Enrichment of Bacteria From Eastern Mediterranean Sea Involved in Lignin Degradation via the Phenylacetyl-CoA Pathway.

The degradation of allochthonous terrestrial organic matter, such as recalcitrant lignin and hemicellulose from plants, occurs in the ocean. We hypothesize that bacteria instead of white-rot fungi, the model organisms of aerobic lignin degradation within terrestrial environments, are responsible for lignin degradation in the ocean due to the ocean's oligotrophy and hypersalinity. Warm oxic seawater from the Eastern Mediterranean Sea was enriched on lignin in laboratory microcosms. Lignin mineralization rates by the lignin-adapted consortia improved after two sequential incubations. Shotgun metagenomic sequencing detected a higher abundance of aromatic compound degradation genes in response to lignin, particularly phenylacetyl-CoA, which may be an effective strategy for marine microbes in fluctuating oxygen concentrations. 16S rRNA gene amplicon sequencing detected a higher abundance of Gammaproteobacteria and Alphaproteobacteria bacteria such as taxonomic families Idiomarinaceae, Alcanivoraceae, and Alteromonadaceae in response to lignin. Meanwhile, fungal Ascomycetes and Basidiomycetes remained at very low abundance. Our findings demonstrate the significant potential of bacteria and microbes utilizing the phenylacetyl-CoA pathway to contribute to lignin degradation in the Eastern Mediterranean where environmental conditions are unfavorable for fungi. Exploring the diversity of bacterial lignin degraders may provide important enzymes for lignin conversion in industry. Enzymes may be key in breaking down high molecular weight lignin and enabling industry to use it as a low-cost and sustainable feedstock for biofuels or other higher-value products.

Distribution of IL-1ß immunoreactive cells in pancreatic biopsies from living volunteers with new-onset type 1 diabetes: comparison with donors without diabetes and with longer duration of disease.

AIMS/HYPOTHESIS: Although IL-1ß is considered a key mediator of beta cell destruction, its cellular expression in islets during early type 1 diabetes remains unclear. We compared its expression in rare pancreatic biopsies from new-onset living volunteers with its expression in cadaveric pancreas sections from non-diabetic autoantibody-positive and -negative individuals and those with long-standing disease. METHODS: Pancreatic biopsy sections from six new-onset living volunteers (group 1) and cadaveric sections from 13 non-diabetic autoantibody-negative donors (group 2), four non-diabetic autoantibody-positive donors (group 3) and nine donors with diabetes of longer duration (0.25-12 years of disease; group 4) were triple-immunostained for IL-1ß, insulin and glucagon. Intra- and peri-islet IL-1ß-positive cells in insulin-positive and -negative islets and in random exocrine fields were enumerated. RESULTS: The mean number of IL-1ß-positive cells per islet from each donor in peri- and intra-islet regions was <1.25 and <0.5, respectively. In all study groups, the percentage of islets with IL-1ß cells in peri- and/or intra-islet regions was highly variable and ranged from 4.48% to 17.59% in group 1, 1.42% to 44.26% in group 2, 7.93% to 17.53% in group 3 and 3.85% to 42.86% in group 4, except in a single case where the value was 75%. In 25/32 donors, a higher percentage of islets showed IL-1ß-positive cells in peri-islet than in intra-islet regions. In sections from diabetic donors (groups 1 and 4), a higher mean number of IL-1ß-positive cells occurred in insulin-positive islets than in insulin-negative islets. In group 2, 70-90% of islets in 3/13 sections had weak-to-moderate IL-1ß staining in alpha cells but staining was virtually absent or substantially reduced in the remaining groups. The mean number of exocrine IL-1ß-positive cells in group 1 was lower than in the other groups. CONCLUSIONS/INTERPRETATION: At onset of type 1 diabetes, the low number of islet-associated IL-1ß-positive cells may be insufficient to elicit beta cell destruction. The variable expression in alpha cells in groups 2-4 suggests their cellular heterogeneity and probable physiological role. The significance of a higher but variable number of exocrine IL-1ß-positive cells seen in non-diabetic individuals and those with long-term type 1 diabetes remains unclear.

Rapid Response of Eastern Mediterranean Deep Sea Microbial Communities to Oil.

Deep marine oil spills like the Deepwater Horizon (DWH) in the Gulf of Mexico have the potential to drastically impact marine systems. Crude oil contamination in marine systems remains a concern, especially for countries around the Mediterranean Sea with off shore oil production. The goal of this study was to investigate the response of indigenous microbial communities to crude oil in the deep Eastern Mediterranean Sea (E. Med.) water column and to minimize potential bias associated with storage and shifts in microbial community structure from sample storage. 16S rRNA amplicon sequencing was combined with GeoChip metagenomic analysis to monitor the microbial community changes to the crude oil and dispersant in on-ship microcosms set up immediately after water collection. After 3 days of incubation at 14 °C, the microbial communities from two different water depths: 824 m and 1210 m became dominated by well-known oil degrading bacteria. The archaeal population and the overall microbial community diversity drastically decreased. Similarly, GeoChip metagenomic analysis revealed a tremendous enrichment of genes related to oil biodegradation, which was consistent with the results from the DWH oil spill. These results highlight a rapid microbial adaption to oil contamination in the deep E. Med., and indicate strong oil biodegradation potential.

High-Quality Draft Genome Sequences of Four Lignocellulose-Degrading Bacteria Isolated from Puerto Rican Forest Soil: Gordonia sp., Paenibacillus sp., Variovorax sp., and Vogesella sp.

Here, we report the high-quality draft genome sequences of four phylogenetically diverse lignocellulose-degrading bacteria isolated from tropical soil (Gordonia sp., Paenibacillus sp., Variovorax sp., and Vogesella sp.) to elucidate the genetic basis of their ability to degrade lignocellulose. These isolates may provide novel enzymes for biofuel production.

Complete genome sequence of Pseudomonas stutzeri strain RCH2 isolated from a Hexavalent Chromium [Cr(VI)] contaminated site.

Hexavalent Chromium [Cr(VI)] is a widespread contaminant found in soil, sediment, and ground water in several DOE sites, including Hanford 100 H area. In order to stimulate microbially mediated reduction of Cr(VI) at this site, a poly-lactate hydrogen release compound was injected into the chromium contaminated aquifer. Targeted enrichment of dominant nitrate-reducing bacteria post injection resulted in the isolation of Pseudomonas stutzeri strain RCH2. P. stutzeri strain RCH2 was isolated using acetate as the electron donor and is a complete denitrifier. Experiments with anaerobic washed cell suspension of strain RCH2 revealed it could reduce Cr(VI) and Fe(III). The genome of strain RCH2 was sequenced using a combination of Illumina and 454 sequencing technologies and contained a circular chromosome of 4.6 Mb and three plasmids. Global genome comparisons of strain RCH2 with six other fully sequenced P. stutzeri strains revealed most genomic regions are conserved, however strain RCH2 has an additional 244 genes, some of which are involved in chemotaxis, Flp pilus biogenesis and pyruvate/2-oxogluturate complex formation.

Near-Complete Genome Sequence of Thalassospira sp. Strain KO164 Isolated from a Lignin-Enriched Marine Sediment Microcosm.

Thalassospira sp. strain KO164 was isolated from eastern Mediterranean seawater and sediment laboratory microcosms enriched on insoluble organosolv lignin under oxic conditions. The near-complete genome sequence presented here will facilitate analyses into this deep-ocean bacterium's ability to degrade recalcitrant organics such as lignin.

Computational Ranking of Yerba Mate Small Molecules Based on Their Predicted Contribution to Antibacterial Activity against Methicillin-Resistant Staphylococcus aureus.

The aqueous extract of yerba mate, a South American tea beverage made from Ilex paraguariensis leaves, has demonstrated bactericidal and inhibitory activity against bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). The gas chromatography-mass spectrometry (GC-MS) analysis of two unique fractions of yerba mate aqueous extract revealed 8 identifiable small molecules in those fractions with antimicrobial activity. For a more comprehensive analysis, a data analysis pipeline was assembled to prioritize compounds for antimicrobial testing against both MRSA and methicillin-sensitive S. aureus using forty-two unique fractions of the tea extract that were generated in duplicate, assayed for activity, and analyzed with GC-MS. As validation of our automated analysis, we checked our predicted active compounds for activity in literature references and used authentic standards to test for antimicrobial activity. 3,4-dihydroxybenzaldehyde showed the most antibacterial activity against MRSA at low concentrations in our bioassays. In addition, quinic acid and quercetin were identified using random forests analysis and 5-hydroxy pipecolic acid was identified using linear discriminant analysis. We also generated a ranked list of unidentified compounds that may contribute to the antimicrobial activity of yerba mate against MRSA. Here we utilized GC-MS data to implement an automated analysis that resulted in a ranked list of compounds that likely contribute to the antimicrobial activity of aqueous yerba mate extract against MRSA.

Genome Sequence of Halomonas sp. Strain KO116, an Ionic Liquid-Tolerant Marine Bacterium Isolated from a Lignin-Enriched Seawater Microcosm.

Halomonas sp. strain KO116 was isolated from Nile Delta Mediterranean Sea surface water enriched with insoluble organosolv lignin. It was further screened for growth on alkali lignin minimal salts medium agar. The strain tolerates the ionic liquid 1-ethyl-3-methylimidazolium acetate. Its complete genome sequence is presented in this report.

High-Quality Draft Genome Sequence of Desulfovibrio carbinoliphilus FW-101-2B, an Organic Acid-Oxidizing Sulfate-Reducing Bacterium Isolated from Uranium(VI)-Contaminated Groundwater.

Desulfovibrio carbinoliphilus subsp. oakridgensis FW-101-2B is an anaerobic, organic acid/alcohol-oxidizing, sulfate-reducing δ-proteobacterium. FW-101-2B was isolated from contaminated groundwater at The Field Research Center at Oak Ridge National Lab after in situ stimulation for heavy metal-reducing conditions. The genome will help elucidate the metabolic potential of sulfate-reducing bacteria during uranium reduction.

Classification of T cell movement tracks allows for prediction of cell function.

Using a unique combination of visual, statistical, and data mining methods, we tested the hypothesis that an immune cell's movement pattern can convey key information about the cell's function, antigen specificity, and environment. We applied clustering, statistical tests, and a support vector machine (SVM) to assess our ability to classify different datasets of imaged flouresently labelled T cells in mouse liver. We additionally saw clusters of different movement patterns of T cells of identical antigenic specificity. We found that the movement patterns of T cells specific and non-specific for malaria parasites are differentiable with 72% accuracy, and that specific cells have a higher tendency to move towards the parasite than non-specific cells. Movements of antigen-specific T cells in uninfected mice vs. infected mice were differentiable with 69.8% accuracy. We additionally saw clusters of different movement patterns of T cells of identical antigenic specificity. We concluded that our combination of methods has the potential to advance the understanding of cell movements in vivo.

Draft Genome Sequence of the Lignin-Degrading Burkholderia sp. Strain LIG30, Isolated from Wet Tropical Forest Soil.

Burkholderia species are common soil Betaproteobacteria capable of degrading recalcitrant aromatic compounds and xenobiotics. Burkholderia sp. strain LIG30 was isolated from wet tropical forest soil and is capable of utilizing lignin as a sole carbon source. Here we report the draft genome sequence of Burkholderia sp. strain LIG30.

Enzyme activities of aerobic lignocellulolytic bacteria isolated from wet tropical forest soils.

Lignocellulolytic bacteria have promised to be a fruitful source of new enzymes for next-generation lignocellulosic biofuel production. Puerto Rican tropical forest soils were targeted because the resident microbes decompose biomass quickly and to near-completion. Isolates were initially screened based on growth on cellulose or lignin in minimal media. 75 Isolates were further tested for the following lignocellulolytic enzyme activities: phenol oxidase, peroxidase, ß-d-glucosidase, cellobiohydrolase, ß-xylopyranosidase, chitinase, CMCase, and xylanase. Cellulose-derived isolates possessed elevated ß-d-glucosidase, CMCase, and cellobiohydrolase activity but depressed phenol oxidase and peroxidase activity, while the contrary was true of lignin isolates, suggesting that these bacteria are specialized to subsist on cellulose or lignin. Cellobiohydrolase and phenol oxidase activity rates could classify lignin and cellulose isolates with 61% accuracy, which demonstrates the utility of model degradation assays. Based on 16S rRNA gene sequencing, all isolates belonged to phyla dominant in the Puerto Rican soils, Proteobacteria, Firmicutes, and Actinobacteria, suggesting that many dominant taxa are capable of the rapid lignocellulose degradation characteristic of these soils. The isolated genera Aquitalea, Bacillus, Burkholderia, Cupriavidus, Gordonia, and Paenibacillus represent rarely or never before studied lignolytic or cellulolytic species and were undetected by metagenomic analysis of the soils. The study revealed a relationship between phylogeny and lignocellulose-degrading potential, supported by Kruskal-Wallis statistics which showed that enzyme activities of cultivated phyla and genera were different enough to be considered representatives of distinct populations. This can better inform future experiments and enzyme discovery efforts.

Complete genome sequence of the lignin-degrading bacterium Klebsiella sp. strain BRL6-2.

In an effort to discover anaerobic bacteria capable of lignin degradation, we isolated Klebsiella sp. strain BRL6-2 on minimal media with alkali lignin as the sole carbon source. This organism was isolated anaerobically from tropical forest soils collected from the Bisley watershed at the Ridge site in the El Yunque National Forest in Puerto Rico, USA, part of the Luquillo Long-Term Ecological Research Station. At this site, the soils experience strong fluctuations in redox potential and are characterized by cycles of iron oxidation and reduction. Genome sequencing was targeted because of its ability to grow on lignin anaerobically and lignocellulolytic activity via in vitro enzyme assays. The genome of Klebsiella sp. strain BRL6-2 is 5.80 Mbp with no detected plasmids, and includes a relatively small arsenal of genes encoding lignocellulolytic carbohydrate active enzymes. The genome revealed four putative peroxidases including glutathione and DyP-type peroxidases, and a complete protocatechuate pathway encoded in a single gene cluster. Physiological studies revealed Klebsiella sp. strain BRL6-2 to be relatively stress tolerant to high ionic strength conditions. It grows in increasing concentrations of ionic liquid (1-ethyl-3-methyl-imidazolium acetate) up to 73.44 mM and NaCl up to 1.5 M.

Global transcriptome response to ionic liquid by a tropical rain forest soil bacterium, Enterobacter lignolyticus.

To process plant-based renewable biofuels, pretreatment of plant feedstock with ionic liquids has significant advantages over current methods for deconstruction of lignocellulosic feedstocks. However, ionic liquids are often toxic to the microorganisms used subsequently for biomass saccharification and fermentation. We previously isolated Enterobacter lignolyticus strain SCF1, a lignocellulolytic bacterium from tropical rain forest soil, and report here that it can grow in the presence of 0.5 M 1-ethyl-3-methylimidazolium chloride, a commonly used ionic liquid. We investigated molecular mechanisms of SCF1 ionic liquid tolerance using a combination of phenotypic growth assays, phospholipid fatty acid analysis, and RNA sequencing technologies. Potential modes of resistance to 1-ethyl-3-methylimidazolium chloride include an increase in cyclopropane fatty acids in the cell membrane, scavenging of compatible solutes, up-regulation of osmoprotectant transporters and drug efflux pumps, and down-regulation of membrane porins. These findings represent an important first step in understanding mechanisms of ionic liquid resistance in bacteria and provide a basis for engineering microbial tolerance.

Complete genome sequence of "Enterobacter lignolyticus" SCF1.

In an effort to discover anaerobic bacteria capable of lignin degradation, we isolated "Enterobacter lignolyticus" SCF1 on minimal media with alkali lignin as the sole source of carbon. This organism was isolated anaerobically from tropical forest soils collected from the Short Cloud Forest site in the El Yunque National Forest in Puerto Rico, USA, part of the Luquillo Long-Term Ecological Research Station. At this site, the soils experience strong fluctuations in redox potential and are net methane producers. Because of its ability to grow on lignin anaerobically, we sequenced the genome. The genome of "E. lignolyticus" SCF1 is 4.81 Mbp with no detected plasmids, and includes a relatively small arsenal of lignocellulolytic carbohydrate active enzymes. Lignin degradation was observed in culture, and the genome revealed two putative laccases, a putative peroxidase, and a complete 4-hydroxyphenylacetate degradation pathway encoded in a single gene cluster.