Sea star wasting disease pathology in Pisaster ochraceus shows a basal-to-surface process affecting color phenotypes differently.

Sea star wasting disease (SSWD) refers to a suite of poorly described non-specific clinical signs including abnormal posture, epidermal ulceration, and limb autotomy (sloughing) causing mortalities of over 20 species of sea stars and subsequent ecological shifts throughout the northeastern Pacific. While SSWD is widely assumed to be infectious, with environmental conditions facilitating disease progression, few data exist on cellular changes associated with the disease. This is unfortunate, because such observations could inform mechanisms of disease pathogenesis and host susceptibility. Here, we replicated SSWD by exposing captive Pisaster ochraceus to a suite of non-infectious organic substances and show that development of gross lesions is a basal-to-surface process involving inflammation (e.g. infiltration of coelomocytes) of ossicles and mutable collagenous tissue, leading to epidermal ulceration. Affected sea stars also manifest increases in a heretofore undocumented coelomocyte type, spindle cells, that might be a useful marker of inflammation in this species. Finally, compared to purple morphs, orange P. ochraceus developed more severe lesions but survived longer. Longer-lived, and presumably more visible, severely-lesioned orange sea stars could have important demographic implications in terms of detectability of lesioned animals in the wild and measures of apparent prevalence of disease.

Paraburkholderia madseniana sp. nov., a phenolic acid-degrading bacterium isolated from acidic forest soil.

RP11T was isolated from forest soil following enrichment with 4-hydroxybenzoic acid. Cells of RP11T are aerobic, non-sporulating, exhibit swimming motility, and are rods (0.8 µm by 1.4 µm) that often occur as diplobacillus or in short chains (3-4 cells). Optimal growth on minimal media containing 4-hydroxybenzoic acid (µ=0.216 hr-1) occurred at 30 °C, pH 6.5 or 7.0 and 0% salinity. Comparative chemotaxonomic, genomic and phylogenetic analyses revealed the isolate was distinct from its closest relative type strains identified as Paraburkholderia aspalathi LMG 27731T, Paraburkholderia fungorum LMG 16225T and Paraburkholderia caffeinilytica CF1T. Strain RP11T is genetically distinct from P. aspalathi, its closest relative, in terms of 16S rRNA gene sequence similarity (98.7%), genomic average nucleotide identity (94%) and in silico DNA-DNA hybridization (56.7 %±2.8). The composition of fatty acids and substrate utilization pattern differentiated strain RP11T from its closest relatives, including growth on phthalic acid. Strain RP11T encoded the greatest number of aromatic degradation genes of all eleven closely related type strains and uniquely encoded a phthalic acid dioxygenase and paralog of the 3-hydroxybenzoate 4-monooxygenase. The only ubiquinone detected in strain RP11T was Q-8, and the major cellular fatty acids were C16 : 0, 3OH-C16 : 0, C17 : 0 cyclo, C19 : 0 cyclo ω8c, and summed feature 8 (C18 : 1 ω7c/ω6c). On the basis of this polyphasic approach, it was determined that strain RP11T represents a novel species from the genus Paraburkholderia for which the name Paraburkholderia madseniana sp. nov. is proposed. The type strain is RP11T (=DSM 110123T=LMG 31517T).

Benzene Degradation by a Variovorax Species within a Coal Tar-Contaminated Groundwater Microbial Community.

Investigations of environmental microbial communities are crucial for the discovery of populations capable of degrading hazardous compounds and may lead to improved bioremediation strategies. The goal of this study was to identify microorganisms responsible for aerobic benzene degradation in coal tar-contaminated groundwater. Benzene degradation was monitored in laboratory incubations of well waters using gas chromatography mass spectrometry (GC-MS). Stable isotope probing (SIP) experiments using [13C]benzene enabled us to obtain 13C-labled community DNA. From this, 16S rRNA clone libraries identified Gammaproteobacteria and Betaproteobacteria as the active benzene-metabolizing microbial populations. Subsequent cultivation experiments yielded nine bacterial isolates that grew in the presence of benzene; five were confirmed in laboratory cultures to grow on benzene. The isolated benzene-degrading organisms were genotypically similar (>97% 16S rRNA gene nucleotide identities) to the organisms identified in SIP experiments. One isolate, Variovorax MAK3, was further investigated for the expression of a putative aromatic ring-hydroxylating dioxygenase (RHD) hypothesized to be involved in benzene degradation. Microcosm experiments using Variovorax MAK3 revealed a 10-fold increase in RHD (Vapar_5383) expression, establishing a link between this gene and benzene degradation. Furthermore, the addition of Variovorax MAK3 to microcosms prepared from site waters accelerated community benzene degradation and correspondingly increased RHD gene expression. In microcosms using uninoculated groundwater, quantitative (q)PCR assays (with 16S rRNA and RDH genes) showed that Variovorax was present and responsive to added benzene. These data demonstrate how the convergence of cultivation-dependent and -independent techniques can boost understandings of active populations and functional genes in complex benzene-degrading microbial communities. IMPORTANCE: Benzene is a human carcinogen whose presence in contaminated groundwater drives environmental cleanup efforts. Although the aerobic biodegradation of benzene has long been established, knowledge of the identity of the microorganisms in complex naturally occurring microbial communities responsible for benzene biodegradation has evaded scientific inquiry for many decades. Here, we applied a molecular biology technique known as stable isotope probing (SIP) to the microbial communities residing in contaminated groundwater samples to identify the community members active in benzene biodegradation. We complemented this approach by isolating and growing in the laboratory a bacterium representative of the bacteria found using SIP. Further characterization of the isolated bacterium enabled us to track the expression of a key gene that attacks benzene both in pure cultures of the bacterium and in the naturally occurring groundwater microbial community. This work advances information regarding the documentation of microbial processes, especially the populations and genes that contribute to bioremediation.

Microbial community dynamics and stability during an ammonia-induced shift to syntrophic acetate oxidation.

Anaerobic digesters rely on the diversity and distribution of parallel metabolic pathways mediated by complex syntrophic microbial communities to maintain robust and optimal performance. Using mesophilic swine waste digesters, we experimented with increased ammonia loading to induce a shift from aceticlastic methanogenesis to an alternative acetate-consuming pathway of syntrophic acetate oxidation. In comparison with control digesters, we observed shifts in bacterial 16S rRNA gene content and in functional gene repertoires over the course of the digesters' 3-year operating period. During the first year, under identical startup conditions, all bioreactors mirrored each other closely in terms of bacterial phylotype content, phylogenetic structure, and evenness. When we perturbed the digesters by increasing the ammonia concentration or temperature, the distribution of bacterial phylotypes became more uneven, followed by a return to more even communities once syntrophic acetate oxidation had allowed the experimental bioreactors to regain stable operation. The emergence of syntrophic acetate oxidation coincided with a partial shift from aceticlastic to hydrogenotrophic methanogens. Our 16S rRNA gene analysis also revealed that acetate-fed enrichment experiments resulted in communities that did not represent the bioreactor community. Analysis of shotgun sequencing of community DNA suggests that syntrophic acetate oxidation was carried out by a heterogeneous community rather than by a specific keystone population with representatives of enriched cultures with this metabolic capacity.

Sugarcane mill mud-induced putative host (soybean (Glycine max))-rhizobia symbiosis in sandy loam soil.

Domestic production of controlled-release, compost-based, and microbe-enhanced fertilizers is being expanded in the U.S. as a part of rural development. Sugarcane mill mud is a sterilized (≈90°C) agricultural byproduct in surplus that has received interests as a soil amendment in several Southern states, because of its high phosphorus and organic carbon contents. Addition of mill mud to sandy loam significantly increased the nodule formation compared to fertilized and unfertilized controls. Mill mud addition also resulted in pod yields similar to the fertilized control. Though not found in mill mud itself, mill mud additions correlated with an increase in soil Rhizobia as determined by deep 16S rRNA gene sequencing. We hypothesize that Firmicutes in sterilized mill mud induced Rhizobia that in turn enhanced soybean (Glycine max) growth. Collectively, mill mud enhanced the plant growth promoting bacteria when applied to a silt loam, although the relative influence of mill mud-derived bacteria, organic carbon, and nutrients is yet to be determined.

Metagenome-assembled genomes from sugarcane mill mud.

The genomes of 11 bacteria and 3 archaea were assembled from metagenomic DNA extracted from sugarcane mill mud. These metagenome-assembled genomes ranged from 1.79 to 6.45 Mb, with 2,263 to 5,551 predicted proteins, 80.65% to 100% genome completeness, and 43.19% to 68.02% G+C content.

Phenolic acid-degrading Paraburkholderia prime decomposition in forest soil.

Plant-derived phenolic acids are catabolized by soil microorganisms whose activity may enhance the decomposition of soil organic carbon (SOC). We characterized whether phenolic acid-degrading bacteria enhance SOC mineralization in forest soils when primed with 13C-labeled p-hydroxybenzoic acid (pHB). We further tested whether pHB-induced priming could explain differences in SOC content among mono-specific tree plantations in a 70-year-old common garden experiment. pHB addition primed significant losses of SOC (3-13 µmols C g-1 dry wt soil over 7 days) compared to glucose, which reduced mineralization (-3 to -8 µmols C g-1 dry wt soil over 7 days). The principal degraders of pHB were Paraburkholderia and Caballeronia in all plantations regardless of tree species or soil type, with one predominant phylotype (RP11ASV) enriched 23-fold following peak pHB respiration. We isolated and confirmed the phenolic degrading activity of a strain matching this phylotype (RP11T), which encoded numerous oxidative enzymes, including secretion signal-bearing laccase, Dyp-type peroxidase and aryl-alcohol oxidase. Increased relative abundance of RP11ASV corresponded with higher pHB respiration and expression of pHB monooxygenase (pobA), which was inversely proportional to SOC content among plantations. pobA expression proved a responsive measure of priming activity. We found that stimulating phenolic-acid degrading bacteria can prime decomposition and that this activity, corresponding with differences in tree species, is a potential mechanism in SOC cycling in forests. Overall, this study highlights the ecology and function of Paraburkholderia whose associations with plant roots and capacity to degrade phenolics suggest a role for specialized bacteria in the priming effect.

Subsurface cycling of nitrogen and anaerobic aromatic hydrocarbon biodegradation revealed by nucleic Acid and metabolic biomarkers.

Microbial processes are crucial for ecosystem maintenance, yet documentation of these processes in complex open field sites is challenging. Here we used a multidisciplinary strategy (site geochemistry, laboratory biodegradation assays, and field extraction of molecular biomarkers) to deduce an ongoing linkage between aromatic hydrocarbon biodegradation and nitrogen cycling in a contaminated subsurface site. Three site wells were monitored over a 10-month period, which revealed fluctuating concentrations of nitrate, ammonia, sulfate, sulfide, methane, and other constituents. Biodegradation assays performed under multiple redox conditions indicated that naphthalene metabolism was favored under aerobic conditions. To explore in situ field processes, we measured metabolites of anaerobic naphthalene metabolism and expressed mRNA transcripts selected to document aerobic and anaerobic microbial transformations of ammonia, nitrate, and methylated aromatic contaminants. Gas chromatography-mass spectrometry detection of two carboxylated naphthalene metabolites and transcribed benzylsuccinate synthase, cytochrome c nitrite reductase, and ammonia monooxygenase genes indicated that anaerobic metabolism of aromatic compounds and both dissimilatory nitrate reduction to ammonia (DNRA) and nitrification occurred in situ. These data link formation (via DNRA) and destruction (via nitrification) of ammonia to in situ cycling of nitrogen in this subsurface habitat, where metabolism of aromatic pollutants has led to accumulation of reduced metabolic end products (e.g., ammonia and methane).

Virome Variation during Sea Star Wasting Disease Progression in Pisaster ochraceus (Asteroidea, Echinodermata).

Sea star wasting disease (SSWD) is a condition that has affected asteroids for over 120 years, yet mechanistic understanding of this wasting etiology remains elusive. We investigated temporal virome variation in two Pisaster ochraceus specimens that wasted in the absence of external stimuli and two specimens that did not experience SSWD for the duration of our study, and compared viromes of wasting lesion margin tissues to both artificial scar margins and grossly normal tissues over time. Global assembly of all SSWD-affected tissue libraries resulted in 24 viral genome fragments represented in >1 library. Genome fragments mostly matched densoviruses and picornaviruses with fewer matching nodaviruses, and a sobemovirus. Picornavirus-like and densovirus-like genome fragments were most similar to viral genomes recovered in metagenomic study of other marine invertebrates. Read recruitment revealed only two picornavirus-like genome fragments that recruited from only SSWD-affected specimens, but neither was unique to wasting lesions. Wasting lesion margin reads recruited to a greater number of viral genotypes (i.e., richness) than did either scar tissue and grossly normal tissue reads. Taken together, these data suggest that no single viral genome fragment was associated with SSWD. Rather, wasting lesion margins may generally support viral proliferation.

Evidence That Microorganisms at the Animal-Water Interface Drive Sea Star Wasting Disease.

Sea star wasting (SSW) disease describes a condition affecting asteroids that resulted in significant Northeastern Pacific population decline following a mass mortality event in 2013. The etiology of SSW is unresolved. We hypothesized that SSW is a sequela of microbial organic matter remineralization near respiratory surfaces, one consequence of which may be limited O2 availability at the animal-water interface. Microbial assemblages inhabiting tissues and at the asteroid-water interface bore signatures of copiotroph proliferation before SSW onset, followed by the appearance of putatively facultative and strictly anaerobic taxa at the time of lesion genesis and as animals died. SSW lesions were induced in Pisaster ochraceus by enrichment with a variety of organic matter (OM) sources. These results together illustrate that depleted O2 conditions at the animal-water interface may be established by heterotrophic microbial activity in response to organic matter loading. SSW was also induced by modestly (∼39%) depleted O2 conditions in aquaria, suggesting that small perturbations in dissolved O2 may exacerbate the condition. SSW susceptibility between species was significantly and positively correlated with surface rugosity, a key determinant of diffusive boundary layer thickness. Tissues of SSW-affected individuals collected in 2013-2014 bore δ15N signatures reflecting anaerobic processes, which suggests that this phenomenon may have affected asteroids during mass mortality at the time. The impacts of enhanced microbial activity and subsequent O2 diffusion limitation may be more pronounced under higher temperatures due to lower O2 solubility, in more rugose asteroid species due to restricted hydrodynamic flow, and in larger specimens due to their lower surface area to volume ratios which affects diffusive respiratory potential.

Cellular antioxidant activity of common vegetables.

The measurement of antioxidant activity using biologically relevant assays is important to screen fruits, vegetables, natural products, and dietary supplements for potential health benefits. The cellular antioxidant activity (CAA) assay quantifies antioxidant activity using a cell culture model and was developed to meet the need for a more biologically representative method than the popular chemistry antioxidant capacity measures. The objective of the study was to determine the CAA, total phenolic contents, and oxygen radical absorbance capacity (ORAC) values of 27 vegetables commonly consumed in the United States. Beets, broccoli, and red pepper had the highest CAA values, whereas cucumber had the lowest. CAA values were significantly correlated to total phenolic content. Potatoes were found to be the largest contributors of vegetable phenolics and CAA to the American diet. Increased fruit and vegetable consumption is an effective strategy to increase antioxidant intake and decrease oxidative stress and may lead to reduced risk of developing chronic diseases, such as cancer and cardiovascular disease.

Stable isotope probing reveals Trichosporon yeast to be active in situ in soil phenol metabolism.

The aim of this study was to extend the results of our previous stable isotope probing (SIP) investigation: we identified a soil fungus involved in phenol biodegradation at an agricultural field site. DNA extracts from our previous study were examined using fungi-specific PCR amplification of the 18S-28S internal transcribed spacer (ITS) region. We prepared an 80-member clone library using PCR-amplified, (13)C-labeled DNA derived from field soil that received 12 daily doses of (13)C-phenol. Restriction-fragment-length-polymorphism screening and DNA sequencing revealed a dominant clone (41% of the clone library), the ITS sequence of which corresponded to that of the fungal genus Trichosporon. We successfully grew and isolated a white, filamentous fungus from site soil samples after plating soil dilutions on mineral salts agar containing 250 p.p.m. phenol. Restreaking on both yeast extract-peptone-galactose and Sabouraud dextrose agar plates led to further purification of the fungus, the morphological characteristics of which matched those of the genus Trichosporon. The ITS sequence of our isolated fungus was identical to that of a clone from our SIP-based library, confirming it to be Trichosporon multisporum. High-performance liquid chromatography and turbidometeric analyses showed that the culture was able to metabolize and grow on 200 p.p.m. phenol in an aqueous mineral salts medium within 24 h at room temperature. Gas chromatography-mass spectrometry analysis of (13)CO(2) respiration from laboratory soil incubations demonstrated accelerated phenol mineralization in treatments inoculated with T. multisporum. These findings show that T. multisporum actively degraded phenol in our field-based, soil experiments.

Use of field-based stable isotope probing to identify adapted populations and track carbon flow through a phenol-degrading soil microbial community.

The goal of this field study was to provide insight into three distinct populations of microorganisms involved in in situ metabolism of phenol. Our approach measured 13CO2 respired from [13C]phenol and stable isotope probing (SIP) of soil DNA at an agricultural field site. Traditionally, SIP-based investigations have been subject to the uncertainties posed by carbon cross-feeding. By altering our field-based, substrate-dosing methodologies, experiments were designed to look beyond primary degraders to detect trophically related populations in the food chain. Using gas chromatography-mass spectrometry (GC/MS), it was shown that (13)C-labeled biomass, derived from primary phenol degraders in soil, was a suitable growth substrate for other members of the soil microbial community. Next, three dosing regimes were designed to examine active members of the microbial community involved in phenol metabolism in situ: (i) 1 dose of [13C]phenol, (ii) 11 daily doses of unlabeled phenol followed by 1 dose of [13C]phenol, and (iii) 12 daily doses of [13C]phenol. GC/MS analysis demonstrated that prior exposure to phenol boosted 13CO2 evolution by a factor of 10. Furthermore, imaging of 13C-treated soil using secondary ion mass spectrometry (SIMS) verified that individual bacteria incorporated 13C into their biomass. PCR amplification and 16S rRNA gene sequencing of 13C-labeled soil DNA from the 3 dosing regimes revealed three distinct clone libraries: (i) unenriched, primary phenol degraders were most diverse, consisting of alpha-, beta-, and gamma-proteobacteria and high-G+C-content gram-positive bacteria, (ii) enriched primary phenol degraders were dominated by members of the genera Kocuria and Staphylococcus, and (iii) trophically related (carbon cross-feeders) were dominated by members of the genus Pseudomonas. These data show that SIP has the potential to document population shifts caused by substrate preexposure and to follow the flow of carbon through terrestrial microbial food chains.