Real-Time PCR Quantification and Diversity Analysis of the Functional Genes aprA and dsrA of Sulfate-Reducing Prokaryotes in Marine Sediments of the Peru Continental Margin and the Black Sea

Sulfate-reducing prokaryotes (SRP) are ubiquitous and quantitatively important members in many ecosystems, especially in marine sediments. However their abundance and diversity in subsurface marine sediments is poorly understood. In this study, the abundance and diversity of the functional genes for the enzymes adenosine 5′-phosphosulfate reductase (aprA) and dissimilatory sulfite reductase (dsrA) of SRP in marine sediments of the Peru continental margin and the Black Sea were analyzed, including samples from the deep biosphere (ODP site 1227). For aprA quantification a Q-PCR assay was designed and evaluated. Depth profiles of the aprA and dsrA copy numbers were almost equal for all sites. Gene copy numbers decreased concomitantly with depth from around 108/g sediment close to the sediment surface to less than 105/g sediment at 5 mbsf. The 16S rRNA gene copy numbers of total bacteria were much higher than those of the functional genes at all sediment depths and used to calculate the proportion of SRP to the total Bacteria. The aprA and dsrA copy numbers comprised in average 0.5–1% of the 16S rRNA gene copy numbers of total bacteria in the sediments up to a depth of ca. 40 mbsf. In the zone without detectable sulfate in the pore water from about 40–121 mbsf (Peru margin ODP site 1227), only dsrA (but not aprA) was detected with copy numbers of less than 104/g sediment, comprising ca. 14% of the 16S rRNA gene copy numbers of total bacteria. In this zone, sulfate might be provided for SRP by anaerobic sulfide oxidation. Clone libraries of aprA showed that all isolated sequences originate from SRP showing a close relationship to aprA of characterized species or form a new cluster with only distant relation to aprA of isolated SRP. For dsrA a high diversity was detected, even up to 121 m sediment depth in the deep biosphere

The Deep Biosphere in Terrestrial Sediments in the Chesapeake Bay Area, Virginia, USA

For the first time quantitative data on the abundance of Bacteria, Archaea, and Eukarya in deep terrestrial sediments are provided using multiple methods (total cell counting, quantitative real-time PCR, Q-PCR and catalyzed reporter deposition–fluorescence in situ hybridization, CARD–FISH). The oligotrophic (organic carbon content of ∼0.2%) deep terrestrial sediments in the Chesapeake Bay area at Eyreville, Virginia, USA, were drilled and sampled up to a depth of 140 m in 2006. The possibility of contamination during drilling was checked using fluorescent microspheres. Total cell counts decreased from 109 to 106 cells/g dry weight within the uppermost 20 m, and did not further decrease with depth below. Within the top 7 m, a significant proportion of the total cell counts could be detected with CARD–FISH. The CARD–FISH numbers for Bacteria were about an order of magnitude higher than those for Archaea. The dominance of Bacteria over Archaea was confirmed by Q-PCR. The down core quantitative distribution of prokaryotic and eukaryotic small subunit ribosomal RNA genes as well as functional genes involved in different biogeochemical processes was revealed by Q-PCR for the uppermost 10 m and for 80–140 m depth. Eukarya and the Fe(III)- and Mn(IV)-reducing bacterial group Geobacteriaceae were almost exclusively found in the uppermost meter (arable soil), where reactive iron was detected in higher amounts. The bacterial candidate division JS-1 and the classes Anaerolineae and Caldilineae of the phylum Chloroflexi, highly abundant in marine sediments, were found up to the maximum sampling depth in high copy numbers at this terrestrial site as well. A similar high abundance of the functional gene cbbL encoding for the large subunit of RubisCO suggests that autotrophic microorganisms could be relevant in addition to heterotrophs. The functional gene aprA of sulfate reducing bacteria was found within distinct layers up to ca. 100 m depth in low copy numbers. The gene mcrA of methanogens was not detectable. Cloning and sequencing data of 16S rRNA genes revealed sequences of typical soil Bacteria. The closest relatives of the archaeal sequences were Archaea recovered from terrestrial and marine environments. Phylogenetic analysis of the Crenarchaeota and Euryarchaeota revealed new members of the uncultured South African Gold Mine Group, Deep Sea Hydrothermal Vent Euryarchaeotal Group 6, and Miscellaneous Crenarcheotic Group clusters

Subsurface microbiology and biogeochemistry of a deep, cold-water carbonate mound from the Porcupine Seabight (IODP Expedition 307)

The Porcupine Seabight Challenger Mound is the first carbonate mound to be drilled (∼270 m) and analyzed in detail microbiologically and biogeochemically. Two mound sites and a non-mound Reference site were analyzed with a range of molecular techniques [catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), quantitative PCR (16S rRNA and functional genes, dsrA and mcrA), and 16S rRNA gene PCR-DGGE] to assess prokaryotic diversity, and this was compared with the distribution of total and culturable cell counts, radiotracer activity measurements and geochemistry. There was a significant and active prokaryotic community both within and beneath the carbonate mound. Although total cell numbers at certain depths were lower than the global average for other subseafloor sediments and prokaryotic activities were relatively low (iron and sulfate reduction, acetate oxidation, methanogenesis) they were significantly enhanced compared with the Reference site. In addition, there was some stimulation of prokaryotic activity in the deepest sediments (Miocene, > 10 Ma) including potential for anaerobic oxidation of methane activity below the mound base. Both Bacteria and Archaea were present, with neither dominant, and these were related to sequences commonly found in other subseafloor sediments. With an estimate of some 1600 mounds in the Porcupine Basin alone, carbonate mounds may represent a significant prokaryotic subseafloor habitat

Phylogenetische und funktionelle Charakterisierung symbiotischer Bakterien in darmlosen Würmern

Symbioses between chemoautotrophic bacteria and eukaryotic hosts are widespread in marine environments. In most chemosynthetic endosymbioses, only a single, or at most two bacterial phylotypes co-occur within a host species.In this study the phylogenetic and metabolic diversity of bacterial endosymbionts in gutless marine worms (Annelida, Oligochaeta) from different environments was investigated. Almost all host species harbor a gammaproteobacterial sulfur oxidizer indicating the importance of these Gamma 1 symbionts for the nutrition of the gutless oligochaetes. A second gammaproteobacterial symbiont and deltaproteobacterial symbionts were detected in hosts from coastal silicate sediments, while in hosts from calcareous sands alphaproteobacterial symbionts were identified. Spirochetes were found in hosts from both types of sediments. The phylogenetic diversity of the bacterial symbionts mirrors their different metabolic capabilities. The Deltaproteobacteria have been identified as sulfate reducers and the secondary gammaproteobacterial symbionts are hypothesized to be sulfur oxidizers. Key genes involved in oxidative and reductive sulfur metabolism, CO2 fixation via the Calvin-Benson-Bassham (CBB) cycle, and nitrogen metabolism were successfully detected. Based on phylogenetic analyses it was possible to make potential assignments of genes to a respective symbiont.The use of comparative metagenomics gave first insights into the genome of a gutless oligochaete symbiont. A contiguous sequence of 51 kb from a bacterial artificial chromosome insert contained genes involved in significant metabolic pathways for these symbioses such as sulfur oxidation and CO2 fixation via the CBB cycle indicating that this sequence originated from a thioautotrophic symbiont. This study showed that the symbiotic community in marine gutless oligochaetes with at least three and as many as six different symbiotic phylotypes is much more complex than previously assumed. Despite the high phylogenetic diversity, these associations are clearly specific and stable for most phylotypes within a given host species

Phylogenetic and functional characterization of symbiotic bacteria in gutless marine worms (Annelida, Oligochaeta)

Symbioses between chemoautotrophic bacteria and eukaryotic hosts are widespread in marine environments. In most chemosynthetic endosymbioses, only a single, or at most two bacterial phylotypes co-occur within a host species.In this study the phylogenetic and metabolic diversity of bacterial endosymbionts in gutless marine worms (Annelida, Oligochaeta) from different environments was investigated. Almost all host species harbor a gammaproteobacterial sulfur oxidizer indicating the importance of these Gamma 1 symbionts for the nutrition of the gutless oligochaetes. A second gammaproteobacterial symbiont and deltaproteobacterial symbionts were detected in hosts from coastal silicate sediments, while in hosts from calcareous sands alphaproteobacterial symbionts were identified. Spirochetes were found in hosts from both types of sediments. The phylogenetic diversity of the bacterial symbionts mirrors their different metabolic capabilities. The Deltaproteobacteria have been identified as sulfate reducers and the secondary gammaproteobacterial symbionts are hypothesized to be sulfur oxidizers. Key genes involved in oxidative and reductive sulfur metabolism, CO2 fixation via the Calvin-Benson-Bassham (CBB) cycle, and nitrogen metabolism were successfully detected. Based on phylogenetic analyses it was possible to make potential assignments of genes to a respective symbiont.The use of comparative metagenomics gave first insights into the genome of a gutless oligochaete symbiont. A contiguous sequence of 51 kb from a bacterial artificial chromosome insert contained genes involved in significant metabolic pathways for these symbioses such as sulfur oxidation and CO2 fixation via the CBB cycle indicating that this sequence originated from a thioautotrophic symbiont. This study showed that the symbiotic community in marine gutless oligochaetes with at least three and as many as six different symbiotic phylotypes is much more complex than previously assumed. Despite the high phylogenetic diversity, these associations are clearly specific and stable for most phylotypes within a given host species

Coexistence of Bacterial Sulfide Oxidizers, Sulfate Reducers, and Spirochetes in a Gutless Worm (Oligochaeta) from the Peru Margin

Olavius crassitunicatus is a small symbiont-bearing worm that occurs at high abundance in oxygen-deficient sediments in the East Pacific Ocean. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization, we examined the diversity and phylogeny of bacterial symbionts in two geographically distant O. crassitunicatus populations (separated by 385 km) on the Peru margin (water depth, ∼300 m). Five distinct bacterial phylotypes co-occurred in all specimens from both sites: two members of the γ-Proteobacteria (Gamma 1 and 2 symbionts), two members of the δ-Proteobacteria (Delta 1 and 2 symbionts), and one spirochete. A sixth phylotype belonging to the δ-Proteobacteria (Delta 3 symbiont) was found in only one of the two host populations. Three of the O. crassitunicatus bacterial phylotypes are closely related to symbionts of other gutless oligochaete species; the Gamma 1 phylotype is closely related to sulfide-oxidizing symbionts of Olavius algarvensis, Olavius loisae, and Inanidrilus leukodermatus, the Delta 1 phylotype is closely related to sulfate-reducing symbionts of O. algarvensis, and the spirochete is closely related to spirochetal symbionts of O. loisae. In contrast, the Gamma 2 phylotype and the Delta 2 and 3 phylotypes belong to novel lineages that are not related to other bacterial symbionts. Such a phylogenetically diverse yet highly specific and stable association in which multiple bacterial phylotypes coexist within a single host has not been described previously for marine invertebrates

Microbial photosynthesis in coral reef sediments (Heron Reef, Australia)

We investigated microphytobenthic photosynthesis at four stations in the coral reef sediments at Heron Reef, Australia. The microphytobenthos was dominated by diatoms, dinoflagellates and cyanobacteria, as indicated by biomarker pigment analysis. Conspicuous algae firmly attached to the sand grains (ca. 100 mm in diameter, surrounded by a hard transparent wall) were rich in peridinin, a marker pigment for dinoflagellates, but also showed a high diversity based on cyanobacterial 16S rDNA gene sequence analysis. Specimens of these algae that were buried below the photic zone exhibited an unexpected stimulation of respiration by light, resulting in an increase of local oxygen concentrations upon darkening. Net photosynthesis of the sediments varied between 1.9 and 8.5 mmol O2 m2 h1 and was strongly correlated with Chl a content, which lay between 31 and 84 mg m2. An estimate based on our spatially limited dataset indicates that the microphytobenthic production for the entire reef is in the order of magnitude of the production estimated for corals. Photosynthesis stimulated calcification at all investigated sites (0.2e1.0 mmol Ca2þ m2 h1). The sediments of at least three stations were net calcifying. Sedimentary N2-fixation rates (measured by acetylene reduction assays at two sites) ranged between 0.9 to 3.9 mmol N2 m2 h1 and were highest in the light, indicating the importance of heterocystous cyanobacteria. In coral fingers no N2-fixation was measurable, which stresses the importance of the sediment compartment for reef nitrogen cycling. 2007 Elsevier Lt