Time­resolved transcriptome analysis during transitions of sulfur nutritional status provides insight into triacylglycerol (TAG) and astaxanthin accumulation in the green alga Chromochloris zofingiensis.

BACKGROUND: Chromochloris zofingiensis, an oleaginous microalga, is a promising feedstock for the co-production of triacylglycerol (TAG)-based biodiesel and the high-value product astaxanthin. To reveal the molecular mechanism of TAG and astaxanthin biosynthesis during transitions of sulfur nutritional status, namely sulfur-starvation (SS) and sulfur-replenishment (SR), the physiological responses and the transcriptomic dynamics of C. zofingiensis were examined. RESULTS: The results revealed a reversible TAG and astaxanthin accumulation under SS, which is correlated with the reduction of cell growth and protein content, indicating the reallocation of carbon. By correlating the data on the physiological and transcriptional responses to different sulfur nutritional status, a model for the underlying mechanism of TAG and astaxanthin accumulation in C. zofingiensis was postulated, which involved up-regulation of key genes including diacylglycerol acyltransferase (DGTT5) and beta-carotene ketolase (BKT1), increased energy and NADPH supply by elevating the tricarboxylic acid (TCA) cycle and the oxidative pentose phosphate (OPP) pathway, and the increased carbon precursors (pyruvate and acetyl-CoA) through central carbon metabolism. In addition, the net enhancement of the de novo biosynthesis of fatty acids and the re-direction of the terpenoid precursors toward the branch catalyzed by lycopene beta cyclase (LCYb) and BKT1 escalated the substrate availability for the biosynthesis of TAG and astaxanthin, respectively. CONCLUSIONS: In this study, the time-resolved transcriptional analysis of C. zofingiensis under SS and SR conditions was reported for the first time to elucidate the regulatory roles of key enzymes, including DGTT5, BKT1 and LCYb, in the underlying mechanisms of TAG and astaxanthin accumulation.

Temporal heterogeneity of microbial communities and metabolic activities during a natural algal bloom.

Elucidating the interactions between algae and associated microbial communities is critical for understanding the mechanisms that mediate the dynamic of harmful algal blooms (HABs) in marine environment. However, the microbial functional profiles and their biogeochemical potential in HABs process remains elusive, especially during a complete natural HAB cycle. Here, we used pyrosequencing and functional gene array (GeoChip) to investigate microbial community dynamics and metabolic potential during a natural dinoflagellate (Noctiluca scintillans) bloom. The results shown that bacterioplankton exhibited significant temporal heterogeneity over the course of the bloom stages. Microbial succession was co-driven by environmental parameters and biotic interactions. The functional analysis revealed significant variations in microbial metabolism during matter cycling. At bloom onset-stage, metabolic potential associated with iron oxidation and transport was elevated. Carbon fixation and degradation, denitrification, phosphorus acquisition, and sulfur transfer/oxidation were significantly enhanced at the plateau stage. During the decline and terminal stages, oxidative stress, lysis of compounds, and toxin degradation & protease synthesis increased. This work reveal phycosphere microorganisms can enhanced organic C decomposition capacity, altered N assimilation rate and S/P turnover efficiency, and balancing of the Fe budget during HAB process. The ecological linkage analysis has further shown that microbial composition and functional potential were significantly linked to algal blooms occurrence. It suggest that structural variability and functional plasticity of microbial communities influence HAB trajectory.

Harnessing C/N balance of Chromochloris zofingiensis to overcome the potential conflict in microalgal production.

Accumulation of high-value products in microalgae is not conducive with rapid cell growth, which is the potential conflict in microalgal production. Overcoming such conflict faces numerous challenges in comprehensively understanding cell behavior and metabolism. Here, we show a fully integrated interaction between cell behavior, carbon partitioning, carbon availability and path rate of central carbon metabolism, and have practically overcome the production conflict of Chromochloris zofingiensis. We demonstrate that elevated carbon availability and active path rate of precursors are determinants for product biosynthesis, and the former exhibits a superior potential. As protein content reaches a threshold value to confer survival advantages, carbon availability becomes the major limiting factor for product biosynthesis and cell reproduction. Based on integrated interaction, regulating the C/N balance by feeding carbon source under excess light increases content of high-value products without inhibiting cell growth. Our findings provide a new orientation to achieve great productivity improvements in microalgal production.

A novel fed-batch strategy enhances lipid and astaxanthin productivity without compromising biomass of Chromochloris zofingiensis.

To improve lipid and astaxanthin productivity without compromising biomass during the whole cultivation period, carbon-dependent kinetics involving nitrogen stress was applied under excess light to elevate intracellular carbon availability and metabolic activity of Chromochloris zofingiensis. Results suggested that fed-batch strategy proposed could increase lipid and astaxanthin productivity to 457.1 and 2.0 mg L-1 d-1, respectively. Biomass productivity at 1084.3 mg L-1 d-1 was comparable with that under suitable condition. Then 13C tracer-based metabolic flux analysis (13C-MFA) demonstrated that central carbon metabolism provided ATP, NADPH and carbon availability for lipid biosynthesis during the strategy. In combination with targeted metabolite analysis, 13C-MFA revealed that the strategy improved precursor content for lipid biosynthesis and elevated path rate to synthesize C16:0 and C18:0. The enhanced lipid content potentially accounted for the high biomass productivity. Therefore, comprehensively understanding relationships between carbon availability and carbon conversion could precisely design strategy for productivity improvements during cultivation.

Boost carbon availability and value in algal cell for economic deployment of biomass.

This study elucidated storage carbon metabolism in a dynamic manner through kinetic model, metabolomics and stable metabolic flux analysis. Results revealed nutrient uptake rate, carbon availability and synthetic path rate accounted for the integration of process-compatible products. The uptake rate could be enhanced by promoting carbohydrate accumulation, inducing high performance of tricarboxylic acid cycle and anaplerotic routes. Values of specific rate for lipid from kinetic model and synthetic path rate from metabolic flux analysis revealed that conversion of carbon sinks occupied a key position in increasing productivities of lipid and astaxanthin to 302.34 and 1.83 mg g-1 d-1, respectively. Additionally, economic estimation was applied to link cultivation factors with market scenario and demonstrated that regulating such carbon metabolism raised 30% increase of biomass value. This study therefore provided a new orientation to boost carbon efficiency that helped to engineer carbon flux from carbon source to targeted products precisely and rapidly.

Profiles of quorum sensing (QS)-related sequences in phycospheric microorganisms during a marine dinoflagellate bloom, as determined by a metagenomic approach.

The complicated relationships among environmental microorganisms are regulated by quorum sensing (QS). Understanding QS-based signals could shed light on the interactions between microbial communities in certain environments. Although QS characteristics have been widely discussed, few studies have been conducted on the role of QS in phycospheric microorganisms. Here, we used metagenomics to examine the profile of AI-1 (AinS, HdtS, LuxI) and AI-2 (LuxS) autoinducers from a deeply sequenced microbial database, obtained from a complete dinoflagellate bloom. A total of 3001 putative AI-1 homologs and 130 AI-2 homologs were identified. The predominant member among the AI groups was HdtS. The abundance of HdtS, AinS, and LuxS increased as the bloom developed, whereas the abundance of LuxI showed the opposite trend. Phylogenetic analysis suggested that HdtS and LuxI synthase originated mainly from alpha-, beta-, and gamma-Proteobacteria, whereas AinS synthase originated solely from Vibrionales. In comparison to AI-1, the sequences related to AI-2 (LuxS) demonstrated a much wider taxonomic coverage. Some significant correlations were found between dominant species and QS signals. In addition to the QS, we also performed parallel analysis of the quorum quenching (QQ) sequences. In comparison to QS, the relative abundance of QQ signals was lower; however, an obvious frequency correlation was observed. These results suggested that QS and QQ signals co-participate in regulating microbial communities during an algal bloom. These data helped to reveal the characteristic behavior of algal symbiotic bacteria, and facilitated a better understanding of microbial dynamics during an algal bloom event from a chemical ecological perspective.

Antibiofilm activity substances derived from coral symbiotic bacterial extract inhibit biofouling by the model strain Pseudomonas aeruginosa PAO1.

The mitigation of biofouling has received significant research attention, with particular focus on non-toxic and sustainable strategies. Here, we investigated quorum sensing inhibitor (QSI) bacteria as a means of controlling biofouling in a laboratory-scale system. Approximately, 200 strains were isolated from coral (Pocillopora damicornis) and screened for their ability to inhibit quorum sensing (QS). Approximately, 15% of the isolates exhibited QSI activity, and a typical coral symbiotic bacterium, H12-Vibrio alginolyticus, was selected in order for us to investigate quorum sensing inhibitory activity further. Confocal microscopy revealed that V. alginolyticus extract inhibited biofilm formation from Pseudomonas aeruginosa PAO1. In addition, the secondary metabolites of V. alginolyticus inhibited PAO1 virulence phenotypes by downregulating motility ability, elastase activity and rhamnolipid production. NMR and MS spectrometry suggested that the potential bioactive compound involved was rhodamine isothiocyanate. Quantitative real-time PCR indicated that the bacterial extract induced a significant downregulation of QS regulatory genes (lasB, lasI, lasR, rhlI, rhlR) and virulence-related genes (pqsA, pqsR). The possible mechanism underlying the action of rhodamine isothiocyanate analogue involves the disruption of the las and/or rhl system of PAO1. Our results highlight coral microbes as a bioresource pool for developing QS inhibitors and identifying novel antifouling agents.

Distribution Patterns of Microbial Community Structure Along a 7000-Mile Latitudinal Transect from the Mediterranean Sea Across the Atlantic Ocean to the Brazilian Coastal Sea.

A central goal in marine microecology is to understand the ecological factors shaping spatiotemporal microbial patterns and the underlying processes. We hypothesized that abiotic and/or biotic interactions are probably more important for explaining the distribution patterns of marine bacterioplankton than environmental filtering. In this study, surface seawater samples were collected about 7000 miles from the Mediterranean Sea, transecting the North Atlantic Ocean, to the Brazilian marginal sea. In bacterial biosphere, SAR11, SAR86, Rhodobacteraceae, and Rhodospiriaceae were predominant in the Mediterranean Sea; Prochlorococcus was more frequent in Atlantic Ocean; whereas in the Brazilian coastal sea, the main bacterial members were Synechococcus and SAR11. With respect to archaea, Euryarchaeota were predominant in the Atlantic Ocean and Thaumarchaeota in the Mediterranean Sea. With respect to the eukaryotes, Syndiniales, Spumellaria, Cryomonadida, and Chlorodendrales were predominant in the open ocean, while diatoms and microzooplankton were dominant in the coastal sea. Distinct clusters of prokaryotes and eukaryotes displayed clear spatial heterogeneity. Among the environmental parameters measured, temperature and salinity were key factors controlling bacterial and archaeal community structure, respectively, whereas N/P/Si contributed to eukaryotic variation. The relative contribution of environmental parameters to the microbial distribution pattern was 45.2%. Interaction analysis showed that Gammaproteobacteria, Alphaproteobacteria, and Flavobacteriia were the keystone taxa within the positive-correlation network, while Thermoplasmata was the main contributor in the negative-correlation network. Our study demonstrated that microbial communities are co-governed by environmental filtering and biotic interactions, which are the main deterministic driving factors modulating the spatiotemporal patterns of marine plankton synergistically at the regional or global levels.

TiO2 nanoparticles in the marine environment: Physical effects responsible for the toxicity on algae Phaeodactylum tricornutum.

Nanoscale titanium dioxide (nTiO2) has been widely used in cosmetics, catalysts, varnishes, etc., which is raising concerns about its potential hazards to the ecosystem, including the marine environment. In this study, the toxicological effect of nTiO2 on the marine phytoplankton Phaeodactylum tricornutum was carefully investigated. The results showed that nTiO2 at concentrations ≥20mg/L could significantly inhibit P. tricornutum growth. The 5-day EC50 of nTiO2 to P. tricornutum growth is 167.71mg/L. Interestingly, nTiO2 was found to exert its most severe inhibition effects on the first day of exposure, at a lower EC50 of 12.65mg/L. During the experiment, nTiO2 aggregates were found to entrap algae cells, which is likely responsible for the observed toxic effects. Direct physical effects such as cell wall damage from the algae entrapment were confirmed by flow cytometry and TEM imaging. Moreover, low indirect effects such as shading and oxidative stress were observed, which supported the idea that direct physical effects could be the dominant factor that causes nTiO2 toxicity in P. tricornutum. Our research provides direct evidence for the toxicological impact of nTiO2 on marine microalgae, which will help us to build a good understanding of the ecological risks of nanoparticles in the marine environment.

Draft Genome Sequence of Providencia sneebia Strain ST1, a Quorum Sensing Bacterium Associated with Marine Microalgae.

Providencia sneebia strain ST1 is a symbiotic bacterium (belonging to phylum gammaproteobacteria) with marine microalgae. This bacterium exhibits the ability to produce N-Acyl homoserine lactone signal molecule. To date, no genome that originates from marine Providencia spp. has been reported. In this study, we present the genome sequence of this strain. It has a genome size of 4.89 M, with 19 contigs and an average G+C of 51.97%. The function of 4,631 proteins was predicted, and 3,652 proteins were assigned to COG functional categories. Among them, 407 genes are involved in carbohydrate metabolism, 306 genes participate in nitrogen utilization and energy conversion, and 185 genes related to signal transduction process. Thus, this strain plays an active role in the biogeochemical cycle in algal life history. The whole-genome of this isolate and annotation will help enhance understanding of bacterial ecological behavior in the phycosphere.

Genome sequence of Enterobacter sp. ST3, a quorum sensing bacterium associated with marine dinoflagellate.

Phycosphere environment is a typical marine niche, harbor diverse populations of microorganisms, which are thought to play a critical role in algae host and influence mutualistic and competitive interactions. Understanding quorum sensing-based acyl-homoserine lactone (AHL) language may shed light on the interaction between algal-associated microbial communities in the native environment. In this work, we isolated an epidermal bacterium (was tentatively named Enterobacter sp. ST3, and deposited in SOA China, the number is MCCC1K02277-ST3) from the marine dinoflagellate Scrippsiella trochoidea, and found it has the ability to produce short-chain AHL signal. In order to better understand its communication information at molecular level, the genomic map was investigated. The genome size was determined to be 4.81 Mb with a G + C content of 55.59%, comprising 6 scaffolds of 75 contigs containing 4647 protein-coding genes. The functional proteins were predicted, and 3534 proteins were assigned to COG functional categories. An AHL-relating gene, LuxR, was found in upstream position at contig 1. This genome data may provide clues to increase understanding of the chemical characterization and ecological behavior of strain ST3 in the phycosphere microenvironment.

Characterization and expression patterns of nitrate reductase from Dunaliella bardawil under osmotic stress and dilution shock.

A complementary DNA (cDNA) of nitrate reductase (NR) from Dunaliella bardawil was isolated using RT-PCR and RACEs techniques. The full-length D. bardawil NR (DbNR) cDNA is 3,107 bp containing a putative open reading frame of 2,670 bp in length which encodes 889 amino acids with a calculated molecular weight (MW) of 98.37 kDa, a 34-bp 5'-untranslated region, and a 3'-untranslated region of 403 bp with a poly (A) tail. BLAST search showed that the nucleotide and putative protein sequence exhibit sequence identities of 92 and 79% with the corresponding gene from Dunaliella tertiolecta, respectively. Protein structural analysis showed a typical NR structure of DbNR with five structural distinctive domains which form three common subparts of eukaryotic NR (Euk-NR). Phylogenetic analysis based on the holo-DbNR and sulfite oxidase (SO) and cytochrome b reductase (CbR) subparts manifested that (1) DbNR has a closer relationship with those counterparts from algae and higher plants than from other species and (2) DbNR might have evolved from ancient SO and CbR in a "domain shuffling" pattern. The glycerol contents and transcriptional expression patterns of DbNR under salt stress and dilution shock treatments were also traced. The results implied an indirect role of NaCl on the induction of DbNR through an osmoregulation pathway.

Hypoosmotic expression of Dunaliella bardawil ζ-carotene desaturase is attributed to a hypoosmolarity-responsive element different from other key carotenogenic genes.

Some key carotenogenic genes (crts) in Dunaliella bardawil are regulated in response to salt stress partly due to salt-inducible cis-acting elements in their promoters. Thus, we isolated and compared the ζ-carotene desaturase (Dbzds) promoter with other crts promoters including phytoene synthase (Dbpsy), phytoene desaturase (Dbpds), and lycopene ß-cyclase1 (DblycB1) to identify salt-inducible element(s) in the Dbzds promoter. In silico analysis of the Dbzds promoter found several potential cis-acting elements, such as abscisic acid response element-like sequence, myelocytomatosis oncogene1 recognition motif, AGC box, anaerobic motif2, and activation sequence factor1 binding site. Remarkably, instead of salt-inducible elements, we found a unique regulatory sequence architecture in the Dbzds promoter: a hypoosmolarity-responsive element (HRE) candidate followed by a potential hypoosmolarity-inducible factor GBF5 binding site. Deletion experiments demonstrated that only HRE, but not the GBF5 binding site, is responsible for hypoosmotic expression of the fusion of Zeocin resistance gene (ble) to the enhanced green fluorescent protein (egfp) chimeric gene under salt stress. Dbzds transcripts were in accordance with those of ble-egfp driven by the wild-type Dbzds promoter. Consequently, Dbzds is hypoosmotically regulated by its promoter, and HRE is responsible for this hypoosmotic response. Finally, the hypoosmolarity mechanism of Dbzds was studied by comparing transcript profiles and regulatory elements of Dbzds with those of Dbpsy, Dbpds, DblycB1, and DblycB2, revealing that different induction characteristics of crts may correlate with regulatory sequence architecture.

Six regulatory elements lying in the promoter region imply the functional diversity of chloroplast GAPDH in Duanliella bardawil.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a well-known proverbial protein involved in various functions in vivo. The functional diversity of GAPDH from Dunaliella bardawil (DbGAPDH) may relate to the regulatory elements lying in the promoter at the transcriptional level. Using RT-PCR and RACE reactions, gapdh cDNA was isolated, and the full-length genomic sequence was obtained by LA-PCR-based genome walking. The full-length cDNA sequence was 1645 bp containing an 1128 bp putative open reading frame (ORF), which coded a 375 amino acids-deduced polypeptide whose molecular weight was 40.27 kDa computationally. Protein conserved domain search and structural computation found that DbGAPDH consists of two structural conserved domains highly homologous in most species; multiple sequence alignment discovered two positive charge residues (Lys164 and Arg 233), which play a critical role in the protein-protein interaction between GAPDH, phosphoribulokinase (PRK), and CP12. Phylogenetic analysis demonstrated that DbGAPDH has a closer relationship with analogues from algae and higher plants than with those from other species. In silico analysis of the promoter region revealed six potential regulatory elements might be involved in four hypothesized functions characterized by chloroplast GAPDH: oxygen-, light-, pathogen-, and cold-induced regulation. These results might supply some hints for the functional diversity mechanisms of DbGAPDH, and fresh information for further research to bridge the gap between our knowledge of DNA and protein structure and our understanding of functional biology in GAPDH regulation.

In silico analysis of phytoene synthase and its promoter reveals hints for regulation mechanisms of carotenogenesis in Duanliella bardawil.

MOTIVATION: Previous researches showed that phytoene synthase (Psy) from Dunaliella bardawil is the first regulatory point in carotenogenesis. We hypothesize certain interactions between the environmental stress factors and the regulatory sequences of Psy in D.bardawil (DbPsy). Consequently, LA PCR-based genomic walking approach was performed for isolation of psy promoter and terminator, respectively. The obtained nucleic acid sequences and the corresponding protein structure of DbPsy were analyzed and predicted using various bioinformatics tools. Finally, we presented some hints for the regulation mechanisms of DbPsy at the molecular level according to the computed results. RESULTS: LA PCR-based genomic walking results showed that the isolated sequences are the promoter and terminator of psy, correspondingly. Computational analysis demonstrated several candidate motifs of the promoter exhibiting hypothetic UV-B-, norglurzon- and salt-induced characteristics, as well as some typical domains universally discovered in promoter sequences, such as TATA-box, CCAAT-box and GATA-box, etc. Furthermore, the structure of Psy was also predicted and aligned along with many counterparts at the protein level. Low homology of N-terminus was found in D.bardawil, while a relatively conserved C-terminus was predicted to be involved in the catalytic activity and substrate recognization/binding. Phylogenic analysis classified the DbPsy into a cluster with other algae. These results implied that Psy may share similar regulation mechanisms among algae with respect to their C-termini; while the diversity in N-terminus among Psys, along with the predicted inducible motifs in psy promoter from D.bardawil, may confer the fine tuning differences between D.bardawil and other algae. CONCLUSION: By means of computer techniques, we found in D.barawali that two interesting conserved motifs of psy promoter may involve in UV-B, norglurzon and salt regulation correspondingly; and that the diversity of Psy protein mainly lies in the N-termini among algae. These results indicate some hints for regulation mechanisms of carotenogenesis in D.bradawil. CONTACT: jgjiang@scut.edu.cn.

Effects of salinities on the gene expression of a (NAD+)-dependent glycerol-3-phosphate dehydrogenase in Dunaliella salina.

Glycerol-3-phosphate dehydrogenase (G3pdh) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. In this study, the effects of salinity changes on variation of cell shape and single cell glycerol content of Dunaliella salina were observed, and the effects of salinity changes on the gene expressions of a (NAD+)-dependent G3pdh (EC1.1.1.8) among G3pdh isozymes in D. salina were detected by real-time quantitative PCR. Results showed that the changes of shape and volume of D. salina cell cultured chronically at various salinities were minor, but when the salinity was changed rapidly, the variations of cell shape and cell volume of D. salina were significant, which were recovered basically after 2h except treating by high salinity. Also, it was found some lipid globules in the surface of D. salina cells when the salinity increased from 2.0 to 4.0-5.0 M NaCl rapidly. When D. salina was cultured chronically at various salinities, the accumulation of single cell glycerol increased with increased salinity, and D. salina also could rapidly decrease or increase single cell glycerol contents to adapt to hypoosmotic or hyperosmotic shock. The expression level of G3pdh in D. salina grown at various salinities was significantly inversely correlated to the salinity, but there was no significant correlation between the expression level of G3pdh and salinity after 2 h of treatment by hyperosmotic or hypoosmotic shock.

Application of metabonomic analytical techniques in the modernization and toxicology research of traditional Chinese medicine.

In the recent years, a wide range of metabonomic analytical techniques are widely used in the modern research of traditional Chinese medicine (TCM). At the same time, the international community has attached increasing importance to TCM toxicity problems. Thus, many studies have been implemented to investigate the toxicity mechanisms of TCM. Among these studies, many metabonomic-based methods have been implemented to facilitate TCM toxicity investigation. At present, the most prevailing methods for TCM toxicity research are mainly single analysis techniques using only one analytical means. These techniques include nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS), etc.; with these techniques, some favourable outcomes have been gained in the toxic reaction studies of TCM, such as the action target organs assay, the establishment of action pattern, the elucidation of action mechanism and the exploration of action material foundation. However, every analytical technique has its advantages and drawbacks, no existing analytical technique can be versatile. Multi-analysed techniques can partially overcome the shortcomings of single-analysed techniques. Combination of GC-MS and LC-MS metabolic profiling approaches has unravelled the pathological outcomes of aristolochic acid-induced nephrotoxicity, which can not be achieved by single-analysed techniques. It is believed that with the further development of metabonomic analytical techniques, especially multi-analysed techniques, metabonomics will greatly promote TCM toxicity research and be beneficial to the modernization of TCM in terms of extending the application of modern means in the TCM safety assessment, assisting the formulation of TCM safety norms and establishing the international standards indicators.