Molecular assessment of heterotrophy and prey digestion in zooxanthellate cnidarians.

Zooxanthellate cnidarians are trophically complex, relying on both autotrophy and heterotrophy. Although several aspects of heterotrophy have been studied in these organisms, information linking prey capture with digestion is still missing. We used prey-specific PCR-based tools to assess feeding and prey digestion of two zooxanthellate cnidarians - the tropical sea anemone Aiptasia sp. and the scleractinian coral Oculina arbuscula. Prey DNA disappeared rapidly for the initial 1-3 days, whereas complete digestion of prey DNA required up to 10 days in O. arbuscula and 5 or 6 days in Aiptasia sp. depending on prey species. These digestion times are considerably longer than previously reported from microscopy-based examination of zooxanthellate cnidarians and prey DNA breakdown in other marine invertebrates, but similar to prey DNA breakdown reported from terrestrial invertebrates such as heteroptera and spiders. Deprivation of external prey induced increased digestion rates during the first days after feeding in O. arbuscula, but after 6 days of digestion, there were no differences in the remaining prey levels in fed and unfed corals. This study indicates that prey digestion by symbiotic corals may be slower than previously reported and varies with the type of prey, the cnidarian species and its feeding history. These observations have important implications for bioenergetic and trophodynamic studies on zooxanthellate cnidarians.

Bioluminescent bacteria as indicators of chemical contamination of coastal waters.

The ratio of bioluminescent to total bacteria (bioluminescent ratio, BLR) as an indicator of a variety of types of anthropogenic contamination of estuarine ecosystems was evaluated through a series of laboratory and field studies. Laboratory studies indicated that the BLR of natural bacterioplankton communities was proportionally reduced in the presence of a number of contaminants including diesel fuel and saltmarsh sediments co-contaminated with mercury and polychlorinated biphenyls (PCBs). Bioluminescent ratio inhibition was observed after short-term exposure to a contaminant suggesting a physiological rather than a population response of native microbial communities. Simulated eutrophication did not suppress the BLR. Field observations of the BLR were conducted weekly for a 2-yr period in the Skidaway River estuary, Georgia, USA. These observations revealed considerable seasonal variability associated with the BLR. Bioluminescent ratios were highest during the summer (25 +/- 15%), lower in the fall (6 +/- 5%) and spring (3 +/- 2%), and near zero during the winter. Although the BLR was not significantly correlated to salinity at a single site (Skidaway River estuary), the BLR was significantly correlated with salinity when sites within the same estuary system were compared (r2 = 0.93). Variation in BLR was not correlated to standard bacteriological indicators of water quality including total and fecal coliform bacteria. Comparison of the BLR from impacted and pristine estuarine sites during the fall suggested that anthropogenically impacted sites exhibited lower BLR than predicted from salinity versus BLR relationships developed in pristine systems. These observations suggest that the BLR could be used as a simple and reliable initial indicator of chemical contamination of estuarine systems resulting from human activity.

Diversity and detection of nitrate assimilation genes in marine bacteria.

A PCR approach was used to construct a database of nasA genes (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 sequenced nasA genes. Our results indicate that several groups of heterotrophic bacterial nasA genes are common and widely distributed in oceanic environments.

A quantitative relationship that demonstrates mercury methylation rates in marine sediments are based on the community composition and activity of sulfate-reducing bacteria.

A quantitative framework was developed which estimates mercury methylation rates (MMR) in sediment cores based on measured sulfate reduction rates (SRR) and the community composition sulfate-reducing bacterial consortia. MMR and SRR as well as group-specific 16S rRNA concentrations (as quantified by probe signal) associated with sulfate-reducing bacteria (SRB) were measured in triplicate cores of saltmarsh sediments. Utilizing previously documented conversion factors in conjunction with field observations of sulfate reduction, MMR were calculated, and the results were compared to experimentally derived measurements of MMR. Using our novel field data collected in saltmarsh sediment where sulfate reduction activity is high, calculated and independently measured MMR results were consistently within an order of magnitude and displayed similar trends with sediment depth. In an estuarine sediment where sulfate reduction activity was low, calculated and observed MMR diverged by greater than an order of magnitude, but again trends with depth were similar. We have expanded the small database generated to date on mercury methylation in sulfur-rich marine sediments. The quantitative frameworkwe have developed further elucidates the coupling of mercury methylation to sulfate reduction by basing calculated rates of mercury methylation on the activity and community composition of sulfate-reducing bacteria. The quantitative framework may also provide a promising alternative to the difficult and hazardous determination of MMR using radiolabeled mercury.

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

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 (r(2) = 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.

Sulfate-reducing bacteria methylate mercury at variable rates in pure culture and in marine sediments.

Differences in methylmercury (CH(3)Hg) 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 or Desulfovibrio 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 family Desulfovibrionaceae 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 CH(3)Hg accumulation is coupled to rapid sulfate reduction. The observations described above have broad implications for understanding the control of CH(3)Hg formation and for developing remediation strategies for Hg-contaminated sediments.

Alteration in plasmid DNA following natural transformation to populations of marine bacteria.

This article examines alterations in a broad-host-range plasmid (pQSR50) that were observed following transfer to indigenous marine bacteria by natural transformation. Plasmid DNA from the transformants had altered restriction profiles. However, with the exception of the EcoRI site from one transformant (BS10), fragments amplified by polymerase chain reaction (PCR) and encompassing the recognition sites were cleaved by the relevant endonucleases, providing the sites were present. Analysis with DpnI and MboI indicated differences in DNA methylation between pQSR50 and the transformants. The missing EcoRI site from BS10 and smaller EcoRI fragments observed in transformants indicated that rearrangements had also occurred. Evolution of novel plasmid molecules following gene transfer may be an important mechanism by which natural genetic diversity is generated.

The naturally transformable marine bacterium WJT-1C formally identified as "Vibrio" is a pseudomonad.

A marine bacterial isolate, previously identified as Vibrio WJT-1C (ATCC 55351) and used as a model for investigating the process of natural transformation in the marine environment, has been further examined to determine its taxonomic identity. API 20E test strips, phenotypic testing, and flagellar staining had previously assigned the strain to the genus Vibrio, most closely related to V. campbelli. 16S rRNA analysis indicated that WJT-1C was in the Pseudomonas subgroup of the gamma proteobacteria. Bacteriophage typing and natural transformation with chromosomal DNA indicated that it was distinct from previously described marine transforming pseudomonads including Pseudomonas stutzeri strain JM300. The importance and abundance of the Pseudomonas subgroup of the gamma proteobacteria in the environment suggest that these marine strains are well suited as model organisms for describing the process and importance of natural transformation in nature.

Differential sensitivity of 16S rRNA targeted oligonucleotide probes used for fluorescence in situ hybridization is a result of ribosomal higher order structure.

The use of 16S rRNA targeted gene probes for the direct analysis of microbial communities has revolutionized the field of microbial ecology, yet a comprehensive approach for the design of such probes does not exist. The development of 16S rRNA targeted oligonucleotide probes for use with fluorescence in situ hybridization (FISH) procedures has been especially difficult as a result of the complex nature of the rRNA target molecule. In this study a systematic comparison of 16S rRNA targeted oligonucleotide gene probes was conducted to determine if target location influences the hybridization efficiency of oligonucleotide probes when used with in situ hybridization protocols for the detection of whole microbial cells. Five unique universal 12-mer oligonucleotide sequences, located at different regions of the 16S rRNA molecule, were identified by a computer-aided sequence analysis of over 1000 partial and complete 16S rRNA sequences. The complements of these oligomeric sequences were chemically synthesized for use as probes and end labeled with either [gamma-32P]ATP or the fluorescent molecule tetramethylrhodamine-5/-6. Hybridization sensitivity for each of the probes was determined by hybridization to heat-denatured RNA immobilized on blots or to formaldehyde fixed whole cells. All of the probes hybridized with equal efficiency to denatured RNA. However, the probes exhibited a wide range of sensitivity (from none to very strong) when hybridized with whole cells using a previously developed FISH procedure. Differential hybridization efficiencies against whole cells could not be attributed to cell wall type, since the relative probe efficiency was preserved when either Gram-negative or -positive cells were used. These studies represent one of the first attempts to systematically define criteria for 16S rRNA targeted probe design for use against whole cells and establish target site location as a critical parameter in probe design.

Intergeneric natural plasmid transformation between E. coli and a marine Vibrio species.

Natural transformation is the mechanism of procaryotic gene transfer that involves the uptake and expression of genetic information encoded in extracellular DNA. This process has been regarded as a mechanism to transfer genes (primarily chromosomal markers) between closely related strains or species. Here we demonstrate the cell-contact-dependent transfer of a non-conjugative plasmid from a laboratory E. coli strain to a marine Vibrio species, the first report of intergeneric natural plasmid transformation involving a marine bacterium. The nucleic acid synthesis inhibitors nalidixic acid and rifampicin inhibited the ability of the E. coli to function as a donor. However, dead cells also served as efficient donors. There was an obligate requirement for cell contact. No transfer occurred in the presence of DNase I, when donors and recipients were separated by a 0.2-micron filter, or when spent medium alone was used as a source of transforming DNA. These results indicate that contact-mediated intergeneric plasmid exchange can occur in the absence of detectable viable donor cells and that small non-conjugative plasmids can be spread through heterogeneous microbial communities by a process previously not recognized, natural plasmid transformation. These findings are important in the assessment of genetic risk to the environment, particularly from wastewater treatment systems and the use of genetically engineered organisms in the environment.

Gene transfer in marine water column and sediment microcosms by natural plasmid transformation.

We investigated the possibility for natural transformation in the marine environment by using broad-host-range plasmid multimers and a high-frequency-of-transformation (HFT) Vibrio strain as the recipient. Water and sediment samples were taken from Tampa Bay, the eastern Gulf of Mexico, the Florida Shelf near Miami, and the Bahamas Bank. In water column microcosms, transformation frequencies ranged from 1.7 x 10 to 2.7 x 10 transformants per recipient, with highest frequencies occurring when low levels of nutrients (peptone and yeast extract) were added. The presence of the ambient community either reduced transformation frequency by an order of magnitude or had no effect. In sterile sediments, nutrient additions had no consistent effect on transformation, with transfer frequencies similar to those observed in the water column. Transformation was not observed in any sediment experiment when the ambient microbial community was present. These findings are the first report of natural plasmid transformation in seawater and in the presence of the ambient microbial community. This process may be a mechanism for the acquisition of small, nonconjugative plasmids, which are commonly found in aquatic bacteria. Our data also suggest that natural transformation may be more likely to occur in the water column than in native marine sediments, contradicting prior conclusions based on studies with sterile sediments.

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain.

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.