Urine proteome profile of firefighters with exposure to emergency fire-induced smoke: A pilot study to identify potential carcinogenic effects.

Firefighters are frequently exposed to a variety of chemicals formed from smoke, which pose a risk for numerous diseases, including cancer. Comparative urine proteome profiling could significantly improve our understanding of the early detection of potential cancer biomarkers. In this study, for the first time, we conducted a comparative protein profile analysis of 20 urine samples collected from ten real-life firefighters prior to and following emergency fire-induced smoke. Using a label-free quantitative proteomics platform, we identified and quantified 1325 unique protein groups, of which 45 proteins showed differential expressions in abundance in response to fire-smoke exposure (post) compared to the control (pre). Pathway analysis showed proteins associated with epithelium development (e.g., RHCG, HEG1, ADAMTSL2) and Alzheimer's disease (SORL1) were significantly increased in response to smoke exposure samples. A protein-protein-network study showed a possible link between these differentially abundant proteins and the known cancer gene (TP53). Moreover, a cross-comparison analysis revealed that seven proteins-ALDH1A1, APCS, POMC, COL2A1, RDX, DDAH2, and SDC4 overlapped with the previously published urine cancer proteome datasets, suggesting a potential cancer risk. Our findings demonstrated that the discovery proteomic platform is a promising analytical technique for identifying potential non-invasive biomarkers associated with fire-smoke exposure in firefighters that may be related to cancer.

idMotif: An Interactive Motif Identification in Protein Sequences.

This article presents a visual analytics framework, idMotif, to support domain experts in identifying motifs in protein sequences. A motif is a short sequence of amino acids usually associated with distinct functions of a protein, and identifying similar motifs in protein sequences helps to predict certain types of disease or infection. idMotif can be used to explore, analyze, and visualize such motifs in protein sequences. We introduce a deep-learning-based method for grouping protein sequences and allow users to discover motif candidates of protein groups based on local explanations of the decision of a deep-learning model. idMotif provides several interactive linked views for between and within protein cluster/group and sequence analysis. Through a case study and experts' feedback, we demonstrate how the framework helps domain experts analyze protein sequences and motif identification.

Emerging variants of SARS-CoV-2 NSP10 highlight strong functional conservation of its binding to two non-structural proteins, NSP14 and NSP16.

The coronavirus SARS-CoV-2 protects its RNA from being recognized by host immune responses by methylation of its 5' end, also known as capping. This process is carried out by two enzymes, non-structural protein 16 (NSP16) containing 2'-O-methyltransferase and NSP14 through its N7 methyltransferase activity, which are essential for the replication of the viral genome as well as evading the host's innate immunity. NSP10 acts as a crucial cofactor and stimulator of NSP14 and NSP16. To further understand the role of NSP10, we carried out a comprehensive analysis of >13 million globally collected whole-genome sequences (WGS) of SARS-CoV-2 obtained from the Global Initiative Sharing All Influenza Data (GISAID) and compared it with the reference genome Wuhan/WIV04/2019 to identify all currently known variants in NSP10. T12I, T102I, and A104V in NSP10 have been identified as the three most frequent variants and characterized using X-ray crystallography, biophysical assays, and enhanced sampling simulations. In contrast to other proteins such as spike and NSP6, NSP10 is significantly less prone to mutation due to its crucial role in replication. The functional effects of the variants were examined for their impact on the binding affinity and stability of both NSP14-NSP10 and NSP16-NSP10 complexes. These results highlight the limited changes induced by variant evolution in NSP10 and reflect on the critical roles NSP10 plays during the SARS-CoV-2 life cycle. These results also indicate that there is limited capacity for the virus to overcome inhibitors targeting NSP10 via the generation of variants in inhibitor binding pockets.

Proteomics evaluation of five economical commercial abundant protein depletion kits for enrichment of diseases-specific biomarkers from blood serum.

Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease-specific biomarkers in human serum using bottom-up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%-19% variation in protein identification among the kits. Immunocapturing-based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non-antibody-based methods (i.e., kits using ion exchange resins) and kits leveraging a multi-antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom-up proteomic results revealed that different SAPD kits enrich distinct disease- and pathway-specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.

Future COVID19 surges prediction based on SARS-CoV-2 mutations surveillance.

COVID19 has aptly revealed that airborne viruses such as SARS-CoV-2 with the ability to rapidly mutate combined with high rates of transmission and fatality can cause a deadly worldwide pandemic in a matter of weeks (Plato et al., 2021). Apart from vaccines and post-infection treatment options, strategies for preparedness will be vital in responding to the current and future pandemics. Therefore, there is wide interest in approaches that allow predictions of increase in infections ('surges') before they occur. We describe here real-time genomic surveillance particularly based on mutation analysis, of viral proteins as a methodology for a priori determination of surge in number of infection cases. The full results are available for SARS-CoV-2 at http://pandemics.okstate.edu/covid19/, and are updated daily as new virus sequences become available. This approach is generic and will also be applicable to other pathogens.

Desulfoferrobacter suflitae gen. nov., sp. nov., a novel sulphate-reducing bacterium in the Deltaproteobacteria capable of autotrophic growth with hydrogen or elemental iron.

A mesophilic sulphate-reducing micro-organism, able to grow chemolithoautotrophically with H2/CO2 (20 : 80) and with elemental iron as a sole electron donor, was isolated from a consortium capable of degrading long-chain paraffins and designated strain DRH4T. Cells were oval shaped often with bright refractile cores and occurred singly or in pairs. The cells formed pili. Strain DRH4T could grow chemolithoautotrophically with H2/CO2 or elemental iron and chemoorganotrophically utilizing a number of organic substrates, such as fatty acids from formate to octanoate (C1-C8). Sulphate and thiosulphate served as terminal electron acceptors, but sulphite and nitrate did not. Optimal growth was observed from 37 to 40 °C and pH from 6.5 to 7.2. Strain DRH4T did not require NaCl for growth and could proliferate under a broad range of salinities from freshwater (1 g l-1 NaCl) to seawater (27 g l-1 NaCl) conditions. The genomic DNA G+C content was 54.46 mol %. Based on 16S rRNA gene sequence analysis. strain DRH4T was distinct from previously described Deltaproteobacteria species exhibiting the closest affiliation to Desulforhabdus amnigena ASRB1T, Syntrophobacterium sulfatireducens TB8106T and Desulfovirga adipica 12016T with 93.35, 93.42 and 92.85 % similarity, respectively. Strain DRH4T showed significant physiological differences with the aforementioned organisms. Based on physiological differences and phylogenetic comparisons, we propose to classify DRH4T as the type strain (=DSM 113 455T=JCM 39 248T) of a novel species of a new genus with the name Desulfoferrobacter suflitae gen. nov., sp. nov.

Lentil allergens identification and quantification: An update from omics perspective.

Among legumes, the lentil (Lens culinaris) is a major dietary component in many Mediterranean and Asian countries due to its high nutritional value, especially protein. However, allergic reactions triggered by lentil consumption have also been documented in many countries. Complete allergens profiling is critical for better management of lentil food allergies. Earlier studies suggested Len c 1, a 47 kDa vicilin, Len c 2, a seed-specific-biotinylated 66-kDa protein, and Len c 3, low molecular weight lipid transfer proteins (LTPs) were major allergenic proteins in lentils. Recently, mass-spectrometry-based proteomic platforms successfully identified proteins from lentil samples homologous to known plant allergens. Furthermore, in silico analysis using 337 protein sequences revealed lentil allergens that have not previously been identified as potential allergens in lentil. Herein, we discuss the feasibility of omics platforms utilized for lentil allergens profiling and quantification. In addition, we propose some future strategies that might be beneficial for profiling and development of precise assays for lentil allergens and could facilitate identification of the low allergen-containing lentil cultivars.

Assessing Microbial Metabolic and Biological Diversity to Inform Natural Product Library Assembly.

The pressing need for novel chemical matter to support bioactive compound discovery has led natural product researchers to explore a wide range of source organisms and environments. One of the implicit guiding principles behind those efforts is the notion that sampling different environments is critical to accessing unique natural products. This idea was tested by comparing fungi from disparate biomes: aquatic sediments from Lake Michigan (USA) and terrestrial samples taken from the surrounding soils. Matched sets of Penicillium brevicompactum, Penicillium expansum, and Penicillium oxalicum from the two source environments were compared, revealing modest differences in physiological performance and chemical output. Analysis of LC-MS/MS-derived molecular feature data showed no source-dependent differences in chemical richness. High levels of scaffold homogeneity were also observed with 78-83% of scaffolds shared among the terrestrial and aquatic Penicillium spp. isolates. A comparison of the culturable fungi from the two biomes indicated that certain genera were more strongly associated with aquatic sediments (e.g., Trichoderma, Pseudeurotium, Cladosporium, and Preussia) versus the surrounding terrestrial environment (e.g., Fusarium, Pseudogymnoascus, Humicola, and Acremonium). Taken together, these results suggest that focusing efforts on sampling the microbial resources that are unique to an environment may have a more pronounced effect on enhancing the sought-after natural product diversity needed for chemical discovery and screening collections.

Building Natural Product Libraries Using Quantitative Clade-Based and Chemical Clustering Strategies.

The success of natural product-based drug discovery is predicated on having chemical collections that offer broad coverage of metabolite diversity. We propose a simple set of tools combining genetic barcoding and metabolomics to help investigators build natural product libraries aimed at achieving predetermined levels of chemical coverage. It was found that such tools aided in identifying overlooked pockets of chemical diversity within taxa, which could be useful for refocusing collection strategies. We have used fungal isolates identified as Alternaria from a citizen-science-based soil collection to demonstrate the application of these tools for assessing and carrying out predictive measurements of chemical diversity in a natural product collection. Within Alternaria, different subclades were found to contain nonequivalent levels of chemical diversity. It was also determined that a surprisingly modest number of isolates (195 isolates) was sufficient to afford nearly 99% of Alternaria chemical features in the data set. However, this result must be considered in the context that 17.9% of chemical features appeared in single isolates, suggesting that fungi like Alternaria might be engaged in an ongoing process of actively exploring nature's metabolic landscape. Our results demonstrate that combining modest investments in securing internal transcribed spacer (ITS)-based sequence information (i.e., establishing gene-based clades) with data from liquid chromatography-mass spectrometry (i.e., generating feature accumulation curves) offers a useful route to obtaining actionable insights into chemical diversity coverage trends in a natural product library. It is anticipated that these outcomes could be used to improve opportunities for accessing bioactive molecules that serve as the cornerstone of natural product-based drug discovery. IMPORTANCE Natural product drug discovery efforts rely on libraries of organisms to provide access to diverse pools of compounds. Actionable strategies to rationally maximize chemical diversity, rather than relying on serendipity, can add value to such efforts. Readily implementable biological (i.e., ITS sequence analysis) and chemical (i.e., mass spectrometry-based feature and scaffold measurements) diversity assessment tools can be employed to monitor and adjust library development tactics in real time. In summary, metabolomics-driven technologies and simple gene-based specimen barcoding approaches have broad applicability to building chemically diverse natural product libraries.

Expression of different ParE toxins results in conserved phenotypes with distinguishable classes of toxicity.

Toxin-antitoxin (TA) systems are found on both chromosomes and plasmids. These systems are unique in that they can confer both fatal and protective effects on bacterial cells-a quality that could potentially be harnessed given further understanding of these TA mechanisms. The current work focuses on the ParE subfamily, which is found throughout proteobacteria and has a sequence identity on average of approximately 12% (similarity at 30%-80%). Our aim is to evaluate the equivalency of chromosomally derived ParE toxin activity depending on its bacterial species of origin. Nine ParE toxins were analyzed, originating from six different bacterial species. Based on the resulting toxicity, three categories can be established: ParE toxins that do not exert toxicity under the experimental conditions, toxins that exert toxicity within the first four hours, and those that exert toxicity only after 10-12 hr of exposure. All tested ParE toxins produce a cellular morphologic change from rods to filaments, consistent with disruption of DNA topology. Analysis of the distribution of filamented cells within a population reveals a correlation between the extent of filamentation and toxicity. No membrane septation is visible along the length of the cell filaments, whereas aberrant lipid blebs are evident. Potent ParE-mediated toxicity is also correlated with a hallmark signature of abortive DNA replication, consistent with the inhibition of DNA gyrase.

Virulence factor landscape of a Staphylococcus aureus sequence type 45 strain, MCRF184.

BACKGROUND: We describe the virulence factors of a methicillin-sensitive Staphylococcus aureus sequence type (ST) 45 strain, MCRF184, (spa type t917), that caused severe necrotizing fasciitis in a 72-year-old diabetic male. The genome of MCRF184 possesses three genomic islands: a relatively large type III νSaα with 42 open reading frames (ORFs) that includes superantigen- and lipoprotein-like genes, a truncated νSaß that consists mostly of the enterotoxin gene cluster (egc), and a νSaγ island with 18 ORFs including α-toxin. Additionally, the genome has two phage-related regions: phage φSa3 with three genes of the immune evasion cluster (IEC), and an incomplete phage that is distinct from other S. aureus phages. Finally, the region between orfX and orfY harbors a putative efflux pump, acetyltransferase, regulators, and mobilization genes instead of genes of SCCmec. RESULTS: Virulence factors included phenol soluble modulins (PSMs) α1 through α4 and PSMs ß1 and ß2. Ten ORFs identified in MCRF184 had not been reported in previously sequenced S. aureus strains. CONCLUSION: The dire clinical outcome in the patient and the described virulence factors all suggest that MCRF184, a ST45 strain is a highly virulent strain of S. aureus.

Conserved DNA motifs in the type II-A CRISPR leader region.

The Clustered Regularly Interspaced Short Palindromic Repeats associated (CRISPR-Cas) systems consist of RNA-protein complexes that provide bacteria and archaea with sequence-specific immunity against bacteriophages, plasmids, and other mobile genetic elements. Bacteria and archaea become immune to phage or plasmid infections by inserting short pieces of the intruder DNA (spacer) site-specifically into the leader-repeat junction in a process called adaptation. Previous studies have shown that parts of the leader region, especially the 3' end of the leader, are indispensable for adaptation. However, a comprehensive analysis of leader ends remains absent. Here, we have analyzed the leader, repeat, and Cas proteins from 167 type II-A CRISPR loci. Our results indicate two distinct conserved DNA motifs at the 3' leader end: ATTTGAG (noted previously in the CRISPR1 locus of Streptococcus thermophilus DGCC7710) and a newly defined CTRCGAG, associated with the CRISPR3 locus of S. thermophilus DGCC7710. A third group with a very short CG DNA conservation at the 3' leader end is observed mostly in lactobacilli. Analysis of the repeats and Cas proteins revealed clustering of these CRISPR components that mirrors the leader motif clustering, in agreement with the coevolution of CRISPR-Cas components. Based on our analysis of the type II-A CRISPR loci, we implicate leader end sequences that could confer site-specificity for the adaptation-machinery in the different subsets of type II-A CRISPR loci.

Crystal structures of two nitroreductases from hypervirulent Clostridium difficile and functionally related interactions with the antibiotic metronidazole.

Nitroreductases (NRs) are flavin mononucleotide (FMN)-dependent enzymes that catalyze the biotransformation of organic nitro compounds (RNO2; R = alkyl, aryl) to the nitroso RN=O, hydroxylamino RNHOH, or amine RNH2 derivatives. Metronidazole (Mtz) is a nitro-containing antibiotic that is commonly prescribed for lower-gut infections caused by the anaerobic bacterium Clostridium difficile. C. difficile infections rank number one among hospital acquired infections, and can result in diarrhea, severe colitis, or even death. Although NRs have been implicated in Mtz resistance of C. difficile, no NRs have been characterized from the hypervirulent R20291 strain of C. difficile. We report the first expression, purification, and three-dimensional X-ray crystal structures of two NRs from the C. difficile R20291 strain. The X-ray crystal structures of the two NRs were solved to 2.1 Å resolution. Their homodimeric structures exhibit the classic NR α+ß fold, with each protomer binding one FMN cofactor near the dimer interface. Functional assays demonstrate that these two NRs metabolize Mtz with associated re-oxidation of the proteins. Importantly, these results represent the first isolation and characterization of NRs from the hypervirulent R20291 strain of relevance to organic RNO2 (e.g., Mtz) metabolism.

Dynamics of an experimental microbial invasion.

The ecological dynamics underlying species invasions have been a major focus of research in macroorganisms for the last five decades. However, we still know little about the processes behind invasion by unicellular organisms. To expand our knowledge of microbial invasions, we studied the roles of propagule pressure, nutrient supply, and biotic resistance in the invasion success of a freshwater invasive alga, Prymnesium parvum, using microcosms containing natural freshwater microbial assemblages. Microcosms were subjected to a factorial design with two levels of nutrient-induced diversity and three levels of propagule pressure, and incubated for 7 d, during which P. parvum densities and microbial community composition were tracked. Successful invasion occurred in microcosms receiving high propagule pressure whereas nutrients or community diversity played no role in invasion success. Invaded communities experienced distinctive changes in composition compared with communities where the invasion was unsuccessful. Successfully invaded microbial communities had an increased abundance of fungi and ciliates, and decreased abundances of diatoms and cercozoans. Many of these changes mirrored the microbial community changes detected during a natural P. parvum bloom in the source system. This role of propagule pressure is particularly relevant for P. parvum in the reservoir-dominated southern United States because this species can form large, sustained blooms that can generate intense propagule pressures for downstream sites. Human impact and global climate change are currently causing widespread environmental changes in most southern US freshwater systems that may facilitate P. parvum establishment and, when coupled with strong propagule pressure, could put many more systems at risk for invasion.

Metatranscriptomic analysis of a high-sulfide aquatic spring reveals insights into sulfur cycling and unexpected aerobic metabolism.

Zodletone spring is a sulfide-rich spring in southwestern Oklahoma characterized by shallow, microoxic, light-exposed spring water overlaying anoxic sediments. Previously, culture-independent 16S rRNA gene based diversity surveys have revealed that Zodletone spring source sediments harbor a highly diverse microbial community, with multiple lineages putatively involved in various sulfur-cycling processes. Here, we conducted a metatranscriptomic survey of microbial populations in Zodletone spring source sediments to characterize the relative prevalence and importance of putative phototrophic, chemolithotrophic, and heterotrophic microorganisms in the sulfur cycle, the identity of lineages actively involved in various sulfur cycling processes, and the interaction between sulfur cycling and other geochemical processes at the spring source. Sediment samples at the spring's source were taken at three different times within a 24-h period for geochemical analyses and RNA sequencing. In depth mining of datasets for sulfur cycling transcripts revealed major sulfur cycling pathways and taxa involved, including an unexpected potential role of Actinobacteria in sulfide oxidation and thiosulfate transformation. Surprisingly, transcripts coding for the cyanobacterial Photosystem II D1 protein, methane monooxygenase, and terminal cytochrome oxidases were encountered, indicating that genes for oxygen production and aerobic modes of metabolism are actively being transcribed, despite below-detectable levels (<1 µM) of oxygen in source sediment. Results highlight transcripts involved in sulfur, methane, and oxygen cycles, propose that oxygenic photosynthesis could support aerobic methane and sulfide oxidation in anoxic sediments exposed to sunlight, and provide a viewpoint of microbial metabolic lifestyles under conditions similar to those seen during late Archaean and Proterozoic eons.

Arhodomonas sp. strain Seminole and its genetic potential to degrade aromatic compounds under high-salinity conditions.

Arhodomonas sp. strain Seminole was isolated from a crude oil-impacted brine soil and shown to degrade benzene, toluene, phenol, 4-hydroxybenzoic acid (4-HBA), protocatechuic acid (PCA), and phenylacetic acid (PAA) as the sole sources of carbon at high salinity. Seminole is a member of the genus Arhodomonas in the class Gammaproteobacteria, sharing 96% 16S rRNA gene sequence similarity with Arhodomonas aquaeolei HA-1. Analysis of the genome predicted a number of catabolic genes for the metabolism of benzene, toluene, 4-HBA, and PAA. The predicted pathways were corroborated by identification of enzymes present in the cytosolic proteomes of cells grown on aromatic compounds using liquid chromatography-mass spectrometry. Genome analysis predicted a cluster of 19 genes necessary for the breakdown of benzene or toluene to acetyl coenzyme A (acetyl-CoA) and pyruvate. Of these, 12 enzymes were identified in the proteome of toluene-grown cells compared to lactate-grown cells. Genomic analysis predicted 11 genes required for 4-HBA degradation to form the tricarboxylic acid (TCA) cycle intermediates. Of these, proteomic analysis of 4-HBA-grown cells identified 6 key enzymes involved in the 4-HBA degradation pathway. Similarly, 15 genes needed for the degradation of PAA to the TCA cycle intermediates were predicted. Of these, 9 enzymes of the PAA degradation pathway were identified only in PAA-grown cells and not in lactate-grown cells. Overall, we were able to reconstruct catabolic steps for the breakdown of a variety of aromatic compounds in an extreme halophile, strain Seminole. Such knowledge is important for understanding the role of Arhodomonas spp. in the natural attenuation of hydrocarbon-impacted hypersaline environments.

Alternative splicing in the fiddler crab cognate ecdysteroid receptor: variation in receptor isoform expression and DNA binding properties in response to hormone.

RXR cDNA cloning from three Uca species led to the identification of 4 conserved isoforms, indicative of alternative splicing in the hinge and ligand binding domains (LBD). Sequencing of overlapping clones from a Ucapugilator genomic library identified EcR isoforms matching previously identified cDNA variants; in addition, a cryptic exon in the LBD was detected and evidence for expression of this new isoform was obtained from next-generation sequencing. RNA-seq analysis also identified a new amino terminal EcR variant. EcR and RXR transcript abundance increases throughout ovarian maturation in U. pugilator, while cognate receptor transcript abundance remains constant in a related Indo-Pacific species with a different reproductive strategy. To examine if crab RXR LBD isoforms have different physical properties in vitro, electromobility shift assays were performed with different EcR isoforms. The cognate crab and fruit fly receptors differ in their responses to hormone. Ecdysteroids did not increase DNA binding for the crab heterodimers, while ecdysteroids stimulate binding for Drosophilamelanogaster EcR/USP heterodimers. In swapping experiments, UpEcR/USP heterodimers did not show ligand-responsive differences in DNA binding; both crab RXR LBD isoforms, however, conferred ligand-responsive increases in DNA binding with DmEcRs. These data indicate that both UpRXR LBD isoforms can heterodimerize with the heterologous DmEcR receptors and promote ligand and DNA binding. Unresponsiveness of the cognate receptors to ecdysteroid, however, suggest additional factors may be required to mediate endogenous, perhaps isoform-specific, differences in EcR conformation, consistent with previously reported effects of UpRXR isoforms on UpEcR ligand-binding affinities.

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

BACKGROUND: 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). RESULTS: RDX accumulated readily in coral soft tissues with bioconcentration factors ranging from 1.1 to 1.5. Next-generation sequencing of a normalized meta-transcriptomic library developed for the eukaryotic components of the A. formosa coral holobiont was leveraged to conduct microarray-based global transcript expression analysis of integrated coral/zooxanthellae responses to the RDX exposure. Total differentially expressed transcripts (DET) increased with increasing RDX exposure concentrations as did the proportion of zooxanthellae DET relative to the coral animal. Transcriptional responses in the coral demonstrated higher sensitivity to RDX compared to zooxanthellae where increased expression of gene transcripts coding xenobiotic detoxification mechanisms (i.e. cytochrome P450 and UDP glucuronosyltransferase 2 family) were initiated at the lowest exposure concentration. Increased expression of these detoxification mechanisms was sustained at higher RDX concentrations as well as production of a physical barrier to exposure through a 40% increase in mucocyte density at the maximum RDX exposure. At and above the 1.8 mg/L exposure concentration, DET coding for genes involved in central energy metabolism, including photosynthesis, glycolysis and electron-transport functions, were decreased in zooxanthellae although preliminary data indicated that zooxanthellae densities were not affected. In contrast, significantly increased transcript expression for genes involved in cellular energy production including glycolysis and electron-transport pathways was observed in the coral animal. CONCLUSIONS: Transcriptional network analysis for central energy metabolism demonstrated highly correlated responses to RDX among the coral animal and zooxanthellae indicative of potential compensatory responses to lost photosynthetic potential within the holobiont. These observations underscore the potential for complex integrated responses to RDX exposure among species comprising the coral holobiont and highlight the need to understand holobiont-species interactions to accurately assess pollutant impacts.

Seasonality and disturbance: annual pattern and response of the bacterial and microbial eukaryotic assemblages in a freshwater ecosystem.

High-throughput pyrosequencing of SSU rDNA genes was used to obtain monthly snapshots of eukaryotic and bacterial diversity and community structure at two locations in Lake Texoma, a low salinity lake in the south central United States, over 1 year. The lake experienced two disturbance events (i) a localized bloom of Prymnesium parvum restricted to one of the locations that lasted from January to April, and (ii) a large (17 cm), global rain event in the beginning of May, overlaid onto seasonal environmental change. Eukaryotic species richness as well as both eukaryotic and bacterial community similarity exhibited seasonal patterns, including distinct responses to the rain event. The P. parvum bloom created a natural experiment in which to directly explore the effects of an Ecosystem Disruptive Algal Bloom (EDAB) on the microbial community separated from seasonal changes. Microbial species richness was unaffected by the bloom, however, the eukaryotic community structure (evenness) and the patterns of both eukaryotic and bacterial community similarity at bloom and non-bloom sites were statistically distinct during the 4 months of the bloom. These results indicate that physical and biological disturbances as well as seasonal environmental forces contribute to the structure of both the eukaryotic and bacterial communities.