A novel phage indirectly regulates diatom growth by infecting diatom-associated biofilm-forming bacterium

Algae and heterotrophic bacteria have close and intricate interactions, which are regulated by multiple factors in the natural environment. Phages are the major factor determining bacterial mortality. However, their impacts on the algae-associated bacteria and thus on the algae-bacteria interactions are poorly understood. Here, we obtained a diatom-associated bacterium Stappia indica SNL01 that could form biofilm and had an inhibitory effect on the growth of diatom Thalassiosira pseudonana . Meanwhile, the phage SI01 with a double-stranded circular DNA genome (44,247 bp) infecting S. indica SNL01 was isolated. Phylogenetic analysis revealed that phage SI01 represents a novel member of the Podoviridae family. The phage contained multiple lysis genes encoding for cell wall lysing muramidase and spore cortex lysing SleB, as well as depolymerase-like tail spike protein. By lysing the host bacterium and inhibiting the formation of biofilm, this phage could indirectly promote the growth of the diatom. Our results shed new insights into how phages indirectly regulate algal growth by infecting bacteria closely associated with algae or in the phycosphere. IMPORTANCE The impact of phage infection on the algae-bacteria relationship in the ocean is poorly understood. Here, a novel phage infecting the diatom-associated bacterium Stappia indica SNL01 was isolated. This bacterium could form biofilm and had a negative effect on diatom growth. We revealed that this phage contained multiple lysis genes and could inhibit the formation of bacterial biofilm, thus indirectly promoting diatom growth. This study implicates that phages are not only important regulators of bacteria but also have substantial indirect effects on algae as well as the algae-bacteria relationship.

Palm Oil Mill Effluent for Lipid Production by the Diatom Thalassiosira pseudonana

Biomass and lipid production by the marine centric diatom Thalassiosira pseudonana were characterized in media based on palm oil mill effluent (POME) as a source of key nutrients. The optimal medium comprised 20% by volume POME, 80 µM Na2SiO3, and 35 g NaCl L−1 in water at pH ~7.7. In 15-day batch cultures (16:8 h/h light–dark cycle; 200 µmol photons m−2 s−1, 26 ± 1 °C) bubbled continuously with air mixed with CO2 (2.5% by vol), the peak concentration of dry biomass was 869 ± 14 mg L−1 corresponding to a productivity of ~58 mg L−1 day−1. The neutral lipid content of the biomass was 46.2 ± 1.1% by dry weight. The main components of the esterified lipids were palmitoleic acid methyl ester (31.6% w/w) and myristic acid methyl ester (16.8% w/w). The final biomass concentration and the lipid content were affected by the light–dark cycle. Continuous (24 h light) illumination at the above-specified irradiance reduced biomass productivity to ~54 mg L−1 day−1 and lipid content to 38.1%.

Structural Identification of Metalloproteomes in Marine Diatoms, an Efficient Algae Model in Toxic Metals Bioremediation

The biosorption of pollutants using microbial organisms has received growing interest in the last decades. Diatoms, the most dominant group of phytoplankton in oceans, are (i) pollution tolerant species, (ii) excellent biological indicators of water quality, and (iii) efficient models in assimilation and detoxification of toxic metal ions. Published research articles connecting proteomics with the capacity of diatoms for toxic metal removal are very limited. In this work, we employed a structural based systematic approach to predict and analyze the metalloproteome of six species of marine diatoms: Thalassiosira pseudonana, Phaeodactylum tricornutum, Fragilariopsis cylindrus, Thalassiosira oceanica, Fistulifera solaris, and Pseudo-nitzschia multistriata. The results indicate that the metalloproteome constitutes a significant proportion (~13%) of the total diatom proteome for all species investigated, and the proteins binding non-essential metals (Cd, Hg, Pb, Cr, As, and Ba) are significantly more than those identified for essential metals (Zn, Cu, Fe, Ca, Mg, Mn, Co, and Ni). These findings are most likely related to the well-known toxic metal tolerance of diatoms. In this study, metalloproteomes that may be involved in metabolic processes and in the mechanisms of bioaccumulation and detoxification of toxic metals of diatoms after exposure to toxic metals were identified and described.

Lowering pO2 Interacts with Photoperiod to Alter Physiological Performance of the Coastal Diatom Thalassiosira pseudonana

Exacerbating deoxygenation is extensively affecting marine organisms, with no exception for phytoplankton. To probe these effects, we comparably explored the growth, cell compositions, photosynthesis, and transcriptome of a diatom Thalassiosira pseudonana under a matrix of pO2 levels and Light:Dark cycles at an optimal growth light. The growth rate (μ) of T. pseudonana under a 8:16 L:D cycle was enhanced by 34% by low pO2 but reduced by 22% by hypoxia. Under a 16:8 L:D cycle, however, the μ decreased with decreasing pO2 level. The cellular Chl a content decreased with decreasing pO2 under a 8:16 L:D cycle, whereas the protein content decreased under a 16:8 L:D cycle. The prolonged photoperiod reduced the Chl a but enhanced the protein contents. The lowered pO2 reduced the maximal PSII photochemical quantum yield (FV/FM), photosynthetic oxygen evolution rate (Pn), and respiration rate (Rd) under the 8:16 or 16:8 L:D cycles. Cellular malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were higher under low pO2 than ambient pO2 or hypoxia. Moreover, the prolonged photoperiod reduced the FV/FM and Pn among all three pO2 levels but enhanced the Rd, MDA, and SOD activity. Transcriptome data showed that most of 26 differentially expressed genes (DEGs) that mainly relate to photosynthesis, respiration, and metabolism were down-regulated by hypoxia, with varying expression degrees between the 8:16 and 16:8 L:D cycles. In addition, our results demonstrated that the positive or negative effect of lowering pO2 upon the growth of diatoms depends on the pO2 level and is mediated by the photoperiod.

The genome of the diatom Chaetoceros tenuissimus carries an ancient integrated fragment of an extant virus

Diatoms are one of the most prominent oceanic primary producers and are now recognized to be distributed throughout the world. They maintain their population despite predators, infections, and unfavourable environmental conditions. One of the smallest diatoms, Chaetoceros tenuissimus, can coexist with infectious viruses during blooms. To further understand this relationship, we sequenced the C. tenuissimus strain NIES-3715 genome. A gene fragment of a replication-associated gene from the infectious ssDNA virus (designated endogenous virus-like fragment, EVLF) was found to be integrated into each 41 Mb of haploid assembly. In addition, the EVLF was transcriptionally active and conserved in nine other C. tenuissimus strains from different geographical areas, although the primary structures of their proteins varied. The phylogenetic tree further suggested that the EVLF was acquired by the ancestor of C. tenuissimus. Additionally, retrotransposon genes possessing a reverse transcriptase function were more abundant in C. tenuissimus than in Thalassiosira pseudonana and Phaeodactylum tricornutum. Moreover, a target site duplication, a hallmark for long interspersed nuclear element retrotransposons, flanked the EVLF. Therefore, the EVLF was likely integrated by a retrotransposon during viral infection. The present study provides further insights into the diatom-virus evolutionary relationship.

Fatty Acid Profiles of Selected Microalgae Used as Live Feeds for Shrimp Postlarvae in Vietnam

The importance of microalgal lipids for the survival and growth of shrimp postlarvae has been recognized in a range of studies. Microalgae with fast growth rates and high levels of polyunsaturated fatty acids (PUFA) are considered vital to maximise production and minimise cost in shrimp larviculture. The lipid content and fatty acid composition of microalgae used in shrimp production varies substantially between the algal classes and species being used in Vietnam. This study aims to characterise microalgal lipid and fatty acid (FA) profiles and evaluate the most promising species under growth conditions that are most suitable for shrimp aquaculture. Here, we report that the highest lipid contents were obtained in the Haptophyta microalgae, Tisochrysis lutea and Isochrysis galbana, at 90.3 and 61.1 mg/g, respectively. In contrast, two of the most popular diatom species being used for shrimp larval cultivation in Vietnam, Thalassiosira pseudonana and T. weissflogii, displayed the lowest lipid contents at 16.1 mg/g. Other microalgal species examined showed lipid contents ranging from 28.6 to 55 mg/g. Eicosapentaenoic acid (EPA, 20:5ω3) ranged from 0.6 to 29.9% across the species, with docosahexaenoic acid (DHA, 22:6ω3) present at 0.01 to 11.1%; the two omega (ω)–3 long-chain (LC, ≥C20) LC-PUFA varied between the microalgae groups. Polar lipids were the main lipid class, ranging from 87.2 to 97.3% of total lipids, and triacylglycerol was detected in the range of 0.01 to 2.5%. Saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) increased and PUFA decreased with increasing growth temperatures. This study demonstrated the differences in the lipid contents and FA profiles across 10 microalgal species and the effect of the higher temperature growing conditions encountered in Vietnam.

Molecular mechanism of oil induced growth inhibition in diatoms using Thalassiosira pseudonana as the model species

The 2010 Deepwater Horizon oil-spill exposed the microbes of Gulf of Mexico to unprecedented amount of oil. Conclusive evidence of the underlying molecular mechanism(s) on the negative effects of oil exposure on certain phytoplankton species such as Thalassiosira pseudonana is still lacking, curtailing our understanding of how oil spills alter community composition. We performed experiments on model diatom T. pseudonana to understand the mechanisms underpinning observed reduced growth and photosynthesis rates during oil exposure. Results show severe impairment to processes upstream of photosynthesis, such as light absorption, with proteins associated with the light harvesting complex damaged while the pigments were unaffected. Proteins associated with photosynthetic electron transport were also damaged, severely affecting photosynthetic apparatus and depriving cells of energy and carbon for growth. Negative growth effects were alleviated when an organic carbon source was provided. Further investigation through proteomics combined with pathway enrichment analysis confirmed the above findings, while highlighting other negatively affected processes such as those associated with ferroxidase complex, high-affinity iron-permease complex, and multiple transmembrane transport. We also show that oxidative stress is not the primary route of negative effects, rather secondary. Overall, this study provides a mechanistic understanding of the cellular damage that occurs during oil exposure to T. pseudonana.

Shedding light on biosilica morphogenesis by comparative analysis of the silica-associated proteomes from three diatom species

Morphogenesis of the intricate patterns of diatom silica cell walls is a protein-guided process, yet to date only very few such silica morphogenetic proteins have been identified. Therefore, it is unknown whether all diatoms share conserved proteins of a basal silica forming machinery, and whether unique proteins are responsible for the morphogenesis of species specific silica patterns. To answer these questions, we extracted proteins from the silica of three diatom species (Thalassiosira pseudonana, Thalassiosira oceanica and Cyclotella cryptica) by complete demineralization of the cell walls. LC-MS/MS analysis of the extracts identified 92 proteins that we name 'Soluble Silicome Proteins' (SSPs). Surprisingly, no SSPs are common to all three species, and most SSPs showed very low similarity to one another in sequence alignments. In depth bioinformatics analyses revealed that SSPs can be grouped into distinct classes bases on short unconventional sequence motifs whose functions are yet unknown. The results from in vivo localization of selected SSPs indicates that proteins, which lack sequence homology but share unconventional sequence motifs may exert similar functions in the morphogenesis of the diatom silica cell wall.

Structural Contour Map of the Iota Carbonic Anhydrase from the Diatom Thalassiosira pseudonana Using a Multiprong Approach

Carbonic anhydrases (CAs) are a family of ubiquitous enzymes that catalyze the interconversion of CO2 and HCO3−. The “iota” class (ι-CA) was first found in the marine diatom Thalassiosira pseudonana (tpι-CA) and is widespread among photosynthetic microalgae and prokaryotes. The ι-CA has a domain COG4875 (or COG4337) that can be repeated from one to several times and resembles a calcium–calmodulin protein kinase II association domain (CaMKII-AD). The crystal structure of this domain in the ι-CA from a cyanobacterium and a chlorarachniophyte has been recently determined. However, the three-dimensional organization of the four domain-containing tpι-CA is unknown. Using biophysical techniques and 3-D modeling, we show that the homotetrameric tpι-CA in solution has a flat “drone-like” shape with a core formed by the association of the first two domains of each monomer, and four protruding arms formed by domains 3 and 4. We also observe that the short linker between domains 3 and 4 in each monomer confers high flexibility, allowing for different conformations to be adopted. We propose the possible 3-D structure of a truncated tpι-CA containing fewer domain repeats using experimental data and discuss the implications of this atypical shape on the activity and metal coordination of the ι-CA.