Journal of Microbiology

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Functional Characterization of Extracellular Chitinase Encoded by the YlCTS1 Gene in a Dimorphic Yeast Yarrowia lipolytica: Jeong-Nam Park , Chang Pyo Han , Dong-Jik Lee , Seon Ah Cheon , Hyun Ah Kang; J. Microbiol. 2014;52(4):284-291. Published online March 29, 2014; DOI: https://doi.org/10.1007/s12275-014-4070-8

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The hemiascomycetes yeast Yarrowia lipolytica is a dimorphic yeast with alternating yeast and mycelia forms. Bioinformatic analysis revealed the presence of three putative chitinase genes, YlCTS1, YlCTS2, and YlCTS3, in the Y. lipolytica genome. Here, we demonstrated that the protein of YlCTS1 (YlCts1p), which contains an N-terminal secretion signal peptide, a long C-terminal Ser/Thr-rich domain, and a chitin-binding domain, is a homologue to Saccharomyces cerevisiae chitinase 1 (ScCts1p). Deletion of YlCTS1 remarkably reduced extracellular endochitinase activity in the culture supernatant of Y. lipolytica and enhanced cell aggregation, suggesting a role of YlCts1p in cell separation as ScCts1p does in S. cerevisiae. However, loss of YlCts1p function did not affect hyphal formation induced by fetal bovine serum addition. The mass of YlCts1p was dramatically decreased by jack bean α-mannosidase digestion but not by PNGase F treatment, indicating that YlCts1p is modified only by Omannosylation without N-glycosylation. Moreover, the O-glycan profile of YlCts1p was identical to that of total cell wall mannoproteins, supporting the notion that YlCts1p can be used as a good model for studying O-glycosylation in this dimorphic yeast.

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The N-Acetylglucosamine Kinase from Yarrowia lipolytica Is a Moonlighting Protein
Carmen-Lisset Flores, Joaquín Ariño, Carlos Gancedo
International Journal of Molecular Sciences.2021; 22(23): 13109. CrossRef
Recovery and valorization of agri-food wastes and by-products using the non-conventional yeast Yarrowia lipolytica
Davide Gottardi, Lorenzo Siroli, Lucia Vannini, Francesca Patrignani, Rosalba Lanciotti
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Functional analysis of recombinant human and Yarrowia lipolytica O-GlcNAc transferases expressed in Saccharomyces cerevisiae
Hye Ji Oh, Hye Yun Moon, Seon Ah Cheon, Yoonsoo Hahn, Hyun Ah Kang
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Journal Articles

Note] Antifungal Chitinase against Human Pathogenic Yeasts from Coprinellus congregatus: Yeeun Yoo Hyoung T. Choi; J. Microbiol. 2014;52(5):441-443. Published online February 17, 2014; DOI: https://doi.org/10.1007/s12275-014-3257-3

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Abstract

The inky cap, Coprinellus congregatus, produces mushrooms which become autolyzed rapidly to generate black liquid droplets, in which no cell wall is detected by microscopy. A chitinase (Chi2) which is synthesized during the autolytic phase of C. congregatus inhibits the growths of Candida al-bicans and Cryptococcus neoformans up to 10% at the con-centration of 10 μg/ml, about 50% at concentration of 20 μg/ml, and up to 95% at the concentration of 70 μg/ml. Upon treatment these yeast cells are observed to be severely de-formed, with the formation of large holes in the cell wall. The two yeast species show no growth inhibition at the concen-tration of 5 μg/ml, which means the minimum inhibitory concentrations for both yeast species are 10 μg/ml under these experimental conditions.

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Analysis of the Antifungal Potential of Macrocybe Titans Extract Against Candida Albicans
Fernanda CBN Pereira, Gabrielle C Peiter, Vivian EMS Justo, Gabrieli M Huff, Pollyanna CV Conrado, Mauro AP da Silva, Patrícia S Bonfim-Mendonça, Terezinha IE Svidzinski, Fabio R Rosado, Adriana Fiorini
Future Microbiology.2023; 18(6): 357. CrossRef
Disarming Fungal Pathogens: Bacillus safensis Inhibits Virulence Factor Production and Biofilm Formation by Cryptococcus neoformans and Candida albicans
François L. Mayer, James W. Kronstad, Yong-Sun Bahn, J. Andrew Alspaugh, Deborah Hogan
mBio.2017;[Epub] CrossRef

Biochemical Characterization of Chitinase 2 Expressed during the Autolytic Phase of the Inky Cap, Coprinellus congregatus: Yuri Kang , Hyewon Kim , Hyoung T. Choi; J. Microbiol. 2013;51(2):189-193. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2535-9

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Abstract: Fungal cell walls consist of various glucans and chitin. The inky cap, Coprinellus congregatus, produces mushrooms at 25°C in a regime of 15 h light/9 h dark, and then the mushroom is autolyzed rapidly to generate black liquid droplets in which no cell walls are detected by microscopy. Chitinase cDNA from the mature mushroom tissues of C. congregatus, which consisted of 1,622 nucleotides (chi2), was successfully cloned using the rapid amplification of cDNA ends polymerase chain reaction technique. The deduced 498 amino acid sequence of Chi2 had a conserved catalytic domain as in other fungal chitinase family 18 enzymes. The Chi2 enzyme was purified from the Pichia pastoris expression system, and its estimated molecular weight was 68 kDa. The optimum pH and temperature of Chi2 was pH 4.0 and 35°C, respectively when 4-nitrophenyl N,N’-diacetyl-β-D-chitobioside was used as the substrate. The Km value and Vmax for the substrate A, 4-nitrophenyl N,N’-diacetyl-β-D-chitobioside, was 0.175 mM and 0.16 OD min-1unit-1, respectively.

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Construction of a Streptomyces lydicus A01 Transformant with a chit42 Gene from Trichoderma harzianum P1 and Evaluation of Its Biocontrol Activity against Botrytis cinerea: Qiong Wu , Linquan Bai , Weicheng Liu , Yingying Li , Caige Lu , Yaqian Li , Kehe Fu , Chuanjin Yu , Jie Chen; J. Microbiol. 2013;51(2):166-173. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2321-8

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Abstract: Streptomyces lydicus A01 and Trichoderma harzianum P1 are potential biocontrol agents of fungal diseases in plants. S. lydicus A01 produces natamycin to bind the ergosterol of the fungal cell membrane and inhibits the growth of Botrytis cinerea. T. harzianum P1, on the other hand, features high chitinase activity and decomposes the chitin in the cell wall of B. cinerea. To obtain the synergistic biocontrol effects of chitinase and natamycin on Botrytis cinerea, this study transformed the chit42 gene from T. harzianum P1 to S. lydicus A01. The conjugal transformant (CT) of S. lydicus A01 with the chit42 gene was detected using polymerase chain reaction (PCR). Associated chitinase activity and natamycin production were examined using the 3, 5-dinitrosalicylic acid (DNS) method and ultraviolet spectrophotometry, respectively. The S. lydicus A01-chit42 CT showed substantially higher chitinase activity and natamycin production than its wild type strain (WT). Consequently, the biocontrol effects of S. lydicus A01-chit42 CT on B. cinerea, including inhibition to spore germination and mycelial growth, were highly improved compared with those of the WT. Our research indicates that the biocontrol effect of Streptomyces can be highly improved by transforming the exogenous resistance gene, i.e. chit42 from Trichoderma, which not only enhances the production of antibiotics, but also provides a supplementary function by degrading the cell walls of the pathogens.

Selection of a Streptomyces Strain Able to Produce Cell Wall Degrading Enzymes and Active against Sclerotinia sclerotiorum: Adriana Fróes , Andrew Macrae , Juliana Rosa , Marcella Franco , Rodrigo Souza , Rosângela Soares , Rosalie Coelho; J. Microbiol. 2012;50(5):798-806. Published online November 4, 2012; DOI: https://doi.org/10.1007/s12275-012-2060-2

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Abstract: Control of plant pathogen Sclerotinia sclerotiorum is an ongoing challenge because of its wide host range and the persistence of its sclerotia in soil. Fungicides are the most commonly used method to control this fungus but these can have ecotoxicity impacts. Chitinolytic Streptomyces strains isolated from Brazilian tropical soils were capable of inhibiting S. sclerotiorum growth in vitro, offering new possibilities for integrated pest management and biocontrol, with a new approach to dealing with an old problem. Strain Streptomyces sp. 80 was capable of irreversibly inhibiting fungal growth. Compared to other strains, its crude enzymes had the highest chitinolytic levels when measured at 25°C and strongly inhibited sclerotia from S. sclerotiorum. It produced four hydrolytic enzymes involved in fungal cell wall degradation when cultured in presence of the fungal mycelium. The best production, obtained after three days, was 0.75 U/ml for exochitinase, 0.9 U/ml for endochitinase, 0.16 U/ml for glucanase, and 1.78 U/ml for peptidase. Zymogram analysis confirmed two hydrolytic bands of chitinolytic activity with apparent molecular masses of 45.8 and 206.8 kDa. One glucanase activity with an apparent molecular mass of 55 kDa was also recorded, as well as seven bands of peptidase activity with apparent molecular masses ranging from 15.5 to 108.4 kDa. Differential interference contrast microscopy also showed alterations of hyphal morphology after co-culture. Streptomyces sp. 80 seems to be promising as a biocontrol agent against S. sclerotiorum, contributing to the development of new methods for controlling plant diseases and reducing the negative impact of using fungicides.

NOTE] Antifungal Activity of Extracellular Hydrolases Produced by Autolysing Aspergillus nidulans Cultures: Melinda Szilágyi , Fruzsina Anton , Katalin Forgács , Jae-Hyuk Yu , István Pócsi , Tamás Emri; J. Microbiol. 2012;50(5):849-854. Published online November 4, 2012; DOI: https://doi.org/10.1007/s12275-012-2001-0

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Abstract: Carbon-starving Aspergillus nidulans cultures produce high activities of versatile hydrolytic enzymes and, among these, ChiB endochitinase and EngA β-1,3-endoglucanase showed significant antifungal activity against various fungal species. Double deletion of engA and chiB diminished the antifungal activity of the fermentation broths and increased conidiogenesis and long-term viability of A. nidulans, but decreased the growth rate on culture media containing weak carbon sources. Production of ChiB and EngA can influence fungal communities either directly due to their antifungal properties or indirectly through their effects on vegetative growth. Our data suggest saprophytic fungi as promising future candidates to develop novel biocontrol technologies.

Molecular Screening of Streptomyces Isolates for Antifungal Activity and Family 19 Chitinase Enzymes: Youssuf Gherbawy , Hesham Elhariry , Abdulla Altalhi , Bahig El-Deeb , Ghada Khiralla; J. Microbiol. 2012;50(3):459-468. Published online June 30, 2012; DOI: https://doi.org/10.1007/s12275-012-2095-4

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Abstract: Thirty soil-isolates of Streptomyces were analyzed to determine their antagonism against plant-pathogenic fungi including Fusarium oxysporum, Pythium aristosporum, Colletotrichum gossypii, and Rhizoctonia solani. Seven isolates showed antifungal activity against one or more strain of the tested fungi. Based on the 16S rDNA sequence analysis, these isolates were identified as Streptomyces tendae (YH3), S. griseus (YH8), S. variabilis (YH21), S. endus (YH24), S. violaceusniger (YH27A), S. endus (YH27B), and S. griseus (YH27C). The identity percentages ranged from 98 to 100%. Although some isolates belonged to the same species, there were many differences in their cultural and morphological characteristics. Six isolates out of seven showed chitinase activity according to a chitinolytic activity test and on colloidal chitin agar plates. Based on the conserved regions among the family 19 chitinase genes of Streptomyces sp. two primers were used for detection of the chitinase (chiC) gene in the six isolates. A DNA fragment of 1.4 kb was observed only for the isolates YH8, YH27A, and YH27C. In conclusion, six Streptomyces strains with potential chitinolytic activity were identified from the local environment in Taif City, Saudi Arabia. Of these isolates, three belong to family 19 chitinases. To our knowledge, this is the first reported presence of a chiC gene in S. violaceusniger YH27A.

Journal Article

Chitinase Production by Bacillus thuringiensis and Bacillus licheniformis: Their Potential in Antifungal Biocontrol: Eman Zakaria Gomaa; J. Microbiol. 2012;50(1):103-111. Published online February 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1343-y

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Abstract: Thirty bacterial strains were isolated from the rhizosphere of plants collected from Egypt and screened for production of chitinase enzymes. Bacillus thuringiensis NM101-19 and Bacillus licheniformis NM120-17 had the highest chitinolytic activities amongst those investigated. The production of chitinase by B. thuringiensis and B. licheniformis was optimized using colloidal chitin medium amended with 1.5% colloidal chitin, with casein as a nitrogen source, at 30°C after five days of incubation. An enhancement of chitinase production by the two species was observed by addition of sugar substances and dried fungal mats to the colloidal chitin media. The optimal conditions for chitinase activity by B. thuringiensis and B. licheniformis were at 40°C, pH 7.0 and pH 8.0, respectively. Na+, Mg2+, Cu2+, and Ca2+ caused enhancement of enzyme activities whereas they were markedly inhibited by Zn2+, Hg2+, and Ag+. In vitro, B. thuringiensis and B. licheniformis chitinases had potential for cell wall lysis of many phytopathogenic fungi tested. The addition of B. thuringiensis chitinase was more effective than that of B. licheniformis in increasing the germination of soybean seeds infected with various phytopathogenic fungi.

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Growth Inhibition of the Yeast Transformant by the Expression of a Chitinase from Coprinellus congregatus: Hyangsoon Lim , Hyoung T. Choi; J. Microbiol. 2010;48(5):706-708. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0272-x

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Abstract: Coprinellus congregatus generates several chitinases during its entire life cycle: at the growing hyphal stage and at the mushroom autolysis stage. We have isolated a chitinase gene (chi1) from the mushroom tissue at the autolysing stage, and constructed a chitinase expression vector to get large amount of enzyme protein. Chitinase 1 (chi1) cDNA was heterologously expressed in Saccharomyces cerevisiae by gal1 promoter. The transformants showed no specific change in growth characteristics under normal growth conditions. However the expression of the gene by the gal1 promoter in the yeast transformants resulted in complete growth inhibition, while laccase expression by the gal1 promoter showed normal growth. The chitinase activities from the transformants were also more than 3 times higher than that of the recipient strain, and the chitinase expression by the real time-PCR also showed increased expression of the chi1 in the yeast transformant. Expression of a chitinase which was produced at the mushroom autolysing stage of C. congregatus resulted in yeast growth inhibition.

Note] Enhanced Expression of Chitinase during the Autolysis of Mushroom in Coprinellus congregatus: Hyangsoon Lim , Hyoung T. Choi; J. Microbiol. 2009;47(2):225-228. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0247-3

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Abstract: Fungal cell walls consist of various glucans and chitin. An inky cap, Coprinellus congregatus, produced mushrooms at 25°C in a regime of 15 h light/9 h dark, and then the mushroom was autolyzed rapidly to generate black liquid droplets where no cell wall was detected by microscopy. A chitinase cDNA from the matured mushroom cells of C. congregatus that consisted of 1,541 nucleotides was successfully cloned using the rapid amplification of cDNA ends (RACE)-PCR technique. Its deduced 441 amino acid sequence had the conserved catalytic domain as in other fungal chitinase family 18. Chitinase activity was higher at the matured mushroom stage than primordial and young mushroom stage. When the expression of the cloned chitinase was examined by real-time PCR using the chitinase-specific primers, it was increased more than twice to 20 times during the autolytic process of mushroom than young mushroom or primordial stages, respectively.

Cloning and Expression Analysis of a Chitinase Gene Crchi1 from the Mycoparasitic Fungus Clonostachys rosea (syn. Gliocladium roseum): Zhongwei Gan , Jinkui Yang , Nan Tao , Zefen Yu , Ke-Qin Zhang; J. Microbiol. 2007;45(5):422-430.; DOI: https://doi.org/2594 [pii]

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Abstract: Clonostachys rosea (syn. Gliocladium roseum) is a well-known biocontrol agent and widely distributed around the world. In this study, an endochitinase gene Crchi1 was isolated from the mycoparasitic fungus C. rosea using the DNA walking strategy. The Crchi1 ORF is 1,746 bp long and interrupted by three introns. The cloned gene Crchi1 encodes 426 amino acid residues and shares a high degree of similarity with other chitinases from entomopathogenic and mycoparasitic fungi. Several putative binding sites for transcriptional regulation of Crchi1 in response to carbon (5''-SYGGRG-3'') and nitrogen (5''-GATA-3'') were identified in the upstream of Crchi1. Expression of Crchi1 gene in different carbon sources was analyzed using real-time PCR (RT-PCR). We found that the Crchi1 expression was suppressed by glucose but strongly stimulated by chitin or solubilized components of the cell wall from Rhizoctonia solani. Phylogenetic analysis of chitinases from entomopathogenic and mycoparasitic fungi suggests that these chitinases have probably evolved from a common ancestor.

The role and characterization of β-1,3-glucanase in biocontrol of fusarium solani by pseudomonas stutzeri YPL-1: Lim, Ho Seong , Kim, Sang Dal; J. Microbiol. 1995;33(4):295-301.

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Abstract: An antifungal Pseudomonas stutzeri YPL-1 produced extracellular chitinase and β-1,3-glucanase that were key enzymes in the decomposition of fungal hyphal walls. These lytic extracellular enzymes markedly inhibited mycelial growth of the phytopathogenic fungus Fusarium solani. A chitinase from P. stutzeri YPL-1 inhibited fungal mycelial growth by 87%, whereas a β-1,3-glucanase from the bacterium inhibited growth by 53%. Furthermore, co-operative action of the enzymes synergistically inhibited 95% of the fungal growth. The lytic enzymes caused abnormal swelling and retreating on the fungal hyphal walls in a dual cultures. Scanning electron microscopy clearly showed hyphal degradation of F. solani in the regions interacting with P. stutzeri YPL-1. In an in vivo pot test, P. stutzeri YPL-1 proved to have biocontrol ability as a powerful agent in controlling plant disease. Planting of kidney bean (Phaseolus vulgaris L.) seedlings with the bacterial suspension in F. solani-infested soil significantly suppressed the development of fusarial root-rot. The characteristics of a crude preparation of β-1,3-glucanase produced from P. stutzeri YPL-1 were investigated. The bacterium detected after 2 hr of incubation. The enzyme had optimum temperature and pH of 40℃ and pH 5.5, respectively. The enzyme was stable in the pH range of 4.5 to 7.0 and at temperatures below 40℃, with a half-life of 40 min at 60℃.

Purification and Characterization of Chitinase from a Marine Bacterium, Vibrio sp. 98CJ11027: Shin Hye Park , Jung-Hyun Lee , Hong Kum Lee; J. Microbiol. 2000;38(4):224-229.

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Abstract: Chitin-degrading marine bacterial strain 98CJ11027 was isolated from bryozoa from the coastal area of Cheju Island, Korea, and identified as a member of the genus Vibrio. The molecular mass of the main extracellular chitinase (chitinase I), purified from strain 98CJ11027, was estimated to be 98 kDa. The optimal condition for chitinase I activity is pH 6.0 and 45 C. The activity was inhibited by Fe^+2 and Cu^+2. Chitinase I displayed the hydrolysis type of chitobiosidase and catalyzed reversed hydrolysis leading to the synthesis of tetraacetylchitotetraose.

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