Unraveling the subtleties of ß-(1→3)-glucan phosphorylase specificity in the GH94, GH149, and GH161 glycoside hydrolase families.

Glycoside phosphorylases (GPs) catalyze the phosphorolysis of glycans into the corresponding sugar 1-phosphates and shortened glycan chains. Given the diversity of natural ß-(1→3)-glucans and their wide range of biotechnological applications, the identification of enzymatic tools that can act on ß-(1→3)-glucooligosaccharides is an attractive area of research. GP activities acting on ß-(1→3)-glucooligosaccharides have been described in bacteria, the photosynthetic excavate Euglena gracilis, and the heterokont Ochromonas spp. Previously, we characterized ß-(1→3)-glucan GPs from bacteria and E. gracilis, leading to their classification in glycoside hydrolase family GH149. Here, we characterized GPs from Gram-positive bacteria and heterokont algae acting on ß-(1→3)-glucooligosaccharides. We identified a phosphorylase sequence from Ochromonas spp. (OcP1) together with its orthologs from other species, leading us to propose the establishment of a new GH family, designated GH161. To establish the activity of GH161 members, we recombinantly expressed a bacterial GH161 gene sequence (PapP) from the Gram-positive bacterium Paenibacillus polymyxa ATCC 842 in Escherichia coli We found that PapP acts on ß-(1→3)-glucooligosaccharide acceptors with a degree of polymerization (DP) ≥ 2. This activity was distinct from that of characterized GH149 ß-(1→3)-glucan phosphorylases, which operate on acceptors with DP ≥ 1. We also found that bacterial GH161 genes co-localize with genes encoding ß-glucosidases and ATP-binding cassette transporters, highlighting a probable involvement of GH161 enzymes in carbohydrate degradation. Importantly, in some species, GH161 and GH94 genes were present in tandem, providing evidence that GPs from different CAZy families may work sequentially to degrade oligosaccharides.

Evolution of heterotrophy in chrysophytes as reflected by comparative transcriptomics.

Shifts in the nutritional mode between phototrophy, mixotrophy and heterotrophy are a widespread phenomenon in the evolution of eukaryotic diversity. The transition between nutritional modes is particularly pronounced in chrysophytes and occurred independently several times through parallel evolution. Thus, chrysophytes provide a unique opportunity for studying the molecular basis of nutritional diversification and of the accompanying pathway reduction and degradation of plastid structures. In order to analyze the succession in switching the nutritional mode from mixotrophy to heterotrophy, we compared the transcriptome of the mixotrophic Poterioochromonas malhamensis with the transcriptomes of three obligate heterotrophic species of Ochromonadales. We used the transcriptome of P. malhamensis as a reference for plastid reduction in the heterotrophic taxa. The analyzed heterotrophic taxa were in different stages of plastid reduction. We investigated the reduction of several photosynthesis related pathways e.g. the xanthophyll cycle, the mevalonate pathway, the shikimate pathway and the tryptophan biosynthesis as well as the reduction of plastid structures and postulate a presumable succession of pathway reduction and degradation of accompanying structures.

A tale of two mixotrophic chrysophytes: Insights into the metabolisms of two Ochromonas species (Chrysophyceae) through a comparison of gene expression.

Ochromonas spp. strains CCMP1393 and BG-1 are phagotrophic phytoflagellates with different nutritional strategies. Strain CCMP1393 is an obligate phototroph while strain BG-1 readily grows in continuous darkness in the presence of bacterial prey. Growth and gene expression of strain CCMP1393 were investigated under conditions allowing phagotrophic, mixotrophic, or phototrophic nutrition. The availability of light and bacterial prey led to the differential expression of 42% or 45-59% of all genes, respectively. Data from strain CCMP1393 were compared to those from a study conducted previously on strain BG-1, and revealed notable differences in carbon and nitrogen metabolism between the 2 congeners under similar environmental conditions. Strain BG-1 utilized bacterial carbon and amino acids through glycolysis and the tricarboxylic acid cycle, while downregulating light harvesting and carbon fixation in the Calvin cycle when both light and bacteria were available. In contrast, the upregulation of genes related to photosynthesis, light harvesting, chlorophyll synthesis, and carbon fixation in the presence of light and prey for strain CCMP1393 implied that this species is more phototrophic than strain BG-1, and that phagotrophy may have enhanced phototrophy. Cellular chlorophyll a content was also significantly higher in strain CCMP1393 supplied with bacteria compared to those without prey. Our results thus point to very different physiological strategies for mixotrophic nutrition in these closely related chrysophyte species.

Autotrophic and heterotrophic acquisition of carbon and nitrogen by a mixotrophic chrysophyte established through stable isotope analysis.

Collectively, phagotrophic algae (mixotrophs) form a functional continuum of nutritional modes between autotrophy and heterotrophy, but the specific physiological benefits of mixotrophic nutrition differ among taxa. Ochromonas spp. are ubiquitous chrysophytes that exhibit high nutritional flexibility, although most species generally fall towards the heterotrophic end of the mixotrophy spectrum. We assessed the sources of carbon and nitrogen in Ochromonas sp. strain BG-1 growing mixotrophically via short-term stable isotope probing. An axenic culture was grown in the presence of either heat-killed bacteria enriched with 15N and 13C, or unlabeled heat-killed bacteria and labeled inorganic substrates (13C-bicarbonate and 15N-ammonium). The alga exhibited high growth rates (up to 2 divisions per day) only until heat-killed bacteria were depleted. NanoSIMS and bulk IRMS isotope analyses revealed that Ochromonas obtained 84-99% of its carbon and 88-95% of its nitrogen from consumed bacteria. The chrysophyte assimilated inorganic 13C-carbon and 15N-nitrogen when bacterial abundances were very low, but autotrophic (photosynthetic) activity was insufficient to support net population growth of the alga. Our use of nanoSIMS represents its first application towards the study of a mixotrophic alga, enabling a better understanding and quantitative assessment of carbon and nutrient acquisition by this species.

Effect of light and prey availability on gene expression of the mixotrophic chrysophyte, Ochromonas sp.

BACKGROUND: Ochromonas is a genus of mixotrophic chrysophytes that is found ubiquitously in many aquatic environments. Species in this genus can be important consumers of bacteria but vary in their ability to perform photosynthesis. We studied the effect of light and bacteria on growth and gene expression of a predominantly phagotrophic Ochromonas species. Axenic cultures of Ochromonas sp. were fed with heat-killed bacteria (HKB) and grown in constant light or darkness. RNA was extracted from cultures in the light or in the dark with HKB present (Light + HKB; Dark + HKB), and in the light after HKB were depleted (Light + depleted HKB). RESULTS: There were no significant differences in the growth or bacterial ingestion rates between algae grown in light or dark conditions. The availability of light led to a differential expression of only 8% of genes in the transcriptome. A number of genes associated with photosynthesis, phagotrophy, and tetrapyrrole synthesis was upregulated in the Light + HKB treatment compared to Dark + HKB. Conversely, the comparison between the Light + HKB and Light + depleted HKB treatments revealed that the presence of HKB led to differential expression of 59% of genes, including the majority of genes involved in major carbon and nitrogen metabolic pathways. Genes coding for unidirectional enzymes for the utilization of glucose were upregulated in the presence of HKB, implying increased glycolytic activities during phagotrophy. Algae without HKB upregulated their expression of genes coding for ammonium transporters, implying uptake of inorganic nitrogen from the culture medium when prey were unavailable. CONCLUSIONS: Transcriptomic results agreed with previous observations that light had minimal effect on the population growth of Ochromonas sp. However, light led to the upregulation of a number of phototrophy- and phagotrophy-related genes, while the availability of bacterial prey led to prominent changes in major carbon and nitrogen metabolic pathways. Our study demonstrated the potential of transcriptomic approaches to improve our understanding of the trophic physiologies of complex mixotrophs, and revealed responses in Ochromonas sp. not apparent from traditional culture studies.