Christopher E. Bagwell

Molecular analysis of diazotroph diversity in the rhizosphere of the smooth cordgrass, Spartina alterniflora.

ABSTRACT N2 fixation by diazotrophic bacteria associated with the roots of the smooth cordgrass, Spartina alterniflora, is an important source of new nitrogen in many salt marsh ecosystems. However, the diversity and phylogenetic affiliations of these rhizosphere diazotrophs are unknown. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified nifH sequence segments was used in previous studies to examine the stability and dynamics of the Spartina rhizosphere diazotroph assemblages in the North Inlet salt marsh, near Georgetown, S.C. In this study, plugs were taken from gel bands from representative DGGE gels, thenifH amplimers were recovered and cloned, and their sequences were determined. A total of 59 sequences were recovered, and the amino acid sequences predicted from them were aligned with sequences from known and unknown diazotrophs in order to determine the types of organisms present in the Spartina rhizosphere. We recovered numerous sequences from diazotrophs in the γ subdivision of the division Proteobacteria(γ-Proteobacteria) and from various anaerobic diazotrophs. Diazotrophs in the α-Proteobacteria were poorly represented. None of the Spartina rhizosphere DGGE band sequences were identical to any known or previously recovered environmental nifH sequences. TheSpartina rhizosphere diazotroph assemblage is very diverse and apparently consists mainly of unknown organisms.

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 Spartinawrack 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 α-Proteobacteria, and 4 were very similar (>99%) to that of Azospirillum brasilense. Seven sequences clustered with those from known γ-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.

Molecular diversity of diazotrophs in oligotrophic tropical seagrass bed communities

Denaturing gradient gel electrophoresis (DGGE) was employed to resolve PCR-amplified nifH sequences from vegetated and unvegetated sediments from two oligotrophic seagrass bed sites on San Salvador Island, Bahamas, in order to assess diazotroph species composition. All DGGE profiles from these sites showed the same prominent bands. These bands were sequenced, yielding 67 different nifH sequences, which were used in phylogenetic reconstructions. Most sequences were from anaerobes, but some were affiliated with the alpha- and (gamma-+beta-) Proteobacteria. Several NifH sequences were nearly identical to those from Azospirillum brasilense and Vibrio diazotrophicus. These seagrass bed sediments support a diverse diazotroph assemblage that is, at least superficially, similar to that associated with an intertidal grass (Spartina alterniflora).

Persistence of Selected Spartina alterniflora Rhizoplane Diazotrophs Exposed to Natural and Manipulated Environmental Variability

ABSTRACT Rhizoplane-rhizosphere nitrogen-fixing microorganisms (diazotrophs) are thought to provide a major source of biologically available nitrogen in salt marshes dominated bySpartina alterniflora. Compositional and functional stability has been demonstrated for this important functional group; however, the quantitative responses of specific diazotroph populations to environmental variability have not been assessed. Changes in the relative abundances of selected rhizoplane diazotrophs in response to long-term fertilization were monitored quantitatively by reverse sample genome probing. Fertilization stimulated Spartina, with plant height nearly tripling after 1 year. Fertilization also resulted in significant changes in interstitial porewater parameters. Diazotrophic activity (acetylene reduction assay) was sensitive to the fertilization treatments and was inhibited in some plots on several sampling dates. However, inhibition was never consistent across all of the replicates within a treatment and activity always recovered. The rhizoplane diazotrophs were quite responsive to environmental variability and to experimental treatments, but none were displaced by either environmental variability or experimental treatments. All strains were detected consistently throughout this study, and extensive spatial heterogeneity in the distribution patterns of these organisms was observed. The physiological traits that differentiate the diazotroph populations presumably support competitiveness and niche specialization, resulting in the observed resilience of the diazotroph populations in the rhizosphere.

Microdiversity of Culturable Diazotrophs from the Rhizoplanes of the Salt Marsh Grasses Spartina alterniflora and Juncus roemerianus

A bstractSalt marshes dominated by Spartina alterniflora (smooth cordgrass) are among the most productive ecosystems known, despite nitrogen limitation. Rhizoplane/rhizosphere diazotrophy (nitrogen fixation) serves as a significant source of combined nitrogen in these systems. Several recent studies have demonstrated remarkable physiological and phylogenetic macro- and microdiversity within this important functional group of organisms. However, the ecological significance of this diversity is presently unknown. The physiological characteristics of the culturable, oxygen-utilizing fraction of the rhizoplane diazotroph assemblages from Spartina alterniflora and from another salt marsh grass, the black needle rush Juncus roemerianus, were examined in combination with an assessment of the phylogenetic relatedness by whole genome DNA–DNA hybridization. Analysis of substrate utilization data permitted quantitative evaluation of fully cross-hybridizing strain groups and physiological clusters. Phylogenetically related strains, defined by DNA homology ≥90% relative to the positive control, displayed extensive physiological diversity. Seven bootstrap-supported physiological clusters, composed largely of phylogenetically dissimilar strains, showed similar utilization patterns for at least one class of ecologically relevant substrates (carbohydrates, carboxylic acids, or amino acids). These diazotrophs appear to be physiologically adapted for utilization of specific substrates or classes of substrates, lending support to diazotrophic functional redundancy. Microenvironmental heterogeneity is credited for promoting this diversity by selecting for physiologically specialized diazotroph populations to occupy defined niches in situ. One outcome of this physiological diversity is maintenance of a crucial environmental function (nitrogen fixation) over a broad range of environmental conditions.

Physiological Diversity of Rhizoplane Diazotrophs of the Saltmeadow Cordgrass, Spartina patens: Implications for Host Specific Ecotypes

Diazotrophic bacteria are important contributors to salt marsh productivity, but the biotic and abiotic factors that influence their distributions and function and the extent of their diversity cannot be understood in the absence of physiological information. Here we examine the physiological diversity and distribution patterns of diazotrophic bacteria associated with the rhizoplane of the saltmeadow cordgrass, Spartina patens, in comparison with diazotrophs from other intertidal grasses (tall and short form Spartina alterniflora and Juncus roemerianus) from the same salt marsh. S. patens plants were collected from two distinct habitats, and a total of 115 strains (111 Gram negative and 4 Gram positive strains) were isolated into pure culture by stab inoculating roots and rhizomes into combined nitrogen-free semisolid media. Most strains were microaerophilic and approximately one-half were motile. API test strips were used to eliminate redundancy within the culture collection, resulting in 21 physiologically different API groups (17 Gram negative and 4 Gram positive groups). A representative strain from each API group was selected for dot blot hybridization with a nifH specific probe and 16 strains (13 Gram negative and 3 Gram positive) were scored as positive. The nifH positive API group representative strains were characterized further using BIOLOG test plates. Substrate utilization potentials defined two S. patens strain clusters, and only one S. patens strain was physiologically similar to any other strain from a different host plant origin. No distinctions could be made based on the different S. patens habitats, suggesting that the host plant may have a greater impact than abiotic environmental conditions on the distributions of the rhizoplane diazotrophs recovered.

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

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.

Influence of host plant-derived and abiotic environmental parameters on the composition of the diazotroph assemblage associated with roots of Juncus roemerianus.

Environmental factors governing the distributions of plant root-associated bacteria are poorly understood. Most plant species occurring in salt marsh estuaries are restricted to very specific habitats within the marsh and plant-derived and abiotic environmental features covary. We examined diazotrophic bacteria inhabiting the rhizoplanes of different populations of the black needlerush, Juncus roemerianus, growing in two different habitats, in order to examine the relative influence of plant-derived and abiotic environmental parameters on diazotroph assemblage composition. Juncus roots were collected from a monotypic Juncus patch in the low intertidal marsh, and from the main monotypic Juncus stand in the high marsh. A total of 235 bacterial pure cultures were isolated from the roots using combined nitrogen-free media. Physiologically similar strains were grouped, producing 58 different groups. Strains representing 49 of these groups tested positive for nifH, and substrate utilization profiles of these strains were compared quantitatively. Three major substrate utilization clusters were identified and all contained both Juncus patch and main stand isolates. Denaturing gradient gel electrophoresis analysis of nifH amplicons recovered from roots and from vegetated sediments taken from the main stand and from two patches was also performed. Juncus root nifH amplicon profiles from all three sampling sites were very similar. Profiles of amplicons from vegetated sediments were also similar across sites, but less similar than the root profiles. Results from two independent methodological approaches indicated a strong impact of the plant host relative to that of the abiotic environment on the composition of the root-associated diazotroph assemblage.

Seasonal Variability of Diazotroph Assemblages Associated with the Rhizosphere of the Salt Marsh Cordgrass, Spartina alterniflora

Nitrogen fixation is the primary N source in the highly productive but N-limited North Inlet, SC, USA salt marsh system. The diverse assemblages of nitrogen-fixing (diazotrophic) bacteria associated with the rhizospheres of the short and tall growth forms of Spartina alterniflora were analyzed at two sites, Crab Haul Creek and Goat Island, which are in different tidal creek drainage systems in this marsh. The sites differed in proximity to the main channel for tidal intrusion and in several edaphic parameters. We hypothesized that either the differing abiotic environmental regimes of the two sites or the variation due to seasonal effects result in differences in the diazotroph assemblage. Rhizosphere samples were collected seasonally during 1999 and 2000. DNA was purified and nifH amplified for denaturing gradient gel electrophoresis (DGGE) analysis of diazotroph assemblage composition. Principal components analysis was used to analyze the binary DGGE band position data. Season strongly influenced assemblage composition and biplots were used to identify bands that significantly affected the seasonal and site-specific clustering. The types of organisms that were most responsive to seasonal or site variability were identified on the basis of DGGE band sequences. Seasonally responsive members of the anaerobic diazotrophs were detected during the winter and postsenescence conditions and may have been responsible for elevated pore water sulfide concentrations. Sequences from a diverse assemblage of Gammaproteobacteria were predominant during growth periods of S. alterniflora. Abiotic environmental parameters strongly influenced both the S. alterniflora and the diazotrophic bacterial assemblages associated with this keystone salt marsh plant species.

Kinetics of 1,2-dichloroethane and 1,2-dibromoethane biodegradation in anaerobic enrichment cultures.

ABSTRACT 1,2-Dichloroethane (1,2-DCA) and 1,2-dibromoethane (ethylene dibromide [EDB]) contaminate groundwater at many hazardous waste sites. The objectives of this study were to measure yields, maximum specific growth rates (μ̂), and half-saturation coefficients (KS ) in enrichment cultures that use 1,2-DCA and EDB as terminal electron acceptors and lactate as the electron donor and to evaluate if the presence of EDB has an effect on the kinetics of 1,2-DCA dehalogenation and vice versa. Biodegradation was evaluated at the high concentrations found at some industrial sites (>10 mg/liter) and at lower concentrations found at former leaded-gasoline sites (1.9 to 3.7 mg/liter). At higher concentrations, the Dehalococcoides yield was 1 order of magnitude higher when bacteria were grown with 1,2-DCA than when they were grown with EDB, while μ̂s were similar for the two compounds, ranging from 0.19 to 0.52 day−1 with 1,2-DCA to 0.28 to 0.36 day−1 for EDB. KS was larger for 1,2-DCA (15 to 25 mg/liter) than for EDB (1.8 to 3.7 mg/liter). In treatments that received both compounds, EDB was always consumed first and adversely impacted the kinetics of 1,2-DCA utilization. Furthermore, 1,2-DCA dechlorination was interrupted by the addition of EDB at a concentration 100 times lower than that of the remaining 1,2-DCA; use of 1,2-DCA did not resume until the EDB level decreased close to its maximum contaminant level (MCL). In lower-concentration experiments, the preferential consumption of EDB over 1,2-DCA was confirmed; both compounds were eventually dehalogenated to their respective MCLs (5 μg/liter for 1,2-DCA, 0.05 μg/liter for EDB). The enrichment culture grown with 1,2-DCA has the advantage of a more rapid transition to 1,2-DCA after EDB is consumed.