Journal of Microbiology

Journal Articles

Non-Mitochondrial Aconitase-2 Mediates the Transcription of Nuclear-Encoded Electron Transport Chain Genes in Fission Yeast: Ho-Jung Kim, Soo-Yeon Cho, Soo-Jin Jung, Yong-Jun Cho, Jung-Hye Roe, Kyoung-Dong Kim; J. Microbiol. 2024;62(8):639-648. Published online June 25, 2024; DOI: https://doi.org/10.1007/s12275-024-00147-8

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Abstract: Aconitase-2 (Aco2) is present in the mitochondria, cytosol, and nucleus of fission yeast. To explore its function beyond the well-known role in the mitochondrial tricarboxylic acid (TCA) cycle, we conducted genome-wide profiling using the aco2ΔNLS mutant, which lacks a nuclear localization signal (NLS). The RNA sequencing (RNA-seq) data showed a general downregulation of electron transport chain (ETC) genes in the aco2ΔNLS mutant, except for those in the complex II, leading to a growth defect in respiratory-prone media. Complementation analysis with non-catalytic Aco2 [aco2ΔNLS + aco2(3CS)], where three cysteines were substituted with serine, restored normal growth and typical ETC gene expression. This suggests that Aco2's catalytic activity is not essential for its role in ETC gene regulation. Our mRNA decay assay indicated that the decrease in ETC gene expression was due to transcriptional regulation rather than changes in mRNA stability. Additionally, we investigated the Php complex's role in ETC gene regulation and found that ETC genes, except those within complex II, were downregulated in php3Δ and php5Δ strains, similar to the aco2ΔNLS mutant. These findings highlight a novel role for nuclear aconitase in ETC gene regulation and suggest a potential connection between the Php complex and Aco2.

Roles of Dhh1 RNA helicase in yeast filamentous growth: Analysis of N-terminal phosphorylation residues and ATPase domains: Eunji Lee , Daehee Jung , Jinmi Kim; J. Microbiol. 2020;58(10):853-858. Published online September 29, 2020; DOI: https://doi.org/10.1007/s12275-020-0431-7

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In yeast Saccharomyces cerevisiae, the Dhh1 protein, a member of the DEAD-box RNA helicase, stimulates Dcp2/Dcp1- mediated mRNA decapping and functions as a general translation repressor. Dhh1 also positively regulates translation of a selected set of mRNAs, including Ste12, a transcription factor for yeast mating and pseudohyphal growth. Given the diverse functions of Dhh1, we investigated whether the putative phosphorylation sites or the conserved motifs for the DEADbox RNA helicases were crucial in the regulatory roles of Dhh1 during pseudohyphal growth. Mutations in the ATPase A or B motif (DHH1-K96R or DHH1-D195A) showed significant defects in pseudohyphal colony morphology and agar invasive phenotypes. The N-terminal phospho-mimetic mutation, DHH1-T16E, showed defects in pseudohyphal phenotypes. Decreased levels of Ste12 protein were also observed in these pseudohyphal-defective mutant cells under filamentous- inducing low nitrogen conditions. We suggest that the ATPase motifs and the Thr16 phosphorylation site of Dhh1 are crucial to its regulatory roles in pseudohyphal growth under low nitrogen conditions.

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Biological implications of decapping: beyond bulk mRNA decay
Fivos Borbolis, Popi Syntichaki
The FEBS Journal.2022; 289(6): 1457. CrossRef
The Complex Genetic Basis and Multilayered Regulatory Control of Yeast Pseudohyphal Growth
Anuj Kumar
Annual Review of Genetics.2021; 55(1): 1. CrossRef

Research Support, Non-U.S. Gov'ts

The effect of the cwf14 gene of fission yeast on cell wall integrity is associated with rho1: Dong-Uk Kim , Shinae Maeng , Hyemi Lee , Miyoung Nam , Sook-Jeong Lee , Kwang-Lae Hoe; J. Microbiol. 2016;54(2):98-105. Published online February 2, 2016; DOI: https://doi.org/10.1007/s12275-016-5569-y

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Abstract

In all eukaryotic organisms, a wide range of morphologies are responsible for critical cellular function and development. In particular, the Rho GTPases, which are highly conserved from yeast to mammals, are key molecules in signaling pathways that control cell polarity processes and cell wall biosynthesis, which are fundamental aspects of morphogenesis. Therefore, using haploinsufficiency deletion mutants of the fission yeast Schizosaccharomyces pombe, we screened the slow-growing mutants and their morphogenesis, specifically focusing on regulation of their Rho GTPases. Based on this screening, we found that the cwf14 mutant of S. pombe exhibited the slow growth and abnormal phenotypes with an elongated cell shape and thicker cell wall when compared with wild-type cells. In particular, cells with the cwf14 deletion showed excessive Rho1 expression. However, the wildtype strain with ectopically expressed Rho1 did not exhibited any significant change in the level of cwf14, suggesting that cwf14 may act on the upstream of Rho1. Furthermore, the cells with a cwf14 deletion also have increased sensitivity to β-glucanase, a cell wall-digesting enzyme, which is also seen in Rho1-overexpressing cells. Overall, our results suggest that the cwf14 plays a key role in fission yeast morphogenesis and cell wall biosynthesis and/or degradation possibly via the regulation of Rho1 expression.

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Transcriptome of Nosema ceranae and Upregulated Microsporidia Genes during Its Infection of Western Honey Bee (Apis mellifera)
Yi-Hsuan Li, Zih-Ting Chang, Ming-Ren Yen, Yu-Feng Huang, Tzu-Han Chen, Ju-Chun Chang, Ming-Cheng Wu, Yu-Liang Yang, Yue-Wen Chen, Yu-Shin Nai
Insects.2022; 13(8): 716. CrossRef
Proteomic profiling and glycomic analysis of the yeast cell wall in strains with Aflatoxin B1 elimination ability
Beatriz García‐Béjar, Rebecca A. Owens, Ana Briones, María Arévalo‐Villena
Environmental Microbiology.2021; 23(9): 5305. CrossRef

NOTE] A Protective Role of Methionine-R-Sulfoxide Reductase against Cadmium in Schizosaccharomyces pombe: Chang-Jin Lim , Hannah Jo , Kyunghoon Kim; J. Microbiol. 2014;52(11):976-981. Published online May 30, 2014; DOI: https://doi.org/10.1007/s12275-014-3512-7

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Abstract

The Schizosaccharomyces pombe cells harboring the methionine- R-sulfoxide reductase (MsrB)-overexpressing recombinant plasmid pFMetSO exhibited better growth than vector control cells, when shifted into fresh medium containing cadmium chloride (abbreviated as Cd). Although both groups of cells contained enhanced reactive oxygen species (ROS) and nitric oxide (NO) levels in the presence of Cd, ROS and NO levels were significantly lower in the S. pombe cells harboring pFMetSO than in vector control cells. Conversely, the S. pombe cells harboring pFMetSO possessed higher total glutathione (GSH) levels and a greater reduced/oxidized GSH ratio than vector control cells under the same conditions.

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Pleurotus pulmonarius Strain: Arsenic(III)/Cadmium(II) Accumulation, Tolerance, and Simulation Application in Environmental Remediation
Yuhui Zhang, Xiaohong Chen, Ling Xie
International Journal of Environmental Research and Public Health.2023; 20(6): 5056. CrossRef
Impact of cadmium and nickel on ion homeostasis in the yeast Schizosaccharomyces pombe
Miroslava Pozgajova, Alica Navratilova, Julius Arvay, Hana Duranova, Anna Trakovicka
Journal of Environmental Science and Health, Part B.2020; 55(2): 166. CrossRef
A methionine-R-sulfoxide reductase, OsMSRB5, is required for rice defense against copper toxicity
Tengwei Xiao, Mengmeng Mi, Changyong Wang, Meng Qian, Yahua Chen, Luqing Zheng, Hongsheng Zhang, Zhubing Hu, Zhenguo Shen, Yan Xia
Environmental and Experimental Botany.2018; 153: 45. CrossRef
Identification and Characterization of a Novel Methionine Sulfoxide Reductase B Gene (AccMsrB) fromApis cerana cerana(Hymenoptera: Apidae)
Feng Liu, Zhihong Gong, Weixing Zhang, Ying Wang, Lanting Ma, Hongfang Wang, Xingqi Guo, Baohua Xu
Annals of the Entomological Society of America.2015; 108(4): 575. CrossRef

Validation Study

Generation of Expression Vectors for High-Throughput Functional Analysis of Target Genes in Schizosaccharomyces pombe: Jiwon Ahn , Chung-Hae Choi , Chang-Mo Kang , Chun-Ho Kim , Hee-Moon Park , Kyung-Bin Song , Kwang-Lae Hoe , Misun Won , Kyung-Sook Chung; J. Microbiol. 2009;47(6):789-795. Published online February 4, 2010; DOI: https://doi.org/10.1007/s12275-009-0010-4

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Abstract: An immediate challenge in the post-genomic era is to assign a biological functions to proteins unraveled by genome analysis. This report is based on studies conducted using Schizosaccharomyces pombe, a simple model organism, and presents various vector systems as tools for high-throughput functional analysis of human genes. We constructed S. pombe expression vectors for efficient cloning of genes via the Gateway system. We modified the pREP and pSLF series vectors, which are widely used for gene expression in S. pombe. The vectors constructed have a uniform backbone of S. pombe autonomously replicating sequence (ARS) elements with different selective markers, namely, ura4+ and Saccharomyces cerevisiae LEU2 complementing leu1. These vectors contain 3 different strengths of the inducible promoter nmt1, which affect the expression levels of the cloned open reading frames (ORFs). Further, target proteins can be fused with an N-terminal or C-terminal tag such as triple hemagglutinin (3× HA), enhanced green fluorescent protein (EGFP), or Discosoma red fluorescent protein (DsRed). We tested the feasibility of the constructed vectors by using 3 human genes, namely, RAB18, SCC-112, and PTEN. Proper expression of tagged RAB18 was confirmed by western blot analysis. Further, localization of RAB18, SCC112, and PTEN was demonstrated. The constructed vectors can be utilized for high-throughput functional analysis of heterologous genes.

Research Support, Non-U.S. Gov'ts

Overexpression of Bacterioferritin Comigratory Protein (Bcp) Enhances Viability and Reduced Glutathione Level in the Fission Yeast Under Stress: Ga-Young Kang , Eun-Hee Park , Kyunghoon Kim , Chang-Jin Lim; J. Microbiol. 2009;47(1):60-67. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0077-3

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Abstract: The structural gene encoding bacterioferritin comigratory protein (Bcp) was amplified using PCR from the genomic DNA of Schizosaccharomyces pombe, and transferred into the shuttle vector pRS316 to generate the recombinant plasmid pBCP10. The bcp+ mRNA level in the pBCP10-containing yeast cells was significantly higher than that in the control yeast cells, indicating that the cloned gene is functioning. The S. pombe cells harboring the plasmid pBCP10 exhibited higher survival on the solid minimal media with hydrogen peroxide, tert-BOOH or cadmium than the control yeast cells. They also exhibited enhanced cellular viability in the liquid media containing the stressful agents. The increased viabilities of the fission yeast cells harboring the plasmid pBCP10 were also obtained with 0.4% glucose or 0.4% sucrose as a sole carbon source, and nitrogen starvation, compared with those of the control yeast cells. The total glutathione (GSH) content and total GSH/GSSG ratio were significantly higher in the yeast cells harboring the plasmid pBCP10 than in the control yeast cells. In brief, the S. pombe Bcp plays a protective role in the defensive response to oxidative stress possibly via up-regulation of total and reduced glutathione levels.

The Role and Regulation of Trx1, a Cytosolic Thioredoxin in Schizosaccharomyces pombe: Ji-Yoon Song , Jung-Hye Roe; J. Microbiol. 2008;46(4):408-414. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0076-4

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Abstract

The genome of fission yeast Schizosaccharomyces pombe harbors two genes for thioredoxins, trx1+ and trx2+, which encode cytosolic and mitochondrial thioredoxins, respectively. The Δtrx1 mutant was found sensitive to diverse external stressors such as various oxidants, heat, and salt, whereas Δtrx2 mutant was not sensitive except to paraquat, a superoxide generator. Both Δtrx1 and Δtrx2 mutants were more resistant to diamide, a thiol-specific oxidant, than the wild type. The trx1+ gene expression was induced by H2O2 and menadione, being mediated through a stress-responsive transcription factor Pap1. In Δtrx1 cells, the basal expression of Pap1-regulated genes were elevated, suggesting a role for Trx1 as a reducer for oxidized (activated) Pap1. The Δtrx1 mutant exhibited cysteine auxotrophy, which can be overcome by adding sulfite. This suggests that Trx1 serves as a primary electron donor for 3’-phosphoadenosine-5’-phosphosulfate (PAPS) reductase and thus is an essential protein for sulfur assimilation in S. pombe. These results suggest that, in contrast to Trx2 whose role is more confined to mitochondrial functions, Trx1 plays a major role in protecting S. pombe against various stressful conditions and enables proper sulfur metabolism.

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Journal Article

Carbon Source-Dependent Regulation of the Schizosaccharomyces pombe pbh1 Gene: Su-Jung Kim , Nam-Chul Cho , In Wang Ryu , Kyunghoon Kim , Eun-Hee Park , Chang-Jin Lim; J. Microbiol. 2006;44(6):689-693.; DOI: https://doi.org/2454 [pii]

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Abstract: Pbh1, from the fission yeast Schizosaccharomyces pombe, is a baculoviral inhibitor of apoptosis (IAP) repeat (BIR) domain-containing protein. Its unique encoding gene was previously found to be regulated by nitric oxide and nitrogen starvation. In the current work, the Pbh1-lacZ fusion gene was used to elucidate the transcriptional regulation of the pbh1 gene under various carbon sources. When fermentable carbon sources, such as glucose (at a low concentration of 0.2%), sucrose (2.0%) and lactose (2.0%), were the sole carbon source, the synthesis of β-galactosidase from the Pbh1-lacZ fusion gene was reasonably enhanced. However, the induction by these fermentable carbon sources was abolished in the Pap1-negative S. pombe cells, implying that this type of induction of the pbh1 gene is mediated by Pap1. Ethanol (2.0%), a nonfermentable carbon source, was also able to enhance the synthesis of β-galactosidase from the fusion gene in wild-type cells but not in Pap1-negative cells. The results indicate that the S. pombe pbh1 gene is up-regulated under metabolic oxidative stress in a Pap1-dependent manner.

Research Support, Non-U.S. Gov'ts

Transcriptional Analysis and Pap1-Dependence of the Unique Gene Encoding Thioredoxin Reductase from the Fission Yeast: Hyun-Jung Kang , Sung-Min Hong , Byung-Chul Kim , Kyunghoon Kim , Eun-Hee Park , Chang-Jin Lim; J. Microbiol. 2006;44(1):35-41.; DOI: https://doi.org/2339 [pii]

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Abstract: The unique gene encoding thioredoxin reductase (TrxR) was previously cloned and characterized from the fission yeast Schizosaccharomyces pombe, and its expression was induced by oxidative stress. To elucidate the regulatory mechanism of the S. pombe TrxR gene, three fusion plasmids were generated using polymerase chain reaction: pYUTR20, pYUTR30, and pYUTR40. Plasmid pYUTR20 has an upstream region of 891 base pairs, pYUTR30 has 499 in this region, and pYUTR40 has an 186 bp upstream region. Negatively acting sequence is located between ‒1,526 ~ ‒891 bp upstream of the gene. The upstream sequence, responsible for the induction of TrxR by menadione (MD), is situated on the ‒499 ~ ‒186 bp region, which is also required for TrxR induction by mercuric chloride. The same region also appeared to be required for Pap1-mediated transcriptional regulation of the TrxR gene, which contains the two plausible Pap1 binding sites, TTACGAAT and TTACGCGA. Consistently, basal and inducible expression of the TrxR gene was markedly lower in the Pap1-negative TP108-3C cells than in wild-type yeast cells. In summary, up-regulation of the S. pombe TrxR gene is mediated by Pap1 via the transcriptional motif( s) located on the ‒499 ~ ‒186 bp region.

Transcriptional Regulation of the Schizosaccharomyces pombe Gene Encoding Glutathione S-Transferase I by a Transcription Factor Pap1: Hong-Gyum Kim , Byung-Chul Kim , Kyunghoon Kim , Eun-Hee Park , Chang-Jin Lim; J. Microbiol. 2004;42(4):353-356.; DOI: https://doi.org/2099 [pii]

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Abstract: In a previous study, a gst gene was isolated from the fission yeast Schizosaccharomyces pombe. This gene was dubbed gst I, and was characterized using the gstI-lacZ fusion plasmid pYSH2000. In this work, four additional fusion plasmids, pYSHSD1, pYSHSD2, pYSHSD3 and pYSHSD4, were constructed, in order to carry (respectively) 770, 551, 358 and 151 bp upstream regions from the translational initiation point. The sequence responsible for induction by aluminum, mercury and hydrogen peroxide was located in the range between -1,088 and -770 bp upstream of the S. pombe gst I gene. The same region was identified to contain the nucleotide sequence responsible for regulation by Pap1, and has one putative Pap1 binding site, TTACGTAT, located in the range between -954 ~ -947 bp upstream of the gst I gene. Negatively acting sequences are located between -1,088 and -151 bp. These findings imply that the Pap1 protein is involved in basal and inducible transcription of the gst I gene in the fission yeast S. pombe.

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.

Cloning and Regulation of Schizosaccharomyces pombe Gene Encoding Ribosomal Protein S20: Yoon-Jong Lee , Kyunghoon Kim , Eun-Hee Park , Ki-Sup Ahn , Daemyung Kim , Chang-Jin Lim; J. Microbiol. 2001;39(1):31-36.

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Abstract: A cDNA clone encoding the ribosomal protein S20 has been isolated from the Schizosaccharomyces pombe cDNA library by colony hybridization. The insert contained in the original plasmid pYJ10 was transferred into shuttle vector pRS316 to generate plasmid pYJ11. The cDNA insert of plasmid pYJ11 contains 484 nucleotides and encodes a protein of 118 amino acids with a calculated mass of 13,544 daltons. The deduced amino acid sequence of S. pombe ribosomal protein S20 is very homologous with fruit fly, rat, and budding yeast counterparts. It is also homologous with Xenopus S22 ribosomal protein. S. pombe ribosomal protein S20 appears to be relatively hydrophobic except the C-terminal region. The 728 bp upstream region of the S20 gene was amplified from chromosomal DNA and transferred into the BamHI/EcoRI site of the promoterless b-galactosidase gene of the vector YEp357R, which resulted in fusion plasmid pYS20. The synthesis of b-galactosidase from the fusion plasmid appeared to be the highest in the mid-exponential phase. The S. pombe cells with the fusion plasmid grown at 35oC gave lower b-galactosidase activity than the cells grown at 30oC. Computer analysis showed the consensus sequence CAGTCACA in the upstream regions of various ribosomal protein genes in S. pombe, which would be involved in the coordinated expression of small ibosomal proteins.

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