Myoung Sug Kim

Isolation and Structural Determination of the Antifouling Diketopiperazines from Marine-Derived Streptomyces praecox 291-11

Marine derived actinomycetes constituting 185 strains were screened for their antifouling activity against the marine seaweed, Ulva pertusa, and fouling diatom, Navicula annexa. Strain 291-11 isolated from the seaweed, Undaria pinnatifida, rhizosphere showed the highest antifouling activity and was identified as Streptomyces praecox based on a 16S rDNA sequence analysis. Strain 291-11 was therefore named S. praecox 291-11. The antifouling compounds from S. praecox 291-11 were isolated, and their structures were analyzed. The chemical constituents representing the antifouling activity were identified as (6S,3S)-6-benzyl-3-methyl-2,5-diketopiperazine (bmDKP) and (6S,3S)-6-isobutyl-3-methyl-2,5-diketopiperazine (imDKP) by interpreting the nuclear magnetic resonance and high-resolution mass spectroscopy data. Approximately 4.8 mg of bmDKP and 3.1 mg of imDKP were isolated from 1.2 g of the S. praecox 291-11 crude extract. Eight different compositions of culture media were investigated for culture, the TBFeC medium being best for bmDKP and TCGC being the optimum for imDKP production. Two compounds respectively showed a 17.7 and 21 therapeutic ratio (LC50/EC50) to inhibit zoospores, and two compounds respectively showed a 263 and 120.2 therapeutic ratio to inhibit diatoms.

Induction of antifouling diterpene production by Streptomyces cinnabarinus PK209 in co-culture with marine-derived Alteromonas sp. KNS-16.

An active antifouling diterpene was isolated from marine actinomycete strain PK209 and productivity was induced in a co-culture experiment. The active constituent was identified as the diterpene lobocompactol by interpretion of nuclear magnetic resonance and mass spectroscopy data. A PK209 co-culture was designed and a lobocompactol-resistant bacterium, KNS-16, was selected as co-culture competitor to induce lobocompactol production. Adding a small volume of 16-h-old KNS-16 culture to the 96-h-old PK209 culture caused rapid induction of lobocompactol production. The final yield was 2.7 mg/L, 10.4-fold higher than that collected from a single PK209 culture. The two bacteria, strains PK209 and KNS-16, were identified as Streptomyces cinnabarinus and Alteromonas sp. based on 16S rDNA sequencing. Lobocompactol showed significant antifouling activity, of 0.18 and 0.43 µg/mL, for EC₅₀ against the macroalga Ulva pertusa and the diatom Navicula annexa respectively. It showed activity with MIC of 61-112 µg/mL against fouling bacteria.

Detection of genetic variation using dual-labeled peptide nucleic acid (PNA) probe-based melting point analysis

BackgroundThermal denaturation of probe-target hybrid is highly reproducible, and which makes probe melting point analysis reliable in the detection of mutations, polymorphisms and epigenetic differences in DNA. To improve resolution of these detections, we used dual-labeled (quencher and fluorescence), full base of peptide nucleic acid (PNA) probe for fluorescence probe based melting point analysis. Because of their uncharged nature and peptide bond-linked backbone, PNA probes have more favorable hybridization properties, which make a large difference in the melting temperature between specific hybridization and partial hybridization.ResultsHere, we have shown that full base dual-labeled PNA is apt material for fluorescence probe-based melting point analysis with large difference in the melting temperature between full specific hybridization and that of partial hybridization, including insertion and deletion. In case of narrowly distributed mutations, PNA probe effectively detects three mutations in a single reaction tube with three probes. Moreover, we successfully diagnose virus analogues with amplification and melting temperature signal. Lastly, Melting temperature of PNA oligomer can be easily adjusted just by adding gamma-modified PNA probe.ConclusionsThe PNA probes offer advantage of improved flexibility in probe design, which could be used in various applications in mutation detection among a wide range of spectrums.

Genotype and virulence of Streptococcus iniae isolated from diseased olive flounder Paralichthys olivaceus in Korea

In the study, we characterized 29 Streptococcus iniae isolates from diseased olive flounder Paralichthys olivaceus in Korea from 2000 to 2005. Biochemical characteristics of 29 isolates using API 20 strep were identical. Through analysis of repetitive sequence-based PCR (rep-PCR) using BoxA primer and random amplified polymorphic DNA using p14 primer, 29 isolates of S. iniae were divided into two genotypes. The isolates were divided into two clusters by comparison of genetic distance using a sequence of the capsular polysaccharide D gene that was consistent with genotyping by the rep-PCR. The isolates belonging to genotype 1 in rep-PCR analysis showed a high virulence in the flounder, while the isolates belonging to genotype 2 were relatively low in virulence. Therefore, a correlation between the genotype and the virulence of S. iniae isolates has been identified.

Draft Genome Sequence of Beta-Hemolytic Streptococcus iniae KCTC 11634

ABSTRACT Streptococcus iniae is a beta-hemolytic, Gram-positive coccus, which affects a broad range of freshwater and marine fish species, causing substantial economic losses in the aquaculture industry worldwide. Thus, it is very important to derive a complete genome sequence of the bacterium to aid in the development of vaccines and methods for preventing fish streptococcosis and zoonotic infections in humans. Here, we present the draft genome sequence of S. iniae KCTC 11634 (1,955,615 bp, with a G+C content of 36.6%), which contains 1,868 putative coding sequences.

An outbreak of Lactococcus garvieae Infection in Cage-cultured Red Lip Mullet Chelon haematocheilus with Green Liver Syndrome

Red lip mullet Chelon haematocheilus (body weight = 468 ± 91 g) which became sick during an outbreak of disease at mariculture facilities at Cheonsu Bay, Korea, during July–August 2013, were examined to identify the cause of the disease. Diseased mullets displayed green liver syndrome, and Lactococcus garvieae were isolated from their internal organs. Argulus sp., Trichodina sp., and/or Vibrio spp. were also discovered in some infected fish. Histopathological examination revealed that fatty liver syndrome with hepatocyte degeneration, reflected in heterokaryons, inflammatory lesions, and melanomacrophage centers (MMC S), had caused fibrosis around the kidney, spleen, and blood vessels. After the outbreak, visceral fat and green liver syndrome in the mul lets were consistently observed throughout the year in the same mariculture facilities, indicating that the cultured mullets suffered a chronic metabolic disorder. Although Vibrio spp. were also isolated from some individuals, L. garvieae, which is known to be a causative agent of red lip mullet mortality, was isolated from all diseased individuals. This is the first report of L. garvieae infection in cultured red lip mullet.

Characteristics and Pathogenicity for Japnaes Eel Anguilla japonica of Vibrio vulnificus Isolated from Oyster, Sediment and Seawater in the Korea Coast

Biotyping of Vibrio vulnificus strains isolated from marine environments along the south coast of Korea showed that the majority of the isolates (94.7%) belonged to biotype 1 and the remaining isolates (5.3%) belonged to biotype 2. Analysis of 16S rRNA V. vulnificus strains isolated from marine environments using a multiplex polymerase chain reaction (PCR) revealed that 78.7% were type A and 21.3% were type B. Random amplified polymorphic DNA (RAPD) was used to analyze the genomic differences in V. vulnificus among the biotype 2 strains isolated from marine environments (newly isolated strains group) and reference strains obtained from infected eels (reference strains group). The two groups had distinctly different profiles of the amplicons produced from RAPD. Additionally, biochemical comparison of these strains revealed that all four strains isolated from marine environments differed from the strains isolated from eels in their ability to promote D-mannitol fermentation. Two (NH 1 and NH 2) out of four isolates of biotype 2 from marine environments showed pathogenicity in eels Anguilla japonica in a challenge test. These isolates did not agglutinate with antisera against V. vulnificus NCIMB 2137 (serovar E), ATCC 27562 (non-serovar E), and ATCC 33816 (atypical serovar E).