Analysis of a diverse assemblage of diazotrophic bacteria from Spartina alterniflora using DGGE and clone library screening

• Published in: Journal of microbiological methods Vol. 73; no. 2; pp. 160 - 171
• Main Authors: Lovell, Charles R., Decker, Peter V., Bagwell, Christopher E., Thompson, Shelly, Matsui, George Y.
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier B.V 01-05-2008; Elsevier Science
• Subjects: Assemblage composition; Bacteria - classification; Bacteria - genetics; Bacteria - isolation & purification; Bacterial Proteins - genetics; Bacteriology; Biodiversity; Biological and medical sciences; Clone library; Cluster Analysis; DGGE; Diazotroph; DNA Primers - genetics; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; Electrophoresis, Gel, Pulsed-Field; Fundamental and applied biological sciences. Psychology; Microbiology; Miscellaneous; Molecular Sequence Data; Oxidoreductases - genetics; Phylogeny; Plant Roots - microbiology; Poaceae - microbiology; Sequence Analysis, DNA; Sequence Homology; Soil Microbiology; Spartina alterniflora; Clone library; DGGE; Diazotroph; Assemblage composition; Halophyte; Monocotyledones; Gramineae; Angiospermae; Bacteria; Spermatophyta; Spartina alterniflora
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2008.02.005

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.