In the present work, current knowledge on the potential fate, microbial degradation, and toxicity of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was thoroughly reviewed, focusing on the toxicological assessment of a variety of potential RDX degradation pathways in bacteria and fungi. The present review on microbial degradation pathways and toxicities of degradation intermediates suggests that, among aerobic RDX degradation pathways, the one via denitration may be preferred in a toxicological perspective, and that among anaerobic pathways, those forming 4-nitro-2,4-diazabutanal (NDAB) via ring cleavage of 1-nitroso-3,5-dinitro-1,3,5-triazinane (MNX) may be toxicologically advantageous owing to its potential mineralization under partial or complete anoxic conditions. These findings provide important information on RDX-degrading microbial pathways, toxicologically most suitable to be stimulated in contaminated fields.

Phosphomannomutase (ManB) is involved in the biosynthesis of GDP-mannose, which is vital for numerous processes such as synthesis of carbohydrates, production of alginates and ascorbic acid, and post-translational modification of proteins. Here, we discovered that a deletion mutant of manB (BG101) in Streptomyces coelicolor (S. coelicolor) showed higher sensitivity to bacteriostatic chloramphenicol (CM) than the wild-type strain (M145), along with decreased production of CM metabolites. Deletion of manB also decreased the mRNA expression level of drug efflux pumps (i.e., cmlR1 and cmlR2) in S. coelicolor, resulting in increased sensitivity to CM. This is the first report on changes in antibiotic sensitivity to CM by deletion of one glycolysis-related enzyme in S. coelicolor, and the results suggest different approaches for studying the antibiotic-resistant mechanism and its regulation.

Foot and mouth disease (FMD) is one of the acute infectious diseases in hoofed and even-toed mammals, including pigs, and it occurs via acute infection by Aphthovirus. When FMD is suspected, animals around the location of origin are typically slaughtered and buried. Other methods such as rendering, composting, and incineration have not been verified in practice in Korea. After the FMD incident, the regular monitoring of the microbial community is required, as microorganisms greatly modify the characteristics of the ecosystem in which they live. This is the result of their metabolic activities causing chemical changes to take place in the surrounding environment. In this study, we investigated changes in the microbial community during a 24 week period with DNA extracts from leachate, formed by the decomposition of buried pigs at a laboratory test site, using denaturing gradient gel electrophoresis (DGGE) with a genomic DNA. Our results revealed that Bacteroides coprosuis, which is common in pig excreta, and Sporanaerobacter acetigenes, which is a sulfur-reduced microbe, were continuously observed. During the early stages (0~2 weeks) of tissue decomposition, Clostridium cochlearium, Fusobacterium ulcerans, and Fusobacterium sp., which are involved in skin decomposition, were also observed. In addition, various microbes such as Turicibacter sanguinis, Clostridium haemolyticum, Bacteroides propionicifaciens, and Comamonas sp. were seen during the later stages (16~24 weeks). In particular, the number of existing microbial species gradually increased during the early stages, including the exponential phase, decreased during the middle stages, and then increased again during the later stages. Therefore, these results indicate that the decomposition of pigs continues for a long period of time and leachate is created continuously during this process. It is known that leachate can easily flow into the neighboring environment, so a long-term management plan is needed in burial locations for FMD-infected animals.

In Burkholderia pseudomallei, the pathogen that causes melioidosis, the gene cluster encoding the capsular polysaccharide, is located on chromosome 1. Among the 19 capsular genes in this cluster, wzm has not been thoroughly studied. To study the function of wzm, we generated a deletion mutant and compared it with the wild-type strain. The mutant produced less biofilm in minimal media and was more sensitive to desiccation and oxidative stress compared with the wild-type strain, indicating that wzm is involved in biofilm formation and membrane integrity. Scanning electron microscopy showed that the bacterial cells of the mutant strain have more defined surfaces with indentations, whereas cells of the wild-type strain do not.

Most of the Helicobacter pylori strains containing the cag pathogenicity island (PAI) have been associated with more severe gastric disease in infected humans. The cag PAI is composed of 27 proteins, and some of the components are required for CagA translocation into host cells as well as induction of proinflammatory cytokines, such as interleukin-8 (IL-8); however, the exact function of most of the components remains unknown or poorly characterized. In this study, we demonstrated that CagT (HP0532), which is an essential structural component of the cag PAI apparatus, plays an important role in the translocation of CagA into host epithelial cells. In addition to being located on the bacterial surface, CagT is also partially localized in the inner membrane, where it acts as a chaperone-like protein and promotes CagA translocation. However, CagT secretion was not detected by immunoprecipitation analysis of cell culture supernatants. Meanwhile, CagT was related to the introduction of IL-8 of the host cell. These results suggest that CagT is expressed on both the inner and outer bacterial membranes, where it serves as a unique type IV secretion system component that is involved in CagA secretion and cag PAI apparatus assembly.

Viral pathogens, alongside other pathogens, have major effects on crustacean aquaculture. Hepatopancreatic parvovirus (HPV) is an emerging virus in the shrimp industry and has been detected in shrimp farms worldwide. The HPV genome has greater diversity than other shrimp viruses owing to its wide host range and geographical distribution. Therefore, developing diagnostic tools is essential to detect even small copy numbers from the target region of native HPV isolates. We have developed two easy to use quantitative real-time PCR kits, called Green Star and Dual Star, which contain all of the necessary components for real-time PCR, including HPV primers, using the primers obtained from the sequences of HPV isolates from Korea, and analyzed their specificity, efficiency, and reproducibility. These two kits could detect from 1 to $1{\times}10^9$ copies of cloned HPV DNA. The minimum detection limits obtained from HPV-infected shrimp were $7.74{\times}10^1$ and $9.06{\times}10^1$ copies in the Green Star and Dual Star assay kits, respectively. These kits can be used for rapid, sensitive, and efficient screening for HPV isolates from Korea before the introduction of postlarval stages into culture ponds, thereby decreasing the incidence of early development of the disease.

A strain of Streptomyces cavourensis subsp. cavourensis (coded as SY224) antagonistic to Colletotrichum gloeosporioides infecting pepper plants was isolated. SY224 produced lytic enzymes such as chitinase, ${\beta}$-1,3-glucanase, lipase, and protease in respective assays. To examine for antifungal activity, the treatments amended with the nonsterilized supernatant resulted in the highest growth inhibition rate of about 92.9% and 87.4% at concentrations of 30% and 10%, respectively. However, the sterilized treatments (autoclaved or chloroform treated) gave a lowered but significant inhibitory effect of about 63.4% and 62.6% for the 10% supernatant concentration, and 75.2% and 74.8% for the of 30% supernatant concentration in the PDA agar medium, respectively, indicative of the role of a non-protein, heat stable compound on the overall effect. This antifungal compound, which inhibited spore germination and altered hyphal morphology, was extracted by EtOAc and purified by ODS, silica gel, Sephadex LH-20 column, and HPLC, where an active fraction was confirmed to be 2-furancarboxaldehyde by GS-CI MS techniques. These results suggested that SY224 had a high potential in the biocontrol of anthracnose in pepper, mainly due to a combined effect of lytic enzymes and a non-protein, heat-stable antifungal compound, 2-furancarboxaldehyde.

Pleurocidin, a 25-mer antimicrobial peptide, has been known to exhibit potent antibacterial activity. To investigate the functional roles in N- and C-terminal regions of pleurocidin on the antibacterial activity, we designed four truncated analogs. The antibacterial susceptibility testing showed that pleurocidin and its analogs exerted antibacterial effect against various bacterial strains and further possessed specific activity patterns corresponding with their hydrophobic scale [pleurocidin > Anal 3 (1-22) > Anal 1 (4-25) > Anal 4 (1-19) > Anal 2 (7-25)]. Fluorescence experiments using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 3,3'-dipropylthiadicarbocyanine iodide [$diSC_3(5)$] indicated that the differences in antibacterial activity of the peptides were caused by its membrane-active mechanisms including membrane disruption and depolarization. Blue shift in tryptophan fluorescence demonstrated that the decrease in net hydrophobicity attenuates the binding affinity of pleurocidin to interact with plasma membrane. Therefore, the present study suggests that hydrophobicity in the N- and C-terminal regions of pleurocidin plays a key role in its antibacterial activity.

We previously isolated a rhizobacterium (Bacillus subtilis IJ-31) and demonstrated that its associated allelochemicals could indicate plant growth retardation. However, little is known about how the growth of plants is regulated by B. subtilis IJ-31 and its allelochemicals. In this study, we investigated whether plant growth retardation in this relationship occurred through the inhibition of gibberellin (GA) biosynthesis. GA $3{\beta}$-hydroxylase activity was found to be inhibited by B. subtilis IJ-31 and hydrocinnamic acid (HCA), which is one of the allelochemicals produced by B. subtilis IJ-31. Additionally, thin layer chromatography (TLC) demonstrated that B. subtilis IJ-31 culture broth and HCA both inhibit GA $3{\beta}$-hydroxylase (MBP-GA4) activity. The retardation of plants by HCA was then confirmed in vivo and in vitro using a Ryegrass and Arabidopsis growth retardation assay. Furthermore, treatment with either B. subtilis IJ-31 culture extract or its allelochemicals resulted in significant down-regulation of XTR9 gene expression in Arabidopsis. Overall, we identified the functional mechanism of plant growth retardation by B. subtilis IJ-31 and its allelochemicals.

We applied enzymatic hydrolysis and tangential flow filtration (TFF) to purify a novel anticancer peptide from Mytilus coruscus (M. coruscus) and investigated its anticancer properties. To prepare the peptide, eight proteases were employed for enzymatic hydrolysis. Pepsin hydrolysates, which showed clearly superior cytotoxic activity on prostate cancer cells, were further purified using a flow filtration system using a TFF and consecutive chromatographic methods. Finally, a novel anticancer peptide was obtained, and the sequence was identified as Ala-Phe-Asn-Ile-His-Asn-Arg-Asn-Leu-Leu. The peptide from M. coruscus effectively induced cell death on prostate, breast and lung cancer cells but not on normal liver cells. This is the first report of an anticancer peptide derived from the hydrolysates of M. coruscus.

A xylanase-producing thermophilic strain, Geobacillus sp. TC-W7, was isolated from a hot spring in Yongtai (Fuzhou, China). Subsequently, the xylanase gene that encoded 407 amino acids was cloned and expressed. The recombinant xylanase was purified by GST affinity chromatography and exhibited maximum activity at $75^{\circ}C$ and a pH of 8.2. The enzyme was active up to $95^{\circ}C$ and showed activity over a wide pH range of 5.2 to 10.2. Additionally, the recombinant xylanase showed high thermostability and pH stability. More than 85% of the enzyme's activity was retained after incubation at $70^{\circ}C$ for 90 min at a pH of 8.2. The activity of the recombinant xylanase was enhanced by treatment with 10 mM enzyme inhibitors (DDT, Tween-20, 2-Me, or TritonX-100) and was inhibited by EDTA or PMSF. Its functionality was stable in the presence of $Li^+$, $Na^+$, and $K^+$, but inhibited by $Hg^{2+}$, $Ni^{2+}$, $Co^{2+}$, $Cu^{2+}$, $Zn^{2+}$, $Pb^{2+}$, $Fe^{3+}$, and $Al^{3+}$. The functionality of the crude xylanase had similar properties to the recombinant xylanase except for when it was treated with $Al^{2+}$ or $Fe^{2+}$. The enzyme might be a promising candidate for various industrial applications such as the biofuel, food, and paper and pulp industries.

In this study, we investigated various cultural and operational factors to enhance electricity generation in a microbial fuel cell (MFC) using Geobacter sulfurreducens. The pure culture of G. sulfurreducens was cultivated using various substrates including acetate, malate, succinate, and butyrate, with fumarate as an electron acceptor. Cell growth was observed only in acetate-fed medium, when the cell concentrations increased 4-fold for 3 days. A high acetate concentration suppressed electricity generation. As the acetate concentration was increased from 5 to 20 mM, the power density dropped from 16 to $13mW/m^2$, whereas the coulombic efficiency (CE) declined by about half. The immobilization of G. sulfurreducens on the anode considerably reduced the enrichment period from 15 to 7 days. Using argon gas to create an anaerobic condition in the anode chamber led to increased pH, and electricity generation subsequently dropped. When the plain carbon paper cathode was replaced by Pt-coated carbon paper (0.5 mg $Pt/cm^2$), the CE increased greatly from 39% to 83%.

A thermotolerant Saccharomyces cerevisiae mutant strain, TT6, was constructed after multi-parental hybridization of five mutant strains obtained by UV or NTG treatment of the original strain, S. cerevisiae KV1. When incubated at $40^{\circ}C$ in YPD broth, TT6 began to grow exponentially in 10 h, but KV1 did not show any noticeable growth even after 22 h. The thermotolerant growth of TT6 was confirmed by serial dilution assay at $42^{\circ}C$; TT6 grew at a cell concentration ($10^{-5}$) 10,000 times lower than that of KV1 ($10^{-1}$). Whereas ethanol production from YP containing 23% (w/v) glucose by KV1 decreased with increasing temperature from $30^{\circ}C$ to $36^{\circ}C$, ethanol production by TT6 did not decrease at temperatures up to $37^{\circ}C$. When TT6 was tested for ethanol production at $36^{\circ}C$ by simultaneous saccharification and fermentation (SSF) from 23% corn, 24 h of fermentation time or 50% of the glucoamylase dose was saved when compared with KV1 at $30^{\circ}C$. The ethanol yield from corn by SSF with TT6 at $36^{\circ}C$ was 91.7% of the theoretical yield, whereas that of KV1 at $30^{\circ}C$ was 90.6%.

5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) was selected as an electron transfer mediator and was covalently immobilized onto high porosity carbon cloth to employ as a working electrode in an electrochemical $NAD^+$-regeneration process, which was coupled to an enzymatic reaction. The voltammetric behavior of DTNB attached to carbon cloth resembled that of DTNB in buffered aqueous solution, and the electrocatalytic anodic current grew continuously upon addition of NADH at different concentrations, indicating that DTNB is immobilized to carbon cloth effectively and the immobilized DTNB is active as a soluble one. The bioelectrocatalytic $NAD^+$ regeneration was coupled to the conversion of L-glutamate into ${\alpha}$-ketoglutarate by L-glutamate dehydrogenase within the same microreactor. The conversion at 3 mM monosodium glutamate was very rapid, up to 12 h, to result in 90%, and then slow up to 24 h, showing 94%, followed by slight decrease. Low conversion was shown when substrate concentration exceeding 4 mM was tested, suggesting that L-glutamate dehydrogenase is inhibited by ${\alpha}$-ketoglutarate. However, our electrochemical $NAD^+$ regeneration procedure looks advantageous over the enzymatic procedure using NADH oxidase, from the viewpoint of reaction time to completion.

The aim of this work was to establish the optimal conditions for production of carboxymethylcellulase (CMCase) by a newly isolated marine bacterium using response surface methodology (RSM). A microorganism producing CMCase, isolated from seawater, was identified as Cellulophaga lytica based 16S rDNA sequencing and the neighborjoining method. The optimal conditions of rice bran, ammonium chloride, and initial pH of the medium for cell growth were 100.0 g/l, 5.00 g/l, and 7.0, respectively, whereas those for production of CMCase were 79.9 g/l, 8.52 g/l, and 6.1. The optimal concentrations of $K_2HPO_4$, NaCl, $MgSO_4{\cdot}7H_2O$, and $(NH_4)_2SO_4$ for cell growth were 6.25, 0.62, 0.28, and 0.42 g/l, respectively, whereas those for production of CMCase were 3.72, 0.54, 0.70, and 0.34 g/l. The optimal temperature for cell growth and the CMCase production by C. lytica LBH-14 were $35^{\circ}C$ and $25^{\circ}C$, respectively. The maximal production of CMCase under optimized condition for 3 days was 110.8 U/ml, which was 5.3 times higher than that before optimization. In this study, rice bran and ammonium chloride were developed as carbon and nitrogen sources for the production of CMCase by C. lytica LBH-14. The time for production of CMCase by a newly isolated marine bacterium with submerged fermentations reduced to 3 days, which resulted in enhanced productivity of CMCase and a decrease in its production cost.

The inhibitory effect of IgY against Vibrio parahaemolyticus and Vibrio vulnificus responsible for seafood-borne diseases was investigated in this study. Water-soluble fractions (WSF) of protein containing IgYs were isolated from the egg yolk of hens initially immunized with formalin-inactivated V. parahaemolyticus or V. vulnificus. Protein, total and specific IgY contents of the WSF were determined. The inhibitory and protective effects of IgYs on the growth of V. parahaemolyticus and V. vulnificus were assayed in liquid medium and in mice. IgYs showed high affinity to their corresponding antigens with high titer from day 28 onwards. Protein contents and total IgY concentrations remained stable throughout the immunization period, whereas specific IgY concentrations increased steadily and reached a plateau at day 49. Specific IgY powder (150 mg/ml) significantly inhibited further multiplication of both V. parahaemolyticus and V. vulnificus in liquid medium as compared with the control IgY. The bacteria count in mice feces was lower in mice pretreated with specific IgYs than in those pretreated with PBS or control IgY. Higher survival of mice was observed in the experimental groups pretreated with either anti-V. parahaemolyticus (75% survival) or anti-V. vulnificus (87% survival) IgYs, compared with those in the control groups pretreated with PBS or nonspecific IgY. All mice in the control groups died within three days after bacteria inoculation; hence, the protective effect of specific IgYs against infection caused by V. parahaemolyticus and V. vulnificus was demonstrated.

Weissella confusa 31, an isolate from human feces, possesses desirable properties as a probiotic strain, including bile salt resistance. W. confusa 31 is not inhibited by bile salts up to 0.3% concentration. Proteins affected by bile salts (0.05%) were examined by 2-D gel electrophoresis. Our proteomic analyses revealed that the intensities of 29 spots were changed, where 17 increased (including 2 spots observed only under the bile salts stress conditions) and 12 decreased. Proteins were identified by MALDI-TOF mass spectrometry. Proteins increased in the band intensities included adenylate kinase (12.75-fold increase), Clp-like ATP-dependent protease (11.91-fold), 6-phosphogluconate dehydrogenase (10.35-fold), and HSP 70 (5.07-fold). Some of the increased or decreased proteins are also known to be involved in other types of stress responses.

The entire nucleotide sequence of the HIS3 gene encoding imidazoleglycerol phosphate dehydratase (IGPD) in yeast Candida milleri CBS 8195 was determined. Sequence analysis revealed an open-reading frame of C. milleri HIS3 that spans 678 bp, encodes 226 amino acids, and shares 80.5% amino acid identity to Torulaspora delbrueckii IGPD, followed by that to Saccharomyces cerevisiae (79.6%). The cloned HIS3 gene complemented a his3 mutation in S. cerevisiae, suggesting that it encodes a functional IGPD in C. milleri CBS 8195. A new auxotrophic marker is now available for acid-tolerant yeast C. milleri.

Levodopa or L-3,4-dihydroxyphenylalanine (L-DOPA) is the precursor of the neurotransmitter dopamine. L-DOPA is a famous treatment for Parkinson's disease symptoms. In this study, electroenzymatic synthesis of L-DOPA was performed in a three-electrode cell, comprising a Ag/AgCl reference electrode, a platinum wire auxiliary electrode, and a glassy carbon working electrode. L-DOPA had an oxidation peak at 376 mV and a reduction peak at -550 mV. The optimum conditions of pH, temperature, and amount of free tyrosinase enzyme were pH 7, $30^{\circ}C$, and 250 IU, respectively. The kinetic constant of the free tyrosinase enzyme was found for both cresolase and catacholase activity to be 0.25 and 0.4 mM, respectively. A cyclic voltammogram was used to investigate the electron transfer rate constant. The mean heterogeneous electron transfer rate ($k_e$) was $5.8{\times}10^{-4}$ cm/s. The results suggest that the electroenzymatic method could be an alternative way to produce L-DOPA without the use of a reducing agent such as ascorbic acid.

The anaerobic oxidation of methane (AOM) in anaerobic sewage sludge was characterized. The net methane oxidation was observed in samples amended with methane plus sulfate or with methane alone, whereas methane formation was observed in the samples without methane, indicating that methane oxidation and formation occurred simultaneously. The ratio of the net methane oxidation rate to $H_2S$ formation was 100:1, suggesting that the AOM was not closely associated with sulfate reduction in the anaerobic sludge. The net AOM was positively associated with the methane concentration and sludge dilution ratio. However, the rate of AOM was negatively correlated with organic substrate (acetate) concentration. Therefore, the production and oxidation of methane could be controlled by environmental conditions and dissolved organic compounds in the bulk solution.