Recovery and phylogenetic analysis of nifH sequences from diazotrophic bacteria associated with dead aboveground biomass of Spartina alterniflora

Abstract

DNA was extracted from dry standing dead Spartina alterniflora stalks as well as dry Spartina wrack from the North Inlet (South Carolina) and Sapelo Island (Georgia) salt marshes. Partial nifH sequences were PCR amplified, the products were separated by denaturing gradient gel electrophoresis (DGGE), and the prominent DGGE bands were sequenced. Most sequences (109 of 121) clustered with those from alpha-Proteobacteria, and 4 were very similar (>99%) to that of Azospirillum brasilense. Seven sequences clustered with those from known gamma-Proteobacteria and five with those from known anaerobic diazotrophs. The diazotroph assemblages associated with dead Spartina biomass in these two salt marshes were very similar, and relatively few major lineages were represented.

FIG. 1

Denaturing gradient gel images showing nifH amplimers from dead aboveground Spartina biomass. PCR and DGGE conditions are described in Materials and Methods. Lanes: A, North Inlet standing dead Spartina; B, North Inlet Spartina wrack; C, Sapelo Island standing dead; D, Sapelo Island wrack; Art., artifact.

FIG. 2

Phylogenetic analysis of dead aboveground Spartina biomass NifH amino acid sequences from presumptive α-Proteobacteria, from various known α-Proteobacteria, and from selected unknown, presumptive α-Proteobacteria from other sources. NIS, North Inlet standing dead Spartina; NIW, North Inlet Spartina wrack; SIS, Sapelo Island standing dead Spartina; SIW, Sapelo Island Spartina wrack. The percentage of 500 bootstrap samples that supported each branch is shown. Bootstrap values below 50% are not shown.

FIG. 3

Phylogenetic analysis of dead aboveground Spartina biomass NifH amino acid sequences from presumptive γ-Proteobacteria, from various known γ-Proteobacteria, and from selected unknown, presumptive γ-Proteobacteria from other sources. NIS, North Inlet standing dead Spartina; NIW, North Inlet Spartina wrack; SIS, Sapelo Island standing dead Spartina; SIW, Sapelo Island Spartina wrack. The percentage of 500 bootstrap samples that supported each branch is shown. Bootstrap values below 50% are not shown.

FIG. 4

Phylogenetic analysis of dead aboveground Spartina biomass NifH amino acid sequences from unknown, presumptive anaerobic bacterial sequences, from various known anaerobic bacteria, and from selected unknown, presumptive anaerobic bacteria from other sources. NIS, North Inlet standing dead Spartina; NIW, North Inlet Spartina wrack; SIS, Sapelo Island standing dead Spartina; SIW, Sapelo Island Spartina wrack. The percentage of 500 bootstrap samples that supported each branch is shown. Bootstrap values below 50% are not shown.