Transformation and mineralization of nitrogenous soil components in the gut of soil-feeding termites

This thesis consists of several studies that focused on the role of soil-feeding termites and termite gut microbiota in the transformation and mineralization of nitrogenous soil components. The results can be summarized into four subject matters, namely: 1. N mineralization and transformation during soil gut passage In order to better understand the role of soil-feeding termites in the dynamics of N in tropical soils, soil microcosms that received 15N tracers were incubated with termites. Here, our results demonstrated the importance of nitrogenous soil components (peptides) in the diet of soilfeeding termites, providing close to 50% of the termite’s carbon flux. The mineralization process, also results in the formation of enormous amounts of ammonia both in the gut (~150 mM) and the nest material. Additionally, we provided the first evidence for a termite-associated nitrification activity during the feeding activities of termites, which is coupled to denitrification and dissimilatory nitrate reduction to ammonia. At the ecosystem level, soil-feeding termites are estimated to contribute more towards N retention than to N loss in tropical soils. 2. Roles of termite gut microbiota in peptide breakdown and amino acids turnover Using gut homogenates, our studies revealed that termite gut microbiota play major roles in the hydrolysis and mineralization of peptidic components of soil organic matter. Both proteolytic and lysozyme activities were associated with termite tissues (i.e., salivary glands) and also the particulate fraction of the gut content. Together with the high alkalinity of the gut, soil peptides and microbial biomass are sequentially subjected to hydrolysis, solubilization, and extraction in the intestinal tract. Amino acids, which accumulate, are either directly absorbed by the insect or turned over by the dense hindgut microbiota, preferably by anaerobic amino-acid-fermenting bacteria. This underscores theimportant role of termite gut microbiota and the in situ physiological gut conditions, in enhancing the mineralization and utilization of peptidic components of soil organic matter by the termite. 3. Intestinal nitrate reduction leads to N2O and N2 emission Nitrate, a product of the nitrification activities in the gut, is reduced by the intestinal microbiota either to N2O and N2, or to ammonia. The reduction of nitrate to ammonia takes place mainly in the anterior gut region whereas denitrification occurs in the posterior hindgut. Virtually, no nitrate-reducing activities were present in the alkaline gut sections. Living termites emit both N2O and N2, but the emission of N2 rather than N2O seems to be the prevalent nitrogenous gas produced by soil-feeding termites. Nitrate reduction via denitrification represents ~26% of the total electrons flowing through methanogenesis in the intestinal tracts of soil-feeding termites. This study documents the first report on intestinal nitrate reduction to N2 and also provides the first evidence of soilfeeding termites as a source of the greenhouse gas N2O. 4. Excretion of ammonia via an “acid-trap” mechanism Soil-feeding termites preferentially utilize the peptidic components of soil organic matter. Consequently, ammonia levels in the hemolymph (~300 μM) and in the gut fluid accumulate to enormous concentrations. Using 15N tracers, we demonstrate that the alkalinity of the gut plays an important role removing ammonia by volatilizing NH4 + to NH3, which then diffuses into the hemolymph. Subsequently, NH3 is entrapped in the posterior hindgut with a circumneutral pH, most likely via an active transport mechanism. Finally, ammonia is egested through feces into the nest material. Also, preliminary evidence alludes to the role of Malpighian tubules in the excretion of ammonia as uric acid, a hitherto unknown function in soil-feeding termites

Rhodococcus opacus strain RW, a resorcinol-degrading bacterium from the gut of Macrotermes michaelseni

The population of resorcinol degrading bacteria in the intestinal tract of fungi-cultivating termite, Macrotermes michaelseni, was estimated to be 6.8 × 102 cells/ml. A gram-positive bacterium designated RW, capable of degrading resorcinol, was isolated from the highest positive dilution. Isolate RW could also degrade phenol and benzoic acid aerobically, and anaerobically using nitrate as an electron acceptor. The isolate is a rod-shaped bacterium that exhibited evolutionary relatedness with the genus Rhodococcus, as determined by phenotypic traits and physiological tests, and a 16S rRNA gene sequence similarity value of 99.6% to the closest Rhodococcus opacus strain. On the basis of these results isolate RW is proposed as a new strain in the species R. opacus. The ability of the isolate to degrade resorcinol, phenol and benzoic acid makes it a potential candidate for use in bioremediation of environments contaminated by such or related compounds.African Journal of Biotechnology Vol. 4 (7), pp. 639-645, 200

High-frequency variability of bacterioplankton in response to environmental drivers in Red Sea coastal waters

Autotrophic and heterotrophic bacterioplankton are essential to the biogeochemistry of tropical ecosystems. However, the processes that govern their dynamics are not well known. We provide here a high-frequency assessment of bacterial community dynamics and concurrent environmental factors in Red Sea coastal waters. Weekly sampling of surface samples during a full annual cycle at an enclosed station revealed high variability in ecological conditions, which reflected in changes of major bacterioplankton communities. Temperature varied between 23 and 34°C during the sampling period. Autotrophic (Synechococcus, 1.7–16.2 × 104 cells mL−1) and heterotrophic bacteria (1.6–4.3 × 105 cells mL−1) showed two maxima in abundance in spring and summer, while minima were found in winter and autumn. Heterotrophic cells with high nucleic acid content (HNA) peaked in July, but their contribution to the total cell counts (35–60%) did not show a clear seasonal pattern. Actively respiring cells (CTC+) contributed between 4 and 51% of the total number of heterotrophic bacteria, while live cells (with intact membrane) consistently accounted for over 90%. Sequenced 16S rRNA amplicons revealed a predominance of Proteobacteria in summer and autumn (>40%) and a smaller contribution in winter (21–24%), with members of the Alphaproteobacteria class dominating throughout the year. The contribution of the Flavobacteriaceae family was highest in winter (21%), while the Rhodobacteraceae contribution was lowest (6%). Temperature, chlorophyll-a, and dissolved organic carbon concentration were the environmental variables with the greatest effects on bacterial abundance and diversity patterns

Postglacial adaptations enabled colonization and quasi-clonal dispersal of ammonia-oxidizing archaea in modern European large lakes

Ammonia-oxidizing archaea (AOA) play a key role in the aquatic nitrogen cycle. Their genetic diversity is viewed as the outcome of evolutionary processes that shaped ancestral transition from terrestrial to marine habitats. However, current genome-wide insights into AOA evolution rarely consider brackish and freshwater representatives or provide their divergence timeline in lacustrine systems. An unbiased global assessment of lacustrine AOA diversity is critical for understanding their origins, dispersal mechanisms, and ecosystem roles. Here, we leveraged continental-scale metagenomics to document that AOA species diversity in freshwater systems is remarkably low compared to marine environments. We show that the uncultured freshwater AOA, "Candidatus Nitrosopumilus limneticus," is ubiquitous and genotypically static in various large European lakes where it evolved 13 million years ago. We find that extensive proteome remodeling was a key innovation for freshwater colonization of AOA. These findings reveal the genetic diversity and adaptive mechanisms of a keystone species that has survived clonally in lakes for millennia

Combined Analyses of the ITS Loci and the Corresponding 16S rRNA Genes Reveal High Micro- and Macrodiversity of SAR11 Populations in the Red Sea

<div><p>Bacteria belonging to the SAR11 clade are among the most abundant prokaryotes in the pelagic zone of the ocean. 16S rRNA gene-based analyses indicate that they constitute up to 60% of the bacterioplankton community in the surface waters of the Red Sea. This extremely oligotrophic water body is further characterized by an epipelagic zone, which has a temperature above 24°C throughout the year, and a remarkable uniform temperature (∼22°C) and salinity (∼41 psu) from the mixed layer (∼200 m) to the bottom at over 2000 m depth. Despite these conditions that set it apart from other marine environments, the microbiology of this ecosystem is still vastly understudied. Prompted by the limited phylogenetic resolution of the 16S rRNA gene, we extended our previous study by sequencing the internal transcribed spacer (ITS) region of SAR11 in different depths of the Red Sea’s water column together with the respective 16S fragment. The overall diversity captured by the ITS loci was ten times higher than that of the corresponding 16S rRNA genes. Moreover, species estimates based on the ITS showed a highly diverse population of SAR11 in the mixed layer that became diminished in deep isothermal waters, which was in contrast to results of the related 16S rRNA genes. While the 16S rRNA gene-based sequences clustered into three phylogenetic subgroups, the related ITS fragments fell into several phylotypes that showed clear depth-dependent shifts in relative abundances. Blast-based analyses not only documented the observed vertical partitioning and universal co-occurrence of specific phylotypes in five other distinct oceanic provinces, but also highlighted the influence of ecosystem-specific traits (e.g., temperature, nutrient availability, and concentration of dissolved oxygen) on the population dynamics of this ubiquitous marine bacterium.</p> </div

Transformation and mineralization of nitrogenous soil components in the gut of soil-feeding termites

This thesis consists of several studies that focused on the role of soil-feeding termites and termite gut microbiota in the transformation and mineralization of nitrogenous soil components. The results can be summarized into four subject matters, namely: 1. N mineralization and transformation during soil gut passage In order to better understand the role of soil-feeding termites in the dynamics of N in tropical soils, soil microcosms that received 15N tracers were incubated with termites. Here, our results demonstrated the importance of nitrogenous soil components (peptides) in the diet of soilfeeding termites, providing close to 50% of the termite’s carbon flux. The mineralization process, also results in the formation of enormous amounts of ammonia both in the gut (~150 mM) and the nest material. Additionally, we provided the first evidence for a termite-associated nitrification activity during the feeding activities of termites, which is coupled to denitrification and dissimilatory nitrate reduction to ammonia. At the ecosystem level, soil-feeding termites are estimated to contribute more towards N retention than to N loss in tropical soils. 2. Roles of termite gut microbiota in peptide breakdown and amino acids turnover Using gut homogenates, our studies revealed that termite gut microbiota play major roles in the hydrolysis and mineralization of peptidic components of soil organic matter. Both proteolytic and lysozyme activities were associated with termite tissues (i.e., salivary glands) and also the particulate fraction of the gut content. Together with the high alkalinity of the gut, soil peptides and microbial biomass are sequentially subjected to hydrolysis, solubilization, and extraction in the intestinal tract. Amino acids, which accumulate, are either directly absorbed by the insect or turned over by the dense hindgut microbiota, preferably by anaerobic amino-acid-fermenting bacteria. This underscores theimportant role of termite gut microbiota and the in situ physiological gut conditions, in enhancing the mineralization and utilization of peptidic components of soil organic matter by the termite. 3. Intestinal nitrate reduction leads to N2O and N2 emission Nitrate, a product of the nitrification activities in the gut, is reduced by the intestinal microbiota either to N2O and N2, or to ammonia. The reduction of nitrate to ammonia takes place mainly in the anterior gut region whereas denitrification occurs in the posterior hindgut. Virtually, no nitrate-reducing activities were present in the alkaline gut sections. Living termites emit both N2O and N2, but the emission of N2 rather than N2O seems to be the prevalent nitrogenous gas produced by soil-feeding termites. Nitrate reduction via denitrification represents ~26% of the total electrons flowing through methanogenesis in the intestinal tracts of soil-feeding termites. This study documents the first report on intestinal nitrate reduction to N2 and also provides the first evidence of soilfeeding termites as a source of the greenhouse gas N2O. 4. Excretion of ammonia via an “acid-trap” mechanism Soil-feeding termites preferentially utilize the peptidic components of soil organic matter. Consequently, ammonia levels in the hemolymph (~300 μM) and in the gut fluid accumulate to enormous concentrations. Using 15N tracers, we demonstrate that the alkalinity of the gut plays an important role removing ammonia by volatilizing NH4 + to NH3, which then diffuses into the hemolymph. Subsequently, NH3 is entrapped in the posterior hindgut with a circumneutral pH, most likely via an active transport mechanism. Finally, ammonia is egested through feces into the nest material. Also, preliminary evidence alludes to the role of Malpighian tubules in the excretion of ammonia as uric acid, a hitherto unknown function in soil-feeding termites

Phylogenetic diversity, distribution, and cophylogeny of giant bacteria (Epulopiscium) with their surgeonfish hosts in the Red Sea.

Epulopiscium is a group of giant bacteria found in high abundance in intestinal tracts of herbivorous surgeonfish. Despite their peculiarly large cell size (can be up to 600 µm), extreme polyploidy (some with over 100,000 genome copies per cell) and viviparity (whereby mother cells produce live offspring), details about their diversity, distribution or their role in the host gut are lacking. Previous studies have highlighted the existence of morphologically distinct Epulopiscium cell types (defined as morphotypes A to J) in surgeonfish genera, but the corresponding genetic diversity and distribution among other surgeonfishes remain mostly unknown. Therefore, we investigated the phylogenetic diversity of Epulopiscium, distribution and co-occurrence in multiple unrelated hosts. Here, we identified eleven new phylogenetic clades, six of which were also morphologically characterized. Three of these novel clades were phylogenetically and morphologically similar to cigar-shaped type A1 cells, found in a wide range of surgeonfishes including Acanthurus nigrofuscus, while three were similar to smaller, rod-shaped type E that has not been phylogenetically classified thus far. Our results also confirmed that biogeography appears to have relatively little influence on Epulopiscium diversity, as clades found in the Great Barrier Reef (GBR) and Hawaii were also recovered from the Red Sea. Although multiple symbiont clades inhabited a given species of host surgeonfish and multiple host species possessed a given symbiont clade, statistical analysis of host and symbiont phylogenies indicated significant cophylogeny, which in turn suggests co-evolutionary relationships. A cluster analysis of Epulopiscium sequences from previously published amplicon sequencing dataset revealed a similar pattern, where specific clades were consistently found in high abundance amongst closely related surgeonfishes. Differences in abundance may indicate specialization of clades to certain gut environments reflected by inferred differences in the host diets. Overall, our analysis identified a large phylogenetic diversity of Epulopiscium (up to 10% sequence divergence of 16S rRNA genes), which lets us hypothesize that there are multiple species that are spread across guts of different host species

16S rRNA gene-based phylogenetic tree of subgroups and their relative abundances in the Red Sea’s water column.

<p>(A) Phylogram showing collapsed nodes (subgroups) that produced significant bootstraps with both neighbor-joining and maximum parsimony approaches in trees generated using PAUP. Numbers in bracket denote intra−/intergenetic divergence within each subgroup; the latter was calculated by comparing the rest with S1a. A detailed phylogenetic tree is provided as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050274#pone.0050274.s004" target="_blank">Figure S4</a>. (B) Shows the shift in the relative abundances of each subgroup at different depths of the Red Sea’s water column.</p