites are located within the Georgia Coastal Ecosystems Long Term Ecological Research (LTER) study area, more specifically the UGAMI site (UGA Marine Institute).
The recent discovery of key genes that mediate competing pathways at a critical juncture in the marine sulfur cycle has allowed biogeochemists to make rapid advances in understanding where and when sulfur transformations occur in the ocean, and most importantly, what factors regulate them. This project describes an environmental functional genomics project that will rapidly increase our knowledge of the role that bacterioplankton play in dimethylsulfoniopropionate (DMSP) cycling in ocean surface waters, focusing particularly on biological controls of volatile sulfur exchange across the ocean/atmosphere boundary.
The investigators have asked three critical hypotheses to explain the regulation of bacterial DMSP degradation: that involve investigations on the energy constraints of DMSP cycling, the role that DMSP concentration in the oceans plays, and the sulfur requirements for bacterial growth. These research areas serve as the focus for hypothesis-driven laboratory and field studies using functional genomics approaches that will track patterns in gene expression in relation to sulfur metabolism. The hypotheses will be tested with:
1) chemostat systems with a model marine bacterium Silicibacter pomeroyi;
2) microcosm experiments with Gulf of Mexico seawater; and
3) field studies at various sites in the Gulf of Mexico. Marine bacterioplankton play a key role in regulating the flux of DMSP-derived sulfur to the atmosphere, a process of great importance for global climate regulation and marine productivity.
The investigators will also be involved in graduate and undergraduate student education, and two post-doctoral associates will be trained to address multidisciplinary challenges in environmental microbiology. High school biology students in Athens, GA will participate in marine microbial biology research that includes bacterial diversity and discovery studies in coastal Georgia, follow-up training in molecular tools and bioinformatics in their own classroom, and summer internships at the University of Georgia and Dauphin Island Sea Laboratory.
(The description above is from the NSF Award Abstract).