Cold adaptation strategies and diversity of Antarctic bacteria

Bacteria have been isolated from virtually every environment on Earth. The Antarctic continent is no exception. In this extremely cold and dry environment bacteria have colonised various refugia and have evolved a number of strategies for coping with the extreme physico-chemical fluctuations they are exposed to within the environment. These psychrophilic adaptations include cold adapted proteins and lipids which are interest for biotechnology in areas such as frozen foods, agriculture and cryogenic storage. One type of cold adapted protein of particular interest is the antifreeze protein (AFP) for its recrystalisation inhibition and thermal hysteresis activity. It was first isolated from Antarctic fish in the 1970, but has since been found in plants, fungi, insects and bacteria. Over 800 bacterial isolates were cultured from lakes of the, Vestfold Hills, Larsemann Hills and MacRobertson Land, Antarctica. Approximately 87% were Gram negative rods. A novel AFP assay designed for high-throughput analysis in Antarctica, demonstrated putative activity in 187 isolates. Subsequent SPLAT analysis (qualification assessment of recrystalisation inhibition activity) of the putative positive isolates showed 19 isolates with significant recrystalisation inhibition activity. These 19 isolates were cultured from five separate lakes with substantial physico-chemical differences. The 19 AFP active isolates were characterised, using amplified ribosomal DNA restriction analysis (ARDRA) and 16S rDNA sequencing, as predominantly belonging to genera from the a- and y-Proteobacteria, although they were more prominent in the gamma subdivision. One of these isolates (213, Halomonas sp.) was shown as dominant within its community by DGGE analysis, indicating a possible selective advantage for AFP active bacteria. This is the first report of the phylogenetic distribution of AFP activity within bacteria, thus providing information which could enable future bacterial AFP assessments to be aimed at specific taxonomic groups

The Phytoplankton Nannochloropsis oculata Enhances the Ability of Roseobacter Clade Bacteria to Inhibit the Growth of Fish Pathogen Vibrio anguillarum

.., and perhaps other phytoplankton species, with certain roseobacters might provide a powerful tool for eliminating fish pathogens from fish-rearing tanks

Plasmid Metagenome Reveals High Levels of Antibiotic Resistance Genes and Mobile Genetic Elements in Activated Sludge

The overuse or misuse of antibiotics has accelerated antibiotic resistance, creating a major challenge for the public health in the world. Sewage treatment plants (STPs) are considered as important reservoirs for antibiotic resistance genes (ARGs) and activated sludge characterized with high microbial density and diversity facilitates ARG horizontal gene transfer (HGT) via mobile genetic elements (MGEs). However, little is known regarding the pool of ARGs and MGEs in sludge microbiome. In this study, the transposon aided capture (TRACA) system was employed to isolate novel plasmids from activated sludge of one STP in Hong Kong, China. We also used Illumina Hiseq 2000 high-throughput sequencing and metagenomics analysis to investigate the plasmid metagenome. Two novel plasmids were acquired from the sludge microbiome by using TRACA system and one novel plasmid was identified through metagenomics analysis. Our results revealed high levels of various ARGs as well as MGEs for HGT, including integrons, transposons and plasmids. The application of the TRACA system to isolate novel plasmids from the environmental metagenome, coupled with subsequent high-throughput sequencing and metagenomic analysis, highlighted the prevalence of ARGs and MGEs in microbial community of STPs

Reducing the Effects of PCR Amplification and Sequencing Artifacts on 16S rRNA-Based Studies

The advent of next generation sequencing has coincided with a growth in interest in using these approaches to better understand the role of the structure and function of the microbial communities in human, animal, and environmental health. Yet, use of next generation sequencing to perform 16S rRNA gene sequence surveys has resulted in considerable controversy surrounding the effects of sequencing errors on downstream analyses. We analyzed 2.7×10[superscript 6] reads distributed among 90 identical mock community samples, which were collections of genomic DNA from 21 different species with known 16S rRNA gene sequences; we observed an average error rate of 0.0060. To improve this error rate, we evaluated numerous methods of identifying bad sequence reads, identifying regions within reads of poor quality, and correcting base calls and were able to reduce the overall error rate to 0.0002. Implementation of the PyroNoise algorithm provided the best combination of error rate, sequence length, and number of sequences. Perhaps more problematic than sequencing errors was the presence of chimeras generated during PCR. Because we knew the true sequences within the mock community and the chimeras they could form, we identified 8% of the raw sequence reads as chimeric. After quality filtering the raw sequences and using the Uchime chimera detection program, the overall chimera rate decreased to 1%. The chimeras that could not be detected were largely responsible for the identification of spurious operational taxonomic units (OTUs) and genus-level phylotypes. The number of spurious OTUs and phylotypes increased with sequencing effort indicating that comparison of communities should be made using an equal number of sequences. Finally, we applied our improved quality-filtering pipeline to several benchmarking studies and observed that even with our stringent data curation pipeline, biases in the data generation pipeline and batch effects were observed that could potentially confound the interpretation of microbial community data.National Institutes of Health (U.S.) (1R01HG005975-01)National Science Foundation (U.S.) (award #0743432)National Institutes of Health (U.S.) (grant NIHU54HG004969

Expression Profiling without Genome Sequence Information in a Non-Model Species, Pandalid Shrimp (Pandalus latirostris), by Next-Generation Sequencing

While the study of phenotypic variation is a central theme in evolutionary biology, the genetic approaches available to understanding this variation are usually limited because of a lack of genomic information in non-model organisms. This study explored the utility of next-generation sequencing (NGS) technologies for studying phenotypic variations between 2 populations of a non-model species, the Hokkai shrimp (Pandalus latirostris; Decapoda, Pandalidae). Before we performed transcriptome analyses using NGS, we examined the genetic and phenotypic differentiation between the populations. Analyses using microsatellite DNA markers suggested that these populations genetically differed from one another and that gene flow is restricted between them. Moreover, the results of our 4-year field observations indicated that the egg traits varied genetically between the populations. Using mRNA extracted from the ovaries of 5 females in each population of Hokkai shrimp, we then performed a transcriptome analysis of the 2 populations. A total of 13.66 gigabases (Gb) of 75-bp reads was obtained. Further, 58,804 and 33,548 contigs for the first and second population, respectively, and 47,467 contigs for both populations were produced by de novo assembly. We detected 552 sequences with the former approach and 702 sequences with the later one; both sets of sequences showed greater than twofold differences in the expression levels between the 2 populations. Twenty-nine sequences were found in both approaches and were considered to be differentially expressed genes. Among them, 9 sequences showed significant similarity to functional genes. The present study showed a de novo assembly approach for the transcriptome of a non-model species using only short-read sequence data, and provides a strategy for identifying sequences showing significantly different expression levels between populations

The O2, pH and Ca2+ Microenvironment of Benthic Foraminifera in a High CO2 World

Ocean acidification (OA) can have adverse effects on marine calcifiers. Yet, phototrophic marine calcifiers elevate their external oxygen and pH microenvironment in daylight, through the uptake of dissolved inorganic carbon (DIC) by photosynthesis. We studied to which extent pH elevation within their microenvironments in daylight can counteract ambient seawater pH reductions, i.e. OA conditions. We measured the O2 and pH microenvironment of four photosymbiotic and two symbiont-free benthic tropical foraminiferal species at three different OA treatments (∼432, 1141 and 2151 µatm pCO2). The O2 concentration difference between the seawater and the test surface (ΔO2) was taken as a measure for the photosynthetic rate. Our results showed that O2 and pH levels were significantly higher on photosymbiotic foraminiferal surfaces in light than in dark conditions, and than on surfaces of symbiont-free foraminifera. Rates of photosynthesis at saturated light conditions did not change significantly between OA treatments (except in individuals that exhibited symbiont loss, i.e. bleaching, at elevated pCO2). The pH at the cell surface decreased during incubations at elevated pCO2, also during light incubations. Photosynthesis increased the surface pH but this increase was insufficient to compensate for ambient seawater pH decreases. We thus conclude that photosynthesis does only partly protect symbiont bearing foraminifera against OA

Arctic Ocean Microbial Community Structure before and after the 2007 Record Sea Ice Minimum

Increasing global temperatures are having a profound impact in the Arctic, including the dramatic loss of multiyear sea ice in 2007 that has continued to the present. The majority of life in the Arctic is microbial and the consequences of climate-mediated changes on microbial marine food webs, which are responsible for biogeochemical cycling and support higher trophic levels, are unknown. We examined microbial communities over time by using high-throughput sequencing of microbial DNA collected between 2003 and 2010 from the subsurface chlorophyll maximum (SCM) layer of the Beaufort Sea (Canadian Arctic). We found that overall this layer has freshened and concentrations of nitrate, the limiting nutrient for photosynthetic production in Arctic seas, have decreased. We compared microbial communities from before and after the record September 2007 sea ice minimum and detected significant differences in communities from all three domains of life. In particular, there were significant changes in species composition of Eukarya, with ciliates becoming more common and heterotrophic marine stramenopiles (MASTs) accounting for a smaller proportion of sequences retrieved after 2007. Within the Archaea, Marine Group I Thaumarchaeota, which earlier represented up to 60% of the Archaea sequences in this layer, have declined to <10%. Bacterial communities overall were less diverse after 2007, with a significant decrease of the Bacteroidetes. These significant shifts suggest that the microbial food webs are sensitive to physical oceanographic changes such as those occurring in the Canadian Arctic over the past decade

Simultaneous simulations of uptake in plants and leaching to groundwater of cadmium and lead for arable land amended with compost or farmyard manure.

he water budget of soil, the uptake in plants and the leaching to groundwater of cadmium (Cd) and lead (Pb) were simulated simultaneously using a physiological plant uptake model and a tipping buckets water and solute transport model for soil. Simulations were compared to results from a ten-year experimental field study, where four organic amendments were applied every second year. Predicted concentrations slightly decreased (Cd) or stagnated (Pb) in control soils, but increased in amended soils by about 10% (Cd) and 6% to 18% (Pb). Estimated plant uptake was lower in amended plots, due to an increase of Kd (dry soil to water partition coefficient). Predicted concentrations in plants were close to measured levels in plant residues (straw), but higher than measured concentrations in grains. Initially, Pb was mainly predicted to deposit from air into plants (82% in 1998); the next years, uptake from soil became dominating (30% from air in 2006), because of decreasing levels in air. For Cd, predicted uptake from air into plants was negligible (1–5%)

Rapid Screening of Complex DNA Samples by Single-Molecule Amplification and Sequencing

Microbial cloning makes Sanger sequencing of complex DNA samples possible but is labor intensive. We present a simple, rapid and robust method that enables laboratories without special equipment to perform single-molecule amplicon sequencing, although in a low-throughput manner, from sub-picogram quantities of DNA. The method can also be used for quick quality control of next-generation sequencing libraries, as was demonstrated for a metagenomic sample

Soil Moisture and Fungi Affect Seed Survival in California Grassland Annual Plants

Survival of seeds in the seed bank is important for the population dynamics of many plant species, yet the environmental factors that control seed survival at a landscape level remain poorly understood. These factors may include soil moisture, vegetation cover, soil type, and soil pathogens. Because many soil fungi respond to moisture and host species, fungi may mediate environmental drivers of seed survival. Here, I measure patterns of seed survival in California annual grassland plants across 15 species in three experiments. First, I surveyed seed survival for eight species at 18 grasslands and coastal sage scrub sites ranging across coastal and inland Santa Barbara County, California. Species differed in seed survival, and soil moisture and geographic location had the strongest influence on survival. Grasslands had higher survival than coastal sage scrub sites for some species. Second, I used a fungicide addition and exotic grass thatch removal experiment in the field to tease apart the relative impact of fungi, thatch, and their interaction in an invaded grassland. Seed survival was lower in the winter (wet season) than in the summer (dry season), but fungicide improved winter survival. Seed survival varied between species but did not depend on thatch. Third, I manipulated water and fungicide in the laboratory to directly examine the relationship between water, fungi, and survival. Seed survival declined from dry to single watered to continuously watered treatments. Fungicide slightly improved seed survival when seeds were watered once but not continually. Together, these experiments demonstrate an important role of soil moisture, potentially mediated by fungal pathogens, in driving seed survival