Young-Ju Jee

Phylogenetic analysis of six species of pacific abalone (Haliotidae) based on DNA sequences of 16s rRNA and cytochrome c oxidase subunit I mitochondrial genes

Six species of abalones (Haliotidae) are found on the Korean coasts. Identification and characterization of these abalones are usually based on morphologic characters. In this research we compared the partial sequences of the mitochondrial 16S ribosomal RNA and cytochrome c oxidase subunit I genes to identify species using molecular data and to determine their phylogenetic relationships. Sequence alignments and phylogenetic analysis revealed that the 6 species fell into 2 distinct groups which were genetically distant from each other and exhibited little internal phylogenetic resolution. One group included Haliotis discus hannai, H. discus discus, H. madaka, and H. gigantea, while the other group contained H. diversicolor supertexta and H. diversicolor diversicolor. The 16S rRNA sequences were relatively more conserved than to the COI sequences, but both gene sequences provided sufficient phylogenetic information to distinguish among the 6 species of Pacific abalone, and thus could be valuable molecular characters for species identification.

Identification and characterization of a bacteriocin produced by an isolated Bacillus sp. SW1-1 that exhibits antibacterial activity against fish pathogens

The selected isolate, Bacillus sp. SW1-1 showed antibacterial activity against both Gram-positive and Gram-negative bacteria involved in fish diseases, including Edwardsiella tarda, Streptococcus iniae, S. parauberis, Vibrio anguillarum, and V. harveyi. The Maximum bacteriocin production was observed at 30°C after 24 h with brain heart infusion medium (pH 7.0). The bacteriocin SW1-1 was purified by 50% ammonium sulfate precipitation, followed by HiPrep diethylaminoethyl 16/10 FF and Sephacryl S-100 High resolution column chromatography. The substance was characterized as a bacteriocin-like inhibitory substance with a molecular mass of 38 kDa. Bacteriocin SW1-1 was sensitive to the proteolytic action of pepsin, trypsin, chymotrypsin, and protease types I and XIV, and relatively heat labile, despite the fact that bacteriocin activity was still detected after heating at 100°C for 30 min. The activity of bacteriocin SW1-1 was stable in the pH range of 2.0–11.0, and relatively unaffected by organic chemicals. The bacteriocin SW1-1 had a bacteriolytic mechanism, resulting in cell wall degradation of E. tarda. These characteristics indicate that this bacteriocin may be a potential candidate for alternative agent to control important pathogens of fish diseases in aquaculture.

Genetic Organization of Two Types of Flounder Warm-Temperature Acclimation-Associated 65-kDa Protein and Their Gene Expression Profiles

We isolated and characterized two cDNA clone encoding warm-temperature acclimation-associated 65-kDa proteins (PoWap65-1 and PoWap65-2) from the olive flounder, Paralichthys olivaceus. The deduced amino acid sequences of PoWap65s showed overall identities of 33–73% with other fish Wap65 and mammalian hemopexin-like proteins. The 5′-flanking regions of both PoWap65-encoding genes contained various putative transcriptional elements. While PoWap65-1 and PoWap65-2 were structurally similar, they exhibited highly differential patterns of expression. PoWap65-1 was expressed only in the liver, whereas PoWap65-2 transcripts were detected in a wide range of tissues. The accumulation of PoWap65s mRNA was expressed differentially during development. Expression of them in warm temperatures also differed in flounder embryonic cells. PoWap65-1 was upregulated by temperature stimulation whereas PoWap65-2 was not detected. PoWap65s were highly regulated by Edwardsiella tarda infection and hypoxia. Pathogen challenge induced PoWap65-2 expression in the liver whereas PoWap65-1 was downregulated. Hypoxia induced the expression of both PoWap65s in the liver of juvenile fish.

Molecular Cloning, Purification, and Characterization of a Cold-Adapted Esterase from Photobacterium sp. MA1-3

The gene encoding an esterase from Photobacterium sp. MA1-3 was cloned in Escherichia coli using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (948 bp) corresponded to a protein of 315 amino acid residues with a molecular weight of 35 kDa and a pI of 6.06. The deduced protein showed 74% and 68% amino acid sequence identities with the putative esterases from Photobacterium profundum SS9 and Photobacterium damselae, respectively. Absence of a signal peptide indicated that it was a cell-bound protein. Sequence analysis showed that the protein contained the signature G-X-S-X-G included in most serine-esterases and lipases. The MA1-3 esterase was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at 18°C. The enzyme was a serine-esterase and was active against C2, C4, C8 and C10 p-nitrophenyl esters. The optimum pH and temperature for enzyme activity were pH 8.0 and 30°C, respectively. Relative activity remained up to 45% even at 5°C with an activation energy of 7.69 kcal/mol, which indicated that it was a cold-adapted enzyme. Enzyme activity was inhibited by Cd 2+ , Cu 2+ , Zn 2+ , and Hg 2+ ions.

Shewanella sp. Ke75 esterase with specificity for p-nitorphenyl butyrate: Gene cloning and characterization

A bacterial strain that produces a cold-adapted esterase was isolated from tidal flats and identified as Shewanella sp. Ke75. In the present study, the corresponding gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (957 bp) corresponded to a protein of 318 amino acid residues with a calculated molecular weight of 34875 Da. The esterase showed 68 and 57% identities with the putative esterases of Shewanella amazonensis SB2B and Colwellia psychrerythraea 34H, respectively. The esterase contained a putative leader sequence, as well as the conserved catalytic triad (Ser, His, Asp), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein Ke75 was produced in both soluble and insoluble forms when Escherichia coli cells harboring the gene were cultured at 30°C. The enzyme showed specificity for C4 (butyrate) as a substrate, with little activity toward the other p-nitrophenyl esters tested. The optimum pH and temperature for enzyme activity were pH 9.0 and 30°C, respectively. Relative activity remained up to 60% even at 5°C with an activation energy of 6.29 kcal/mol, which indicated that it was a cold-adapted enzyme. Enzyme activity was enhanced in the presence of Mn2+ ions, but inhibited by Cd2+, Cu2+, Hg2+, and Zn2+ ions.

Analysis of Hemolytic Microflora from the Ark Shell (Scapharca broughtonii)

The southern coast of Korea is important for the ark shell (Scapharca broughtonii) aquaculture, but the productivity was rapidly reduced during the previous decade by mass mortality. To overcome this economic loss, investigations only focused on environmental factors, and microbiological researches were performed insufficiently. In this study, two sites (Gangjin and Jinhae bay) were selected for their high and low rate of mortality, respectively, and the existence of microflora from underwater sediments in the bodies of S. broughtonii was analyzed. We screened the whole body of each sample and chose unique colonies, which exhibit alpha- and beta-hemolytic activity, for identification. The microflora in S. broughtonii was less variable than sediments, and restricted species were isolated. We identified 17 genera of 88 species and 16 genera of 64 species from the two bays, respectively. A major proportion was comprised of Bacillus species, with the Bacillus cereus group being the most common species among the Bacillus strains, while Paenibacillus, Lynsilbacillus, and Vibrio species were the second most abundant species. At the genus level, there were no significant microbial differences between the two coastal regions. 64 species were isolated from rare site (Jinhae bay), but more species (88) with greater variety were isolated from the frequent site (Gangjin bay). Therefore, it was assumed that the cause of mass mortality lay in the difference in specie-level diversity, and conducting investigations on the diagnosis of pathogenic species by challenging tests using isolated unique species.

Molecular characterization and gene expression analysis of a metalloprotease from Pacific abalone Haliotis discus hannai

We isolated a metalloprotease (MP) homologue from an abalone muscle cDNA library. A 3284-kb full-length cDNA encoding a predicted polypeptide of 667 amino acids was sequenced. The abalone MP Haliotis discus hannai (HdMP) exhibited a domain structure typical of the peptidase M4 family, a 21-amino acid N-terminal hydrophobic signal sequence followed by a long propeptide sequence of 347 amino acids and the mature protease domain comprising 299 amino acids. The mature region contains features characteristic of a zinc protease, including a zinc-binding motif (HEXXH) and an active site. The protein showed 32–38% amino acid sequence identity with other known MP sequences and with a hypothetical protein from owl limpet. The mRNA transcript is expressed in almost all tissues, with high expression in the mantle and adductor muscle of healthy abalones, and is expressed constitutively during the early developmental stages after fertilization. Lipopolysaccharide or poly I:C stimulation induced the expression of the HdMP transcript in the digestive track and gills of abalones. Collectively, these results suggest that HdMP could play multiple roles in defense, the immune response, growth, and regulation of reproduction.

Development and characterization of microsatellite markers in the sea squirt, Halocynthia roretzi

We characterized nine new microsatellite markers isolated from (GT)n and (CT)n microsatellite-enriched genomic libraries of the sea squirt (Halocynthia roretzi). All markers were polymorphic in 92 individuals from a single natural population with 2–21 (mean 9.22) alleles per locus. The observed and expected heterozygosity of these markers were 0.086–0.886 and 0.102–0.870, respectively. One marker (Hr2004) significantly deviated from Hardy–Weinberg equilibrium. These new microsatellite markers should be useful for assessing the genetic diversity and population structure in H. roretzi.