• Journal of Microbiology and Biotechnology
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• v.34 no.6
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• pp.1348-1355
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• 2024

The eukaryotic translation initiation factor eIF5B is a bacterial IF2 ortholog that plays an important role in ribosome joining and stabilization of the initiator tRNA on the AUG start codon during the initiation of translation. We identified the fluorophenyl oxazole derivative 2,2-dibromo-1-(2-(4-fluorophenyl)benzo[d]oxazol-5-yl)ethanone quinolinol as an inhibitor of fungal protein synthesis using an in vitro translation assay in a fungal system. Mutants resistant to this compound were isolated in Saccharomyces cerevisiae and were demonstrated to contain amino acid substitutions in eIF5B that conferred the resistance. These results suggest that eIF5B is a target of potential antifungal compound and that mutation of eIF5B can confer resistance. Subsequent identification of 16 other mutants revealed that primary mutations clustered mainly on domain 2 of eIF5B and secondarily mainly on domain 4. Domain 2 has been implicated in the interaction with the small ribosomal subunit during initiation of translation. The tested translation inhibitor could act by weakening the functional contact between eIF5B and the ribosome complex. This data provides the basis for the development of a new family of antifungals.

• Journal of Microbiology and Biotechnology
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• v.17 no.10
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• pp.1727-1732
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• 2007

Phenylacetic acid (PAA) is produced by many bacteria as an antifungal agent and also appears to be an environmentally toxic chemical. The object of this study was to detect PAA using Pseudomonas putida harboring a reporter plasmid that has a PAA-inducible promoter fused to a green fluorescent protein (GFP) gene. Pseudomonas putida KT2440 was used to construct a green fluorescent protein-based reporter fusion using the paaA promoter region to detect the presence of PAA. The reporter strain exhibited a high level of gfp expression in minimal medium containing PAA; however, the level of GFP expression diminished when glucose was added to the medium, whereas other carbon sources, such as succinate and pyruvate, showed no catabolic repression. Interestingly, overexpression of a paaF gene encoding PAA-CoA ligase minimized catabolic repression. The reporter strain could also successfully detect PAA produced by other PAA-producing bacteria. This GFP-based bioreporter provides a useful tool for detecting bacteria producing PAA.

• Korean Journal of Plant Tissue Culture
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• v.27 no.4
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• pp.339-343
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• 2000

When plants are exposed to subfreezing temperatures ice crystals are forming within extracelluar space in leaves. The growth of ice crystal is closely related to the degree of freezing injury. It was shown that an antifreeze protein binds to an ice nucleator through hydrogen bonds to prevent growth of ice crystal and also reduces freezing damage. The antifreeze proteins in plants are similar to PR proteins but only the PR proteins induced upon cold acclimation were shown to have dual functions in antifreezing as well as antifungal activities. Three of the genes encoded for CLP, GLP, and TLP were isolated from barley and Kentucky bluegrass based on amino acid sequence revealed after purification and low temperature-inducibility as shown in analysis of the protein. The deduced amino acid of the genes cloned showed a signal for secretion into extracellular space where the antifreezing activity sup-posed to work. The western analysis using the antisera raised against the antifreeze proteins showed a positive correlation between the amount of the protein and the level of freeze tolerance among different cultivars of barely. Besides it was revealed that TLP is responsible for a freeze tolerance induced by a treatment of trinexapac ethyl in Kentucky bluegrass. Analysis of an overwintering wild rice, Oryza rufipogon also showed that an acquisition of freeze tolerance relied on accumulation of the protein similar to CLP. The more direct evidence for the role of CLP in freeze tolerance was made with the analysis of the transgenic tobacco showing extracellular accumulation of CLP and enhanced freeze tolerance measured by amount of ion leakage and rate of photosynthetic electron transport upon freezing. These antifreeze proteins genes will be good candidates for transformation into crops such as lettuce and strawberry to develop into the new crops capable of freeze-storage and such as rose and grape to enhance a freeze tolerance for a safe survival during winter.

• KSBB Journal
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• v.25 no.3
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• pp.289-296
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• 2010

Lectins are carbohydrate-binding and a cell-agglutinating proteins, and are concerted with a plants defence mechanism. In particular, chitin-binding lectins in locular fluid of cherry tomato fruit seemed to have a role in defending plants against fungi. The antifungal activity using lectin isolated from locular fluid of cherry tomato fruit was measured in the plant pathogen Cladosporium cucumerinum, Monosporascus cannonballus, Fusarium oxysporum, and Rhizoctonia solani. Amoung the four strains, a potent antifungal activity was detected in Cladosporium cucumerinum and Monosporascus cannonballus, not in Fusarium oxysporum, and Rhizoctonia solani. The molecular weight of this lectin isolated as double protein bands by SDS-PAGE was calculated to be 87 kDa and 47 kDa from the relative mobilities compared with those of reference molecular weight markers. The isolated lectin agglutinated human red blood cells (A, B, AB, O) treated with trypsin, and the most activity was found at B. The optimal temperature of isolated lectin was at $30^{\circ}C$. For the thermal stability, lectin was stable at $20-80^{\circ}C$. The optimal pH of this lectin was at 7.2, and showed complete loss below pH 9.0.

• Microbiology and Biotechnology Letters
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• v.49 no.3
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• pp.305-315
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• 2021

The cotton leafworm, Spodoptera littoralis, is a major pest in Egypt and many countries worldwide, and causes heavy economic losses. As a result, management measures to control the spread of the worm are required. S. littoralis nucleopolyhedrovirus (SpliNPV) is one of the most promising bioagents for the efficient control of insect pests. In this study, a chitinase gene (chitA) of a 1.8 kb DNA fragment was cloned and fully characterized from SpliNPV-EG1, an Egyptian isolate. A sequence of 601 amino acids was deduced when the gene was completely sequenced with a predicted molecular mass of 67 kDa for the preprotein. Transcriptional analyses using reverse transcription polymerase chain reaction (RT-PCR) revealed that chitA transcripts were detected first at 12 h post infection (hpi) and remained detectable until 168 hpi, suggesting their transcriptional regulation from a putative late promoter motif. In addition, quantitative analysis using quantitative RT-PCR showed a steady increase of 7.86-fold at 12 hpi in chitA transcription levels, which increased up to 71.4-fold at 120 hpi. An approximately 50 kDa protein fragment with chitinolytic activity was purified from ChitA-induced bacterial culture and detected by western blotting with an anti-recombinant SpliNPV chitinase antibody. Moreover, purification of the expressed ChitA recombinant protein showed in vitro growth inhibition of two different fungi species, Fusarium solani and F. oxysporum, confirming that the enzyme assembly and activity was correct. The results supported the potential role and application of the SpliNPV-ChitA protein as a synergistic agent in agricultural fungal and pest control programs.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.301-310
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• 2010

Bacillus subtilis strains produce a broad spectrum of bioactive peptides. The lipopeptide surfactin belongs to one well-known class, which includes amphiphilic membrane-active biosurfactants and peptide antibiotics. Both the srfA promoter and the ComP-ComA signal transduction system are an important part of the factor that results in the production of surfactin. Bs-M49, obtained by means of low-energy ion implantation in wild-type Bs-916, produced significantly lower levels of surfactin, and had no obvious effects against R. solani. Occasionally, we found strain Bs-M49 decreased spore formation and the development of competence. Blast comparison of the sequences from Bs-916 and M49 indicate that there is no difference in the srfA operon promoter PsrfA, but there are differences in the coding sequence of the comA gene. These differences result in three missense mutations within the M49 ComA protein. RT-PCR analyses results showed that the expression levels of selected genes involved in competence and sporulation in both the wild-type Bs-916 and mutant M49 strains were significantly different. When we integrated the comA ORF into the chromosome of M49 at the amyE locus, M49 restored hemolytic activity and antifungal activity. Then, HPLC analyses results also showed the comA-complemented strain had a similar ability to produce surf actin with wild-type strain Bs-916. These data suggested that the mutation of three key amino acids in ComA greatly affected the biological activity of Bacillus subtilis. ComA protein 3D structure prediction and motif search prediction indicated that ComA has two obvious motifs common to response regulator proteins, which are the N-terminal response regulator receiver motif and the C-terminal helix-turn-helix motif. The three residues in the ComA N-terminal portion may be involved in phosphorylation activation mechanism. These structural prediction results implicate that three mutated residues in the ComA protein may play an important role in the formation of a salt-bridge to the phosphoryl group keeping active conformation to subsequent regulation of the expression of downstream genes.

• Mycobiology
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• v.37 no.3
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• pp.161-170
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• 2009

Cryptococcus neoformans is a basidiomycete human fungal pathogen that causes meningoencephalitis in both immunocompromised and immunocompetent individuals. The ability to sense and respond to diverse extracellular signals is essential for the pathogen to infect and cause disease in the host. Four major stress-activated signaling (SAS) pathways have been characterized in C. neoformans, including the HOG (high osmolarity glycerol response), PKC/Mpk1 MAPK (mitogen-activated protein kinase), calcium-dependent calcineurin, and RAS signaling pathways. The HOG pathway in C. neoformans not only controls responses to diverse environmental stresses, including osmotic shock, UV irradiation, oxidative stress, heavy metal stress, antifungal drugs, toxic metabolites, and high temperature, but also regulates ergosterol biosynthesis. The PKC(protein kinase C)/Mpk1 pathway in C. neoformans is involved in a variety of stress responses, including osmotic, oxidative, and nitrosative stresses and breaches of cell wall integrity. The $Ca^{2+}$/calmodulin- and Ras-signaling pathways also play critical roles in adaptation to certain environmental stresses, such as high temperature and sexual differentiation. Perturbation of the SAS pathways not only impairs the ability of C. neoformans to resist a variety of environmental stresses during host infection, but also affects production of virulence factors, such as capsule and melanin. A drug(s) capable of targeting signaling components of the SAS pathway will be effective for treatment of cryptococcosis.

• Animal cells and systems
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• v.9 no.4
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• pp.239-244
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• 2005

A cDNA clone for a wound- and pathogen-induced gene in Chinese cabbage (Brassica rapa subsp. pekinensis) was isolated and characterized. The cabbage gene, designated BrPR4, encodes a pathogenesis-related protein 4 (PR4) of 140 amino acids. The BrPR4 protein shows high similarity with wound-inducible antifungal proteins of tobacco, potato, barley, and wheat. The BrPR4 gene is locally induced by a nonhost pathogen, Pseudomonas syringae pv. tomato, that elicits a hypersensitive response in Chinese cabbage. Treatment of the cabbage leaves with benzothiadiazole (BTH), methyl jasmonate or ethephon showed that the BrPR4 gene expression is strongly induced by ethylene, but not by methyl jasmonate or BTH. The BrPR4 gene is also activated by wounding. Interestingly, however, the wound-inducible BrPR4 gene expression is repressed by salicylic acid or BTH, suggesting that there is cross-talk between salicylate-dependent and -independent signaling pathways.

• Korean Journal of Agricultural Science
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• v.15 no.1
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• pp.76-81
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• 1988

This study was done to find out the antifungal activity of two antibacterial antibiotics, Chlorampenicol and Streptomycin sulfate, against Phytophthora n. var. Parasitica, the causal agent of Phytophthora blight of sesame, growing on artificial media. On PDA medium, Chlorampenicol at 10 ppm, Streptomycin sulfate at 25ppm highly inhibited mycerial growth and completely inhibited zoosporangial formation of Phytophthora n. var. parasitica, and Chlorampenicol at 5 ppm, Streptomycin sulfate at 10 ppm slightly inhibited the mycerial growth and zoosporangial formation of the fungus. These antibiotics showed considerably increased inhibitory effect on the fungal growth when they were mixed with other chemical. Protein content in myceria of the fungus was decreased and abnormal growth of mycerial apex was observed by treatment of these antibiotics. Phytotoxicity on sesame seedlings was not observed by application of them.

• International Journal of Industrial Entomology and Biomaterials
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• v.17 no.1
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• pp.131-135
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• 2008

A cDNA encoding a defensin-like peptide (Protaetiamycine) from the larvae of a beetle, Protaetia brevitarsis was cloned. The DNAs encoded the deduced propeptide of 79 amino acid residues with the predicted molecular weight of 8.4 kDa and PI of 8.24. Overall amino acid sequence of this protein has 39% similarity to that of Rhodnius prolixus defensin, 43% similarity to that of Acalolepta luxuriosa defensin, and 72% similarity to that of Oryctes rhinoceros defensin, suggesting that this gene is an insect defensin. In an attempt to apply the anti-bacterial peptide to the development of therapeutic agents, a 12-mer peptide amidated at its C-terminus, ACAAHCLAIGRG-$NH_2$ (Ala55-Lys66-$NH_2$, 12Pbn) was synthesized. This peptide showed some antifungal activity against Candida albicans. To increase antifungal activity, six 9-mer peptides were synthesized by modifying amino acid sequences of 12Pbn fragment. Among these peptides, 9Pbm3-9Pbm6 exhibited strong activity compared with Cecropin B and mellitin.