Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues ...

University of Illinois at Urbana-Champaign

Distinct partitioning has been observed in the composition and diversity of bacterial communities inhabiting the surface and overlying seawater of three coral species infected with black band disease (BBD) on the southern Caribbean island of Curaçao, Netherlands Antilles. PCR amplification and sequencing of bacterial 16S rRNA genes (rDNA) with universally conserved primers have identified over 524 unique bacterial sequences affiliated with 12 bacterial divisions. The molecular sequences exhibited less than 5% similarity in bacterial community composition between seawater and the healthy, black band diseased, and dead coral surfaces. The BBD bacterial mat rapidly migrates across and kills the coral tissue. Clone libraries constructed from the BBD mat were comprised of eight bacterial divisions and 13% unknowns. Several sequences representing bacteria previously found in other marine and terrestrial organisms (including humans) were isolated from the infected coral surfaces, including Clostridium spp., Arcobacter spp., Campylobacter spp., Cytophaga fermentans, Cytophaga columnaris, and Trichodesmium tenue.

Partitioning of bacterial communities between seawater and healthy, black band diseased, and dead coral surfaces.

For 30 years it has been assumed that a single species of cyanobacteria, Phormidium corallyticum, is the volumetrically dominant component of all cases of black band disease (BBD) in coral. Cyanobacterium-specific 16S rRNA gene primers and terminal restriction fragment length polymorphism analyses were used to determine the phylogenetic diversity of these BBD cyanobacteria on coral reefs in the Caribbean and Indo-Pacific Seas. These analyses indicate that the cyanobacteria that inhabit BBD bacterial mats collected from the Caribbean and Indo-Pacific Seas belong to at least three different taxa, despite the fact that the corals in each case exhibit similar signs and patterns of BBD mat development.

Cyanobacteria associated with coral black band disease in Caribbean and Indo-Pacific Reefs.

RNA-arbitrarily primed PCR techniques have been applied for the first time to identify differential gene expression in black band disease (BBD), a virulent coral infection that affects reef ecosystems worldwide. The gene activity for the BBD mat on infected surfaces of the brain coral Diploria strigosa was compared with that for portions of the BBD mat that were removed from the coral and suspended nearby in the seawater column. The results obtained indicate that three genes (DD 95-2, DD 95-4, and DD 99-9) were up-regulated in the BBD bacterial mat on the coral surface compared to the transcript base levels observed in the BBD mat suspended in seawater. Clone DD 95-4 has homology with known amino acid ABC transporter systems in bacteria, while clone DD 99-9 exhibits homology with chlorophyll A apoprotein A1 in cyanobacteria. This protein is essential in the final conformation of photosystem I P700. DD 95-2, the only gene that was fully repressed in the BBD mat samples suspended in seawater, exhibited homology with the AraC-type DNA binding domain-containing proteins. These transcriptional activators coordinate the expression of genes essential for virulence in many species of gram-negative bacteria.

Identification of differential gene expression in bacteria associated with coral black band disease by using RNA-arbitrarily primed PCR.

Black band disease (BBD) is a virulent polymicrobial disease primarily affecting massive-framework-building species of scleractinian corals. While it has been well established that the BBD bacterial mat is dominated by a cyanobacterium, the quantitative composition of the BBD bacterial mat community has not described previously. Terminal-restriction fragment length polymorphism (T-RFLP) analysis was used to characterize the infectious bacterial community of the bacterial mat causing BBD. These analyses revealed that the bacterial composition of the BBD mat does not vary between different coral species but does vary when different species of cyanobacteria are dominant within the mat. On the basis of the results of a new method developed to identify organisms detected by T-RFLP analysis, our data show that besides the cyanobacterium, five species of the division Firmicutes, two species of the Cytophaga-Flexibacter-Bacteroides (CFB) group, and one species of delta-proteobacteria are also consistently abundant within the infectious mat. Of these dominant taxa, six were consistently detected in healthy corals. However, four of the six were found in much higher numbers in BBD mats than in healthy corals. One species of the CFB group and one species of Firmicutes were not always associated with the bacterial communities present in healthy corals. Of the eight dominant bacteria identified, two species were previously found in clone libraries obtained from BBD samples; however, these were not previously recognized as important. Furthermore, despite having been described as an important component of the pathogenetic mat, a Beggiatoa species was not detected in any of the samples analyzed. These results will permit the dominant BBD bacteria to be targeted for isolation and culturing experiments aimed at deciphering the disease etiology.

Bacterial community associated with black band disease in corals.

The high incidence of coral disease in shallow coastal marine environments suggests seawater depth and coastal pollution have an impact on the microbial communities inhabiting healthy coral tissues. A study was undertaken to determine how bacterial communities inhabiting tissues of the coral Montastraea annularis change at 5 m, 10 m and 20 m water depth in varying proximity to the urban centre and seaport of Willemstad, Curaçao, Netherlands Antilles. Analyses of terminal restriction fragment length polymorphisms (TRFLP) of 16S rRNA gene sequences show significant differences in bacterial communities of polluted and control localities only at the shallowest seawater depth. Furthermore, distinct differences in bacterial communities were found with increasing water depth. Comparisons of TRFLP peaks with sequenced clone libraries indicate the black band disease cyanobacterium clone CD1C11 is common and most abundant on healthy corals in less than 10 m water depth. Similarly, sequences belonging to a previously unrecognized group of likely phototrophic bacteria, herein referred to as CAB-I, were also more common in shallow water. To assess the influence of environmental and physiologic factors on bacterial community structure, canonical correspondence analysis was performed using explanatory variables associated with: (i) light availability; (ii) seawater pollution; (iii) coral mucus composition; (iv) the community structure of symbiotic algae; and (v) the photosynthetic activity of symbiotic algae. Eleven per cent of the variation in bacterial communities was accounted for by covariation with these variables; the most important being photosynthetically active radiation (sunlight) and the coral uptake of sewage-derived compounds as recorded by the delta(15)N of coral tissue.

Coral microbial communities, zooxanthellae and mucus along gradients of seawater depth and coastal pollution.

The Yellowstone caldera contains the most numerous and diverse geothermal systems on Earth, yielding an extensive array of unique high-temperature environments that host a variety of deeply-rooted and understudied Archaea, Bacteria and Eukarya. The combination of extreme temperature and chemical conditions encountered in geothermal environments often results in considerably less microbial diversity than other terrestrial habitats and offers a tremendous opportunity for studying the structure and function of indigenous microbial communities and for establishing linkages between putative metabolisms and element cycling. Metagenome sequence (14-15,000 Sanger reads per site) was obtained for five high-temperature (>65 degrees C) chemotrophic microbial communities sampled from geothermal springs (or pools) in Yellowstone National Park (YNP) that exhibit a wide range in geochemistry including pH, dissolved sulfide, dissolved oxygen and ferrous iron. Metagenome data revealed significant differences in the predominant phyla associated with each of these geochemical environments. Novel members of the Sulfolobales are dominant in low pH environments, while other Crenarchaeota including distantly-related Thermoproteales and Desulfurococcales populations dominate in suboxic sulfidic sediments. Several novel archaeal groups are well represented in an acidic (pH 3) Fe-oxyhydroxide mat, where a higher O2 influx is accompanied with an increase in archaeal diversity. The presence or absence of genes and pathways important in S oxidation-reduction, H2-oxidation, and aerobic respiration (terminal oxidation) provide insight regarding the metabolic strategies of indigenous organisms present in geothermal systems. Multiple-pathway and protein-specific functional analysis of metagenome sequence data corroborated results from phylogenetic analyses and clearly demonstrate major differences in metabolic potential across sites. The distribution of functional genes involved in electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, Fe, O2) control microbial community structure and function in YNP geothermal springs.

Metagenomes from high-temperature chemotrophic systems reveal geochemical controls on microbial community structure and function.

A microfluidic pore structure etched into a silicon wafer was used as a two-dimensional model subsurface sedimentary system (i.e., micromodel) to study mineral precipitation and permeability reduction relevant to groundwater remediation and geological carbon sequestration. Solutions containing CaCl(2) and Na(2)CO(3) at four different saturation states (Ω = [Ca(2+)][CO(3)(2-)]/K(spCaCO(3))) were introduced through two separate inlets, and they mixed by diffusion transverse to the main flow direction along the center of the micromodel resulting in CaCO(3) precipitation. Precipitation rates increased and the total amount of precipitates decreased with increasing saturation state, and only vaterite and calcite crystals were formed (no aragonite). The relative amount of vaterite increased from 80% at the lowest saturation state (Ω(v) = 2.8 for vaterite) to 95% at the highest saturation state (Ω(v) = 4.5). Fluorescent tracer tests conducted before and after CaCO(3) precipitation indicate that pore spaces were occluded by CaCO(3) precipitates along the transverse mixing zone, thus substantially reducing porosity and permeability, and potentially limiting transformation from vaterite to the more stable calcite. The results suggest that mineral precipitation along plume margins can decrease both reactant mixing during groundwater remediation, and injection and storage efficiency during CO(2) sequestration.

Pore-scale study of transverse mixing induced CaCO₃ precipitation and permeability reduction in a model subsurface sedimentary system.

The Aquificales are thermophilic microorganisms that inhabit hydrothermal systems worldwide and are considered one of the earliest lineages of the domain Bacteria. We analyzed metagenome sequence obtained from six thermal "filamentous streamer" communities (∼40 Mbp per site), which targeted three different groups of Aquificales found in Yellowstone National Park (YNP). Unassembled metagenome sequence and PCR-amplified 16S rRNA gene libraries revealed that acidic, sulfidic sites were dominated by Hydrogenobaculum (Aquificaceae) populations, whereas the circum-neutral pH (6.5-7.8) sites containing dissolved sulfide were dominated by Sulfurihydrogenibium spp. (Hydrogenothermaceae). Thermocrinis (Aquificaceae) populations were found primarily in the circum-neutral sites with undetectable sulfide, and to a lesser extent in one sulfidic system at pH 8. Phylogenetic analysis of assembled sequence containing 16S rRNA genes as well as conserved protein-encoding genes revealed that the composition and function of these communities varied across geochemical conditions. Each Aquificales lineage contained genes for CO2 fixation by the reverse-TCA cycle, but only the Sulfurihydrogenibium populations perform citrate cleavage using ATP citrate lyase (Acl). The Aquificaceae populations use an alternative pathway catalyzed by two separate enzymes, citryl-CoA synthetase (Ccs), and citryl-CoA lyase (Ccl). All three Aquificales lineages contained evidence of aerobic respiration, albeit due to completely different types of heme Cu oxidases (subunit I) involved in oxygen reduction. The distribution of Aquificales populations and differences among functional genes involved in energy generation and electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, H2, O2) have resulted in niche specialization among members of the Aquificales.

Metagenome sequence analysis of filamentous microbial communities obtained from geochemically distinct geothermal channels reveals specialization of three aquificales lineages.

A low-diversity microbial community, dominated by the γ-proteobacterium Halomonas sulfidaeris, was detected in samples of warm saline formation porewater collected from the Cambrian Mt. Simon Sandstone in the Illinois Basin of the North American Midcontinent (1.8 km/5872 ft burial depth, 50°C, pH 8, 181 bars pressure). These highly porous and permeable quartz arenite sandstones are directly analogous to reservoirs around the world targeted for large-scale hydrocarbon extraction, as well as subsurface gas and carbon storage. A new downhole low-contamination subsurface sampling probe was used to collect in situ formation water samples for microbial environmental metagenomic analyses. Multiple lines of evidence suggest that this H. sulfidaeris-dominated subsurface microbial community is indigenous and not derived from drilling mud microbial contamination. Data to support this includes V1-V3 pyrosequencing of formation water and drilling mud, as well as comparison with previously published microbial analyses of drilling muds in other sites. Metabolic pathway reconstruction, constrained by the geology, geochemistry and present-day environmental conditions of the Mt. Simon Sandstone, implies that H. sulfidaeris-dominated subsurface microbial community may utilize iron and nitrogen metabolisms and extensively recycle indigenous nutrients and substrates. The presence of aromatic compound metabolic pathways suggests this microbial community can readily adapt to and survive subsurface hydrocarbon migration.

Halomonas sulfidaeris-dominated microbial community inhabits a 1.8 km-deep subsurface Cambrian Sandstone reservoir.

Corals represent symbiotic meta-organisms that require harmonization among the coral animal, photosynthetic zooxanthellae and associated microbes to survive environmental stresses. We investigated integrated-responses among coral and zooxanthellae in the scleractinian coral Acropora formosa in response to an emerging marine pollutant, the munitions constituent, 1,3,5-trinitro-1,3,5 triazine (RDX; 5 day exposures to 0 (control), 0.5, 0.9, 1.8, 3.7, and 7.2 mg/L, measured in seawater).

Coral-zooxanthellae meta-transcriptomics reveals integrated response to pollutant stress.

A fundamental knowledge of microbial community structure in petroleum reservoirs can improve predictive modeling of these environments. We used hydrocarbon profiles, stable isotopes, and high-density DNA microarray analysis to characterize microbial communities in produced water from four Alaskan North Slope hydrocarbon reservoirs. Produced fluids from Schrader Bluff (24-27°C), Kuparuk (47-70°C), Sag River (80°C), and Ivishak (80-83°C) reservoirs were collected, with paired soured/non-soured wells sampled from Kuparuk and Ivishak. Chemical and stable isotope data suggested Schrader Bluff had substantial biogenic methane, whereas methane was mostly thermogenic in deeper reservoirs. Acetoclastic methanogens (Methanosaeta) were most prominent in Schrader Bluff samples, and the combined δD and δ(13)C values of methane also indicated acetoclastic methanogenesis could be a primary route for biogenic methane. Conversely, hydrogenotrophic methanogens (e.g., Methanobacteriaceae) and sulfide-producing Archaeoglobus and Thermococcus were more prominent in Kuparuk samples. Sulfide-producing microbes were detected in all reservoirs, uncoupled from souring status (e.g., the non-soured Kuparuk samples had higher relative abundances of many sulfate-reducers compared to the soured sample, suggesting sulfate-reducers may be living fermentatively/syntrophically when sulfate is limited). Sulfate abundance via long-term seawater injection resulted in greater relative abundances of Desulfonauticus, Desulfomicrobium, and Desulfuromonas in the soured Ivishak well compared to the non-soured well. In the non-soured Ivishak sample, several taxa affiliated with Thermoanaerobacter and Halomonas predominated. Archaea were not detected in the deepest reservoirs. Functional group taxa differed in relative abundance among reservoirs, likely reflecting differing thermal and/or geochemical influences.

Bruce W. Fouke

Institute for Genomic Biology,
Department of Geology,
Department of Microbiology

University of Illinois at Urbana-Champaign

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

Bruce W. Fouke

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute for Genomic Biology,Department of Geology,Department of Microbiology

University of Illinois at Urbana-Champaign

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

Institute for Genomic Biology,
Department of Geology,
Department of Microbiology