Annotated Genome Sequence of the High-Biomass-Producing Yellow-Green Alga Tribonema minus.

Here, we report the annotated genome sequence for a heterokont alga from the class Xanthophyceae. This high-biomass-producing strain, Tribonema minus UTEX B 3156, was isolated from a wastewater treatment plant in California. It is stable in outdoor raceway ponds and is a promising industrial feedstock for biofuels and bioproducts.

Transposon mutagenesis in Mycobacterium kansasii links a small RNA gene to colony morphology and biofilm formation and identifies 9,885 intragenic insertions that do not compromise colony outgrowth.

Mycobacterium kansasii (Mk) is a resilient opportunistic human pathogen that causes tuberculosis-like chronic pulmonary disease and mortality stemming from comorbidities and treatment failure. The standard treatment of Mk infections requires costly, long-term, multidrug courses with adverse side effects. The emergence of drug-resistant isolates further complicates the already challenging drug therapy regimens and threatens to compromise the future control of Mk infections. Despite the increasingly recognized global burden of Mk infections, the biology of this opportunistic pathogen remains essentially unexplored. In particular, studies reporting gene function or generation of defined mutants are scarce. Moreover, no transposon (Tn) mutagenesis tool has been validated for use in Mk, a situation limiting the repertoire of genetic approaches available to accelerate the dissection of gene function and the generation of gene knockout mutants in this poorly characterized pathogen. In this study, we validated the functionality of a powerful Tn mutagenesis tool in Mk and used this tool in conjunction with a forward genetic screen to establish a previously unrecognized role of a conserved mycobacterial small RNA gene of unknown function in colony morphology features and biofilm formation. We also combined Tn mutagenesis with next-generation sequencing to identify 12,071 Tn insertions that do not compromise viability in vitro. Finally, we demonstrated the susceptibility of the Galleria mellonella larva to Mk, setting the stage for further exploration of this simple and economical infection model system to the study of this pathogen.

Reconstruction and analysis of a carbon-core metabolic network for Dunaliella salina.

BACKGROUND: The green microalga Dunaliella salina accumulates a high proportion of ß-carotene during abiotic stress conditions. To better understand the intracellular flux distribution leading to carotenoid accumulation, this work aimed at reconstructing a carbon core metabolic network for D. salina CCAP 19/18 based on the recently published nuclear genome and its validation with experimental observations and literature data. RESULTS: The reconstruction resulted in a network model with 221 reactions and 212 metabolites within three compartments: cytosol, chloroplast and mitochondrion. The network was implemented in the MATLAB toolbox CellNetAnalyzer and checked for feasibility. Furthermore, a flux balance analysis was carried out for different light and nutrient uptake rates. The comparison of the experimental knowledge with the model prediction revealed that the results of the stoichiometric network analysis are plausible and in good agreement with the observed behavior. Accordingly, our model provides an excellent tool for investigating the carbon core metabolism of D. salina. CONCLUSIONS: The reconstructed metabolic network of D. salina presented in this work is able to predict the biological behavior under light and nutrient stress and will lead to an improved process understanding for the optimized production of high-value products in microalgae.

Draft Nuclear Genome Sequence of the Halophilic and Beta-Carotene-Accumulating Green Alga Dunaliella salina Strain CCAP19/18.

The halotolerant alga Dunaliella salina is a model for stress tolerance and is used commercially for production of beta-carotene (=pro-vitamin A). The presented draft genome of the genuine strain CCAP19/18 will allow investigations into metabolic processes involved in regulation of stress responses, including carotenogenesis and adaptations to life in high-salinity environments.

Physiological responses of three species of Antarctic mixotrophic phytoflagellates to changes in light and dissolved nutrients.

Antarctic phototrophs are challenged by extreme temperatures, ice cover, nutrient limitation, and prolonged periods of darkness. Yet this environment may also provide niche opportunities for phytoplankton utilizing alternative nutritional modes. Mixotrophy, the combination of photosynthesis and particle ingestion, has been proposed as a mechanism for some phytoplankton to contend with the adverse conditions of the Antarctic. We conducted feeding experiments using fluorescent bacteria-sized tracers to compare the effects of light and nutrients on bacterivory rates in three Antarctic marine photosynthetic nanoflagellates representing two evolutionary lineages: Cryptophyceae (Geminigera cryophila) and Prasinophyceae (Pyramimonas tychotreta and Mantoniella antarctica). Only G. cryophila had previously been identified as mixotrophic. We also measured photoautotrophic abilities over a range of light intensities (P vs. I) and used dark survival experiments to assess cell population dynamics in the absence of light. Feeding behavior in these three nanoflagellates was affected by either light, nutrient levels, or a combination of both factors in a species-specific manner that was not conserved by evolutionary lineage. The different responses to environmental factors by these mixotrophs supported the idea of tradeoffs in the use of phagotrophy and phototrophy for growth.

Photosynthetic carbon from algal symbionts peaks during the latter stages of embryonic development in the salamander Ambystoma maculatum.

BACKGROUND: It was recently discovered that symbiotic algae in the eggs of the salamander Ambystoma maculatum translocate fixed carbon from photosynthesis to developing embryos. Fixed carbon translocation was shown in embryos at one time point during development, however, it was unknown if fixed carbon translocation occurs throughout all developmental stages. FINDINGS: In this study, fixed carbon translocation was measured in salamander eggs at six time points over the latter half of development. Fixed carbon translocation did not occur until the middle tailbud portion of development (stages 26-30), and translocation was measured in 20% or less of eggs sampled. Peak carbon translocation occurred during the late tailbud phase of development (stages 31-35), where as much as 87% of eggs sampled showed translocation, and average percent translocation was 6.5%. During the final stages of development, fixed carbon translocation declined, and translocation was not detected in embryos five days prior to hatching. CONCLUSIONS: The onset of fixed carbon translocation from Oophila to A. maculatum embryos during the second half of embryonic development is likely due to the corresponding settlement and concentration of Oophila in the inner egg envelope. In addition, carbon translocation ceases in late stage embryos as the inner egg envelope thins and ruptures in preparation for hatching.

Antarctic mixotrophic protist abundances by microscopy and molecular methods.

Protists are traditionally described as either phototrophic or heterotrophic, but studies have indicated that mixotrophic species, organisms that combine both strategies, can have significant impacts on prey populations in marine microbial food webs. While estimates of active mixotroph abundances in environmental samples are determined microscopically by fluorescent particle ingestion, species identification is difficult. We developed SYBR-based qPCR strategies for three Antarctic algal species that we identified as mixotrophic. This method and traditional ingestion experiments were applied to determine the total mixotroph abundance in Antarctic water samples, to ascertain the abundance of known mixotrophic species, and to identify environmental variables that impact the distribution and abundance of these species. Despite differences in sampling locations and years, mixotroph distribution was strongly influenced by season. Environmental variables that best explained variation in the individual mixotroph species abundances included temperature, oxygen, date, fluorescence, conductivity, and latitude. Phosphate was identified as an additional explanatory variable when nutrients were included in the analysis. Utilizing culture-based grazing rates and qPCR abundances, the estimated summed impact on bacterial populations by the three mixotrophs was usually < 2% of the overall mixotrophic grazing, but in one sample, Pyramimonas was estimated to contribute up to 80% of mixotrophic grazing.

Phagotrophy by the picoeukaryotic green alga Micromonas: implications for Arctic Oceans.

Photosynthetic picoeukaryotes (PPE) are recognized as major primary producers and contributors to phytoplankton biomass in oceanic and coastal environments. Molecular surveys indicate a large phylogenetic diversity in the picoeukaryotes, with members of the Prymnesiophyceae and Chrysophyseae tending to be more common in open ocean waters and Prasinophyceae dominating coastal and Arctic waters. In addition to their role as primary producers, PPE have been identified in several studies as mixotrophic and major predators of prokaryotes. Mixotrophy, the combination of photosynthesis and phagotrophy in a single organism, is well established for most photosynthetic lineages. However, green algae, including prasinophytes, were widely considered as a purely photosynthetic group. The prasinophyte Micromonas is perhaps the most common picoeukaryote in coastal and Arctic waters and is one of the relatively few cultured representatives of the picoeukaryotes available for physiological investigations. In this study, we demonstrate phagotrophy by a strain of Micromonas (CCMP2099) isolated from Arctic waters and show that environmental factors (light and nutrient concentration) affect ingestion rates in this mixotroph. In addition, we show size-selective feeding with a preference for smaller particles, and determine P vs I (photosynthesis vs irradiance) responses in different nutrient conditions. If other strains have mixotrophic abilities similar to Micromonas CCMP2099, the widespread distribution and frequently high abundances of Micromonas suggest that these green algae may have significant impact on prokaryote populations in several oceanic regimes.