Characterization of the basic leucine zipper transcription factor family of Neoporphyra haitanensis and its role in acclimation to dehydration stress.

BACKGROUND: Neoporphyra haitanensis, a major marine crop native to southern China, grows in the harsh intertidal habitats of rocky coasts. The thallus can tolerate fluctuating and extreme environmental stresses, for example, repeated desiccation/rehydration due to the turning tides. It is also a typical model system for investigating stress tolerance mechanisms in intertidal seaweed. The basic leucine zipper (bZIP) transcription factors play important roles in the regulation of plants' responses to environmental stress stimuli. However, little information is available regarding the bZIP family in the marine crop Nh. haitanensis. RESULTS: We identified 19 bZIP genes in the Nh. haitanensis genome and described their conserved domains. Based on phylogenetic analysis, these 19 NhhbZIP genes, distributed unevenly on the 11 superscaffolds, were divided into four groups. In each group, there were analogous exon/intron numbers and motif compositions, along with diverse exon lengths. Cross-species collinearity analysis indicated that 17 and 9 NhhbZIP genes were orthologous to bZIP genes in Neopyropia yezoensis and Porphyra umbilicalis, respectively. Evidence from RNA sequencing (RNA-seq) data showed that the majority of NhhbZIP genes (73.68%) exhibited transcript abundance in all treatments. Furthermore, genes NN 2, 4 and 5 showed significantly altered expression in response to moderate dehydration, severe dehydration, and rehydration, respectively. Gene co-expression network analysis of the representative genes was carried out, followed by gene set enrichment analysis. Two NhhbZIP genes collectively responding to dehydration and rehydration and their co-expressing genes mainly participated in DNA repair, DNA metabolic process, and regulation of helicase activity. Two specific NhhbZIP genes responding to severe dehydration and their corresponding network genes were mainly involved in macromolecule modification, cellular catabolic process, and transmembrane transport. Three specific NhhbZIP genes responding to rehydration and their co-expression gene networks were mainly involved in the regulation of the cell cycle process and defense response. CONCLUSIONS: This study provides new insights into the structural composition, evolution, and function of the NhhbZIP gene family. Our results will help us to further study the functions of bZIP genes in response to dehydration and rehydration in Nh. haitanensis and improve Nh. haitanensis in southern China.

Quantification of Photosynthetic Pigments in Neopyropia yezoensis Using Hyperspectral Imagery.

Phycobilisomes and chlorophyll-a (Chla) play important roles in the photosynthetic physiology of red macroalgae and serve as the primary light-harvesting antennae and reaction center for photosystem II. Neopyropia is an economically important red macroalga widely cultivated in East Asian countries. The contents and ratios of 3 main phycobiliproteins and Chla are visible traits to evaluate its commercial quality. The traditional analytical methods used for measuring these components have several limitations. Therefore, a high-throughput, nondestructive, optical method based on hyperspectral imaging technology was developed for phenotyping the pigments phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), and Chla in Neopyropia thalli in this study. The average spectra from the region of interest were collected at wavelengths ranging from 400 to 1000 nm using a hyperspectral camera. Following different preprocessing methods, 2 machine learning methods, partial least squares regression (PLSR) and support vector machine regression (SVR), were performed to establish the best prediction models for PE, PC, APC, and Chla contents. The prediction results showed that the PLSR model performed the best for PE (R Test 2 = 0.96, MAPE = 8.31%, RPD = 5.21) and the SVR model performed the best for PC (R Test 2 = 0.94, MAPE = 7.18%, RPD = 4.16) and APC (R Test 2 = 0.84, MAPE = 18.25%, RPD = 2.53). Two models (PLSR and SVR) performed almost the same for Chla (PLSR: R Test 2 = 0.92, MAPE = 12.77%, RPD = 3.61; SVR: R Test 2 = 0.93, MAPE = 13.51%, RPD =3.60). Further validation of the optimal models was performed using field-collected samples, and the result demonstrated satisfactory robustness and accuracy. The distribution of PE, PC, APC, and Chla contents within a thallus was visualized according to the optimal prediction models. The results showed that hyperspectral imaging technology was effective for fast, accurate, and noninvasive phenotyping of the PE, PC, APC, and Chla contents of Neopyropia in situ. This could benefit the efficiency of macroalgae breeding, phenomics research, and other related applications.

Metagenome-Assembled Genomes From Pyropia haitanensis Microbiome Provide Insights Into the Potential Metabolic Functions to the Seaweed.

Pyropia is an economically important edible red alga worldwide. The aquaculture industry and Pyropia production have grown considerably in recent decades. Microbial communities inhabit the algal surface and produce a variety of compounds that can influence host adaptation. Previous studies on the Pyropia microbiome were focused on the microbial components or the function of specific microbial lineages, which frequently exclude metabolic information and contained only a small fraction of the overall community. Here, we performed a genome-centric analysis to study the metabolic potential of the Pyropia haitanensis phycosphere bacteria. We reconstructed 202 unique metagenome-assembled genomes (MAGs) comprising all major taxa present within the P. haitanensis microbiome. The addition of MAGs to the genome tree containing all publicly available Pyropia-associated microorganisms increased the phylogenetic diversity by 50% within the bacteria. Metabolic reconstruction of the MAGs showed functional redundancy across taxa for pathways including nitrate reduction, taurine metabolism, organophosphorus, and 1-aminocyclopropane-1-carboxylate degradation, auxin, and vitamin B12 synthesis. Some microbial functions, such as auxin and vitamin B12 synthesis, that were previously assigned to a few Pyropia-associated microorganisms were distributed across the diverse epiphytic taxa. Other metabolic pathways, such as ammonia oxidation, denitrification, and sulfide oxidation, were confined to specific keystone taxa.

Heat Shock Protein 20 Gene Superfamilies in Red Algae: Evolutionary and Functional Diversities.

Heat shock protein 20 (Hsp20) genes play important roles in plant growth, development, and response to environmental stress. However, the Hsp20 gene family has not yet been systematically investigated, and its function in red algae (Rhodophyta) remains poorly understood. Herein, we characterized Hsp20 gene families in red algae by studying gene structure, conserved motifs, phylogenetic relationships, chromosome location, gene duplication, cis-regulatory elements, and expression profiles. In this study, 97 Hsp20 genes were identified using bioinformatic methods and classified into 13 subfamilies based on phylogenetic relationships. Phylogenetic analysis revealed that Hsp20 genes might have a polyphyletic origin and a complex evolutionary pattern. Gene structure analysis revealed that most Hsp20 genes possessed no introns, and all Hsp20 genes contained a conserved α-crystalline domain in the C-terminal region. Conserved motif analysis revealed that Hsp20 genes belonging to the same subfamily shared similar motifs. Gene duplication analysis demonstrated that tandem and segmental duplication events occurred in these gene families. Additionally, these gene families in red algae might have experienced strong purifying selection pressure during evolution, and Hsp20 genes in Pyropia yezoensis, Pyropia haitanensis, and Porphyra umbilicalis were highly evolutionarily conserved. The cis-elements of phytohormone-, light-, stress-responsive, and development-related were identified in the red algal Hsp20 gene promoter sequences. Finally, using Py. yezoensis, as a representative of red algae, the Hsp20 gene expression profile was explored. Based on the RNA-seq data, Py. yezoensis Hsp20 (PyyHsp20) genes were found to be involved in Py. yezoensis responses against abiotic and biotic stresses and exhibited diverse expression patterns. Moreover, PyyHsp20 is involved in Py. yezoensis growth and development and revealed spatial and temporal expression patterns. These results provide comprehensive and valuable information on Hsp20 gene families in red algae and lay a foundation for their functional characterization. In addition, our study provides new insights into the evolution of Hsp20 gene families in red algae and will help understand the adaptability of red algae to diverse environments.

Comparative Gene Expression and Physiological Analyses Reveal Molecular Mechanisms in Wound-Induced Spore Formation in the Edible Seaweed Nori.

Genetic reprogramming of differentiated cells is studied broadly in multicellular Viridiplantae as an adaptation to herbivory or damage; however, mechanisms underlying cell development and redifferentiation are largely unknown in red algae, their nearest multicellular relatives. Here we investgate cell reprogramming in the widely cultivated, edible seaweed Neopyropia yezoesis ("nori"), where vegetative cells in wounded blades differentiate and release as large numbers of asexual spores. Based upon physiological changes and transcriptomic dynamics after wound stress in N. yezoensis and its congener Neoporphyra haitanensis, another cultivar that does not differentiate spores after wounding, we propose a three-phase model of wound-induced spore development in N. yezoensis. In Phase I, propagation of ROS by RBOH and SOD elicites systematic transduction of the wound signal, while Ca2+ dependent signaling induces cell reprogramming. In Phase II, a TOR signaling pathway and regulation of cyclin and CDK genes result in cell divisions that spread inward from the wound edge. Once sporangia form, Phase III involves expression of proteins required for spore maturation and cell wall softening. Our analyses not only provide the first model for core molecular processes controlling cellular reprogramming in rhodophytes, but also have practical implications for achieving greater control over seeding in commercial nori farming.

Genome-wide analysis of HSP70 gene superfamily in Pyropia yezoensis (Bangiales, Rhodophyta): identification, characterization and expression profiles in response to dehydration stress.

BACKGROUND: Heat shock proteins (HSPs) perform a fundamental role in protecting plants against abiotic stresses. Individual family members have been analyzed in previous studies, but there has not yet been a comprehensive analysis of the HSP70 gene family in Pyropia yezoensis. RESULTS: We investigated 15 putative HSP70 genes in Py. yezoensis. These genes were classified into two sub-families, denoted as DnaK and Hsp110. In each sub-family, there was relative conservation of the gene structure and motif. Synteny-based analysis indicated that seven and three PyyHSP70 genes were orthologous to HSP70 genes in Pyropia haitanensis and Porphyra umbilicalis, respectively. Most PyyHSP70s showed up-regulated expression under different degrees of dehydration stress. PyyHSP70-1 and PyyHSP70-3 were expressed in higher degrees compared with other PyyHSP70s in dehydration treatments, and then expression degrees somewhat decreased in rehydration treatment. Subcellular localization showed PyyHSP70-1-GFP and PyyHSP70-3-GFP were in the cytoplasm and nucleus/cytoplasm, respectively. Similar expression patterns of paired orthologs in Py. yezoensis and Py. haitanensis suggest important roles for HSP70s in intertidal environmental adaptation during evolution. CONCLUSIONS: These findings provide insight into the evolution and modification of the PyyHSP70 gene family and will help to determine the functions of the HSP70 genes in Py. yezoensis growth and development.

Identification and application of T3SS translocation signal in Edwardsiella piscicida attenuated carrier as a bivalent vaccine.

Type III secretion system (T3SS)-dependent translocation has been used to deliver heterologous antigens by vaccine carriers into host cells. In this research, we identified the translocation signal of Edwardsiella piscicida T3SS effector EseG and constructed an antibiotic resistance-free balanced-lethal system as attenuated vaccine carrier to present antigens by T3SS. Edwardsiella piscicida LSE40 asd gene deletion mutant was constructed and complemented with pYA3342 harbouring the asd (aspartate ß-semialdehyde dehydrogenase) gene from Salmonella. Fusion proteins composed of EseG N-terminal 1-108 amino acids and the TEM1-ß-lactamase reporter were inserted in plasmid pYA3342. The fusion protein could secrete into the cell culture, translocate into HeLa cells, and localize in the membrane fraction. Then, the double gene deletion mutant LSE40ΔasdΔpurA was constructed as an attenuated vaccine carrier, and Aeromonas hydrophila GapA (glyceraldehyde-3-phosphate dehydrogenase) was fused with the translocation signal, instead of the TEM1-ß-lactamase reporter. The bivalent vaccine could protect blue gourami (Trichogaster trichopterus) against E. piscicida and A. hydrophila, with the relative per cent survival of 80.77% and 63.83%, respectively. These results indicated that EseG N-terminal 1-108 amino acid peptide was the translocation signal of E. piscicida T3SS, which could be used to construct bivalent vaccines based on an attenuated E. piscicida carrier.

Comparative Transcriptome Analysis Provides Insights into Response of Ulva compressa to Fluctuating Salinity Conditions.

Ulva compressa, a green tide-forming species, can adapt to hypo-salinity conditions, such as estuaries and brackish lakes. To understand the underlying molecular mechanisms of hypo-salinity stress tolerance, transcriptome-wide gene expression profiles in U. compressa were created using digital gene expression profiles. The RNA-seq data were analyzed based on the comparison of differently expressed genes involved in specific pathways under hypo-salinity and recovery conditions. The up-regulation of genes in photosynthesis and glycolysis pathways may contribute to the recovery of photosynthesis and energy metabolism, which could provide sufficient energy for the tolerance under long-term hyposaline stress. Multiple strategies, such as ion transportation and osmolytes metabolism, were performed to maintain the osmotic homeostasis. Additionally, several long noncoding RNA were differently expressed during the stress, which could play important roles in the osmotolerance. Our work will serve as an essential foundation for the understanding of the tolerance mechanism of U. compressa under the fluctuating salinity conditions.

Biomass estimation of cultivated red algae Pyropia using unmanned aerial platform based multispectral imaging.

BACKGROUND: Pyropia is an economically advantageous genus of red macroalgae, which has been cultivated in the coastal areas of East Asia for over 300 years. Realizing estimation of macroalgae biomass in a high-throughput way would great benefit their cultivation management and research on breeding and phenomics. However, the conventional method is labour-intensive, time-consuming, manually destructive, and prone to human error. Nowadays, high-throughput phenotyping using unmanned aerial vehicle (UAV)-based spectral imaging is widely used for terrestrial crops, grassland, and forest, but no such application in marine aquaculture has been reported. RESULTS: In this study, multispectral images of cultivated Pyropia yezoensis were taken using a UAV system in the north of Haizhou Bay in the midwestern coast of Yellow Sea. The exposure period of P. yezoensis was utilized to prevent the significant shielding effect of seawater on the reflectance spectrum. The vegetation indices of normalized difference vegetation index (NDVI), ratio vegetation index (RVI), difference vegetation index (DVI) and normalized difference of red edge (NDRE) were derived and indicated no significant difference between the time that P. yezoensis was completely exposed to the air and 1 h later. The regression models of the vegetation indices and P. yezoensis biomass per unit area were established and validated. The quadratic model of DVI (Biomass = - 5.550DVI2 + 105.410DVI + 7.530) showed more accuracy than the other index or indices combination, with the highest coefficient of determination (R2), root mean square error (RMSE), and relative estimated accuracy (Ac) values of 0.925, 8.06, and 74.93%, respectively. The regression model was further validated by consistently predicting the biomass with a high R2 value of 0.918, RMSE of 8.80, and Ac of 82.25%. CONCLUSIONS: This study suggests that the biomass of Pyropia can be effectively estimated using UAV-based spectral imaging with high accuracy and consistency. It also implied that multispectral aerial imaging is potential to assist digital management and phenomics research on cultivated macroalgae in a high-throughput way.

Pyropia yezoensis genome reveals diverse mechanisms of carbon acquisition in the intertidal environment.

Changes in atmospheric CO2 concentration have played a central role in algal and plant adaptation and evolution. The commercially important red algal genus, Pyropia (Bangiales) appears to have responded to inorganic carbon (Ci) availability by evolving alternating heteromorphic generations that occupy distinct habitats. The leafy gametophyte inhabits the intertidal zone that undergoes frequent emersion, whereas the sporophyte conchocelis bores into mollusk shells. Here, we analyze a high-quality genome assembly of Pyropia yezoensis to elucidate the interplay between Ci availability and life cycle evolution. We find horizontal gene transfers from bacteria and expansion of gene families (e.g. carbonic anhydrase, anti-oxidative related genes), many of which show gametophyte-specific expression or significant up-regulation in gametophyte in response to dehydration. In conchocelis, the release of HCO3- from shell promoted by carbonic anhydrase provides a source of Ci. This hypothesis is supported by the incorporation of 13C isotope by conchocelis when co-cultured with 13C-labeled CaCO3.

Transcriptomic Insights into Innate Immunity Responding to Red Rot Disease in Red Alga Pyropia yezoensis.

Pyropia yezoensis, one of the most economically important marine algae, suffers from the biotic stress of the oomycete necrotrophic pathogen Pythium porphyrae. However, little is known about the molecular defensive mechanisms employed by Pyr. yezoensis during the infection process. In the present study, we defined three stages of red rot disease based on histopathological features and photosynthetic physiology. Transcriptomic analysis was carried out at different stages of infection to identify the genes related to the innate immune system in Pyr. yezoensis. In total, 2139 up-regulated genes and 1672 down-regulated genes were identified from all the infected groups. Pathogen receptor genes, including three lectin genes (pattern recognition receptors (PRRs)) and five genes encoding typical plant R protein domains (leucine rich repeat (LRR), nucleotide binding site (NBS), or Toll/interleukin-1 receptor (TIR)), were found to be up-regulated after infection. Several defense mechanisms that were typically regarded as PAMP-triggered immunity (PTI) in plants were induced during the infection. These included defensive and protective enzymes, heat shock proteins, secondary metabolites, cellulase, and protease inhibitors. As a part of the effector-triggered immunity (ETI), the expression of genes related to the ubiquitin-proteasome system (UPS) and hypersensitive cell death response (HR) increased significantly during the infection. The current study suggests that, similar to plants, Pyr. yezoensis possesses a conserved innate immune system that counters the invasion of necrotrophic pathogen Pyt. porphyrae. However, the innate immunity genes of Pyr. yezoensis appear to be more ancient in origin compared to those in higher plants.

Compositional Shifts of Bacterial Communities Associated With Pyropia yezoensis and Surrounding Seawater Co-occurring With Red Rot Disease.

Pyropia yezoensis is commercially the most important edible red alga in China, and red rot disease is viewed as one of the major constraints for its cultivation. Microbes within the oomycetic genus Pythium have been reported as the causative agents for this disease; however, little is known about the interactions between the disease and the epiphytic and planktonic bacterial communities. In the present study, bacterial communities associated with uninfected, locally infected, and seriously infected thalli collected from cultivation farms, and within seawater adjacent to the thalli, were investigated using in-depth 16S ribosomal RNA (rRNA) gene sequencing in conjunction with assessing multiple environmental factors. For both thalli and seawater, uninfected and infected communities were significantly different though alpha diversity was similar. Phylogenetic differences between epiphytic bacterial communities associated with P. yezoensis were mainly reflected by the relative changes in the dominant operational taxonomic units (OTUs) assigned as genus Flavirhabdus, genus Sulfitobacter, and family Rhodobacteraceae. The prevalent OTUs in seawater also differed in relative abundance across the communities and were affiliated with diverse taxa, including the phyla Actinobacteria, Verrucomicrobia, and Bacteroidetes, and the classes Alpha- and Gamma-proteobacteria. The differentiation of bacterial communities associated with P. yezoensis and seawater was primarily shaped by reactive silicate (RS) content and salinity, respectively. In particular, 14 potential indicators (two OTUs on P. yezoensis and twelve OTUs in seawater) were identified that significantly differentiated P. yezoensis health statuses and correlated with environmental changes. Overall, the present study provides insights into the alterations of bacterial communities associated with P. yezoensis and surrounding seawater co-occurring with red rot disease. Observed changes were closely associated with health status of algal host, and highlight the potential of using community differentiation to forecast disease occurrence.

Expression, secretion and bactericidal activity of type VI secretion system in Vibrio anguillarum.

The type VI secretion system (T6SS) was recently shown to modulate quorum sensing and the stress response in Vibrio anguillarum serotype O1 strain NB10. It is not known whether there is a functionally active T6SS in other serotypes of V. anguillarum. Here, homologues to T6SS cluster VtsEFGH and hemolysin-coregulated protein (Hcp)-encoding genes were found to be prevalent and conserved in clinical isolates of V. anguillarum from fish, including four O1 and five non-O1 serotype strains. Unexpectedly, only the non-O1 serotype strains expressed VtsEFGH and Hcp under laboratory and marine-like conditions, in contrast to the serotype O1 strains. This suggested that the V. anguillarum non-O1 serotype strains tested have constitutive expression of T6SS. Examination of a representative non-O1 strain, MHK3, showed that Hcp production was growth phase dependent and that maximum Hcp production was observed in the exponential growth phase. Moreover, Hcp production by MHK3 was most active under warm marine-like conditions. Further examination revealed a correlation of the constitutive expression of T6SS with bactericidal activity against Escherichia coli and Edwardsiella tarda. The work presented here suggests that the constitutive expression of T6SS provides V. anguillarum with advantage in microbial competition in marine environments.

Construction and characterization of the angR mutant of Vibrio anguillarum via insertional inactivation.

Vibrio anguillarum strain M3 was isolated from cultured diseased flounder Paralichthys olivaceus. In order to determine whether chromosomal angR gene plays an important role in infecting flounder host, part fragment of a virulence regulatory gene angR from V. anguillarum M3 genome was cloned and ligated with suicide plasmids pNQ705 in the present study. The conserved fragment of angR gene was amplified from M3 genome and inserted into suicide plasmid pNQ705, a kind of conditional replicon. The recombinant plasmid was transferred into Vibrio anguillarum M3 strain genomic DNA through bacterial conjugation and homologous recombination. A mutant V. anguillarum strain with angR gene mutation was constructed and screened successfully using TCBS medium containing chloramphenicol. PCR identification and sequencing showed that the recombinant plasmid inserted into Vibrio anguillarum genome DNA, as expected design, resulted in insertional inactivation of angR gene. Results of artificial infection experiment showed that virulence of mutant Vibrio anguillarum strain reduced dramatically as compared with the wild strain M3.

Correction: De Novo Assembly of the Japanese Flounder (Paralichthys olivaceus) Spleen Transcriptome to Identify Putative Genes Involved in Immunity.

[This corrects the article DOI: 10.1371/journal.pone.0117642.].

De Novo assembly of the Japanese flounder (Paralichthys olivaceus) spleen transcriptome to identify putative genes involved in immunity.

BACKGROUND: Japanese flounder (Paralichthys olivaceus) is an economically important marine fish in Asia and has suffered from disease outbreaks caused by various pathogens, which requires more information for immune relevant genes on genome background. However, genomic and transcriptomic data for Japanese flounder remain scarce, which limits studies on the immune system of this species. In this study, we characterized the Japanese flounder spleen transcriptome using an Illumina paired-end sequencing platform to identify putative genes involved in immunity. METHODOLOGY/PRINCIPAL FINDINGS: A cDNA library from the spleen of P. olivaceus was constructed and randomly sequenced using an Illumina technique. The removal of low quality reads generated 12,196,968 trimmed reads, which assembled into 96,627 unigenes. A total of 21,391 unigenes (22.14%) were annotated in the NCBI Nr database, and only 1.1% of the BLASTx top-hits matched P. olivaceus protein sequences. Approximately 12,503 (58.45%) unigenes were categorized into three Gene Ontology groups, 19,547 (91.38%) were classified into 26 Cluster of Orthologous Groups, and 10,649 (49.78%) were assigned to six Kyoto Encyclopedia of Genes and Genomes pathways. Furthermore, 40,928 putative simple sequence repeats and 47, 362 putative single nucleotide polymorphisms were identified. Importantly, we identified 1,563 putative immune-associated unigenes that mapped to 15 immune signaling pathways. CONCLUSIONS/SIGNIFICANCE: The P. olivaceus transciptome data provides a rich source to discover and identify new genes, and the immune-relevant sequences identified here will facilitate our understanding of the mechanisms involved in the immune response. Furthermore, the plentiful potential SSRs and SNPs found in this study are important resources with respect to future development of a linkage map or marker assisted breeding programs for the flounder.

Generation and evaluation of virulence attenuated mutants of Edwardsiella tarda as vaccine candidates to combat edwardsiellosis in flounder (Paralichthys olivaceus).

Edwardsiella tarda is an intracellular pathogen that causes edwardsiellosis in fish. The development of a live attenuated vaccine may be an effective approach for preventing this disease in fish. In this study, we introduced deletions of esrB, esaC, evpH, rpoS, and purA into the E. tarda LSE40ΔaroA strain, thereby generating five double-gene mutants (ΔaroAΔesrB, ΔaroAΔesaC, ΔaroAΔrpoS, ΔaroAΔevpH, and ΔaroAΔpurA) and two triple-gene mutants (ΔaroAΔesrBΔevpH and ΔaroAΔesaCΔevpH). When blue gourami (Trichogaster trichopterus) was used as a fish model for the primary screening and evaluation of the vaccine candidates, all mutants were attenuated significantly by more than 2 to 3 logs in terms of the 50% lethal dose (LD(50)). Five double-gene mutants yielded relative percentage survival (RPS) rates of 26.1-82.6% after challenge with wild-type E. tarda. The ΔaroAΔesrB mutant that conferred the highest RPS (82.6%) in blue gourami was also evaluated in flounder (Paralichthys olivaceus). After vaccination via intramuscular (i.m.) injection or immersion, this mutant could persist in the flounder for 14-35 days and it induced higher serum antibody titers than the control fish (P < 0.01). Flounder vaccinated via i.m. injection at doses of 10(3)-10(7) CFU/fish had RPS rates of 14.3-66.7% after i.m. challenge with 10(4) CFU/fish using wild-type E. tarda. Flounder vaccinated via immersion at a dose of 10(7) CFU/ml exhibited 100% RPS against immersion challenge with 10(7) CFU/ml using wild-type E. tarda. These results indicate that the ΔaroAΔesrB mutant could be used as an effective live vaccine to combat edwardsiellosis in flounder.

Complete nucleotide sequence of Klebsiella phage P13 and prediction of an EPS depolymerase gene.

The complete genome of Klebsiella phage P13 was sequenced and analyzed. Bacteriophage P13 has a double-stranded linear DNA with a length of 45,976 bp and a G+C content of 51.7 %, which is slightly lower than that of Klebsiella pneumoniae KCTC 2242. The codon biases of phage P13 are very similar to those of SP6-like phages and K. pneumoniae KCTC 2242. Bioinformatics analysis shows that the phage P13 genome has 282 open reading frames (ORFs) that are greater than 100 bp in length, and 50 of these ORFs were identified as predicted genes with an average length of 833 bp. Among these genes, 41 show homology to known proteins in the GenBank database. The functions of the 24 putative proteins were investigated, and 13 of these were found to be highly conserved. According to the homology analysis of the 50 predicted genes and the whole genome, phage P13 is homologous to SP6-like phages. Furthermore, the morphological characteristics of phage P13 suggest that it belongs to the SP6-like viral genus of the Podoviridae subfamily Autographivirinae. Two hypothetical genes encoding an extracellular polysaccharide depolymerase were predicted using PSI-BLAST. This analysis serves as groundwork for further research and application of the enzyme.

Molecular cloning, characterization, and heterologous expression of a new κ-carrageenase gene from marine bacterium Zobellia sp. ZM-2.

κ-Carrageenases exhibit apparent distinctions in gene sequence, molecular weight, enzyme properties, and posttranslational processes. In this study, a new κ-carrageenase gene named cgkZ was cloned from the marine bacterium Zobellia sp. ZM-2. The gene comprised an open reading frame of 1,638 bp and encoded 545 amino acids. The natural signal peptide of κ-carrageenase was used successfully for the secretory production of the recombinant enzyme in Escherichia coli. A posttranslational process that removes an amino acid sequence of about 20 kDa from the C-terminal end of κ-carrageenase was first discovered in E. coli. An increase in enzyme activity by 167.3% in the presence of 5 mM DTT was discovered, and Na(+) at a certain concentration range was positively correlated with enzyme activity. The κ-carrageenase production of E. coli was 9.0 times higher than that of ZM-2. These results indicate the potential use of the enzyme in the biotechnological industry.

Complete Genome Sequence of Vibrio anguillarum M3, a Serotype O1 Strain Isolated from Japanese Flounder in China.

Vibrio anguillarum is an important bacterial pathogen that causes vibriosis in marine fish. We present the complete genome sequence of V. anguillarum M3, a serotype O1 clinical strain isolated from Japanese flounder (Paralichthys olivaceus) in Shandong, China.