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.

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.

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.

BackgroundThe green microalga Dunaliella salina accumulates a high proportion of β-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.ResultsThe 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.ConclusionsThe 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.

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.

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.

Mixotrophic flagellates can comprise significant proportions of plankton biomass in marine ecosystems. Despite the growing recognition of the importance of this ecological strategy, and the identification of major environmental factors controlling phagotrophic behavior (light and nutrients), the physiological and molecular mechanisms underlying mixotrophic behavior are still unclear. In this study, we performed RNA-Seq transcriptomic analysis for two mixotrophic prasinophytes, Micromonas polaris and Pyramimonas tychotreta, under dissolved nutrient regimes that altered their ingestion of bacteria prey. Though the strains examined were polar isolates, both belong to genera with widespread distribution. Our aim was to characterize the transcriptomes of these two non-model phytoflagellates, identify transcripts consistent with phagotrophic activity and assess their differential expression in response to nutrient stress. De novo assembly of the transcriptomes yielded large numbers of novel coding transcripts with no known match within public databases. A summary of the transcripts by Gene Ontology terms showed many expected expression patterns, including genes involved in photosynthetic pathways and enzymes implicated in nutrient uptake pathways. Searches of KEGG databases identified several genes associated with intra-cellular digestive pathways actively transcribed in both prasinophytes. Differential expression analysis showed a larger response in P. tychotreta, where 23,373 genes were up-regulated and 1,752 were down-regulated in the low nutrient treatment when phagotrophy was enhanced. In contrast, in M. polaris, low nutrient treatments resulted in up-regulation of 314 transcripts while down-regulating 371. With respect to phagotrophic-related expression, 37 genes were co-expressed in both P. tychotreta and M. polaris, and although the response was less pronounced in M. polaris, it is consistent with differences in observed ingestion behavior. This study presents the first genomic data for Pyramimonas tychotreta, and also contributes to the limited available data for Micromonas polaris. Furthermore, it provides insight into the presence of genes associated with phagocytosis within the Prasinophyceae and contributes to the understanding of potential target genes required for the construction of a complete model of gene regulation of phagocytic behavior in algae.

The green alga Scenedesmus obliquus is an emerging platform species for the industrial production of biofuels. Here, we report the draft assembly and annotation for the nuclear, plastid, and mitochondrial genomes of S. obliquus strain DOE0152z.