Data-Independent-Acquisition-Based Proteomic Approach towards Understanding the Acclimation Strategy of Oleaginous Microalga Microchloropsis gaditana CCMP526 in Hypersaline Conditions.

Salinity is one of the significant factors that affect growth and cellular metabolism, including photosynthesis and lipid accumulation, in microalgae and higher plants. Microchloropsis gaditana CCMP526 can acclimatize to different salinity levels by accumulating compatible solutes, carbohydrates, and lipids as energy storage molecules. We used proteomics to understand the molecular basis for acclimation of M. gaditana to increased salinity levels [55 and 100 PSU (practical salinity unit)]. Correspondence analysis was used for the identification of salinity-responsive proteins (SRPs). The highest number of salinity-induced proteins was observed in 100 PSU. Gene ontology enrichment analysis revealed a separate path of acclimation for cells exposed to 55 and 100 PSU. Osmolyte and lipid biosynthesis were upregulated in hypersaline conditions. Concomitantly, lipid oxidation pathways were also upregulated in hypersaline conditions, providing acetyl-CoA for energy metabolism through the tricarboxylic acid cycle. Carbon fixation and photosynthesis were tightly regulated, while chlorophyll biosynthesis was affected in hypersaline conditions. Importantly, temporal proteome analysis of salinity-induced M. gaditana revealed vital SRPs which could be used for engineering salinity resilient microalgal strains for improved productivity in hypersaline culture conditions.

Temporal acclimation of Microchloropsis gaditana CCMP526 in response to hypersalinity.

Evaporation from culture ponds and raceways can subject algae to hypersalinity stress, and this is exacerbated by global warming. We investigated the effect of salinity on a marine microalga, Microchloropsis gaditana, which is of industrial significance because of its high lipid-accumulating capability. Both short-term (hours) and medium-term (days) effects of salinity were studied across various salinities (37.5, 55, 70 and 100 PSU). Salinity above 55 PSU suppressed cell growth and specific growth rate was significantly reduced at 100 PSU. Photosynthesis (Fv/Fm, rETRmax and Ik) was severely affected at high salinity conditions. Total carbohydrate per cell increased ∼1.7-fold after 24 h, which is consistent with previous findings that salinity induces osmolyte production to counter osmotic shock. In addition, accumulation of lipid increased by ∼4.6-fold in response to salinity. Our findings indicate a possible mechanism of acclimation to salinity, opening up new frontiers for osmolytes in pharmacological and cosmetics applications.

Time for Multiple Extraction Methods in Proteomics? A Comparison of Three Protein Extraction Methods in the Eustigmatophyte Alga Microchloropsis gaditana CCMP526.

Proteomics is a crucial postgenomic biotechnology for functional and systems scale analyses in cell and integrative biology, not to mention clinical and precision medicine research. However, a fundamental requirement for an accurate examination of the protein complement of cells is an efficient method for extracting the proteins. This study reports on the evaluation of three protein extraction methods: trichloroacetic acid (TCA)-acetone, phenol, and TRIzol, in the eustigmatophyte alga Microchloropsis gaditana CCMP526 for proteomic analysis. M. gaditana is a potential candidate for algal-based biofuels. This industrially important strain is also rich in dietary oil and pigments and is used as feed in the aquaculture industry. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis was performed for proteins obtained using the three extraction methods and their effects were examined by the abundance ratio. Protein yield was higher using the TCA-acetone and phenol methods than with the TRIzol method. The TCA method was superior than other methods examined here in terms of protein coverage and abundance. Subcellular localization of the identified proteins revealed no significant difference among the extraction methods. Importantly, each method revealed a unique set of proteins. To the best of our knowledge, this is the first report on evaluation of protein extraction methods for the proteomic analysis of M. gaditana CCMP526. These observations underscore the importance of using multiple protein extraction methods for comprehensive proteome coverage, as the field of proteomics edges toward diverse applications in biofuels, aquaculture industry, marine biology, and agriculture.

Multi-omics Frontiers in Algal Research: Techniques and Progress to Explore Biofuels in the Postgenomics World.

Current momentum of microalgal research rests extensively in tapping the potential of multi-omics methodologies in regard to sustainable biofuels. Microalgal biomass is fermented to bioethanol; while lipids, particularly triacylglycerides (TAGs), are transesterified to biodiesels. Biodiesel has emerged as an ideal biofuel candidate; hence, its commercialization and use are increasingly being emphasized. Abiotic stresses exaggerate TAG accumulation, but the precise mechanisms are yet to be known. More recently, comprehensive multi-omics studies in microalgae have emerged from the biofuel perspective. Genomics and transcriptomics of microalgae have provided crucial leads and basic understanding toward lipid biosynthesis. Proteomics and metabolomics are now complementing "algal omics" and offer precise functional insights into the attendant static and dynamic physiological contexts. Indeed, the field has progressed from shotgun to targeted approaches. Notably, targeted proteomics studies in microalga are not yet reported. Several multi-omics tools and technologies that may be used to dig deeper into the microalgal physiology are examined and highlighted in this review. The article therefore aims to both introduce various available high-throughput biotechnologies and applications of "omics" in microalgae, and enlists a compendium of the emerging cutting edge literature. We suggest that a strategic and thoughtful combination of data streams from different omics platforms can provide a system-wide overview. The algal omics warrants closer attention in the future, with a view to technical, economic, and societal impacts that are anticipated in the current postgenomics era.

Evolution of the RNase P RNA structural domain in Leptospira spp.

We have employed the RNase P RNA (RPR) gene, which is present as single copy in chromosome I of Leptospira spp. to investigate the phylogeny of structural domains present in the RNA subunit of the tRNA processing enzyme, RNase P. RPR gene sequences of 150 strains derived from NCBI database along with sequences determined from 8 reference strains were examined to fathom strain specific structural differences present in leptospiral RPR. Sequence variations in the RPR gene impacted on the configuration of loops, stems and bulges found in the RPR highlighting species and strain specific structural motifs. In vitro transcribed leptospiral RPR ribozymes are demonstrated to process pre-tRNA into mature tRNA in consonance with the positioning of Leptospira in the taxonomic domain of bacteria. RPR sequence datasets used to construct a phylogenetic tree exemplified the segregation of strains into their respective lineages with a (re)speciation of strain SH 9 to Leptospira borgpetersenii, strains Fiocruz LV 3954 and Fiocruz LV 4135 to Leptospira santarosai, strain CBC 613 to Leptospira kirschneri and strain HAI 1536 to Leptospira noguchii. Furthermore, it allowed characterization of an isolate P2653, presumptively characterized as either serovar Hebdomadis, Kremastos or Longnan to Leptospira weilii, serovar Longnan.