Cloning, Bioinformatic Characterization And Expression Of SPT Gene From Marine Coccolithovirus And Its Relationship With Host Ceramide Production

Coccolithophores are unicellular marine microalgae. One prominent feature of it is the abilityto produce “the coccoliths”. Emiliania huxleyi is one of the only two bloom formingcoccolithophores. It is now the most abundant coccolithophore in the marine system especial insub-polar waters and ranks among the ten most important coccolithophores in terms of calciteexport and DMSP production. Consequently it is a key species for current studies on globalbiogeochemical cycles and climate modeling.In addition, E. huxleyi is known to play an important role in biotechnology research due to itscapable of production abundant bioactive metabolites. E. huxleyi virus (Coccolithovirus, EhV) isconsidered to responsible for the demise of an E.huxleyi bloom and is the key factor of E.huxleyinatural mortality.Ceramide is the key intermediate in the biosynthesis of all complexsphingolipids and exhibited a wide variety of bioactivity Therefore, ceramide have raised a largeinterest as active components both in the food and cosmetic industries.Along with thesequencing of EhV genome came the discovery, for one of the largeest algal virus to date, of agreat number of protein-conding sequences (CDSs).). A novel single-chain serine palmitoyltransferase (SPT), one of the sphingolipid biosynthesis pathway (SBP) encoded by EhV，is thefirst and rate limiting enzyme of ceramide biosynthesis.It was hypothesized that viral SPT genecould contribute to ceramide synthetize in E.huxleyi cell. However, nothing is known whetherthe viral SPT expression was related to host ceramide production or not. If it was the case, couldEhV be used as one natural vector carrying SPT gene to explore a new way for ceramideproduction? Therefore, in this study we investigated the relationship between E. huxleyiceramide production and SPT gene expression.Here, E. huxleyi EhBOF92and its specific virus EhV99B1were used for study. Weinvestigated the cloning and bioinformatic characterization of SPT from EhV99B1isolate. Fora functional identification, EhV99B1-LCB2gene (the catalytic subunit of SPT) was expressedin Escherichia coli BL21cells and the activity was analysised with thin layer chromatography(TLC) in vivo. A high-performance liquid chromatograph (HPLC) was used to assay theceramides in E.huxleyi cells. Real-time, quantitative PCR (RTFQ-PCR) following2-ΔΔCtmethod and western immunoblot analysis were used to estimate relative EhV99B1-SPT mRNA expression values and protein expression levels during different stages along EhV99B1infection. The main results of this paper were as follows:(1) We cloned a novel SPT-like gene in EhV-99B1.The open-reading frame (ORF) ofEhV99B1-SPT encoded a protein of496amino acids with a calculated molecular mass of96kDa and Ip6.01. The results of sequence analysis showed that there was about31%-45%identity in amino acid sequence with other organisms. The maximum likelihood phylogenetictree suggested that the EhV99B1-SPT gene possibly horizontally transferred from theeukaryote. Hydrophobic profiles of deduced amino acid sequences suggested SPT was ahydrophobic, globular and membrane-associated protein with5transmembrane domains(TMDs) motifs. Several potential N-linked glycosylation sites were presented in SPT. Theseresults suggested that EhV99B1-SPT was an integral endoplasmic reticulum membraneprotein. Despite lower sequence identity, the secondary and three-dimensional structurespredicted showed that the “pocket” structure element composed of2α-helices and4β-sheetswas the catalytic center of this enzyme, with a typical conserved “TFTKSFG” active site inthe N-terminal region and was very close to those of prokaryotic organisms. However, theN-terminal domain of EhV99B1-SPT most closely resembled the LCB2catalysis subunit andthe C-terminal domain most closely resembled the LCB1regulatory subunit of otherorganisms which together formed a spherical molecule. This “chimera” was highly similar toα-oxoamine synthase (AOS) members.(2) The EhV99B1-LCB2gene was amplified by PCR with specific primers and subcloned intothe prokaryotic expression vector pGEX-4T-3to get the recombinant plasmid pGEX-4T-3/EhV99B1-LCB2. A positive plasmid was transformed into E. coli BL21and the target genewas successfully expressed by IPTG-induction. For a functional identification, the totalsphingoid bases of E. coli were extracted and subjected to TLC plate. It resulted insignificant accumulation of new sphingolipid in E. coli cells. It indicated that therecombinant EhV99B1-LCB2exhibited catalytic function.(3) HPLC method was used to measure the concentration of two ceramides (NFA-Cer andPS-Cer) in E.huxleyi. The total lids of E.huxleyi cultures were extracted and subjected toTLC plate. NFA-Cer and PS-Cer were developed with chloroform/methanol/glacial aceticacid (19:0.9:0.1, by vol.) as a solvent system, showing Rf with0.38and0.4, respectively.Ceramides were derivatized with benzoylation reagent and diluted with hexane-ethanol (99:1)(flow-rate1.0ml/min) on normal phase CN column. The derivative NFA-Cer and PS-Cerwere detected at230nm and254nm thought UV-visible full wave scanning. Under thiscondition, chromatograph peak of two ceramides were fine without tailing. The logarithmiccalibration on curves for NFA-Cer was linear among84-840μg/ml (R2=0.99248) and for PS-Cer was linear among14-280μg/ml (R2=0.99381). The average recovery of this methodwas above95%with RSD=3.016%(n=5). Therefore, this HPLC method was suitable forquantitation of ceramides in E.huxleyi.(4) EhV99B1-SPT mRNA transcription levels increased slowly from0to12h post infection andthen it decreased gradually and falled into minimal levels (63.4%to initial level) at24h.From24h, it started increase significantly and finally reached to the maximum level (5.383times to initial level) at30h. During6to18after infection, EhV99B1-SPT proteinexpression levels decreased and then it increased to maximum (about1.06time to initiallevel) at30h. From30h, it displayed rapidly declines and falled into minimum (about60toinitial level) at36. In brief, it seemed that the dynamic of EhV99B1-SPT gene transcriptionalevents and protein expression were not coinstantaneous. Compared with control, EhVinfection resulted in the accumulation of ceramid in E. huxleyi. The concentration ofNFA-cer increased gradually during0-18h post-infection, and then reached the maximum(2.24times to control) at30h. The concentration of PS-cer remains light increase duringwhole infection process. The levels of EhV99B1-SPT mRNA transcription and translationshowed a notable postitive correlation with host ceramide production and the correlationcoefficient were0.582and0.481. These results indicated that EhV infection led to theincrease of ceramides in E. huxleyi and EhV99B1-SPT gene expression was coupled toceramide production in host in some extent. However, EhV99B1-SPT gene may not be theonly one member for the regulation of host ceramide synthesis. Some more virus-hostfunctional genes should be involved in ceramide metabolism in E. huxleyi.