Nonclassical autophagy activation pathways are essential for production of infectious Influenza A virus in vitro.

Autophagy is a critical mechanism deployed by eukaryotic cells in response to stress, including viral infection, to boost the innate antimicrobial responses. However, an increasing number of pathogens hijack the autophagic machinery to facilitate their own replication. Influenza A virus (IAV), responsible for several global pandemics, has an intricate dependence on autophagy for successful replication in mammalian cells. To elucidate key chokepoints in the host stress responses facilitating IAV replication, we constructed a meta-transcriptome of IAV and host gene expression dynamics during early (1-3 hpi), mid (4-6 hpi), and late (8-12 hpi) stages of the viral replication cycle at two multiplicities of infection (MOI): 1 and 5. We supplemented the global transcriptome study with phosphoproteomic analysis of stress-activated protein kinase (SAPK/JNK) signaling in lung carcinoma (predominantly used as an in vitro model of IAV replication) and normal human bronchial epithelial cells. We report significant differences in the activation profiles of autophagy regulating genes upon IAV infection at the two MOI as well as divergent dependence on ULK1 signaling within the normal and cancer cells. Regardless of the cell model, JNK-Thr187 signaling was crucial for the production of infectious viral particles.

Merging Fungal and Bacterial Community Profiles via an Internal Control.

Integrated measurements of fungi and bacteria are critical to understand how interactions between these taxa drive key processes in ecosystems ranging from soils to animal guts. High-throughput amplicon sequencing is commonly used to census microbiomes, but the genetic markers targeted for fungi and bacteria (typically ribosomal regions) are domain-specific so profiling must be performed separately, obscuring relationships between these groups. To solve this problem, we developed a spike-in method with an internal control (IC) construct containing primer sites commonly used for bacterial and fungal taxonomic profiling. The internal control offers several advantages: estimation of absolute abundances, estimation of fungal to bacterial ratios (F:B), integration of bacterial and fungal profiles for holistic community analysis, and lower costs compared to other quantitation methods. To validate the IC as a scaling method, we compared IC-derived measures of F:B to measures from quantitative PCR (qPCR) using a commercial mock community (the ZymoBiomic Microbial Community DNA Standard II, containing two fungi and eight bacteria) and complex environmental samples. For both the mock community and the environmental samples, the IC produced F:B values that were statistically consistent with qPCR. Merging the environmental fungal and bacterial profiles based on the IC-derived F:B values revealed new relationships among samples in terms of community similarity. This IC method is the first spike-in method to employ a single construct for cross-domain amplicon sequencing, offering more reliable measurements.

Microbial community changes during a toxic cyanobacterial bloom in an alkaline Hungarian lake.

The Carpathian Basin is a lowland plain located mainly in Hungary. Due to the nature of the bedrock, alluvial deposits, and a bowl shape, many lakes and ponds of the area are characterized by high alkalinity. In this study, we characterized temporal changes in eukaryal and bacterial community dynamics with high throughput sequencing and relate the changes to environmental conditions in Lake Velence located in Fejér county, Hungary. The sampled Lake Velence microbial populations (algal and bacterial) were analyzed to identify potential correlations with other community members and environmental parameters at six timepoints over 6 weeks in the Spring of 2012. Correlations between community members suggest a positive relationship between certain algal and bacterial populations (e.g. Chlamydomondaceae with Actinobacteria and Acidobacteria), while other correlations allude to changes in these relationships over time. During the study, high nitrogen availability may have favored non-nitrogen fixing cyanobacteria, such as the toxin-producing Microcystis aeruginosa, and the eutrophic effect may have been exacerbated by high phosphorus availability as well as the high calcium and magnesium content of the Carpathian Basin bedrock, potentially fostering exopolymer production and cell aggregation. Cyanobacterial bloom formation could have a negative environmental impact on other community members and potentially affect overall water quality as well as recreational activities. To our knowledge, this is the first prediction for relationships between photoautotrophic eukaryotes and bacteria from an alkaline, Hungarian lake.

Sources and resources: importance of nutrients, resource allocation, and ecology in microalgal cultivation for lipid accumulation.

Regardless of current market conditions and availability of conventional petroleum sources, alternatives are needed to circumvent future economic and environmental impacts from continued exploration and harvesting of conventional hydrocarbons. Diatoms and green algae (microalgae) are eukaryotic photoautotrophs that can utilize inorganic carbon (e.g., CO2) as a carbon source and sunlight as an energy source, and many microalgae can store carbon and energy in the form of neutral lipids. In addition to accumulating useful precursors for biofuels and chemical feed stocks, the use of autotrophic microorganisms can further contribute to reduced CO2 emissions through utilization of atmospheric CO2. Because of the inherent connection between carbon, nitrogen, and phosphorus in biological systems, macronutrient deprivation has been proven to significantly enhance lipid accumulation in different diatom and algae species. However, much work is needed to understand the link between carbon, nitrogen, and phosphorus in controlling resource allocation at different levels of biological resolution (cellular versus ecological). An improved understanding of the relationship between the effects of N, P, and micronutrient availability on carbon resource allocation (cell growth versus lipid storage) in microalgae is needed in conjunction with life cycle analysis. This mini-review will briefly discuss the current literature on the use of nutrient deprivation and other conditions to control and optimize microalgal growth in the context of cell and lipid accumulation for scale-up processes.

Diurnal transcriptional variation is reduced in a nitrogen-fixing diatom endosymbiont.

Many organisms have formed symbiotic relationships with nitrogen (N)-fixing bacteria to overcome N limitation. Diatoms in the family Rhopalodiaceae host unicellular, N-fixing cyanobacterial endosymbionts called spheroid bodies (SBs). Although this relationship is relatively young, SBs share many key features with older endosymbionts, including coordinated cell division and genome reduction. Unlike free-living relatives that fix N exclusively at night, SBs fix N largely during the day; however, how SB metabolism is regulated and coordinated with the host is not yet understood. We compared four SB genomes, including those from two new host species (Rhopalodia gibba and Epithemia adnata), to build a genome-wide phylogeny which provides a better understanding of SB evolutionary origins. Contrary to models of endosymbiotic genome reduction, the SB chromosome is unusually stable for an endosymbiont genome, likely due to the early loss of all mobile elements. Transcriptomic data for the R. gibba SB and host organelles addressed whether and how the allocation of transcriptional resources depends on light and nitrogen availability. Although allocation to the SB was high under all conditions, relative expression of chloroplast photosynthesis genes increased in the absence of nitrate, but this pattern was suppressed by nitrate addition. SB expression of catabolism genes was generally greater during daytime rather than at night, although the magnitude of diurnal changes in expression was modest compared to free-living Cyanobacteria. We conclude that SB daytime catabolism likely supports N-fixation by linking the process to host photosynthetic carbon fixation.

A multi-omic characterization of temperature stress in a halotolerant Scenedesmus strain for algal biotechnology.

Microalgae efficiently convert sunlight into lipids and carbohydrates, offering bio-based alternatives for energy and chemical production. Improving algal productivity and robustness against abiotic stress requires a systems level characterization enabled by functional genomics. Here, we characterize a halotolerant microalga Scenedesmus sp. NREL 46B-D3 demonstrating peak growth near 25 °C that reaches 30 g/m2/day and the highest biomass accumulation capacity post cell division reported to date for a halotolerant strain. Functional genomics analysis revealed that genes involved in lipid production, ion channels and antiporters are expanded and expressed. Exposure to temperature stress shifts fatty acid metabolism and increases amino acids synthesis. Co-expression analysis shows that many fatty acid biosynthesis genes are overexpressed with specific transcription factors under cold stress. These and other genes involved in the metabolic and regulatory response to temperature stress can be further explored for strain improvement.

A Lipid-Accumulating Alga Maintains Growth in Outdoor, Alkaliphilic Raceway Pond with Mixed Microbial Communities.

Algal biofuels and valuable co-products are being produced in both open and closed cultivation systems. Growing algae in open pond systems may be a more economical alternative, but this approach allows environmental microorganisms to colonize the pond and potentially infect or outcompete the algal "crop." In this study, we monitored the microbial community of an outdoor, open raceway pond inoculated with a high lipid-producing alkaliphilic alga, Chlorella vulgaris BA050. The strain C. vulgaris BA050 was previously isolated from Soap Lake, Washington, a system characterized by a high pH (∼9.8). An outdoor raceway pond (200 L) was inoculated with C. vulgaris and monitored for 10 days and then the culture was transferred to a 2,000 L raceway pond and cultivated for an additional 6 days. Community DNA samples were collected over the 16-day period in conjunction with water chemistry analyses and cell counts. Universal primers for the SSU rRNA gene sequences for Eukarya, Bacteria, and Archaea were used for barcoded pyrosequence determination. The environmental parameters that most closely correlated with C. vulgaris abundance were pH and phosphate. Community analyses indicated that the pond system remained dominated by the Chlorella population (93% of eukaryotic sequences), but was also colonized by other microorganisms. Bacterial sequence diversity increased over time while archaeal sequence diversity declined over the same time period. Using SparCC co-occurrence network analysis, a positive correlation was observed between C. vulgaris and Pseudomonas sp. throughout the experiment, which may suggest a symbiotic relationship between the two organisms. The putative relationship coupled with high pH may have contributed to the success of C. vulgaris. The characterization of the microbial community dynamics of an alkaliphilic open pond system provides significant insight into open pond systems that could be used to control photoautotrophic biomass productivity in an open, non-sterile environment.