Journal of Microbiology

Journal Articles

Phylogenetic Assessment of Understudied Families in Hymenochaetales (Basidiomycota, Fungi)-Reporting Uncovered Species and Reflecting the Recent Taxonomic Updates in the Republic of Korea.: Yoonhee Cho, Dohye Kim, Young Woon Lim; J. Microbiol. 2024;62(6):429-447. Published online May 16, 2024; DOI: https://doi.org/10.1007/s12275-024-00120-5

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Abstract: Hymenochaetales Oberw. is an order classified in Basidiomycota of Fungi, and species in this order display notable diversity. They exhibit various fruiting body shapes, including clavarioid, effused-reflexed, and resupinate basidiomes. Few mycorrhizal species have been reported in Hymenochaetales, but wood-decaying species dominate the order. Hymenochaetaceae Imazeki & Toki and Schizoporaceae Jülich are the most species-rich families within Hymenochaetales, and most species in the Republic of Korea belong to these two families. As such, current taxonomic classification and nomenclature are not reflected upon species in the remaining Hymenochaetales families. For this study, a multifaceted morphological and multigenetic marker-based phylogenetic investigation was conducted to, firstly, comprehensively identify understudied Hymenochaetales specimens in Korea and, secondly, reflect the updates on the species classification. Five genetic markers were assessed for the phylogenetic analysis: nuclear small subunit ribosomal DNA (nSSU), internal transcribed spacer (ITS), nuclear large subunit ribosomal DNA (nLSU), RNA polymerase II subunit 2 gene (RPB2), and translation elongation factor 1 gene (TEF1). The results from phylogenetic analysis supported 18 species classified under eight families (excluding Hymenochaetaceae and Schizoporaceae) in Korea. Species formerly placed in Rickenellaceae and Trichaptum sensu lato have been systematically revised based on recent taxonomic reconstructions. In addition, our findings revealed one new species, Rickenella umbelliformis, and identified five formerly nationally unreported species classified under five understudied families. Our findings contribute to a better understanding of Hymenochaetales diversity and highlight the need for continued research.

Heterologous expression and enzymatic characterization of γ-glutamyltranspeptidase from Bacillus amyloliquefaciens: Jung-Min Lee , Jaejung Lee , Gyeong-Hwa Nam , Byung-Sam Son , Myoung-Uoon Jang , So-Won Lee , Byung-Serk Hurh , Tae-Jip Kim; J. Microbiol. 2017;55(2):147-152. Published online January 26, 2017; DOI: https://doi.org/10.1007/s12275-017-6638-6

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Abstract: γ-Glutamyltranspeptidase (GGT) catalyzes the cleavage of γ- glutamyl compounds and the transfer of γ-glutamyl moiety to water or to amino acid/peptide acceptors. GGT can be utilized for the generation of γ-glutamyl peptides or glutamic acid, which are used as food taste enhancers. In the present study, Bacillus amyloliquefaciens SMB469 with high GGT activity was isolated from Doenjang, a traditional fermented soy food of Korea. The gene encoding GGT from B. amyloliquefaciens SMB469 (BaGGT469) was cloned from the isolate, and heterologously expressed in E. coli and B. subtilis. For comparison, three additional GGT genes were cloned from B. subtilis 168, B. licheniformis DSM 13, and B. amyloliquefaciens FZB42. The BaGGT469 protein was composed of 591 amino acids. The final protein comprises two separate polypeptide chains of 45.7 and 19.7 kDa, generated via autocatalytic cleavage. The specific activity of BaGGT469 was determined to be 17.8 U/mg with γ-L-glutamyl-p-nitroanilide as the substrate and diglycine as the acceptor. GGTs from B. amyloliquefaciens showed 1.4- and 1.7-fold higher transpeptidase activities than those from B. subtilis and B. licheniformis, respectively. Especially, recombinant B. subtilis expressing BaGGT469 demonstrated 11- and 23-fold higher GGT activity than recombinant E. coli and the native B. amyloliquefaciens, respectively, did. These results suggest that BaGGT469 can be utilized for the enzymatic production of various γ- glutamyl compounds.

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Role of bacterial γ-glutamyltranspeptidase as a novel virulence factor in bone-resorbing pathogenesis: Jinmoon Kim , Sungil Jang , Aeryun Kim , Hanfu Su , Niluka Gunawardhana , Yeong-Eui Jeon , Eun Jung Bak , Ji-Hye Kim , Jeong-Heon Cha; J. Microbiol. 2016;54(5):396-402. Published online April 20, 2016; DOI: https://doi.org/10.1007/s12275-016-6137-1

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Abstract: Mammalian γ-glutamyltranspeptidase (GGT) has been identified as a bone-resorbing factor. Since GGT of Bacillus subtilis exhibits similarity in their primary structure and enzymatic characteristics with mammalian GGTs, the bone-resorbing activity of bacterial GGT was examined in this study. Osteoclastogenesis was performed in a co-culture system of mouse calvaria-derived osteoblasts and bone marrow cells. A conditioned medium from GGT-overproducing B. subtilis culture showed significantly higher activity of osteoclast formation than a conditioned medium from wild-type B. subtilis culture. Recombinant GGT (rGGT) of wild-type B. subtilis and an enzymatic activity-defected rGGT of B. subtilis 2288 mutant were expressed in Escherichia coli and purified using His tag. Both purified rGGTs induced similar levels of osteoclastogenesis, suggesting that B. subtilis GGT possesses virulent boneresorbing activity and its activity is probably independent of its enzymatic activity. Furthermore, a recombinant protein of B. subtilis GGT heavy subunit (Bs rGGT/H) showed strong activity of osteoclastogenesis while the light subunit failed to show strong activity, suggesting that the bone-resorbing activity is mainly located at the heavy subunit. More importantly, the GGT enzymatic activity may not be required for this virulence activity since the light subunit contains the catalytic pocket. In addition, B. subtilis rGGT stimulated mRNA expressions of receptor activator of nuclear factor kappa-B ligand (RANKL) and cyclooxygenase-2 (COX-2), while an osteoprotegerin inhibited the osteoclast formation induced by Bs rGGT/H. This is the first demonstration that bacterial GGT itself is sufficient to act as a bone-resorbing virulence factor via RANKL-dependent pathway. Therefore, it can be hypothesized that GGT of periodontopathic bacteria may play an important role as a virulence factor in bone destruction.

Crystal Structure of XoLAP, a Leucine Aminopeptidase, from Xanthomonas oryzae pv. oryzae: Jin-Kwang Kim , Sampath Natarajan , Hanseul Park , Kim-Hung Huynh , Sang Hee Lee , Jeong-Gu Kim , Yeh-Jin Ahn , Lin-Woo Kang; J. Microbiol. 2013;51(5):627-632. Published online October 31, 2013; DOI: https://doi.org/10.1007/s12275-013-3234-2

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Abstract: Aminopeptidases are metalloproteinases that degrade N-terminal residues from protein and play important roles in cell growth and development by controlling cell homeostasis and protein maturation. We determined the crystal structure of XoLAP, a leucyl aminopeptidase, at 2.6 Å resolution from Xanthomonas oryzae pv. oryzae, causing the destructive rice disease of bacterial blight. It is the first crystal structure of aminopeptidase from phytopathogens as a drug target. XoLAP existed as a hexamer and the monomer structure consisted of an N-terminal cap domain and a C-terminal peptidase domain with two divalent zinc ions. XoLAP structure was compared with BlLAP and EcLAP (EcPepA) structures. Based on the structural comparison, the molecular model of XoLAP in complex with the natural aminopeptidase inhibitor of microginin FR1 was proposed. The model structure will be useful to develop a novel antibacterial drug against Xoo.

Clades of γ-Glutamyltransferases (GGTs) in the Ascomycota and Heterologous Expression of Colletotrichum graminicola CgGGT1, a Member of the Pezizomycotina-only GGT Clade: Marco H. Bello , Lynn Epstein; J. Microbiol. 2013;51(1):88-99. Published online March 2, 2013; DOI: https://doi.org/10.1007/s12275-013-2434-0

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Abstract: Gamma-glutamyltransferase (GGT, EC 2.3.2.2) cleaves the γ-glutamyl linkage in glutathione (GSH). Ascomycetes in either the Saccharomycotina or the Taphrinomycotina have one to three GGTs, whereas members of the Pezizomycotina have two to four GGTs. A Bayesian analysis indicates there are three well-supported main clades of GGTs in the Ascomycota. 1) A Saccharomycotina and a Taphrinomycotinaspecific GGT sub-clade form a yeast main clade. This clade has the three relatively well-characterized fungal GGTs: (Saccharomyces cerevisiae CIS2 and Schizosaccharomyces pombe Ggt1 and Ggt2) and most of its members have all 14 of the highly conserved and critical amino acids that are found in GGTs in the other kingdoms. 2) In contrast, a main clade (GGT3) differs in 11 of the 14 highly conserved amino acids that are found in GGTs in the other kingdoms. All of the 44 Pezizomycotina analyzed have either one or two GGT3s. 3) There is a Pezizomycotina-only GGT clade that has two wellsupported sub-clades (GGT1 and GGT2); this clade differs in only two of the 14 highly conserved amino acids found in GGTs in the other kingdoms. Because the Pezizomycotina GGTs differ in apparently critical amino acids from the crosskingdom consensus, a putative GGT from Colletotrichum graminicola, a member of the Pezizomycotina, was cloned and the protein product was expressed as a secreted protein in Pichia pastoris. A GGT enzyme assay of the P. pastoris supernatant showed that the recombinant protein was active, thereby demonstrating that CgGGT1 is a bona fide GGT.

Characterization of Thermostable Deblocking Aminopeptidases of Archaeon Thermococcus onnurineus NA1 by Proteomic and Biochemical Approaches: Yeol Gyun Lee , Sun-Hee Leem , Young-Ho Chung , Seung Il Kim; J. Microbiol. 2012;50(5):792-797. Published online November 4, 2012; DOI: https://doi.org/10.1007/s12275-012-2461-2

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Abstract: Thermococcus onnurineus NA1 is a hyperthermophilic archaeon that grows optimally at >80°C. The deblocking aminopeptidase (DAP) (TNA1-DAP1) encoded in Ton_1032 of T. onnurineus NA1 is considered a major DAP. However, four genes encoding putative DAP have been identified from a genomic analysis of T. onnurineus NA1. A proteomic analysis revealed that all four DAPs were differentially induced in YPS culture medium and, particularly, two DAPs (TNA1-DAP1 and TNA1-DAP2) were dominantly expressed in T. onnurineus NA1. The biochemical properties and enzyme activity of DAPs induced in an E. coli expression system suggested that the two major DAPs play complementary roles in T. onnurineus NA1.

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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19 Citations

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.

Screening, Purification, and Characterization of an Extracellular Prolyl Oligopeptidase from Coprinopsis clastophylla: Jen-Tao Chen , Mei-Li Chao , Chiou-Yen Wen , Wen-Shen Chu; J. Microbiol. 2012;50(4):652-659. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2099-0

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Abstract: Culture filtrates of 22 mushrooms were screened for extracellular prolyl oligopeptidase activity. Four strains with relatively high enzyme activity were all from inky cap mushrooms. The production of Coprinopsis clastophylla prolyl oligopeptidase was associated with the growth of the fungus and the enzyme was not released by cell lysis. The enzyme was purified 285-fold to a specific activity of 52.05 U/mg. It was purified to a single band on a native polyacrylamide gel. However, the enzyme separated into three bands on a sodium dodecyl sulfate-polyacrylamide gel with mobility corresponding to molecular weights of approximately 84, 60, and 26 kDa. The results of tandem mass spectrometric analysis revealed that the 60 kDa protein was likely a degradation product of the 84 kDa protein. The isoelectric point of the purified enzyme was 5.2. The purified enzyme had an optimal pH and temperature of 8.0 and 37°C, respectively. Diisopropyl fluorophosphate (DFP), p-chloromercuribenzoaic acid (PCMB), Hg2+, and Cu2+ strongly inhibited C. clastophylla prolyl oligopeptidase. This enzyme is a serine peptidase and one or more cysteine residues of the enzyme are close to the active site.

Helicobacter pylori γ-Glutamyltranspeptidase Induces Cell Cycle Arrest at the G1-S Phase Transition: Kyung-Mi Kim , Seung-Gyu Lee , Jung-Min Kim , Do-Su Kim , Jea-Young Song , Hyung-Lyun Kang , Woo-Kon Lee , Myung-Je Cho , Kwang-Ho Rhee , Hee-Shang Youn , Seung-Chul Baik; J. Microbiol. 2010;48(3):372-377. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9293-8

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38 Citations

Abstract: In our previous study, we showed that Helicobacter pylori γ-glutamyltranspeptidase (GGT) is associated with H. pylori-induced apoptosis through a mitochondrial pathway. To better understand the role of GGT in apoptosis, we examined the effect of GGT on cell cycle regulation in AGS cells. To determine the effect of recombinant GGT (rGGT) on cell cycle distribution and apoptosis, rGGT-treated and untreated AGS cells were analyzed in parallel by flow cytometry using propidium iodide (PI). We found that rGGT inhibited the growth of AGS cells in a time-dependent manner, and that the pre-exposure of cells to a caspase-3 inhibitor (z-DEVD-fmk) effectively blocked GGT-induced apoptosis. Cell cycle analysis showed G1 phase arrest and apoptosis in AGS cells following rGGT treatment. The rGGT-mediated G1 phase arrest was found to be associated with down-regulation of cyclin E, cyclin A, Cdk 4, and Cdk 6, and the up-regulation of the cyclindependent kinase (Cdk) inhibitors p27 and p21. Our results suggest that H. pylori GGT induces cell cycle arrest at the G1-S phase transition.

Characterization of Calcium-Activated Bifunctional Peptidase of the Psychrotrophic Bacillus cereus: Jong-Il Kim , Sun-Min Lee , Hyun-Joo Jung; J. Microbiol. 2005;43(3):237-243.; DOI: https://doi.org/2219 [pii]

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Abstract: The protease purified from Bacillus cereus JH108 has the function of leucine specific endopeptidase. When measured by hydrolysis of synthetic substrate (N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide), the enzyme activity exhibited optimal activity at pH 9.0, 60^oC. The endopeptidase activity was stimulated by Ca^+^+, Co^+^+, Mn^+^+, Mg^+^+, and Ni^+^+, and was inhibited by metal chelating agents such as EDTA, 1,10-phenanthroline, and EGTA. Addition of serine protease inhibitor, PMSF, resulted in the elimination of the activity. The endopeptidase activity was fully recovered from the inhibition of EDTA by the addition of 1 mM Ca^+^+, and was partially restored by Co^+^+ and Mn^+^+, indicating that the enzyme was stabilized and activated by divalent cations and has a serine residue at the active site. Addition of Ca^+^+ increased the pH and heat stability of endopeptidase activity. These results show that endopeptidase requires calcium ions for activity and/or stability. A Lineweaver-Burk plot analysis indicated that the K_m value of endopeptidase is 0.315 mM and V_max is 0.222 mmol of N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide per min. Bestatin was shown to act as a competitive inhibitor to the endopeptidase activity.

The Schizosaccharomyces pombe Gene Encoding [gamma]-Glutamyl Transpeptidase I Is Regulated by Non-fermentable Carbon Sources and Nitrogen Starvation: Hong-Gyum Kim , Hey-Jung Park , Hyun-Jung Kang , Hye-Won Lim , Kyunghoon Kim , Eun-Hee Park , Kisup Ahn , Chang-Jin Lim; J. Microbiol. 2005;43(1):44-48.; DOI: https://doi.org/2139 [pii]

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Abstract: In our previous study, the first structural gene (GGTI) encoding g-glutamyl transpeptidase was cloned and characterized from the fission yeast Schizosaccharomyces pombe, and its transcription, using the GGTI-lacZ fusion gene, containing the 1,085 bp upstream region from the translational initiation point, was found to be enhanced by sodium nitroprusside and L-buthionine-(S,R)-sulfoximine (BSO). In the present work, regulation of the GGTI gene was further elucidated. Non-fermentable carbon sources, such as acetate and ethanol, markedly enhanced the synthesis of [beta]-galactosidase from the GGTI-lacZ fusion gene. However, its induction by non-fermentable carbon sources appeared to be independent of the presence of the Pap1 protein. Nitrogen starvation also gave rise to induction of GGTI gene expression in a Pap1-independent manner. The three additional fusion plasmids, carrying 754, 421 and 156 bp regions, were constructed. The sequence responsible for the induction by non-fermentable carbon sources and nitrogen starvation was identified to exist within a -421 bp region of the GGTI gene. Taken together, the S. pombe GGTI gene is regulated by non-fermentable carbon sources and nitrogen starvation.

Nitrogen Depletion Causes Up-Regulation of Glutathione Content and γ-Glutamyltranspeptidase in Schizosaccharomyces pombe: Seung-Hyun Song , Chang-Jin Lim; J. Microbiol. 2008;46(1):70-74.; DOI: https://doi.org/10.1007/s12275-007-0244-y

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Abstract: This work aims to elucidate the relationship between nitrogen depletion and Glutathione (GSH) level in Schizosaccharomyces pombe. The total GSH level was much higher in the Pap1-positive KP1 cells than in the Pap1-negative TP108-3C cells, suggesting that synthesis of GSH is dependent on Pap1. When the Pap1-positive KP1 cells were transferred to the nitrogen-depleted medium, total GSH level significantly increased up to 6 h and then slightly declined after 9 h. Elevation of the total GSH level was observed to be much less with the Pap1-negative cells. However, glucose deprivation was not able to enhance the GSH level in the KP1 cells. Activity of γ-glutamyltranspeptidase (γ-GT), an enzyme in the first step of GSH catabolism, also increased during nitrogen depletion. The total GSH level was more significantly enhanced in the KP1 cells overexpressing γ-GT2 than γ-GT1 during nitrogen starvation. Reactive oxygen species (ROS) levels were not changed during nitrogen starvation in both Pap1-positive and Pap1-negative cells. Collectively, nitrogen depletion causes up-regulation of GSH synthesis and γ-GT in a Pap1-dependent manner.

Multicatalytic Alkaline Serine Protease from the Psychrotrophic Bacillus amyloliquefaciens S94: Eui-Sun Son , Jong-Il Kim; J. Microbiol. 2003;41(1):58-62.

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Abstract: An extracellular protease of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine protease. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. The maximum proteolytic activity against different protein substrates occurred at pH 10, 45℃ (protein substrate) and pH 8, 45℃ (synthetic substrate). The purified enzyme was specific in that it readily hydrolyzed substrates with Leu or Lys residues at P1 site. The protease had characteristics of a cold-adapted protein, which was more active for the hydrolysis of synthetic substrate in the range of 15℃ to 45℃, specially at low temperature.

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