title: Analysis of a diverse assemblage of diazotrophic bacteria from Spartina alterniflora using DGGE and clone library screening.
abstract: Methods to assess the diversity of the diazotroph assemblage in the rhizosphere of the salt marsh cordgrass, Spartina alterniflora were examined. The effectiveness of nifH PCR-denaturing gradient gel electrophoresis (DGGE) was compared to that of nifH clone library analysis. Seventeen DGGE gel bands were sequenced and yielded 58 nonidentical nifH sequences from a total of 67 sequences determined. A clone library constructed using the GC-clamp nifH primers that were employed in the PCR-DGGE (designated the GC-Library) yielded 83 nonidentical sequences from a total of 257 nifH sequences. A second library constructed using an alternate set of nifH primers (N-Library) yielded 83 nonidentical sequences from a total of 138 nifH sequences. Rarefaction curves for the libraries did not reach saturation, although the GC-Library curve was substantially dampened and appeared to be closer to saturation than the N-Library curve. Phylogenetic analyses showed that DGGE gel band sequencing recovered nifH sequences that were frequently sampled in the GC-Library, as well as sequences that were infrequently sampled, and provided a species composition assessment that was robust, efficient, and relatively inexpensive to obtain. Further, the DGGE method permits a large number of samples to be examined for differences in banding patterns, after which bands of interest can be sampled for sequence determination.
authors.affiliation: Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, United States. lovell@biol.sc.edu
authors.forename: Charles R
authors.initials: CR
authors.lastname: Lovell
authors.forename: Peter V
authors.initials: PV
authors.lastname: Decker
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Bagwell
authors.forename: Shelly
authors.initials: S
authors.lastname: Thompson
authors.forename: George Y
authors.initials: GY
authors.lastname: Matsui
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics; D044822:Biodiversity; D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D016521:Electrophoresis, Gel, Pulsed-Field; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics; D010802:Phylogeny; D018517:Plant Roots / Q000382:microbiology; D006109:Poaceae / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D012988:Soil Microbiology
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D010088:Oxidoreductases; C029130:nitrogenase reductase