Paenibacillus hexagrammi sp. nov., a novel bacterium isolated from the gut content of Hexagrammos agrammus.

A Gram-stain-positive, aerobic bacterium, designated as YPD9-1T, was isolated from the gut contents of a spotty belly greenling, Hexagrammos agrammus, collected near Dokdo island, South Korea. The rod-shaped cells were oxidase-positive, and catalase-negative. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0, C16 : 0, iso-C16 : 0 and iso-C17: 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and two unidentified lipids. The DNA G+C content was 47.6 mol% and the predominant respiratory quinone was menaquinone MK-7. The 16S rRNA gene sequence of YPD9-1T showed low sequence similarities to species of the genus Paenibacillus, Paenibacillus pocheonensis Gsoil 1138T (97.21 % of sequence similarity), Paenibacillus aestuarii CJ25T (97.12 %) and Paenibacillus allorhizoplanae JJ-42T (96.89 %). The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that YPD9-1T formed a distinct branch among other species of the genus Paenibacillus. The digital DNA-DNA hybridisation, average nucleotide identity, and average amino acid identity values between YPD9-1T and the related species were in the ranges of 15.3-16.2 %, 74.1-78.4 %, and 71.1-71.9 %, respectively, which are below the species cutoff values. On the basis of the results of the polyphasic analysis, we conclude that strain YPD9-1T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus hexagrammi sp. nov. is proposed. The type strain of Paenibacillus hexagrammi is YPD9-1T (=KCTC 43424T =LMG 32988T).

Fermentative aminopyrrolnitrin production by metabolically engineered Corynebacterium glutamicum.

Aminopyrrolnitrin (APRN), a natural halogenated phenylpyrrole derivative (HPD), has strong antifungal and antiparasitic activities. Additionally, it showed 2.8-fold increased photostability compared to pyrrolnitrin, a commercially available HPD with antimicrobial activity. For microbial production of APRN, we first engineered anthranilate phosphoribosyltransferase encoded by trpD from Corynebacterium glutamicum, resulting in a TrpDA162D mutation that exhibits feedback-resistant against L-tryptophan and higher substrate affinity compared to wild-type TrpD. Plasmid-borne expression of trpDA162D in C. glutamicum TP851 strain with two copies of trpDA162D in the genome led to the production of 3.1 g/L L-tryptophan in flask culture. Subsequent step for L-tryptophan chlorination into 7-chloro-L-tryptophan was achieved by introducing diverse sources of genes encoding tryptophan 7-halogenase (PrnA or RebH) and flavin reductase (Fre, PrnF, or RebF). The combined expression of prnA from Serratia grimesii or Serratia plymuthica with flavin reductase gene from Escherichia coli, Pseudomonas fluorescens, or Lechevalieria aerocolonigenes yielded higher production of 7-chloro-L-tryptophan in comparison to other sets of two-component systems. In the next step, production of putative monodechloroaminopyrrolnitrin (MDAP) from 7-chloro-L-tryptophan was achieved through the expression of prnB encoding MDAP synthase from S. plymuthica or P. fluorescens. Finally, an artificial APRN biosynthetic pathway was constructed by simultaneously expressing genes coding for tryptophan 7-halogenase, flavin reductase, MDAP synthase, and MDAP halogenase (PrnC) from different microbial sources within the L-tryptophan-producing TP851 strain. As prnC from S. grimesii or S. plymuthica was introduced into the host strain, which carried plasmids expressing prnA from S. plymuthica, fre from E. coli, and prnB from S. plymuthica, APN3639 and APN3638 accumulated 29.5 mg/L and 28.1 mg/L of APRN in the culture broth. This study represents the first report on the fermentative APRN production by metabolically engineered C. glutamicum.

The extracellular matrix protein EFEMP2 is involved in the response to VHSV infection in the olive flounder Paralichthys olivaceus.

The EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) is involved in connective tissue development, elastic fiber formation, and tumor growth. In this study, we characterized the cDNA of EFEMP2 (PoEFEMP2), a member of the fibulin family of ECM proteins, in the olive flounder Paralichthys olivaceus. The coding region of PoEFEMP2 encodes a protein that contains six calcium-binding EGF-like (EGF-CA) domains and four complement Clr-like EGF-like (cEGF) domains. PoEFEMP2 shows 67.51-96.77 % similarities to orthologs in a variety of fish species. PoEFEMP2 mRNA was detected in all tissues examined; the highest levels of PoEFEMP2 mRNA expression were observed in the heart, testis, ovary and muscle. The PoEFEMP2 mRNA level increases during early development. In addition, the PoEFEMP2 mRNA level increased at 3 h post-infection (hpi) and decreased from 6 to 48 hpi in flounder Hirame natural embryo (HINAE) cells infected with viral hemorrhagic septicemia virus (VHSV). Disruption of PoEFEMP2 using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system resulted in a significant upregulation of VHSV G mRNA levels and immune-related genes expression in knockout cells. These findings implicate PoEFEMP2 in antiviral responses in P. olivaceus.

Antifungal activity of aminopyrrolnitrin against Trichophyton verrucosum in a guinea pig model of dermatophytosis.

BACKGROUND: Dermatophytosis is a common and major public health concern worldwide. Despite the increasing availability of antifungal drugs, relapses and untreated cases of dermatophyte infections are reported. Therefore, novel antifungal agents are required. Aminopyrrolnitrin (APRN) shows promise for dermatophytosis treatment because of its antifungal activity. OBJECTIVES: This study aimed to assess the antifungal properties of APRN against Trichophyton verrucosum (T. verrucosum), in both laboratory settings and a guinea pig model. METHODS: The minimum inhibitory concentrations (MICs) of APRN and enilconazole against T. verrucosum were determined according to the CLSI M38 method. The skins of 16 male guinea pigs were infected with 1.0 × 108 conidia of T. verrucosum and the animals were grouped into sets of four: negative control group (NC) received normal saline; positive control group (PC) received 2 µg/mL of enilconazole; and APRN4 and APRN8 received 4 and 8 µg/mL of APRN, respectively. Clinical, mycological and histological efficacies were measured after 10 days. RESULTS: The MIC90 of APRN and enilconazole against T. verrucosum was 4 and 2 µg/mL, respectively. The clinical scores of PC, APRN4, and APRN8 were significantly lower than those of NC. Clinical and mycological efficacies were higher for APRN8, APRN4 and PC. No fungi were observed in the skin tissues of APRN4 and APRN8, while fungi were observed in 50% of the PC. CONCLUSION: APRN showed antifungal activity against T. verrucosum in vitro and in vivo and is a potential candidate for the treatment of dermatophytosis.

Molecular cytogenetic analysis of the olive flounder embryonic cell line FGBC8 and its applicability to biotechnology.

We explored the biotechnological applicability of a previously established olive flounder (Paralichthys olivaceus) embryonic cell line (FGBC8). FGBC8 was transfected with pEGFP-c1 and pluripotency-related genes, then infected with viral hemorrhagic septicemia virus (VHSV), and the expression of immune-related genes was observed through quantitative real-time polymerase chain reaction. Transfected cells showed strong green fluorescence 48 h after transfection, and pluripotency-related genes were successfully transfected. In addition, FGBC8 cells were highly susceptible to VHSV and the expression of immune-related genes was induced during infection. Our results demonstrate that FGBC8 cells are valuable research tools for assessing host-pathogen interactions and biotechnological applications.

Afebrile benign convulsions with or without a reversible splenial lesion in two pediatric patients with COVID-19.

BACKGROUND: Seizures in children with coronavirus disease 2019 (COVID-19) were markedly increased during the Omicron variant surge. Most seizures occurred with fever. New-onset afebrile seizures were rarely reported; therefore, their courses are not well-known. CASE PRESENTATION: Two patients (7 and 26 months of age, respectively) with COVID-19 showed recurrent afebrile seizures immediately after resolution of a fever lasting for 2-3 days. Bilateral convulsive seizures lasted for approximately 1 min/episode (6 of 7 total episodes) and occurred 3-4 times within 2-3 h. However, the patients were alert between seizures, which is in contrast to seizures occurring with encephalopathy or encephalitis. Only one episode required acute antiseizure medication. Brain magnetic resonance imaging showed a reversible splenial lesion in one patient. The serum uric acid level was slightly increased (7.8 mg/dL) in this patient. Electroencephalography findings were all normal. During the follow-up period, no seizures or developmental problems have been observed. CONCLUSIONS: COVID-19-associated, afebrile benign convulsions with or without a reversible splenial lesion are similar to 'benign convulsions with mild gastroenteritis'; therefore, continuation of antiseizure medication does not seem necessary.

Enterovibrio paralichthyis sp. nov., isolated from the gut of an olive flounder Paralichthys olivaceus.

A Gram-stain-negative, facultatively anaerobic, flagellated and coccoid, ovoid or rod-shaped bacterial strain, NIFS-20-8T, was isolated from the intestine of an olive flounder (Paralichthys olivaceus) from the East Sea, Republic of Korea. The neighbour-joining phylogenetic tree of 16S rRNA gene sequences showed that strain NIFS-20-8T fell within the clade comprising the type strains of Enterovibrio species. Strain NIFS-20-8T exhibited 16S rRNA gene sequence similarities of 97.2 and 97.1 % to the type strains of Enterovibrio nigricans and Enterovibrio norvegicus, respectively, and of 96.6-97.0 % to the type strains of the other Enterovibrio species. The average nucleotide identity and digital DNA-DNA hybridization values between the genomic sequence of strain NIFS-20-8T and those of the type strains of four Enterovibrio species were 73.8-75.0 and 19.8-21.1 %, respectively. The DNA G+C content of strain NIFS-20-8T from genomic sequence data was 50.55 mol%. Strain NIFS-20-8T contained Q-8 as the predominant ubiquinone and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0 and C18 : 1 ω7c as the major fatty acids. The major polar lipids detected in stain NIFS-20-8T were phosphatidylethanolamine and phosphatidylglycerol. Distinguishing phenotypic properties, together with phylogenetic and genetic distinctiveness, revealed that strain NIFS-20-8T is separated from recognized Enterovibrio species. On the basis of the data presented here, strain NIFS-20-8T is considered to represent a novel species of the genus Enterovibrio, for which the name Enterovibrio paralichthyis sp. nov. is proposed. The type strain is NIFS-20-8T (= KCTC 82873T=NBRC 115237T).

Characterization of TRIM16, a member of the fish-specific finTRIM family, in olive flounder Paralichthys olivaceus.

Tripartite motif-containing (TRIM) proteins are conserved throughout the metazoan kingdom, and the TRIM subset finTRIM is highly diversified in fish. We isolated TRIM16 cDNA, a member of the finTRIM family, from the olive flounder Paralichthys olivaceus (PoTRIM16). PoTRIM16 contained a 1,725-bp coding sequence encoding a 574-amino acid polypeptide, which in turn contained a really interesting new gene (RING) finger domain, B-box-type zinc finger (B-BOX), nuclease SbcCD subunit C (SbcC), structural maintenance of chromosome (SMC prok B), and stonustoxin (SNTX) subunit alpha (SPRY-PRY-SNTX). Multiple alignment of related sequences revealed that PoTRIM16 showed 86.63-97.40% identity with fish orthologues, and a phylogenetic tree was constructed of vertebrates. PoTRIM16 mRNA was detected in all tissues examined; levels were highest in the eye and ovary. PoTRIM16 mRNA expression was investigated during early development. Under VHSV infection, PoTRIM16 mRNA was downregulated in the liver of P. olivaceus. This is the first study to characterize fish-specific finTRIM in P. olivaceus, which may play a role in the immune response against virus infection.

Twelve quick steps for genome assembly and annotation in the classroom.

Eukaryotic genome sequencing and de novo assembly, once the exclusive domain of well-funded international consortia, have become increasingly affordable, thus fitting the budgets of individual research groups. Third-generation long-read DNA sequencing technologies are increasingly used, providing extensive genomic toolkits that were once reserved for a few select model organisms. Generating high-quality genome assemblies and annotations for many aquatic species still presents significant challenges due to their large genome sizes, complexity, and high chromosome numbers. Indeed, selecting the most appropriate sequencing and software platforms and annotation pipelines for a new genome project can be daunting because tools often only work in limited contexts. In genomics, generating a high-quality genome assembly/annotation has become an indispensable tool for better understanding the biology of any species. Herein, we state 12 steps to help researchers get started in genome projects by presenting guidelines that are broadly applicable (to any species), sustainable over time, and cover all aspects of genome assembly and annotation projects from start to finish. We review some commonly used approaches, including practical methods to extract high-quality DNA and choices for the best sequencing platforms and library preparations. In addition, we discuss the range of potential bioinformatics pipelines, including structural and functional annotations (e.g., transposable elements and repetitive sequences). This paper also includes information on how to build a wide community for a genome project, the importance of data management, and how to make the data and results Findable, Accessible, Interoperable, and Reusable (FAIR) by submitting them to a public repository and sharing them with the research community.

Paenihalocynthiibacter styelae gen. nov., sp. nov., isolated from stalked sea squirt Styela clava.

A Gram-stain-negative, strictly aerobic, non-motile and rod-shaped bacterial strain, MYP1-1T, was isolated from the intestine of a stalked sea squirt (Styela clava) of the South Sea in the Republic of Korea. The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain MYP1-1T clustered with the type strains of Halocynthiibacter species and Pseudohalocynthiibacter aestuariivivens. Strain MYP1-1T exhibited 16S rRNA gene sequence similarity values of 97.0-97.6 % to the type strains of Halocynthiibacter namhaensis, Halocynthiibacter arcticus and P. aestuariivivens. The phylogenetic tree based on genomic sequences showed that strain MYP1-1T formed a distinct branch separating it from the type strains of two Halocynthiibacter species and P. aestuariivivens and other taxa. The DNA G+C content of strain MYP1-1T from its genomic sequence was 55.0 mol%. Strain MYP1-1T contained Q-10 as the predominant ubiquinone and C18 : 1 ω7c as the major fatty acid. The major polar lipids of strain MYP1-1T were phosphatidylcholine, phosphatidylglycerol, one unidentified lipid and one unidentified aminolipid. The differences in fatty acid and polar lipid profiles and other differential phenotypic properties made it reasonable to distinguish strain MYP1-1T from the genera Halocynthiibacter and Pseudohalocynthiibacter. On the basis of the polyphasic taxonomic investigations, we conclude that strain MYP1-1T constitutes a new genus and species within the class Alphaproteobacteria, for which the name Paenihalocynthiibacter styelae gen. nov., sp. nov. is proposed. The type strain is MYP1-1T (=KCTC 82143T=NBRC 114355T).

Antimicrobial Activity and Action Mechanisms of Arg-Rich Short Analog Peptides Designed from the C-Terminal Loop Region of American Oyster Defensin (AOD).

American oyster defensin (AOD) was previously purified from acidified gill extract of the American oyster, Crassostrea virginica. AOD is composed of 38 amino acids with three disulfide bonds and exhibits strong antimicrobial activity against Gram-positive bacteria as well as significant activity against Gram-negative bacteria. Here, to develop promising peptides into antibiotic candidates, we designed five arginine-rich analogs (A0, A1, A2, A3, and A4), predicted their loop and extended strand/random structures-including nine amino acids and a disulfide bond derived from the C-terminus of AOD-and described their antimicrobial and cytotoxic effects, as well as their modes of action. In our experimental results, the A3 and A4 analogs exhibited potent antimicrobial activity against all test organisms-including four Gram-positive bacteria, six Gram-negative bacteria, and Candida albicans-without cell toxicity. A sequence of experiments, including a membrane permeabilization assay, DNA binding study, and DNA polymerization inhibition test, indicated that the two analogs (A3 and A4) possibly did not act directly on the bacterial membrane but instead interacted with intracellular components such as DNA or DNA amplification reactions. AOD analogs also showed strong bacterial inhibition activity in the plasma environment. In addition, analog-treated microbial cells clearly exhibited membrane disruption, damage, and leakage of cytoplasmic contents. Collectively, our results suggest that two analogs, A3 and A4, have potent antimicrobial activity via DNA interaction and have the potential for development into novel antimicrobial agents.

Molecular Characterization of Paralichthys olivaceus MAF1 and Its Potential Role as an Anti-Viral Hemorrhagic Septicaemia Virus Factor in Hirame Natural Embryo Cells.

MAF1 is a global suppressor of RNA polymerase III-dependent transcription, and is conserved from yeast to human. Growing evidence supports the involvement of MAF1 in the immune response of mammals, but its biological functions in fish are unknown. We isolated and characterized Maf1 from the olive flounder Paralichthys olivaceus (PoMaf1). The coding region of PoMaf1 comprised 738 bp encoding a 245-amino-acid protein. The deduced PoMAF1 amino acid sequence shared features with those of MAF1 orthologues from vertebrates. PoMaf1 mRNA was detected in all tissues examined, and the levels were highest in eye and muscle tissue. The PoMaf1 mRNA level increased during early development. In addition, the PoMaf1 transcript level decreased during viral hemorrhagic septicemia virus (VHSV) infection of flounder hirame natural embryo (HINAE) cells. To investigate the role of PoMaf1 in VHSV infection, single-cell-derived PoMaf1 knockout HINAE cells were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system, and cell clones with complete disruption of PoMaf1 were selected. PoMaf1 disruption increased the VHSV glycoprotein (G) mRNA levels during VHSV infection of HINAE cells, implicating PoMAF1 in the immune response to VSHV infection. To our knowledge, this is the first study to characterize fish Maf1, which may play a role in the response to viral infection.

Alteromonas ponticola sp. nov., a gammaproteobacterium isolated from seawater.

A Gram-stain-negative, aerobic, non-spore-forming, non-motile and ovoid or rod-shaped bacterial strain, MYP5T, was isolated from seawater in Jeju island of South Korea. MYP5T grew optimally at 30-35 °C and in the presence of 2.0 % (w/v) NaCl. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that MYP5T fell within the clade enclosed by the type strains of species of the genus Alteromonas, clustering with the type strains of Alteromonas confluentis and Alteromonas halophila. MYP5T exhibited the highest 16S rRNA gene sequence similarity value (98.0 %) to the type strain of A. confluentis and similarities of 95.1-97.9 % to the type strains of the other species of the genus Alteromonas. ANI and dDDH values of genomic sequences between MYP5T and the type strains of 22 species of the genus Alteromonas were 66.8-70.5 % and 18.6-27.5 %, respectively. The DNA G+C content of MYP5T, determined from the genome sequence, was 46.1 %. MYP5T contained Q-8 as the predominant ubiquinone and C18 : 1 ω7c, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0 and 10-methyl C17 : 0 as the major fatty acids. The major polar lipids of MYP5T were phosphatidylethanolamine and phosphatidylglycerol. Distinguishing phenotypic properties, along with the phylogenetic and genetic distinctiveness, revealed that MYP5T is separated from species of the genus Alteromonas. On the basis of the data presented, MYP5T is considered to represent a novel species of the genus Alteromonas, for which the name Alteromonas ponticola sp. nov. is proposed. The type strain is MYP5T (=KCTC 82144T=NBRC 114354T).

Myticusin-beta, antimicrobial peptide from the marine bivalve, Mytilus coruscus.

We isolated and purified an antimicrobial peptide (AMP) from the mantle of the hard-shelled mussel, Mytilus coruscus. The peptide was purified through C18 reversed-phase high-performance liquid chromatography, and displayed antibacterial activity. Total molecular mass of 11,182 Da was determined using matrix-assisted laser desorption ionization time-of-flight mass spectrophotometry. The N-terminal 23-amino acid sequence of its purified peak was obtained through Edman degradation, revealing 82% identity with myticusin-1 of M. coruscus. Complete sequence of the target peptide was determined through cDNA cloning and rapid amplification of cDNA ends. The complete sequence comprised 574 bp with a 387-bp open reading frame (ORF) encoding 24 amino acids of a signal peptide and 104 amino acids of a mature peptide, which was named myticusin-beta. Furthermore, we discovered two novel isoforms of myticusin-beta. We constructed and expressed recombinant myticusin-beta, which displayed antimicrobial activity against gram-positive (Bacillus cereus, Bacillus subtilis, Clostridium perfringens, Staphylococcus aureus, Streptococcus iniae, Streptococcus mutans) and gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Vibrio alginolyticus, Klebsiella pneumoniae). Purified recombinant myticusin-beta also showed anti-parasitic activity at various concentrations. A short AMP analog was designed and synthesized based on the sequence of myticusin-beta, with markedly improved antimicrobial activity. Expression of myticusin-beta was detected in the mantle at the highest level, followed by hemocytes. The results obtained in this work suggest that myticusin-beta is an immune-related AMP of M. coruscus and an effective alternative to antibiotics.

Association between Blood Pressure and Renal Progression in Korean Adults with Normal Renal Function.

BACKGROUND: Although hypertension (HTN) is a well-established major risk factor for renal progression in patients with chronic kidney disease (CKD), few studies investigating its role in renal deterioration in the general population with normal renal function (NRF) have been published. Here, we analyzed the correlation between blood pressure (BP) and impaired renal function (IRF) in Korean adults with NRF. METHODS: Data for the study were collected from the national health screening database of the Korean National Health Insurance Service. Patients whose baseline estimated glomerular filtration rate (eGFR) was less than 60 mL/min/1.73 m² or whose baseline urinalysis showed evidence of proteinuria were excluded. IRF was defined as an eGFR below 60 mL/min/1.73 m². We performed follow up for eGFR for 6 years from 2009 to 2015 and investigated IRF incidence according to baseline BP status. We categorized our study population into two groups of IRF and NRF according to eGFR level in 2015. RESULTS: During 6 years of follow-up examinations, IRF developed in 161,044 (2.86%) of 5,638,320 subjects. The IRF group was largely older, and the incidence was higher in females and patients with low income, HTN, diabetes mellitus, dyslipidemia, and obesity compared with the NRF group. Subjects whose systolic BP was more than 120 mmHg or whose diastolic BP was more than 70 mmHg had an increased risk of developing IRF compared with subjects with lower BP (odds ratio [OR], 1.037; 95% confidence interval [CI], 1.014-1.061 vs. OR, 1.021; 95% CI, 1.004-1.038). CONCLUSION: BP played a major role in renal progression in the general population with NRF. Strict BP control may help prevent CKD in the general population.

Characterization and mutation anaylsis of a cold-active bacterial hormone-sensitive lipase from Salinisphaera sp. P7-4.

In mammals, hormone sensitive lipase (EC 3.1.1.79, HSL) catalyzes the hydrolysis of triacylglycerols as well as the modifications of a broad range of hydrophobic substrates containing ester linkages. HSLs are composed of an N-terminal ligand-binding domain and a C-terminal catalytic domain. Bacterial hormone-sensitive lipases (bHSLs), which are homologous to the C-terminal domain of mammalian HSLs, have a catalytic triad composed of Ser, His, and Asp. Here, a novel cold-active hormone-sensitive lipase (SaHSL) from Salinisphaera sp. P7-4 was identified, functionally characterized, and subjected to site-directed mutations. The enzymatic properties of SaHSL were investigated using several biochemical and biophysical methods. Interestingly, SaHSL exhibited the ability to act on a broad range of substrates including glyceryl tributyrate and glucose pentaacetate. Homology modeling and site-directed mutagenesis indicated that hydrophobic residues (Leu156, Phe164, and Val204) around the substrate-binding pocket were involved in substrate recognition. In addition, highly conserved amino acids (Glu201, Arg207, Leu208, and Asp227) in the regulatory regions were found to be responsible for substrate specificity, thermostability, and enantioselectivity. In summary, this work provides new insights into the understanding of the C-terminal domain of HSL family and evidence that SaHSL can be used in a wide range of industrial applications.

Efficient production of poly γ-d-glutamic acid from the bloom-forming green macroalgae, Ulva sp., by Bacillus sp. SJ-10.

Numerous studies on poly γ-d-glutamicacid (γ-PGA) production have investigated terrestrial renewable sources for reducing production costs, but there are no studies using waste marine resources so far. We aimed to develop a cost-effective production method of γ-d-PGA by Bacillus sp. SJ-10 using green macroalgae (Ulva sp.) as a major substrate without hydrolysis pretreatment. The SJ-10 was shown to not only cause immediate tissue degradation of the Ulva membrane but also grew well as a sole substrate. The γ-d-PGA yield was 6.29 ± 0.34 g/L under optimized conditions via the response surface method, and the produced γ-d-PGA had a thermal decomposition temperature of 310°C and molecular weight of 250-1780 kDa. The calculated cost efficiency for the final yield was 32% when compared with complex media. Therefore, the present study provided a strategy for promoting an ecofriendly and cost-effective means to produce γ-d-PGA via a marine renewable resource.

Empedobacter tilapiae sp. nov., isolated from an intestine of Nile tilapia (Oreochromis niloticus).

A Gram-stain-negative, aerobic, non-motile and ovoid or rod-shaped bacterial strain, MRS2T, was isolated from an intestine of Nile tilapia (Oreochromisniloticus) collected from the Republic of Korea. Strain MRS2T grew optimally at 30 °C and in the presence of 0-2.0 % (w/v) NaCl. A neighbour-joining phylogenetic tree of 16S rRNA gene sequences showed that strain MRS2T clustered with the type strains of Empedobacter species. It exhibited the highest 16S rRNA gene sequence similarity (98.5 %) to the type strain of Empedobacter falsenii and sequence similarities of 97.4-97.6 % to the type strains of two other Empedobacter species. Strain MRS2T contained MK-6 as the predominant ubiquinone and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), iso-C17 : 0 3-OH and iso-C15 : 0 as the major fatty acids. The major polar lipids of strain MRS2T were phosphatidylethanolamine, one unidentified lipid and one unidentified aminolipid. The DNA G+C contents of strain MRS2T were 32.2 mol% or 30.65 mol%. Strain MRS2T exhibited DNA-DNA relatedness values of 12-20 % to the type strains of Empedobacter falsenii, Empedobacter brevis and Empedobacter stercoris. The average nucleotide identity values between strain MRS2T and five strains of E. falsenii and E. brevis were 84.8-91.0 %. The phylogenetic, genetic and differential phenotypic properties indicated that strain MRS2T is separated from Empedobacter species. On the basis of the data presented here, strain MRS2T is considered to represent a novel species of the genus Empedobacter, for which the name Empedobactertilapiae sp. nov. is proposed. The type strain is MRS2T (=KCTC 62904T=NBRC 113550T).

Aliishimia ponticola gen. nov., sp. nov., isolated from seawater.

A Gram-stain-negative, aerobic, non-motile and coccoid, ovoid or rod-shaped bacterial strain, designated MYP11T, was isolated from seawater around Jeju island, Republic of Korea and identified by polyphasic taxonomic study. A neighbour-joining phylogenetic tree of 16S rRNA gene sequences showed that strain MYP11T joined the cluster comprising the type strains of Shimiaabyssi, Shimiaaestuarii and Shimiaaquaeponti, showing 16S rRNA gene sequence similarities of 96.3-96.8 %. Strain MYP11T exhibited 16S rRNA gene sequence similarity values of 94.2-94.9 % to the type strains of other Shimia species. In the upgma dendrogram based on the average nucleotide identity values of genomic sequences, strain MYP11T formed an evolutionary lineage independent of those of Shimia species and other taxa. Strain MYP11T contained Q-10 as the predominant ubiquinone and C18 : 1 ω7c and cyclo C19 : 0 ω8c as the major fatty acids. The major polar lipids of strain MYP11T were phosphatidylcholine, phosphatidylglycerol, two unidentified lipids and one unidentified aminolipid. The DNA G+C content of strain MYP11T was 63.1 or 61.5 mol%. The differences in the fatty acid and polar lipid profiles and DNA G+C contents made it reasonable to distinguish strain MYP11T from the type strains of S. abyssi, S. aestuarii and S. aquaeponti and those of other Shimia species. On the basis of the polyphasic data presented here, strain MYP11T is considered to constitute a new genus and species within the class Alphaproteobacteria, for which the name Aliishimia ponticola gen. nov., sp. nov. is proposed. The type strain is MYP11T (=KCTC 62899T=NBRC 113544T).

Development of novel antimicrobial peptides derived from anti-lipopolysaccharide factor of the swimming crab, Portunus trituberculatus.

Anti-lipopolysaccharide factors (ALFs) are a representative host defense protein in crustaceans. In this study, we successfully developed two novel antimicrobial peptides (AMPs), named crab-ALF2A and crab-ALF6A, which contain changes to the amino acid sequences of the lipopolysaccharide binding domain and signal peptide, respectively, of the ALF of the swimming crab Portunus trituberculatus. The crab-ALF2A peptide showed potent antimicrobial activity against the Gram-positive bacteria Bacillus cereus, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentration [MEC] 1.51-1.93 µg/mL) and the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli (MEC 1.87-1.98 µg/mL), with maximal bactericidal activity at a peptide concentration of 5 µg/mL. The crab-ALF6A peptide also showed potent antimicrobial activity against B. cereus, S. aureus, and S. iniae (MEC 1.49-2.3 µg/mL) and P. aeruginosa and E. coli (MEC 1.72-1.19 µg/mL) at a peptide concentration of 5 µg/mL. Notably, the crab-ALF2A and crab-ALF6A peptides exhibited strong activity against Candida albicans (MECs of 2.11 and 1.95 µg/mL, respectively). These activities were stable following heat treatment. Moreover, the effect of crab-ALF2A and crab-ALF6A peptide treatment on microbe cell morphology was confirmed by scanning electron microscopy. Membrane disruption and damage, and the leakage of cytoplasmic content were clearly observed. A downsizing peptide approach illustrated that the hexapeptide ALF6A8 (RVLLRL) was the shortest peptide showing significant antimicrobial activity. Our approach allows for the generation of novel antimicrobial peptides in a cost effective manner as potential next-generation antibiotics.