Although insects lack the adaptive immunity characteristic of vertebrates, certain species exhibit enhanced subsequent immune responses upon re-encountering a pathogen, a phenomenon known as immune priming. The underlying mechanism of this phenomenon is still elusive. This study evaluated the immune priming of the diamondback moth, Plutella xylostella, induced by a nonpathogenic and commensal bacterium, Bacillus subtilis. Prior exposure of P. xylostella to B. subtilis significantly increased survival against a pathogenic bacterium, Bacillus thuringiensis, compared to larvae without pre-exposure. To extend the effect of the microbial commensals, two antibiotics, ampicillin and kanamycin, were treated to suppress their populations. In the axenic-like condition in the gut, cellular and humoral immune responses were significantly suppressed. An addition of B. subtilis to the diet of P. xylostella significantly enhanced the immune responses. Apolipoprotein D, known as a lipid carrier, acts like a vertebrate lipocalin in the immune priming of the other insect, Spodoptera exigua. The ortholog of this gene has been identified in P. xylostella, and its expression was induced upon B. subtilis treatment. This study sheds light on the potential role of commensal gut microbes, including B. subtilis, in the immune priming of these insects.

Two bacterial genera, Xenorhabdus and Photorhabdus, are mutually symbiotic to the entomopathogenic nematodes, Steinernema and Heterorhabditis, respectively. Success parasitism of the nematode-bacterial complex depends on the host immunosuppression by the bacteria via their secondary metabolites. Lrp (Leucine-responsive regulatory protein) is a global transcriptional factor of the bacteria and play a crucial role in the parasitism. However, its regulatory targets to suppress the insect immunity were not clearly determined. This study investigated the regulatory target genes and subsequent secondary metabolites by Lrp in Xenorhabdus hominickii. Lrp expression occurred at the early infection stage in a target insect, Spodoptera exigua. Among eight non-ribosomal peptide synthetase (NRPS1-NRPS8) genes, six gene (NRPS3-NRPS8) expressions were positively correlated with Lrp expression in the infected larvae of S. exigua. Exchange of the Lrp promoter with an inducible promoter altered the production of the secondary metabolites along with alteration of the NRPS expression levels. The immunosuppressive activities of X. hominickii depended on the Lrp expression level. The metabolites produced by Lrp expression possessed the eicosanoid-biosynthesis inhibitors and hemolytic factors. A cyclic dipeptide (= cPF) was produced under Lrp control and identified to inhibit phospholipase A2 activity of S. exigua in a competitive inhibitory manner. These results suggest that Lrp is a global transcriptional factor of X. hominickii and plays crucial role in insect immunosuppression by modulating NRPS expressions.

Bis(2-ethylhexyl) phthalate (DEHP) is one of the plasticizers used in the polyvinyl chloride (PVC) industry. It is known to be easily released into the environment. In this study, we investigated effects of DEHP on growth, metabolic pathway, and virulence gene expression in soil-borne bacterial plant pathogen, Pectobacterium carotovorum SCC1 using in vitro assays. As a result, DEHP at 20 μg mL-1 did not affect the growth, cell membrane permeability, or ATPase activity of P. carotovorum SCC1. However, it decreased succinyl-CoA synthase (SCS) activity in the tricarboxylic acid (TCA) cycle. Relative expression levels of virulence genes encoding pectate lyase and pectin were differentially influenced by DEHP treatment. These results suggest that biological characteristics of P. carotovorum might be influenced by DEHP in soil.

The aim of this study was to isolate and identify marine bacterium with anti-methicillinresistant Staphylococcus aureus (MRSA) activity, and to purify the anti-MRSA compound, as well as to determine its activity and synergistic effects. Among the marine bacteria isolated in this study, the YJ-1 isolate had the strongest anti-MRSA activity. The YJ-1 isolate was identified on the basis of its biochemical characteristics and an analysis of 16S rRNA gene sequences. The YJ-1 isolate showed over 99.2% homology with Pseudomonas stutzeri, and was designated as a Pseudomonas sp. YJ-1. The optimal culture conditions were 25℃ and initial pH 7.0. For the purification of the anti-MRSA compounds, the YJ-1 was cultured in Pa PES-II medium, and the culture filtrates were extracted by ethyl acetate, hexane, and 80% MeOH. The 80% MeOH fraction was separated by a C18 ODS column, silica gel chromatography and a reverse phase HPLC, to yield three anti-MRSA agents, the MR1, MR2, and MR3 compounds. When the MR1 compound of 250 μg mL-1 concentration was applied to the MRSA cells, over 95% of bacterial cells was killed within 48 hr. Compared with vancomycin and ampicillin, the MR1 compound showed significant anti-MRSA activity. In addition, the anti-MRSA activity was increased by dose and time dependent manners. Furthermore, the combination of an MR1 compound with vancomycin produced a more rapid decrease in the MRSA cells than did the MR1 compound alone. Taken together, our results suggest that the Pseudomonas sp. YJ-1 and its anti-MRSA compounds could be employed as a natural antibacterial agent in MRSA infections.

An organophosphorus pesticide, ethoprophos, has been widely used in agriculture to control major insect pests. As ethoprophos is a well-known neurotoxin, its accumulation in soils and groundwater is concerning worldwide. In this study, we constructed an artificial ethoprophos-enrichment soil system, and its active concentration in soils was measured by gas chromatography on 15-days intervals during 90 days after ethoprophos treatment. Furthermore, the changes in bacterial community and microorganisms responsible for efficient bioremediation were investigated while ethoprophos was degraded in soils. From 15 to 60 days after the treatment, ethoprophos was actively degraded in soils and members of genera Collimonas and Sphingobium appeared dominantly in a natural microbial community especially in 60-days-after-treatment soil. We isolated a bacterium EP60845 from this soil sample, showing significant ethoprophos biodegradation activity in vitro. When we challenged EP60845 strain into ethoprophos-enrichment soils (250 mg/kg of soil), most ethoprophos was removed within 5-days. Phylogenetic 16S rRNA gene sequence analysis and biochemical properties by API 20GN kit demonstrated that the EP60845 strain was a novel Sphingobium sp., which could be used as an efficient ethoprophos- degrading agents for bioremediation purposes.

An auxin-producing bacterial strain, designated 4-3, was isolated from waste button mushroom compost in Boryeong-si, Chungnam. The strain 4-3 was classified as a novel strain of Leucobacter tardus, based on chemotaxonomic and phylogenetic analyses. TLC and HPLC the isolated L. tardus strain 4-3 produced indole-3-acetic acid (IAA), the auxin. Maximum IAA productionof 94.3 mg L-1 was detected for bacteria cultured in R2A medium with 0.1% l-tryptophan, incubated for 24 h at 35oC. Negative correlationwas observed between IAA production and pH of the culture medium, indicating that the increase inIAA caused acidification ofthe medium. The effect of supplementation with varying concentrations of l-tryptophan, a known precursor of IAA, was also assessed. production was maximal at 0.1% l, but decreased at lconcentrations above 0.2%. To investigate the plant growth-promoting effects of the bacterium, L. tardus strain 4-3 culture broth was used to inoculate water cultures and seed pots of mung bean. We found thatadventitious root induction and root growth were 2.2-times higher in thethan in the non-inoculated plants.

This study was conducted to investigate optimum conditions for mass production of ntagonistic microbes Alcaligenes sp. HC12. Alcaligenes sp. HC12 had a potent biological control agent to control browning disease caused by Pseudomonas agarici. Alcaligenes sp. HC12 markedly showed the antagonistic activity against Pseudomonas agarici, the most destructive pathogen of cultivated mushrooms. To define the optimum conditions for the mass production of the Alcaligenes sp. HC12, we have investigated optimum culture conditions and effects of various nutrient source on the bacterial growth. The optimum initial pH and temperature were determined as pH 9.0 and 30o, respectively. The optimal concentration of medium elements for the growth of pathogen inhibitor bacterium(Alcaligenes sp. HC12) was determined as follows: 0.5% dextrine, 1.5% yest extract, 1.0% NaNO3, 0.5% KH2PO4, and 1.5% asparagine.

An alkalophilic microorganism, strain DK1122 producing an alkaline protease was identified. DK1122 was isolated from soil collected in central Korea. The strain DK1122 was Gram-positive, 0.7×2-4 μm in size, and its colony was yellowish white, The strain DK1122 was found to be spore-forming, catalase positive, oxidase positive, caseinolytic, and reduce nitrate to nitrite. The protease was produced aerobically on Horikoshi I agar medium (pH 9.0) with 1% (w/v) skim milk at 40°C for 24 h. Through 16S rRNA gene partial sequencing, the strain DK1122 had the 99.7% sequence similarity to 16S rRNA gene sequence of Bacillus pseudofirmus. Based on the biochemical and physiological properties as well as phylogenetic analysis, the isolated strain was named as Bacillus sp. DK1122. It is expected that Bacillus sp. DK1122 may be a promising candidate for a proudcer of an alkaline protease applicable to the food and detergent industries.

버섯 세균갈성색무늬병원균인 Pseudomonas tolaasii에 대한 길항미생물로 보고된 Pseudomonas azotoformans HC5 균주의 배양적 특성과 대량 배양을 위한 최적배양조건을 설정하였다. HC5 균주의 생육온도는 10~20oC, pH는 6.0~9.0의 범위에서 왕성한 생육을 보였다. 대량배양을 위한 효율적인 영양원 선발을 위하여 기본배지에 탄소원 fructose 등 18종, 무기질소원 NH4Cl 등 6종, 유기질소원 peptone 등 6종 그리고 아미노산 asparagine 등 11종을 각각 1%씩 첨가하였고, 무기염류 13종을 1 mM 농도로 첨가하여 각각에 대한 생육에 미치는 영향을 조사하였다. 또한 선발된 각각의 영양성분들에 대한 최적 농도를 조사하기 위하여 각각의 성분을 최소 0.1%에서 최대 4.0%까지 배지에 첨가하여 배양 후 생육정도를 조사하였다. 그 결과, 대량배양을 위한 생육최적조건은 온도 15oC, pH 6, 탄소원 0.6% adonitol, 유기질소원 1.5% yeast extract, 무기질소원 0.8% NH4H2PO4,아미노산 0.2% asparagine 그리고 무기염류는 5 mM MgSO4에서 왕성한 생육을 보였다.

Oral toxicity of double-stranded RNA (dsRNA) specific to integrin β1 subunit (SeINT) was known in a polyphagous insect pest, Spodoptera exigua. For an application of the dsRNA to control the insect pest, this study prepared a recombinant Escherichia coli expressing dsRNA specific to SeINT. The dsRNA expression was driven by T7 RNA polymerase overexpressed by an inducer in the transformed E. coli. The produced dsRNA amount was proportional to the number of the cultured bacteria. The bacteria gave a significant oral feeding mortality to S. exigua larvae with a significant reduction of the SeINT expression. The resulting insect mortality increased with the fed number of the bacteria. Pretreatment with a sonication to disrupt bacteria cell wall membrane significantly increased the insecticidal activity of the transformed bacteria. Compared to the control bacteria transformed by non-recombinant vector, the larvae fed the bacteria expressing dsRNA specific to SeINT suffered tissue damage in the midgut epithelium, which was characterized by a loose cell-cell contact and a significant cell death. The dsRNA-treated larvae were significantly more susceptible to a Cry toxin derived from Bacillus thuringinesis (Bt) than the larvae treated only with Cry toxin. This study demonstrates that a transformed bacterium expressing dsRNA specific to SeINT has a significant insecticidal activity by oral application against S. exigua and makes the target insects to be highly susceptible to Bt toxin.

버섯 세균갈성색무늬병원균인 Pseudomonas tolaasii에대한 독소저해균으로 보고된 Pseudomonas sp. HC1 균주의 배양적 특성과 최적 배양을 위한 대량배지를 선발하였다. CH1균주의 생육온도는 20~40oC, pH는 5.0~11.0까지넓은 범위에서 왕성한 생육을 보였다. 대량배양을 위한효율적인 영양원 선발을 위하여 기본배지에 탄소원fructose 등 18종, 무기질소원 NH4Cl 등 6종, 유기질소원peptone 등 6종 그리고 아미노산 asparagine 등 11종을 각각 1%씩 첨가하였고, 무기염류 13종을 1mM 농도로 첨가하여 각각에 대한 생육에 미치는 영향을 조사하였다.또한 선발된 각각의 영양성분들에 대한 최적 농도를 조사하기 위하여 각각의 성분을 0.1%에서 4.0%까지 배지에첨가하여 배양후 생육정도를 조사하였다. 그 결과, 대량배양을 위한 생육최적조건은 온도 20oC, pH5, 탄소원 0.9%dextrine, 유기질소원 1.5% yeast extract, 무기질소원0.5% (NH4)2HPO4, 아미노산 3.0% cysteine, 무기염류4mM FeCl3에서 왕성한 생육을 보였다.

Y-Aminobutyric acid(GABA) is a four carbon non-protein amino acid that has several well-known phys-iological functions, such as a postsynaptic inhibitory neurotransmitter in the brain and induction of hypotensive and tran-quilizer effects. A lactic acid bacterium was isolated from button mushroom bed, which is showing high GABAproductivity by TLC or HPLC analysis. The strain was identified as Lactobacillus hilgardii by analysis of 16S rDNAgene sequence. When the maximum production of GABA by L. hilgardii was investigated with various concentration ofmonosodium glutamate, the yield of GABA reached to be 53.65mM at 1% mono sodium glutamate (MSG) in flask cul-tivation. A Glutamate decarboxylase (GAD) enzyme, which was known to convert MSG to GABA, was purified froma cell-free extract of L. hilgardii and the molecular weights of purified GAD was estimated to 60,000 by SDS-PAGE.The optimum pH and temperature of GAD were at pH4.6 and at 37°C, respectively. The GAD activity was increasedby the addition of sulfate ions such as ammonium sulfate, sodium sulfate and magnesium sulfate, indicating that theincrease of hydrophobic interaction causes the increase of GAD activity.

A xylanolytic microorganism, strain DY-7, was isolated from the gut of the mole cricket, Gryllotalpa orientalis. The result of phylogenetic analysis based on its 16S rDNA sequence revealed that the isolate was a Gram-positive bacterium belonging to the genus Streptomyces. The cloned gene (1350-bp) encoding a GH family 10 β -1,4-xylanase (XylA) from Streptomyces sp. strain DY-7 was overexpressed in Escherichia coli BL21 and its gene products were characterized. The hydrolysis activities of rXylA and rXylAΔCBD II against xylosidic materials were maximum at pH 5.5 and 65oC. However, deletion of CBD II in the C-terminus region of XylA significantly increased the thermal stability of the enzyme at high temperatures above 50oC. The xylanolytic activity of rXylA was slightly enhanced in the presence of 1 mM Mn2+ and 5 mM sodium azide but it was completely inactivated by 1 mM Hg2+ and 5 mM N-bromosuccinimide. rXylA was capable of efficiently decomposing various xylosidic compounds, PNP-cellobioside, and PNP-xylopyranoside, whereas other hexose-based compounds were insensitive to the enzyme. The specific activities of rXylA toward oat spelts xylan and PNP-cellobioside were 649.8 U/mg and 328.1 U/mg, respectively. Enzymatic degradation of birchwood xylan and xylooligosaccharides (xylotriose to xylohexaose) resulted in the production of xylobiose (>75%) as the main hydrolysis product together with a small amount (4%<) of xylose as the final hydrolysis product.

A phase variation has been reported in an entomopathogenic bacterium, Xenorhabdus nematophila. Compared to a wild type primary form, a secondary form usually lose several physiological and biochemical characters. This study showed that the phase variation of X. nematophila caused a significant alteration in its immunosuppressive activity and subsequent entomopathogenicity. A secondary form of X. nematophila was detected in laboratory colonies and exhibited significant differences in dye absorption and entomopathogenicity. In addition, the secondary form was different in production of eicosanoid-biosynthesis inhibitors (EBIs) compared to the primary form of X. nematophila. Production of oxindole and p-hydroxypropionic acid was significantly reduced in the culture broth of the secondary form of X. nematophila. The reduced EBI production resulted in significant suppression in the inhibitory effects on a cellular nodule formation and phenoloxidase activity. Culture broth of the primary form of X. nematophila significantly more enhanced the pathogenicity of Bacillus thuringiensis (Bt) than the culture broth of the secondary form. Furthermore, this study developed a high efficient ‘Dual Bt-Plus’ to control both lepidopteran insect pests of Plutella xylostella and Spodoptera exigua by mixing two effective Bt strains along with the addition of potent bacterial metabolites or 100-fold concentrated X. nematophila culture broth.

환경시료에서 마이크로시스틴 분해능을 나타낸 균주 1종을 분리하였고 16S rRNA gene sequence 분석 결과, Microbacterium sp.로 동정되어 Microbacterium sp. MA21로 명명하였다. R2A배지를 기본 배지로 하여 50 μg L-1 microcystin-LR을 첨가하여 30􀆆C, 12시간 동안 배양한 후 PPIA를 통해 microcystin이 80% 이상 분해되는 것을 확인하였다. Microcystin-LR의 분해를 HPLC 분석을 통해 재확인하였고, microcystin 분해산물로 추정되는 두 개의 peak를 확인하였다. 16S rRNA 염기서열을 이용한 계통분류 분석 결과, 본 연구에서 분리한 Microbacterium sp. MA21은 Alphaproteobacteria의 Sphingomonas 속에 속하지 않는 것은 물론 Actinobacteria에는 속하지만 기존에 보고되지 않은, 새로운 genus로 확인되었다.

An entomopathogenic bacterium, Xenorhabdus nematophila, secretes at least eight bacterial metabolites, which have suppressive effects on insect immunity. This study quantified their sequential production during bacterial growth and analyzed their individual immunosuppressive activities against an insect host, Spodoptera exigua. X. nematophila exhibited a typical bacterial growth in both insect host and culture medium, in which eight metabolites were secreted in different time points. At early growth phase (6 to 12 h), Ac-FGV, Cis-cPY, PHPP and indole metabolites were detected in the culture broth. During early growth phase, PHPP was highly potent to inhibit phenoloxidase activity as well as nodule formation. At late growth phase (24 to 48 h), BZA, HPA, PY were detected at 10 – 140 ppm in the culture broth, their metabolites were highly potent to inhibit phospholipase A2 and to induce cytotoxicity to hemocytes. These results suggest that X. nematophila sequentially produces the immune suppressive metabolites, which cooperatively inhibit different steps of insect immune responses.