Identification and Characterization of a Periplasmic α-Carbonic Anhydrase (CA) in the Gametophytes of Saccharina japonica (Phaeophyceae).

Periplasmic or external carbonic anhydrases (CAs) have been well accepted as playing a crucial role in the acquisition of dissolved inorganic carbon; however, no cytological evidence or molecular information on these enzymes has been reported in seaweeds to date. In this study, the full-length cDNA sequence coding for a putative periplasmic Sjα-CA2 was cloned from the gametophytes of Saccharina japonica, an industrial brown seaweed. It was 1,728 bp in length and included a 263-bp 5'-untranslated region (UTR), a 577-bp 3'-UTR, and an 888-bp open reading frame encoding a protein precursor consisting of 295 amino acids. The mature protein, after removal of a predicted 28-residue signal peptide, was composed of 267 amino acids with a relative molecular weight of 29.27 kDa. Multisequence alignment and phylogenetic analysis indicated that it was a member of the α-CA family. Enzyme activity assays showed that the recombinant Sjα-CA2 in Escherichia coli possessed CO2 hydration and esterase activities, thus identifying this gene Sjα-CA2 in function. Immunogold electron microscopic observations with the prepared anti-Sjα-CA2 polyclonal antibody illustrated that Sjα-CA2 was located in periplasmic space of the kelp gametophyte cells. Quantitative real-time PCR results revealed that the transcription of Sjα-CA2 was induced by elevated HCO3- levels, but it was little changed while the kelp gametophytes were subjected to elevated CO2 concentrations. This study suggests that the periplasmic Sjα-CA2 might play a role in adapting to elevated environmental levels of HCO3- by dehydration of HCO3- to generate CO2 , which could be readily taken up by S. japonica gametophytes.

Integrated proteomic and metabolomic analysis of a reconstructed three-species microbial consortium for one-step fermentation of 2-keto-L-gulonic acid, the precursor of vitamin C.

Microbial consortia, with the merits of strong stability, robustness, and multi-function, played critical roles in human health, bioenergy, and food manufacture, etc. On the basis of 'build a consortium to understand it', a novel microbial consortium consisted of Gluconobacter oxydans, Ketogulonicigenium vulgare and Bacillus endophyticus was reconstructed to produce 2-keto-L-gulonic acid (2-KGA), the precursor of vitamin C. With this synthetic consortium, 73.7 g/L 2-KGA was obtained within 30 h, which is comparable to the conventional industrial method. A combined time-series proteomic and metabolomic analysis of the fermentation process was conducted to further investigate the cell-cell interaction. The results suggested that the existence of B. endophyticus and G. oxydans together promoted the growth of K. vulgare by supplying additional nutrients, and promoted the 2-KGA production by supplying more substrate. Meanwhile, the growth of B. endophyticus and G. oxydans was compromised from the competition of the nutrients by K. vulgare, enabling the efficient production of 2-KGA. This study provides valuable guidance for further study of synthetic microbial consortia.

Characterization of a novel γ-type carbonic anhydrase, Sjγ-CA2, in Saccharina japonica: Insights into carbon concentration mechanism in macroalgae.

Carbonic anhydrase (CA) is a crucial component of CO2-concentrating mechanism (CCM) in macroalgae. In Saccharina japonica, an important brown seaweed, 11 CAs, including 5 α-, 3 ß-, and 3 γ-CAs, have been documented. Among them, one α-CA and one ß-CA were localized in the periplasmic space, one α-CA was found in the chloroplast, and one γ-CA was situated in mitochondria. Notably, the known γ-CAs have predominantly been identified in mitochondria. In this study, we identified a chloroplastic γ-type CA, Sjγ-CA2, in S. japonica. Based on the reported amino acid sequence of Sjγ-CA2, the epitope peptide for monoclonal antibody production was selected as 165 Pro-305. After purification and specificity identification, anti-SjγCA2 monoclonal antibody was employed in immunogold electron microscopy. The results illustrated that Sjγ-CA2 was localized in the chloroplasts of both gametophytes and sporophytes of S. japonica. Subsequently, immunoprecipitation coupled with LC-MS/MS analysis revealed that Sjγ-CA2 mainly interacted with photosynthesis-related proteins. Moreover, the first 65 amino acids at N-terminal of Sjγ-CA2 was identified as the chloroplast transit peptide by the transient expression of GFP-SjγCA2 fused protein in tabacco. Real-time PCR results demonstrated an up-regulation of the transcription of Sjγ-CA2 gene in response to high CO2 concentration. These findings implied that Sjγ-CA2 might contribute to minimizing the leakage of CO2 from chloroplasts and help maintaining a high concentration of CO2 around Rubisco.

The enzyme encoded by Myrmecia incisa, a green microalga, phospholipase A2 gene preferentially hydrolyzes arachidonic acid at the sn-2 position of phosphatidylcholine.

The enzyme phospholipase A2 (PLA2) plays a crucial role in acyl remodeling of phospholipids via the Lands' cycle, and consequently alters fatty acid compositions in triacylglycerol (TAG). In this study, a full-length cDNA sequence coding Myrmecia incisa phospholipase A2 (MiPLA2) was cloned using the technique of rapid amplification of cDNA ends. Comparison of the 1082-bp cDNA with its corresponding cloned DNA sequence revealed that MiPLA2 contained 3 introns. Mature MiPLA2 (mMiPLA2) had a conserved Ca2+-binding loop and a catalytic site motif that has been recognized in plant secretory PLA2 (sPLA2) proteins. Correspondingly, phylogenetic analysis illustrated that MiPLA2 was clustered within GroupXIA of plant sPLA2 proteins. To ascertain the function of MiPLA2, the cDNA coding for mMiPLA2 was subcloned into the vector pET-32a to facilitate the production of recombinant mMiPLA2 in Escherichia coli. Recombinant mMiPLA2 was purified and used for the in vitro enzyme reaction. Thin-layer chromatography profiles of the catalytic products generated by recombinant mMiPLA2 indicated a specificity for cleaving sn-2 acyl chains from phospholipids, thereby functionally characterizing MiPLA2. Although recombinant mMiPLA2 displayed a strong preference for phosphatidylethanolamine, it preferentially hydrolyzes arachidonic acid (ArA) at the sn-2 position of phosphatidylcholine. Results from the fused expression of p1300-sp-EGFP-mMiPLA2 illustrated that MiPLA2 was localized in the intercellular space of onion epidermis. Furthermore, the positive correlation between MiPLA2 transcription and free ArA levels were established. Consequently, the role of mMiPLA2 in the biosynthesis of ArA-rich TAG was elucidated. This study helps to understand how M. incisa preferentially uses ArA to synthesize TAG.

Nuclear-encoded CbbX located in chloroplast is essential for the activity of red-type Rubisco in Saccharina japonica.

Saccharina japonica (Laminariales, Phaeophyta) is a brown alga and the major component of algae beds on the northwest coast of the Pacific Ocean. Rubisco, the key enzyme of CO2 fixation in photosynthesis, is inhibited by nonproductive binding of its substrate RuBP and other sugar phosphates. The inhibited Rubisco in eukaryotic phytoplankton of the red plastid lineage was reactivated by CbbXs, the red-type Rubisco activases, through the process of ATP-hydrolysis-powered remodeling. As well documented, CbbXs had two types of subunits encoded by the plastid or nuclear genome respectively. In this study, both proteins of S. japonica (SjCbbX-n and SjCbbX-p) were localized in the chloroplast illustrated by immuno-electron microscopy technique. GST pull-down detection verified SjCbbX-n could interact with SjCbbX-p. Two-dimensional electrophoresis-based Western blot analysis illustrated that the endogenous SjCbbXs could form heterohexamer in the ratio of 1:1. Activase activity assays showed that although both the recombinant proteins of SjCbbXs were functional, SjCbbX-n illustrated the significantly higher activase activity than SjCbbX-p. Notably, when the two proteins were mixed, the highest specific efficiencies of Rubisco were obtained. These results implied SjCbbX-n may be essential for Rubisco activation. Molecular evolutionary analysis of cbbx genes revealed that cbbx-n originated from the duplication of cbbx-p and then evolved independently under the positive selection pressure. This is the first report about the functional relationship between the two types of CbbXs in macroalge with the red-type Rubisco and provides useful information for revealing the mechanism of high photosynthetic efficiency of this important kelp.

Isolation and characterization of a γ-carbonic anhydrase localized in the mitochondria of Saccharina japonica.

Saccharina japonica is an ecologically and economically important seaweed that is dominant in the rocky shores of cold-temperate regions, forms the major component of productive beds, and affects marine environments. S. japonica exhibits a high photosynthetic efficiency in natural seawater with low dissolved CO2 concentration, thus suggesting the presence of its carbon-concentrating mechanism (CCM). However, the genes, proteins, and pathways involved in the CCM of S. japonica have not been fully identified and characterized. Carbonic anhydrase (CA) is a crucial component of CCM in macroalgae. In this study, the cloning, characterization, and subcellular localization of a specific CA were described. Multisequence alignment and phylogenetic analysis indicated that this CA belonged to the gamma (Sjγ-CA) class. This enzyme has a full-length cDAN of 1370 bp, encodes a protein with 246 amino acids (aa; ca. 25.7 kDa), and contains the mitochondrial transit peptide of 16 aa and LbH_gama_CA_like domain of 159 aa that defined the γ-CA region. The Sjγ-CA was successfully expressed in E. coli BL21 and purified as an active recombinant CA. Immunogold electron microscopy and fluorescence localization illustrated that this enzyme is localized in the mitochondria, and its transcription level is up-regulated by low CO2 concentration. These findings showed that Sjγ-CA is a possible component of the CCM in S. japonica. This work is the first to report about the mtCA of macroalgae and provides a basis for further analysis on seaweed CCM.

Transcription profiles of skin and head kidney from goldfish suffering hemorrhagic septicemia with an emphasis on the TLR signaling pathway.

Hemorrhagic septicemia is an acute, highly fatal disease that affects goldfish (Carassius auratus). To gain a better understanding of related immune genes, the transcriptomes of the skin and head kidney of goldfish suffering hemorrhagic septicemia were sequenced, assembled, and characterized. Based on functional annotation, an extensive and diverse catalog of expressed genes were identified in both the skin and head kidney. As two different organs, pair-wise comparison identified 122/77 unigenes up/down-regulated (two-fold change with P<0.05) in the skin and head kidney. Most genes of the immune pathways were expressed and isolated in both skin and head kidney, including interferon (IFN) transcription factors 1-10 and Toll-like receptors (TLRs). Interferon regulatory factor 3 (IRF3), a key IFN transcription factor, was up-regulated at the transcriptional level by polyriboinosinic: polyribocytidylic acid (poly I:C) challenge and regulated the IFN response by increasing the activity of IFN-ß and IFN-stimulated response element (ISRE)-containing promoter. This study will benefit the identification and understanding of novel genes that play important roles in the immunological reactions of fish suffering from hemorrhagic septicemia.

Identification and functional analysis of interferon regulatory factor 3 in Lateolabrax maculatus.

The interferon (IFN) regulatory factor 3 (IRF3) is a member of the IFN regulatory transcription factor family, which binds to the IFN-stimulated response element (ISRE) within the promoter of IFN genes and IFN-stimulated genes. In this study, the IRF3 cDNA of sea perch Lateolabrax maculatus (SpIRF3) was identified, which contained 1781 bp with an open reading frame of 1398 bp that coded a 465 amino acid protein. The SpIRF3 protein shared conserved characterizations with its homologues and displayed the conserved DNA-binding domain, IRF association domain, serine-rich C-terminal domain, and tryptophan residue cluster. Phylogenetic analysis illustrated that SpIRF3 belonged to the IRF3 subfamily. Subcellular localization analysis showed that SpIRF3 mainly resided in the cytoplasm without stimuli but translocated into nuclei in the presence of poly I:C. Real-time PCR data indicated that SpIRF3 was transcriptionally up-regulated by poly I:C stimulation in various organs. Moreover, reporter assay revealed that SpIRF3 functioned as a modulator in triggering the IFN response by inducing the activity of IFN and ISRE-containing promoter. These data revealed that SpIRF3 was a potential molecule in the IFN immune defense system against viral infection.

Mitochondrial genome of the Japanese seabass Lateolabrax japonicus (Teleostei, Perciformes, and Moronidae).

The complete mitochondrial genome of Japanese seabass Lateolabrax japonicus has been determined. The total length of the mitogenome was 16,601 bp and had the gene content of 13 protein-coding, 22 tRNA, and 2 rRNA genes. Except for the eight tRNA and Nd6 genes, all other mitochondrial genes are encoded on the heavy strand. The overall base compositions of the heavy strand are 27.27% A, 25.97% T, 29.35% C, and 17.41% G, with an AT content of 53.24%. The DNA sequence of L. japonicus shared 69.3%, 81%, 80.4%, and 79.7% sequence identity with that of Morone saxatilis, Histiopterus typus, Kyphosus cinerascens, and Siniperca scherzeri. Molecular data presented here provide a useful toll for evolutionary as well as population genetic studies.

Mitochondrial genome of the American shad Alosa sapidissima.

The complete mitochondrial genome of Alosa sapidissima has been determined. The total length of the mitogenome was 16,697 bp and had a gene content (13 protein-coding, 22 tRNAs and 2 rRNAs. Except for the seven tRNA and Nd6 genes, all other mitochondrial genes are encoded on the heavy strand. The overall base composition of the heavy strand is 28.3% A, 24.8% T, 28.9% C, 17.9% G, with an AT content of 53.1%. The DNA sequence of Alosa. sapidissima shared 97.1, 93.9, 88.8 and 82.3% sequence identity with that of Alosa alosa, Alosa pseudoharengus. Molecular data here presented provide a useful toll for evolutionary as well as population genetic studied.