• Journal of Microbiology and Biotechnology
• /
• 제20권8호
• /
• pp.1196-1203
• /
• 2010

In the present work, methanethiol and dimethyldisulfide were investigated as sulfur sources for methionine synthesis in Corynebacterium glutamicum. In silico pathway analysis predicted a high methionine yield for these reduced compounds, provided that they could be utilized. Wild-type cells were able to grow on both methanethiol and dimethyldisulfide as sole sulfur sources. Isotope labeling studies with mutant strains, exhibiting targeted modification of methionine biosynthesis, gave detailed insight into the underlying pathways involved in the assimilation of methanethiol and dimethyldisulfide. Both sulfur compounds are incorporated as an entire molecule, adding the terminal S-$CH_3$ group to O-acetylhomoserine. In this reaction, methionine is directly formed. MetY (O-acetylhomoserine sulfhydrylase) was identified as the enzyme catalyzing the reaction. The deletion of metY resulted in methionine auxotrophic strains grown on methanethiol or dimethyldisulfide as sole sulfur sources. Plasmid-based overexpression of metY in the ${\Delta}$metY background restored the capacity to grow on methanethiol or dimethyldisulfide as sole sulfur sources. In vitro studies with the C. glutamicum wild type revealed a relatively low activity of MetY for methanethiol (63 mU/mg) and dimethyldisulfide (61 mU/mg). Overexpression of metY increased the in vitro activity to 1,780 mU/mg and was beneficial for methionine production, since the intracellular methionine pool was increased 2-fold in the engineered strain. This positive effect was limited by a depletion of the metY substrate O-acetylhomoserine, suggesting a need for further metabolic engineering targets towards competitive production strains.

• 한국미생물생명공학회:학술대회논문집
• /
• 한국미생물생명공학회 2000년도 Proceedings of 2000 KSAM International Symposium and Spring Meeting
• /
• pp.157-167
• /
• 2000

A thermostable chitosanase gene from the isolated strain, Bacillus sp. CK4, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30 kDa enzyme in size. The deduced amino acid sequence of the chitosanase from Bacillus sp. CK4 exhibits 76.6%, 15.3%, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. CK4 belongs to the Cluster III group with Bacillus subtilis. The size of the gene was similar to that of a mesophile, Bacillus subtilis showing a higher preference for codons ending in G or C. The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues were changed to E50D/Q, E62D/Q, and D66N/E by site-directed mutagenesis. The D66N/E mutants enzymes had remarkably decreased kinetic parameters such as $V_{max}$ and k$\sub$cat/, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three cysteine residues at position 49, 72, and 211. Titration of the Cys residues with DTNB showed that none of them were involved in disulfide bond. The C49S and C72S mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However the half-life of the C211S mutant enzyme was less than 60 min at 80$^{\circ}C$, while that of the wild type enzyme was about 90 min. Moreover, the residual activity of C211S was substantially decreased by 8 M urea, and fully lost catalytic activity by 40% ethanol. These results show that the substitution of Cys with Ser at position 211 seems to affect the conformational stability of the chitosanase.

• The Plant Pathology Journal
• /
• 제36권4호
• /
• pp.314-322
• /
• 2020

Interplay between histone acetylation and deacetylation is one of the key components in epigenetic regulation of transcription. Here we report the requirement of MoHDA1-mediated histone deacetylation during asexual development and pathogenesis for the rice blast fungus, Magnaporthe oryzae. Structural similarity and phylogenetic analysis suggested that MoHDA1 is an ortholog of Saccharomyces cerevisiae Hda1, which is a representative member of class II histone deacetylases. Targeted deletion of MoHDA1 caused a little decrease in radial growth and large reduction in asexual sporulation. Comparison of acetylation levels for H3K9 and H3K14 showed that lack of MoHDA1 gene led to significant increase in H3K9 and H3K14 acetylation level, compared to the wild-type and complementation strain, confirming that it is a bona fide histone deacetylase. Expression analysis on some of the key genes involved in asexual reproduction under sporulation-promoting condition showed almost no differences among strains, except for MoCON6 gene, which was up-regulated more than 6-fold in the mutant than wild-type. Although the deletion mutant displayed little defects in germination and subsequent appressorium formation, the mutant was compromised in its ability to cause disease. Wound-inoculation showed that the mutant is impaired in invasive growth as well. We found that the mutant was defective in appressorium-mediated penetration of host, but did not lose the ability to grow on the media containing H₂O₂. Taken together, our data suggest that MoHDA1-dependent histone deacetylation is important for efficient asexual development and infection of host plants in M. oryzae.

• Journal of Microbiology and Biotechnology
• /
• 제16권2호
• /
• pp.247-255
• /
• 2006

Yeast cell wall matrix particles are composed entirely of mannoprotein and ${\beta}-glucan$. The mannoproteins of yeast cell wall can systemically enhance the immune system. We previously purified and analyzed alkali-soluble ${\beta}-glucans$ [${\beta}$-(1,3)- and ${\beta}$-(1,6)-glucans] [10]. In the present study, a wild-type strain was first mutagenized with ultraviolet light, and the cell wall mutants were then selected by treatment with 1.0 mg/ml laminarinase (endo-${\beta}$-(1,3)-D-glucanase). Mannoproteins of Saccharomyces cerevisiae were released by laminarinase, purified by concanavalin-A affinity and ion-exchange chromatography. The results indicated that the mutants yielded 3-fold more mannoprotein than the wild-type. The mannoprotein mass of mutant K48L3 was 2.25 mg/100 mg of yeast cell dry mass. Carbohydrate analysis revealed that they contained mannose, glucose, and N-acetylglucosamine. Saccharomyces cerevisiae cell wall components, mannoproteins, are known to interact with macrophages through receptors, thereby inducing release of tumor necrosis factor alpha ($TNF-{\alpha}$) and nitric oxide. Mannoprotein tractions in the present study had a higher macrophage activity of secretion of $TNF-{\alpha}$ and nitric oxide and direct phagocytosis than positive control ($1{\mu}g$ of lipopolysaccharide). In particular, F1 and F3 fractions in mannoproteins of K48L3 enhanced and upregulated the activity of nitric oxide secretion and macrophage phagocytosis by approximately two- and four-fold, respectively.

• Parasites, Hosts and Diseases
• /
• 제53권4호
• /
• pp.385-394
• /
• 2015

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-${\gamma}$/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.

• 생명과학회지
• /
• 제15권2호
• /
• pp.202-210
• /
• 2005

살모넬라는 질병을 일으키기 위해서 장내의 상피세포에 먼저 부착하여 집락을 이루게 된다. 이에 따라 살모넬라는 부착에 관여하는 세포내 몇 몇 소기관들을 합성한다. 이러한 소기관에는 type 1 fimbriae, plasmid-encoded fimbriae, long polar fimbriae 그리고 thin aggregative fimbriae 등이 있다. 본 논문에서는 type 1 fimbriae, thin aggregative fimbriae, LP fimbriae, 그리고 PE fimbriae 각각을 결손시킨 변이주와 4종류를 모두 결손시킨 변이주를 만들 수 있었다. 변이주들에 대해서 세포배양 부착성 실험을 한 결과, 각각을 결손시킨 변이주들은 몇몇 mammalian 세포주에서 야생형 살모넬라와 동일한 양상으로 부착성을 나타내었고 마우스 실험에서 야생형과 거의 마찬가지로 독성을 가지고 있었다. 반면, 4종류의 fimbriae가 모두 결손된 균주는 닭에서는 독성을 나타내었으나 마우스에서는 매우 약독화되어있음을 확인할 수 있었다. 이상의 결과로부터 오는 차이점은 닭과 비교하여 마우스에서 살모넬라의 부착을 매개하는 표면 구조에 연관성이 있음을 제시하였다.

• 한국식품영양과학회지
• /
• 제20권4호
• /
• pp.395-400
• /
• 1991

식육 및 야생쥐의 장으로부터 식품매개성 질병에 관여하는 Enterobacter를 분리하여 그 특징을 구명하므로 공중위생 및 식품위생학적으로 기초자료를 제시하고저 본 연구를 실시하여 다음과 같은 결론을 얻었다. Enterobacter는 우육에서 1주(E. aerogenes), 돈육에서 2주(E. aerogenes, E. intermedium), 야생쥐의 소장에서 2주(E. aerogenes, E. cloacae)가 동정되어 총 분리된 89균주에 대한 분리율은 5.62%이었다. 분리된 Enterobacter를 각종 화학기질이 첨가된 배지에서 $55^{\circ}C$, 35분간 열처리한 결과, 전반적으로 0.1M glycine, 5% mannitol 그리고 5% sorbitol이 첨가된 배지에서는 열저항성이 강하였고 1% sodium citrate, 1% casein, 10% NaCl 그리고 0.1M cystein이 첨가된 배지에서는 열저항성이 약하였다.

• 한국양식학회지
• /
• 제21권1호
• /
• pp.13-18
• /
• 2008

Marine birnavirus(MABV)는 중요한 어류 병원체로서 일본 양식산 방어서 처음 분리되어 이후 다양한 해산어종에서 MABV 분리가 보고되었으며 환경적인 샘플인 저질, 해양의 동물성 플랑크톤과 해수에서도 MABV 유전자가 검출되고 있다. 본 연구는 Marine birnavirus disease 에 대한 예방학적 접근의 일환으로서 자연산 어류로부터의 MABV 검출 및 지리적 분포, 보균 어종에 대한 유전학적인 조사를 목적으로 2003년과 2005년도에 동중국해역 3지점, 서해안 5지점 그리고 남해안 5지점에서 어류를 채집하였다. RT-PCR(Reverse transcriptase-Polymerase Chain Reaction)을 이용한 연구결과 자연산 해산어류 160마리 중 13종 17마리에서 MABV가 거출되어 감염율10.6%를 보였다. 조사된 13종의 어류 중 특히 농어목에서 가장 높는 검출율을 보였다. 자연산 어류에서 분리한 MABV 분리균주는 염기서열 분석에서 94.7%-100%, 아미노산 분석에서 97.2-100% 유사성을 나타내었으며 IPNV strain과는 구분되며 MABV에 속하였다.

• Journal of Microbiology
• /
• 제33권4호
• /
• pp.339-343
• /
• 1995

We isolated a 10 kb Bam HI fragment originated from the chromosome of a $H_2O$$^2$-resistant mutant strain of Streptomyces coelicolor, which confer $H_2O$$^2$-resistance to S. lividance upon transformation. Among various subclones ot 10kb Bam HI fragment tested for their $H_2O$$^2$-resistant phenotype in S. lividans, a subclone containing 5.2 kb Bam HI-BglII fragment was found to be responsible for $H_2O$$^2$-resistance. The plasmid containing this 5.2 kb fragment was then transformed into S. coellicolor A3(2) at early and tested for their phenotype of $H_2O$$^2$-resistance and the change in various enzymes whose activity can be stained in the gel. We found out that the 5.2 kb insert DNA conferred $H_2O$$^2$-resisstance in S. coelicolor A3(2) at early phase of cell growth. The presence of this DNA also resulted in higher level of peroxidase compared with the wild type cell containing parental vector (pIJ702) only. Esterase activity was also higher in this clone. However, alcohol dehydrogenase activity decreased compared with the wild type. These results suggest that the presence of a gene in 5.2 kb BamHI-BglII DNA fragment causes multiple changes in S. coelicolor related to its response against hydrogen peroxide. The result also implies that not only peroxidase but also esterase may function in the defencse meahsnism agianst $H_2O$$^2$-.

• Journal of Microbiology and Biotechnology
• /
• 제29권3호
• /
• pp.367-372
• /
• 2019

Deactivation of aminoglycosides by their modifying enzymes, including a number of aminoglycoside O-phosphotransferases, is the most ubiquitous resistance mechanism in aminoglycoside-resistant pathogens. Nonetheless, in a couple of biosynthetic pathways for gentamicins, fortimicins, and istamycins, phosphorylation of aminoglycosides seems to be a unique and initial step for the creation of a natural defensive structural feature such as a 3',4'-dideoxy scaffold. Our aim was to elucidate the biochemical details on the beginning of these C3',4'-dideoxygenation biosynthetic steps for aminoglycosides. The biosynthesis of istamycins must surely involve these 3',4'-didehydroxylation steps, but much less has been reported in terms of characterization of istamycin biosynthetic genes, especially about the phosphotransferase-encoding gene. In the disruption and complementation experiments pointing to a putative gene, istP, in the genome of wild-type Streptomyces tenjimariensis, the function of the istP gene was proved here to be a phosphotransferase. Next, an in-frame deletion of a known phosphotransferase-encoding gene forP from the genome of wild-type Micromonospora olivasterospora resulted in the appearance of a hitherto unidentified fortimicin shunt product, namely 3-O-methyl-FOR-KK1, whereas complementation of forP restored the natural fortimicin metabolite profiles. The bilateral complementation of an istP gene (or forP) in the ${\Delta}forP$ mutant (or ${\Delta}istP$ mutant strain) successfully restored the biosynthesis of 3',4'-dideoxy fortimicins and istamycins, thus clearly indicating that they are interchangeable launchers of the biosynthesis of 3',4'-dideoxy types of 1,4-diaminocyclitol antibiotics.