Marc E. Frischer

Sulfate-Reducing Bacteria Methylate Mercury at Variable Rates in Pure Culture and in Marine Sediments

ABSTRACT Differences in methylmercury (CH3Hg) production normalized to the sulfate reduction rate (SRR) in various species of sulfate-reducing bacteria (SRB) were quantified in pure cultures and in marine sediment slurries in order to determine if SRB strains which differ phylogenetically methylate mercury (Hg) at similar rates. Cultures representing five genera of the SRB (Desulfovibrio desulfuricans, Desulfobulbus propionicus,Desulfococcus multivorans, Desulfobacter sp. strain BG-8, and Desulfobacterium sp. strain BG-33) were grown in a strictly anoxic, minimal medium that received a dose of inorganic Hg 120 h after inoculation. The mercury methylation rates (MMR) normalized per cell were up to 3 orders of magnitude higher in pure cultures of members of SRB groups capable of acetate utilization (e.g., the family Desulfobacteriaceae) than in pure cultures of members of groups that are not able to use acetate (e.g., the family Desulfovibrionaceae). Little or no Hg methylation was observed in cultures of Desulfobacterium orDesulfovibrio strains in the absence of sulfate, indicating that Hg methylation was coupled to respiration in these strains. Mercury methylation, sulfate reduction, and the identities of sulfate-reducing bacteria in marine sediment slurries were also studied. Sulfate-reducing consortia were identified by using group-specific oligonucleotide probes that targeted the 16S rRNA molecule. Acetate-amended slurries, which were dominated by members of the Desulfobacterium and Desulfobacter groups, exhibited a pronounced ability to methylate Hg when the MMR were normalized to the SRR, while lactate-amended and control slurries had normalized MMR that were not statistically different. Collectively, the results of pure-culture and amended-sediment experiments suggest that members of the family Desulfobacteriaceae have a greater potential to methylate Hg than members of the familyDesulfovibrionaceae have when the MMR are normalized to the SRR. Hg methylation potential may be related to genetic composition and/or carbon metabolism in the SRB. Furthermore, we found that in marine sediments that are rich in organic matter and dissolved sulfide rapid CH3Hg accumulation is coupled to rapid sulfate reduction. The observations described above have broad implications for understanding the control of CH3Hg formation and for developing remediation strategies for Hg-contaminated sediments.

Diversity and Detection of Nitrate Assimilation Genes in Marine Bacteria

ABSTRACT A PCR approach was used to construct a database of nasAgenes (called narB genes in cyanobacteria) and to detect the genetic potential for heterotrophic bacterial nitrate utilization in marine environments. A nasA-specific PCR primer set that could be used to selectively amplify the nasA gene from heterotrophic bacteria was designed. Using seawater DNA extracts obtained from microbial communities in the South Atlantic Bight, the Barents Sea, and the North Pacific Gyre, we PCR amplified and sequencednasA genes. Our results indicate that several groups of heterotrophic bacterial nasA genes are common and widely distributed in oceanic environments.

Grazing of phytoplankton by microzooplankton in the Barents Sea during early summer

Abstract Phytoplankton growth rates and grazing losses to microzooplankton were determined in surface waters of the central Barents Sea during a cruise in June/July 1999. Five stations were occupied which had been studied repeatedly over the past 15–20 years. Dilution experiments using chlorophyll a (chl a) as a tracer were used to estimate daily rates in three size fractions; image-analyzed fluorescence microscopy provided quantitative estimates of standing stocks of auto- and heterotrophic nano- and microplankton. Phytoplankton contributed the largest share of protistan biomass, followed by bacteria and microzooplankton. On average, nanophytoplankton (

Bacterial Community Structure of Acid-Impacted Lakes: What Controls Diversity?†

ABSTRACT Although it is recognized that acidification of freshwater systems results in decreased overall species richness of plants and animals, little is known about the response of aquatic microbial communities to acidification. In this study we examined bacterioplankton community diversity and structure in 18 lakes located in the Adirondack Park (in the state of New York in the United States) that were affected to various degrees by acidic deposition and assessed correlations with 31 physical and chemical parameters. The pH of these lakes ranged from 4.9 to 7.8. These studies were conducted as a component of the Adirondack Effects Assessment Program supported by the U.S. Environmental Protection Agency. Thirty-one independent 16S rRNA gene libraries consisting of 2,135 clones were constructed from epilimnion and hypolimnion water samples. Bacterioplankton community composition was determined by sequencing and amplified ribosomal DNA restriction analysis of the clone libraries. Nineteen bacterial classes representing 95 subclasses were observed, but clone libraries were dominated by representatives of the Actinobacteria and Betaproteobacteria classes. Although the diversity and richness of bacterioplankton communities were positively correlated with pH, the overall community composition assessed by principal component analysis was not. The strongest correlations were observed between bacterioplankton communities and lake depth, hydraulic retention time, dissolved inorganic carbon, and nonlabile monomeric aluminum concentrations. While there was not an overall correlation between bacterioplankton community structure and pH, several bacterial classes, including the Alphaproteobacteria, were directly correlated with acidity. These results indicate that unlike more identifiable correlations between acidity and species richness for higher trophic levels, controls on bacterioplankton community structure are likely more complex, involving both direct and indirect processes.

Quantification of copepod gut content by differential length amplification quantitative PCR (dla-qPCR)

Quantification of feeding rates and selectivity of zooplankton is vital for understanding the mechanisms structuring marine ecosystems. However, methodological limitations have made many of these studies difficult. Recently, molecular based methods have demonstrated that DNA from prey species can be used to identify zooplankton gut contents, and further, quantitative gut content estimates by quantitative PCR (qPCR) assays targeted to the 18S rRNA gene have been used to estimate feeding rates in appendicularians and copepods. However, while standard single primer based qPCR assays were quantitative for the filter feeding appendicularian Oikopleura dioica, feeding rates were consistently underestimated in the copepod Calanus finmarchicus. In this study, we test the hypothesis that prey DNA is rapidly digested after ingestion by copepods and describe a qPCR-based assay, differential length amplification qPCR (dla-qPCR), to account for DNA digestion. The assay utilizes multiple primer sets that amplify different sized fragments of the prey 18S rRNA gene and, based on the differential amplification of these fragments, the degree of digestion is estimated and corrected for. Application of this approach to C. finmarchicus fed Rhodomonas marina significantly improved quantitative feeding estimates compared to standard qPCR. The development of dla-qPCR represents a significant advancement towards a quantitative method for assessing in situ copepod feeding rates without involving cultivation-based manipulation.

Importance of heterotrophic bacterial assimilation of ammonium and nitrate in the Barents Sea during summer

Abstract In a transect across the Barents Sea into the marginal ice zone (MIZ), five 24-h experimental stations were visited, and uptake rates of NH4+ and NO3− by bacteria were measured along with their contribution to total dissolved inorganic nitrogen (DIN) assimilation. The percent bacterial DIN uptake of total DIN uptake increased substantially from 10% in open Atlantic waters to 40% in the MIZ. The percentage of DIN that accounted for total bacterial nitrogen production also increased from south to north across the transect. On average, at each of the five 24-h stations, bacteria accounted for 16–40% of the total NO3− uptake and 12–40% of the total NH4+ uptake. As a function of depth, bacteria accounted for 17%, 23%, and 26% of the total NH4+ assimilation and 17%, 37%, and 36% of the total NO3− assimilation at 5, 30, and 80 m, respectively. Bacteria accounted for a higher percentage of total NO3− uptake compared to total NH4+ uptake in 12 out of 15 samples. Bacterial productivity explains a substantial amount of the variability associated with bacterial DIN uptake, but the relationship between bacterial production and bacterial DIN uptake is best explained when the data from the open Atlantic water stations are grouped separately from the MIZ stations. The percentage of DIN that accounts for bacterial N production is approximately four-fold higher in 24 h MIZ stations compared with open Atlantic stations. This suggests that bacteria play a larger role in NO3− utilization, particularly in the MIZ, than previously hypothesized and that bacterial uptake of NO3− should not be ignored in estimates of new production. Understanding processes that affect autotrophic based new production, such as heterotrophic bacterial utilization of NO3−, in polar oceans is of particular significance because of the role these regions may play in sequestering CO2.

Whole-cell versus total RNA extraction for analysis of microbial community structure with 16S rRNA-targeted oligonucleotide probes in salt marsh sediments.

ABSTRACT rRNA-targeted oligonucleotide probes have become powerful tools for describing microbial communities, but their use in sediments remains difficult. Here we describe a simple technique involving homogenization, detergents, and dispersants that allows the quantitative extraction of cells from formalin-preserved salt marsh sediments. Resulting cell extracts are amenable to membrane blotting and hybridization protocols. Using this procedure, the efficiency of cell extraction was high (95.7% ± 3.7% [mean ± standard deviation]) relative to direct DAPI (4′,6′-diamidino-2-phenylindole) epifluorescence cell counts for a variety of salt marsh sediments. To test the hypothesis that cells were extracted without phylogenetic bias, the relative abundance (depth distribution) of five major divisions of the gram-negative mesophilic sulfate-reducing delta proteobacteria were determined in sediments maintained in a tidal mesocosm system. A suite of six 16S rRNA-targeted oligonucleotide probes were utilized. The apparent structure of sulfate-reducing bacteria communities determined from whole-cell and RNA extracts were consistent with each other (r2 = 0.60), indicating that the whole-cell extraction and RNA extraction hybridization approaches for describing sediment microbial communities are equally robust. However, the variability associated with both methods was high and appeared to be a result of the natural heterogeneity of sediment microbial communities and methodological artifacts. The relative distribution of sulfate-reducing bacteria was similar to that observed in natural marsh systems, providing preliminary evidence that the mesocosm systems accurately simulate native marsh systems.

Distribution of bacterial biomass and activity in the marginal ice zone of the central Barents Sea during summer

Abstract The purpose of this study was to determine bacterioplankton abundance and activity in the Barents Sea using the novel modified vital stain and probe (mVSP) method. The mVSP is a protocol that combines DAPI and propidium iodide staining with 16S rRNA eubacterial-specific oligonucleotide probes to determine the physiological status of individual microbial cells. Bacterial abundance and metabolic activity were measured in near-surface waters and with depth at stations in the central Barents Sea during a cruise in June/July 1999. Viral abundance was also determined for 19 transect stations and at depth (2–200 m) for five intensive 24-h stations. In general, bacterial and viral abundances varied across the transect, but showed peaks of abundance (6×10 9 cells l −1 , 9×10 9 viruses l −1 ) in Polar Front water masses. Viruses were abundant in seawater and exceeded bacterial abundance. Metabolic activity was determined for individual cells using 16S rRNA eubacterial-specific oligonucleotide probes, and for the total community with 3 H-leucine incorporation. Activity measured by oligonucleotide probes increased from south to north. The fraction of cells that were active was lowest in the southern Barents Sea (20%) and highest in the Polar Front (53%). The proportion of cells at the 24-h stations that were determined to be active decreased with depth, but not with distance from ice cover. Leucine incorporation rates varied significantly and did not always correlate with probe measurements. The proportion of total cells that had compromised membranes and were therefore considered dead remained relatively constant ( −3 day −1 . Growth rates ranged from −1 , implying turnover rates of 2.5 to >200 days. Our results demonstrate that bacterioplankton and viruses are dynamic but ubiquitous features of Arctic microbial communities. The contribution of bacteria and viruses to Arctic food webs is discussed.

Development of an Argopecten-Specific 18S rRNA Targeted Genetic Probe.

Abstract: Comparison of 18S ribosomal RNA gene sequences between diverse bivalve species, including eight scallop species, allowed the design of an 18S rRNA targeted oligonucleotide probe (BS-1364) that was specific for scallops belonging to the genus Argopecten (bay and calico scallops). The high sequence similarity of the 18S rRNA gene between Argopecten irradians and Argopecten gibbus (98.8%) prevented the design of an A. irradians species-specific probe. Hybridization studies using amplified 18S rDNA from a diverse collection of bivalve species demonstrated that the specificity of the digoxygenin-labeled probe was consistent with the predicted specificity indicated by sequence comparison. Hybridization studies using laboratory-spawned bay scallop veligers indicated that a single veliger could be detected by probe hybridization in a blot format, and that probe hybridization signal was proportional (r2= .99) to the abundance of veligers. Methods for rRNA extraction and blotting were developed that allowed bay scallop veligers to be specifically and quantitatively identified in natural plankton samples. Preliminary studies conducted in Tampa Bay, Florida, suggest that introduced scallops can successfully spawn and produce veligers under in situ conditions. The Argopecten-specific probe and methods developed in this study provide the means to study the production and fate of bay scallop larvae in nature and provide evidence that scallops introduced into Tampa Bay have the potential for successful reproduction and enhancement of scallop stocks.

Detection and Discovery of Crustacean Parasites in Blue Crabs (Callinectes sapidus) by Using 18S rRNA Gene-Targeted Denaturing High-Performance Liquid Chromatography

ABSTRACT Recently, we described a novel denaturing high-performance liquid chromatography (DHPLC) approach useful for initial detection and identification of crustacean parasites. Because this approach utilizes general primers targeted to conserved regions of the 18S rRNA gene, a priori genetic sequence information on eukaryotic parasites is not required. This distinction provides a significant advantage over specifically targeted PCR assays that do not allow for the detection of unknown or unsuspected parasites. However, initial field evaluations of the DHPLC assay suggested that because of PCR-biased amplification of dominant host genes it was not possible to detect relatively rare parasite genes in infected crab tissue. Here, we describe the use of a peptide nucleic acid (PNA) PCR hybridization blocking probe in association with DHPLC (PNA-PCR DHPLC) to overcome inherent PCR bias associated with amplification of rare target genes by use of generic primers. This approach was utilized to detect infection of blue crabs (Callinectes sapidus) by the parasitic dinoflagellate Hematodinium sp. Evaluation of 76 crabs caught in Wassaw Sound, GA, indicated a 97% correspondence between detection of the parasite by use of a specific PCR diagnostic assay and that by use of PNA-PCR DHPLC. During these studies, we discovered one crab with an association with a previously undescribed protist symbiont. Phylogenetic analysis of the amplified symbiont 18S rRNA gene indicated that it is most closely related to the free-living kinetoplastid parasite Procryptobia sorokini. To our knowledge, this is the first report of this parasite group in a decapod crab and of this organism exhibiting a presumably parasitic life history.