Journal of Microbiology

Journal Articles

Whole genome and RNA sequencing of oral commensal bacterium Streptococcus anginosus subsp. anginosus with vancomycin tolerance: Kyu Hwan Kwack , Jae-Hyung Lee , Ji-Hoi Moon; J. Microbiol. 2022;60(2):167-176. Published online January 7, 2022; DOI: https://doi.org/10.1007/s12275-022-1425-4

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“Antibiotic tolerance” promotes the rapid subsequent evolution of “antibiotic resistance,” however, it is often overlooked because it is difficult to distinguish between tolerant and susceptible organisms. A commensal bacterium S. anginosus subsp. anginosus strain KHUD_S1, isolated from dental biofilm was found to exhibit a high MBC/MIC ratio of 32 against vancomycin. We observed KHUD_S1 cells exposed to vancomycin did not grow but maintained viability. Transmission electron microscope showed KHUD_S1 cells possessed a dense, thick capsule and maintained the cell wall integrity upon vancomycin exposure. To infer the underlying mechanisms of the vancomycin tolerance in KHUD_S1, we performed whole genome sequencing and RNA sequencing. The KHUD_S1 genome carried three genes encoding branching enzymes that can affect peptidoglycan structure through interpeptide bridge formation. Global gene expression profiling revealed that the vancomycin-induced downregulation of carbohydrate and inorganic ion transport/metabolism as well as translation is less prominent in KHUD_S1 than in the vancomycin susceptible strain KHUD_S3. Based on the transcriptional levels of genes related to peptidoglycan synthesis, KHUD_S1 was determined to have a 3D peptidoglycan architecture distinct from KHUD_S3. It was found that, under vancomycin exposure, the peptidoglycan was remodeled through changes in the interpeptide bridge and transpeptidation reactions. Collectively, these features of S. anginosus KHUD_S1, including a dense capsule and differential gene expression in peptidoglycan synthesis, may contribute to vancomycin tolerance. Our results showing the occurrence of vancomycin tolerance amongst oral commensal bacteria highlight the need for considering future strategies for screening of antibiotic tolerance as an effort to reduce antibiotic resistance.

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Gut resistome profiling reveals high diversity and fluctuations in pancreatic cancer cohorts
Xudong Liu, Kexin Li, Yun Yang, Dingyan Cao, Xinjie Xu, Zilong He, Wenming Wu
Frontiers in Cellular and Infection Microbiology.2024;[Epub] CrossRef
The Sexome ‐ A proof of concept study into microbial transfer between heterosexual couples after sexual intercourse
Ruby Dixon, Siobhon Egan, Sheree Hughes, Brendan Chapman
Forensic Science International.2023; 348: 111711. CrossRef

The type II histidine triad protein HtpsC facilitates invasion of epithelial cells by highly virulent Streptococcus suis serotype 2: Yunjun Lu , Shu Li , Xiaodong Shen , Yan Zhao , Dongming Zhou , Dan Hu , Xushen Cai , Lixia Lu , Xiaohui Xiong , Ming Li , Min Cao; J. Microbiol. 2021;59(10):949-957. Published online September 7, 2021; DOI: https://doi.org/10.1007/s12275-021-1129-1

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Abstract

Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen that presents a significant threat both to pigs and to workers in the pork industry. The initial steps of S. suis 2 pathogenesis are unclear. In this study, we found that the type II histidine triad protein HtpsC from the highly virulent Chinese isolate 05ZYH33 is structurally similar to internalin A (InlA) from Listeria monocytogenes, which plays an important role in mediating listerial invasion of epithelial cells. To determine if HtpsC and InlA function similarly, an isogenic htpsC mutant (ΔhtpsC) was generated in S. suis by homologous recombination. The htpsC deletion strain exhibited a diminished ability to adhere to and invade epithelial cells from different sources. Double immunofluorescence microscopy also revealed reduced survival of the ΔhtpsC mutant after cocultivation with epithelium. Adhesion to epithelium and invasion by the wild type strain was inhibited by a monoclonal antibody against E-cadherin. In contrast, the htpsC-deficient mutant was unaffected by the same treatment, suggesting that E-cadherin is the host-cell receptor that interacts with HtpsC and facilitates bacterial internalization. Based on these results, we propose that HtpsC is involved in the process by which S. suis 2 penetrates host epithelial cells, and that this protein is an important virulence factor associated with cell adhesion and invasion.

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Genomic and phenotypic analysis of invasive Streptococcus suis isolated in Spain reveals genetic diversification and associated virulence traits
Cristina Uruén, Ana Fernandez, José Luis Arnal, Mateo del Pozo, Maria Casas Amoribieta, Ignacio de Blas, Paula Jurado, Jorge Hugo Calvo, Marcelo Gottschalk, Luis Daniel González-Vázquez, Miguel Arenas, Clara M. Marín, Jesús Arenas
Veterinary Research.2024;[Epub] CrossRef
A rapid colloidal gold immunochromatographic assay based on polyclonal antibodies against HtpsC protein for the detection of Streptococcus suis
Yawei Lu, Sibo Wang, Xushen Cai, Min Cao, Qingyu Lu, Dan Hu, Qiong Chen, Xiaohui Xiong
Frontiers in Microbiology.2023;[Epub] CrossRef

Identification of a novel phospholipase D gene and effects of carbon sources on its expression in Bacillus cereus ZY12: Yu Zhao , Yinfeng Xu , Fang Yu , Chunzhi Zhang; J. Microbiol. 2018;56(4):264-271. Published online April 2, 2018; DOI: https://doi.org/10.1007/s12275-018-7529-1

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Abstract

In the present study, a new strain, Bacillus cereus ZY12, producing phospholipase D (PLD) was identified. The expression of PLD in this strain was found to be induced by its substrate, phosphatidylcholine (PC), and completely silenced by other carbon sources, such as glucose, fructose, and maltose, which are generally used in microbial growth cultures, thus presenting a unique expression pattern different from other PLD-producing microorganisms. This study is the first to report on the ability of B. cereus to produce PLD, and successfully clone its PLD-coding gene and identify its function, extending the knowledge on PLD distribution and evolution in microorganisms.

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Structural insights into PA3488-mediated inactivation of Pseudomonas aeruginosa PldA
Xiaoyun Yang, Zongqiang Li, Liang Zhao, Zhun She, Zengqiang Gao, Sen-Fang Sui, Yuhui Dong, Yanhua Li
Nature Communications.2022;[Epub] CrossRef
Construction of a Super-Folder Fluorescent Protein-Guided Secretory Expression System for the Production of Phospholipase D in Bacillus subtilis
Haiyang Zhang, Xuehan Li, Qi Liu, Jianan Sun, Francesco Secundo, Xiangzhao Mao
Journal of Agricultural and Food Chemistry.2021; 69(24): 6842. CrossRef
Microbial phospholipase D: Identification, modification and application
Zhenxia Zhang, Ming Chen, Wei Xu, Wenli Zhang, Tao Zhang, Cuie Guang, Wanmeng Mu
Trends in Food Science & Technology.2020; 96: 145. CrossRef

Research Support, U.S. Gov't, Non-P.H.S.

Multiple cellular roles of Neurospora crassa plc-1, splA2, and cpe-1 in regulation of cytosolic free calcium, carotenoid accumulation, stress responses, and acquisition of thermotolerance§: Ananya Barman , Ranjan Tamuli; J. Microbiol. 2015;53(4):226-235. Published online January 31, 2015; DOI: https://doi.org/10.1007/s12275-015-4465-1

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Abstract

Phospholipase C1 (PLC1), secretory phospholipase A2 (sPLA2) and Ca2+/H+ exchanger proteins regulate calcium signaling and homeostasis in eukaryotes. In this study, we investigate functions for phospholipase C1 (plc-1), sPLA2 (splA2) and a Ca2+/H+ exchanger (cpe-1) in the filamentous fungus Neurospora crassa. The Δplc-1, ΔsplA2, and Δcpe-1 mutants exhibited a growth defect on medium supplemented with the divalent ionophore A23187, suggesting that these genes might play a role in regulation of cytosolic free Ca2+ concentration ([Ca2+]c) in N. crassa. The strains lacking plc-1, splA2, and cpe-1 possessed higher carotenoid content than wild type at 8°C, 22°C, and 30°C, and showed increased ultraviolet (UV)- survival under conditions that induced carotenoid accumulation. Moreover, Δplc-1, ΔsplA2, and Δcpe-1 mutants showed reduced survival rate under hydrogen peroxide-induced oxidative stress and induced thermotolerance after exposure to heat shock temperatures. Thus, this study revealed multiple cellular roles for plc-1, splA2, and cpe-1 genes in regulation of [Ca2+]c, carotenoid accumulation, survival under stress conditions, and acquisition of thermotolerance induced by heat shock.

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PacC mediates spatial regulation of the phospholipid metabolism in the apple fruit-Penicillium expansum interaction
Yatong Zhu, Yuanyuan Zong, Di Gong, Xuexue Wang, William Oyom, Yang Bi, Dov Prusky
Postharvest Biology and Technology.2024; 208: 112666. CrossRef
Methods for the detection of intracellular calcium in filamentous fungi
Megha Rasaily, Serena Ngiimei D, Rahul Kumar Thaosen, Surabhi Gupta, Sangeeta Deka, Ranjan Tamuli
MethodsX.2024; 12: 102570. CrossRef
Thiourea Application Increases Seed and Oil Yields in Camelina Under Heat Stress by Modulating the Plant Water Relations and Antioxidant Defense System
Muhammad Ahmad, Ejaz Ahmad Waraich, Usman Zulfiqar, Aman Ullah, Muhammad Farooq
Journal of Soil Science and Plant Nutrition.2023; 23(1): 290. CrossRef
Trichosporon asahii PLA2 Gene Enhances Drug Resistance to Azoles by Improving Drug Efflux and Biofilm Formation
Xiaoping Ma, Hong Liu, Zhen Liu, Ya Wang, Zhijun Zhong, Guangneng Peng, Yu Gu
International Journal of Molecular Sciences.2023; 24(10): 8855. CrossRef
Interaction of calcium responsive proteins and transcriptional factors with the PHO regulon in yeasts and fungi
Juan F. Martín
Frontiers in Cell and Developmental Biology.2023;[Epub] CrossRef
The cell functions of phospholipase C-1, Ca2+/H+ exchanger-1, and secretory phospholipase A2 in tolerance to stress conditions and cellulose degradation in Neurospora crassa
Darshana Baruah, Ranjan Tamuli
Archives of Microbiology.2023;[Epub] CrossRef
Multiple calcium signaling genes play a role in the circadian period of Neurospora crassa
Darshana Baruah, Christy Noche K Marak, Avishek Roy, Dibakar Gohain, Ajeet Kumar, Pallavi Das, Katherine A Borkovich, Ranjan Tamuli
FEMS Microbiology Letters.2023;[Epub] CrossRef
Phospholipase C: Diverse functions in plant biotic stress resistance and fungal pathogenicity
Yuanpeng Fang, Junmei Jiang, Haixia Ding, Xiangyang Li, Xin Xie
Molecular Plant Pathology.2023; 24(9): 1192. CrossRef
A secretory phospholipase A2 of a fungal pathogen contributes to lipid droplet homeostasis, assimilation of insect‐derived lipids, and repression of host immune responses
Juan Deng, Zhuoyue Lu, Huifang Wang, Ning Li, Guimei Song, Qiankuan Zhu, Jingxin Sun, Yongjun Zhang
Insect Science.2022; 29(6): 1685. CrossRef
Phospholipase C (AoPLC2) regulates mycelial development, trap morphogenesis, and pathogenicity of the nematode-trapping fungus Arthrobotrys oligospora
Meihua Xie, Ni Ma, Na Bai, Meichen Zhu, Ke-Qin Zhang, Jinkui Yang
Journal of Applied Microbiology.2022; 132(3): 2144. CrossRef
Disrupting a phospholipase A 2 gene increasing lipid accumulation in the oleaginous yeast Yarrowia lipolytica
J.X. Li, J. Xu, J.C. Ruan, H.M. Meng, H. Su, X.F. Han, M. Lu, F.L. Li, S.A. Wang
Journal of Applied Microbiology.2021; 130(1): 100. CrossRef
Flexible online in-droplet cell/synthetic particle concentration utilizing alternating current electrothermal-flow field-effect transistor
Haizhen Sun, Yukun Ren, Ye Tao, Tianyi Jiang, Hongyuan Jiang
Lab on a Chip.2021; 21(10): 1987. CrossRef
Calcium signaling is involved in diverse cellular processes in fungi
Avishek Roy, Ajeet Kumar, Darshana Baruah, Ranjan Tamuli
Mycology.2021; 12(1): 10. CrossRef
Dominant mutants of the calcineurin catalytic subunit (CNA-1) showed developmental defects, increased sensitivity to stress conditions, and CNA-1 interacts with CaM and CRZ-1 in Neurospora crassa
Ajeet Kumar, Avishek Roy, Mandar V. Deshmukh, Ranjan Tamuli
Archives of Microbiology.2020; 202(4): 921. CrossRef
Calcineurin responsive zinc‐finger‐1 binds to a unique promoter sequence to upregulate neuronal calcium sensor‐1, whose interaction with MID‐1 increases tolerance to calcium stress in Neurospora crassa
Dibakar Gohain, Ranjan Tamuli
Molecular Microbiology.2019; 111(6): 1510. CrossRef
The NcZrg-17 gene of Neurospora crassa encodes a cation diffusion facilitator transporter required for vegetative development, tolerance to endoplasmic reticulum stress and cellulose degradation under low zinc conditions
Anand Tiwari, Serena Daniel Ngiilmei, Ranjan Tamuli
Current Genetics.2018; 64(4): 811. CrossRef
Phospholipases play multiple cellular roles including growth, stress tolerance, sexual development, and virulence in fungi
Ananya Barman, Dibakar Gohain, Utpal Bora, Ranjan Tamuli
Microbiological Research.2018; 209: 55. CrossRef
The pleiotropic vegetative and sexual development phenotypes of Neurospora crassa arise from double mutants of the calcium signaling genes plc-1, splA2, and cpe-1
Ananya Barman, Ranjan Tamuli
Current Genetics.2017; 63(5): 861. CrossRef
Effects of heat stress on changes in physiology and anatomy in two cultivars of Rhododendron
H.F. Shen, B. Zhao, J.J. Xu, W. Liang, W.M. Huang, H.H. Li
South African Journal of Botany.2017; 112: 338. CrossRef
Phenotypic abnormalities of fr , sp , and och-1 single mutants are suppressed by loss of putative GPI-phospholipase A2 in Neurospora crassa
Masayuki Kamei, Yuko Tsukagoshi, Shinpei Banno, Akihiko Ichiishi, Fumiyasu Fukumori, Makoto Fujimura
Mycoscience.2017; 58(3): 137. CrossRef
Calcineurin Subunits A and B Interact to Regulate Growth and Asexual and Sexual Development in Neurospora crassa
Ranjan Tamuli, Rekha Deka, Katherine A. Borkovich, Stefanie Pöggeler
PLOS ONE.2016; 11(3): e0151867. CrossRef

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

Surface Display Expression of Bacillus licheniformis Lipase in Escherichia coli Using Lpp’OmpA Chimera: Jae-Hyung Jo , Chan-Wook Han , Seung-Hwan Kim , Hyuk-Jin Kwon , Hyune-Hwan Lee; J. Microbiol. 2014;52(10):856-862. Published online August 27, 2014; DOI: https://doi.org/10.1007/s12275-014-4217-7

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Abstract

The lipase from Bacillus licheniformis ATCC14580 was displayed on the cell surface of Escherichia coli using Lpp’OmpA as the anchoring protein. The expressed Lpp’OmpA-lipase fusion protein has a molecular weight of approximately 35 kDa, which was confirmed by SDS-PAGE and western blot analysis. The Lpp’OmpA-lipase fusion protein was located on the cell surface, as determined by immunofluorescence confocal microscopy and flow cytometry. The enzyme activity of the surface-displayed lipase showed clear halo around the colony. The cell surface-displayed lipase showed the highest activity of 248.12 ± 9.42 U/g (lyophilized cell) at the optimal temperature of 37°C and pH 8.0. The enzyme exhibited the highest activity toward the substrate p-nitrophenyl caprylate (C8). These results suggest that E. coli, which displayed the lipase on its surface, could be used as a whole cell biocatalyst.

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Recent advances in bioinspired multienzyme engineering for food applications
Xianhan Chen, Yujin Chen, Dandan Tang, Mengyu Li, Yuting Lu, Yi Cao, Quanyu Zhao, Shuai Jiang, Wei Liu, Ling Jiang
Trends in Food Science & Technology.2025; 156: 104840. CrossRef
Surface Engineering of Escherichia coli to Display Its Phytase (AppA) and Functional Analysis of Enzyme Activities
Patricia L. A. Muñoz-Muñoz, Celina Terán-Ramírez, Rosa E. Mares-Alejandre, Ariana B. Márquez-González, Pablo A. Madero-Ayala, Samuel G. Meléndez-López, Marco A. Ramos-Ibarra
Current Issues in Molecular Biology.2024; 46(4): 3424. CrossRef
Characterization of a novel subfamily 1.4 lipase from Bacillus licheniformis IBRL-CHS2: Cloning and expression optimization
Ammar Khazaal Kadhim Almansoori, Nidyaletchmy Subba Reddy, Mustafa Abdulfattah, Sarah Solehah Ismail, Rashidah Abdul Rahim, Estibaliz Sansinenea
PLOS ONE.2024; 19(12): e0314556. CrossRef
Surface Display of Multiple Metal-Binding Domains in Deinococcus radiodurans Alleviates Cadmium and Lead Toxicity in Rice
Liangyan Wang, Yudong Wang, Shang Dai, Binqiang Wang
International Journal of Molecular Sciences.2024; 25(23): 12570. CrossRef
A bacterial outer membrane vesicle-based click vaccine elicits potent immune response against Staphylococcus aureus in mice
Jingjing Sun, Xuansheng Lin, Yige He, Baozhong Zhang, Nan Zhou, Jian-dong Huang
Frontiers in Immunology.2023;[Epub] CrossRef
Establishment of a soluble expression and rapid purification system for self-assembling protein nanoparticle and characterization of its physiochemical properties
Dan Wang, Linwei Duan, Min Wei, Baizhu Chen, Zhipeng Li, Qingyou Liu
Biochemical Engineering Journal.2022; 186: 108580. CrossRef
A Modular System for the Rapid Comparison of Different Membrane Anchors for Surface Display on Escherichia coli
Sabrina Gallus, Esther Mittmann, Kersten S. Rabe
ChemBioChem.2022;[Epub] CrossRef
Decorating the surface of Escherichia coli with bacterial lipoproteins: a comparative analysis of different display systems
Sonia Nicchi, Maria Giuliani, Fabiola Giusti, Laura Pancotto, Domenico Maione, Isabel Delany, Cesira L. Galeotti, Cecilia Brettoni
Microbial Cell Factories.2021;[Epub] CrossRef
Recombinant expression and surface display of a zearalenone lactonohydrolase from Trichoderma aggressivum in Escherichia coli
Shurong Chen, Li Pan, Siying Liu, Lijie Pan, Xuejie Li, Bin Wang
Protein Expression and Purification.2021; 187: 105933. CrossRef
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Eszter Csibra, Marleen Renders, Vitor B. Pinheiro
ChemBioChem.2020; 21(19): 2844. CrossRef
Surface Display of Complex Enzymes by in Situ SpyCatcher‐SpyTag Interaction
Sabrina Gallus, Theo Peschke, Malte Paulsen, Teresa Burgahn, Christof M. Niemeyer, Kersten S. Rabe
ChemBioChem.2020; 21(15): 2126. CrossRef
Shaking Rate during Production Affects the Activity of Escherichia coli Surface-Displayed Candida antarctica Lipase A
Chen-Fu Chung, Shih-Che Lin, Tzong-Yuan Juang, Yung-Chuan Liu
Catalysts.2020; 10(4): 382. CrossRef
Functional Display of an Amoebic Chitinase in Escherichia coli Expressing the Catalytic Domain of EhCHT1 on the Bacterial Cell Surface
Ricardo Torres-Bañaga, Rosa E. Mares-Alejandre, Celina Terán-Ramírez, Ana L. Estrada-González, Patricia L.A. Muñoz-Muñoz, Samuel G. Meléndez-López, Ignacio A. Rivero, Marco A. Ramos-Ibarra
Applied Biochemistry and Biotechnology.2020; 192(4): 1255. CrossRef
Heterologous expression of antigenic peptides in Bacillus subtilis biofilms
Cédric M. Vogt, Elisabeth M. Schraner, Claudio Aguilar, Catherine Eichwald
Microbial Cell Factories.2016;[Epub] CrossRef
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Current Microbiology.2015; 70(6): 779. CrossRef

Isolation and Characterization of Novel Lipase Gene LipHim1 from the DNA Isolated from Soil Samples: Pavan Kumar Pindi , Raja Srinath Rebba , Theetha L. Pavankumar; J. Microbiol. 2014;52(5):384-388. Published online April 11, 2014; DOI: https://doi.org/10.1007/s12275-014-3302-2

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Abstract

Metagenomics is a magnificent tool to isolate genes from unknown/uncharacterized species and also from organisms that cannot be cultured. In this study, we constructed a meta-genomic library from isolated DNA of soil samples collected from Palamuru University campus premises, in Mahabub-nagar district of Andhra Pradesh, India. We isolated a novel lipase gene LipHim1, which has an open reading frame of 591 base pairs and encodes ~23 kDa protein consisting of 196 amino acids. The Lipase LipHim1 showed maximum 32% homology at the protein level with the extracellular Aeromonas hydrophila lipase (Class II, GDSL family) and was significantly different from all other known lipases. The isolated lipase catalyzed the hydrolysis of fatty acid esters of polyoxyethylene sorbitan such as Tween 60. Our results in-dicate that the isolated lipase gene is novel.

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High-Throughput, Fluorescence-Based Esterase Activity Assay for Assessing Polysorbate Degradation Risk during Biopharmaceutical Development
Adithi C. Bhargava, Kathryn Mains, Albert Siu, Jie Gu, Jonathan Zarzar, Li Yi, Inn H. Yuk
Pharmaceutical Research.2021; 38(3): 397. CrossRef
Isolation and characterization of novel and efficient protease producing bacteria from drinking water resources
S. Jeevan Chandra, A. Shiva Shanker, Pavan Kumar Pindi
Water Supply.2020; 20(1): 157. CrossRef
Integrative computational approach for genome-based study of microbial lipid-degrading enzymes
Tayvich Vorapreeda, Chinae Thammarongtham, Kobkul Laoteng
World Journal of Microbiology and Biotechnology.2016;[Epub] CrossRef

NOTE] The Activity of Phosphoinositide-Specific Phospholipase C Is Required for Vegetative Growth and Cell Wall Regeneration in Coprinopsis cinerea: Young Taek Oh , Chun-Seob Ahn , Kyung-Jin Lee , Jeong-Geun Kim , Hyeon-Su Ro , Jae Won Kim , Chang-Won Lee; J. Microbiol. 2012;50(4):689-692. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2004-x

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Abstract: Three isotypes of phosphoinositide-specific phospholipase C designated CcPLC1, CcPLC2, and CcPLC3 were identified in Coprinopsis cinerea, through a search of the genome sequence database. The functional role of the PI-PLCs were studied by using U73122, which specifically inhibits the activity of PI-PLC. The specificity of the inhibitor effect was confirmed by using an inactive structural analog U73433. The inhibition of PI-PLCs activity resulted in severely retarded germination of basidiospores and oidia, reduced hyphal growth, knobbly hyphal tips with many irregular side branches, and aberrant (branch-like structure) clamp cells. Furthermore, U73122 definitely inhibited cell wall formation. Here we report that PI-PLCs play important roles in various aspects of C. cinerea biology.

Isolation and Characterization of a Family VII Esterase Derived from Alluvial Soil Metagenomic Library: Weixin Tao , Myung Hwan Lee , Jing Wu , Nam Hee Kim , Seon-Woo Lee; J. Microbiol. 2011;49(2):178-185. Published online May 3, 2011; DOI: https://doi.org/10.1007/s12275-011-1102-5

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Abstract: A novel esterase gene, estDL30, was isolated from an alluvial metagenomic library using function-driven screening. estDL30 consisted of 1,524 nucleotides and encoded a 507-amino acid protein. Sequence analysis revealed that EstDL30 is similar to many type B carboxylesterases, containing a G-E-S-A-G pentapeptide with a catalytic Ser residue. Phylogenetic analysis suggested that EstDL30 belongs to the family VII lipases, together with esterases from Bacillus subtilis (P37967), Streptomyces coelicolor A3(2) (CAA22794), and Arthrobacter oxydans (Q01470). Purified EstDL30 showed its highest catalytic efficiency toward p-nitrophenyl butyrate, with a kcat of 229.3 s-1 and kcat/Km of 176.4 s-1mM-1; however, little activity was detected when the acyl chain length exceeded C8. Biochemical characterization of EstDL30 revealed that it is an alkaline esterase that possesses maximal activity at pH 8 and 40°C. The effects of denaturants and divalent cations were also investigated. EstDL30 tolerated well the presence of methanol and Tween 20. Its activity was strongly inhibited by 1 mM Cu2+ and Zn2+, but stimulated by Fe2+. The unique properties of EstDL30, its high activity under alkaline conditions and stability in the presence of organic solvents, may render it applicable to organic synthesis.

Biochemical Characteristics of Immune-Associated Phospholipase A2 and Its Inhibition by an Entomopathogenic Bacterium, Xenorhabdus nematophila: Sony Shrestha , Yonggyun Kim; J. Microbiol. 2009;47(6):774-782. Published online February 4, 2010; DOI: https://doi.org/10.1007/s12275-009-0145-3

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Abstract: An entomopathogenic bacterium, Xenorhabdus nematophila, induces an immunosuppression of target insects by inhibiting phospholipase A2 (PLA2) activity. Recently, an immune-associated PLA2 gene was identified from the red flour beetle, Tribolium castaneum. This study cloned this PLA2 gene in a bacterial expression vector to produce a recombinant enzyme. The recombinant T. castaneum PLA2 (TcPLA2) exhibited its characteristic enzyme activity with substrate concentration, pH, and ambient temperature. Its biochemical characteristics matched to a secretory type of PLA2 (sPLA2) because its activity was inhibited by dithiothreitol (a reducing agent of disulfide bond) and bromophenacyl bromide (a specific sPLA2 inhibitor) but not by methylarachidonyl fluorophosphonate (a specific cytosolic type of PLA2). The X. nematophila culture broth contained PLA2 inhibitory factor(s), which was most abundant in the media obtained at a stationary bacterial growth phase. The PLA2 inhibitory factor(s) was heat-resistant and extracted in both aqueous and organic fractions. Effect of a PLA2-inhibitory fraction on the immunosuppression of T. castaneum was equally comparable with that resulted from inhibition of the TcPLA2 gene expression by RNA interference.

Journal Article

Response Surface Analysis for the Production of an Enantioselective Lipase from Aspergillus niger by Solid-State Fermentation: Fabiano Jares Contesini , Vania Castriani Fernades da Silva , Rafael Ferreira Maciel , Rosemary Joana de Lima , Francisco Fábio Cavalcante Barros , Patrícia de Oliveira Carvalho; J. Microbiol. 2009;47(5):563-571. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-008-0279-8

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Abstract: The lipase produced by the Aspergillus niger strain AC-54 has been widely studied due to its enantioselectivity for racemic mixtures. This study aimed to optimize the production of this enzyme using statistical methodology. Initially a Plackett-Burman (PB) design was used to evaluate the effects of the culture medium components and the culture conditions. Twelve factors were screened: water content, glucose, yeast extract, peptone, olive oil, temperature, NaH2PO4, KH2PO4, MgSO4․7H2O, CaCl2, NaCl, and MnSO4. The screening showed that the significant factors were water content, glucose, yeast extract, peptone, NaH2PO4, and KH2PO4, which were optimized using response surface methodology (RSM) and a mathematical model obtained to explain the behavioral process. The best lipase activity was attained using the following conditions: water content (20%), glucose (4.8%), yeast extract (4.0%), and NaH2PO4 (4.0%). The predicted lipase activity was 33.03 U/ml and the experimental data confirmed the validity of the model. The enzymatic activity was expressed as µmoles of oleic acid released per minute of reaction (µmol/min).

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

Molecular Cloning of the Phospholipase D Gene from Streptomyces sp. YU100 and Its Expression in Escherichia coli: Ji-Seon Lee , Munkhtsetseg Bat-Ochir , Atanas V. Demirev , Doo Hyun Nam; J. Microbiol. 2009;47(1):116-122. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0161-8

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Abstract: The gene for phospholipase D (PLD) of Streptomyces sp. YU100 was cloned from λ phage library and heterologously expressed in Escherichia coli. Using an amplified gene fragment based on the consensus sequences of streptomycetes PLDs, λ phage library of Streptomyces sp. YU100 chromosomal DNA was screened. The sequencing result of BamHI-digested 3.8 kb fragment in a positive phage clone revealed the presence of an open reading frame of a full sequence of PLD gene encoding a 540-amino acid protein including 33-amino acid signal peptide. The deduced amino acid sequence showed a high homology with other Streptomyces PLDs, having the highly conserved ‘HKD’ motifs. The PLD gene excluding signal peptide sequence was amplified and subcloned into a pET-32b(+) expression vector in E. coli BL21(DE3). The recombinant PLD was purified by nickel affinity chromatography and compared the enzyme activity with wild-type PLD. The results imply that the recombinant PLD produced by E. coli had the nearly same enzyme activity as PLD from Streptomyces sp. YU100.

Characterization of an Extracellular Lipase in Burkholderia sp. HY-10 Isolated from a Longicorn Beetle: Doo-Sang Park , Hyun-Woo Oh , Sun-Yeon Heo , Woo-Jin Jeong , Dong Ha Shin , Kyung Sook Bae , Ho-Young Park; J. Microbiol. 2007;45(5):409-417.; DOI: https://doi.org/2596 [pii]

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Abstract: Burkholderia sp. HY-10 isolated from the digestive tracts of the longicorn beetle, Prionus insularis, produced an extracellular lipase with a molecular weight of 33.5 kDa estimated by SDS-PAGE. The lipase was purified from the culture supernatant to near electrophoretic homogenity by a one-step adsorption-desorption procedure using a polypropylene matrix followed by a concentration step. The purified lipase exhibited highest activities at pH 8.5 and 60°C. A broad range of lipase substrates, from C4 to C18 ρ-nitrophenyl esters, were hydrolyzed efficiently by the lipase. The most efficient substrate was ρ-nitrophenyl caproate (C6). A 2485 bp DNA fragment was isolated by PCR amplification and chromosomal walking which encoded two polypeptides of 364 and 346 amino acids, identified as a lipase and a lipase foldase, respectively. The N-terminal amino acid sequence of the purified lipase and nucleotide sequence analysis predicted that the precursor lipase was proteolytically modified through the secretion step and produced a catalytically active 33.5 kDa protein. The deduced amino acid sequence for the lipase shared extensive similarity with those of the lipase family I.2 of lipases from other bacteria. The deduced amino acid sequence contained two Cystein residues forming a disulfide bond in the molecule and three, well-conserved amino acid residues, Ser131, His330, and Asp308, which composed the catalytic triad of the enzyme.

Phosphate and Carbon Source Regulation of Alkaline Phosphatase and Phospholipase in Vibrio vulnificus: Wan-Seok Oh , Young-Sun Im , Kyu-Young Yeon , Young-Jun Yoon , Jung-Wan Kim; J. Microbiol. 2007;45(4):311-317.; DOI: https://doi.org/2567 [pii]

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Abstract: In this study, the effects of phosphate concentration and carbon source on the patterns of alkaline phosphatase (APase) and phospholipase (PLase) expression in Vibrio vulnificus ATCC 29307 were assessed under various conditions. The activities of these enzymes were repressed by excess phosphate (4 mM) in the culture medium, but this repression was reversed upon the onset of phosphate starvation in low phosphate defined medium (LPDM) containing 0.2 mM of phosphate at approximately the end of the exponential growth phase. The expressions of the two enzymes were also influenced by different carbon sources, including glucose, fructose, maltose, glycerol, and sodium acetate at different levels. The APase activity was derepressed most profoundly in LPDM containing fructose as a sole carbon source. However, the repression/derepression of the enzyme by phosphate was not observed in media containing glycerol or sodium acetate. In LPDM-glycerol or sodium acetate, the growth rate was quite low. The highest levels of PLase activity were detected in LPDMsodium acetate, followed by LPDM-fructose. PLase was not fully repressed by high phosphate concentrations when sodium acetate was utilized as the sole carbon source. These results showed that multiple regulatory systems, including the phosphate regulon, may perform a function in the expression of both or either APase and PLC, in the broader context of the survival of V. vulnificus.

Journal Article

S5 Lipase : An Organic Solvent Tolerant Enzyme: Raja Noor Zaliha Raja Abdul Rahman , Syarul Nataqain Baharum , Abu Bakar Salleh , Mahiran Basri; J. Microbiol. 2006;44(6):583-590.; DOI: https://doi.org/2470 [pii]

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Abstract: In this study, an organic solvent tolerant bacterial strain was isolated. This strain was identified as Pseudomonas sp. strain S5, and was shown to degrade BTEX (Benzene, Toluene, Ethyl-Benzene, and Xylene). Strain S5 generates an organic solvent-tolerant lipase in the late logarithmic phase of growth. Maximum lipase production was exhibited when peptone was utilized as the sole nitrogen source. Addition of any of the selected carbon sources to the medium resulted in a significant reduction of enzyme production. Lower lipase generation was noted when an inorganic nitrogen source was used as the sole nitrogen source. This bacterium hydrolyzed all tested triglycerides and the highest levels of production were observed when olive oil was used as a natural triglyceride. Basal medium containing Tween 60 enhanced lipase production to the most significant degree. The absence of magnesium ions (Mg2+) in the basal medium was also shown to stimulate lipase production. Meanwhile, an alkaline earth metal ion, Na+, was found to stimulate the production of S5 lipase.

Isolation and Characterization of Fatty Acid Derivatives from an Actinomycetes and Examination of the Effects on Activities of Phospholipase C and Protein Kinase C: Ko, Hack Ryong , Kim, Bo Yeon , Lee, Hyun Sun , Kang, Dae Ook , Ryu, Sung Ho , Suh, Pann Ghill , Mheen, Tae Ick , Ahnm Jong Seog; J. Microbiol. 1998;36(4):316-321.

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Abstract: In our screening to search inhibitors of phosphoinositide(PI)-specific phospholipase C (PI-PLC), two inhibitors, MT965-A and-B were isolated from a culture broth of an actinomycetes. MT965-A and-B were identified as fatty acid deribatives, 14-methylpentadecanoic acid and 16-methyllinoleic acid methyl ester, respectively, based on the spectral data including NMR and MS. Both inhibitors directly inhibited not only in vitro PLCγ1 activity but also the platelet-derived growth factor(PDGF)-induced inositol phosphates(IPt) formation in NIH 3T3γ1 cells ocerexpressing PLCγ1. However, the inhibitors enhanced in vitro protein kinase C (PKC) activity. On examination of the effects of various fatty acids(FAs) on activities of PLC, PKC, and PDGF-induced IPt formation, the unsaturated FAs(UFAs) showed the same activities like the inhibitors, but the saturated FAs(SFAs) did not show similar activities. It was inferred that the chain length, degree of unsaturation, methyl esterification, branching with a methyl group, and cis-configuration were important for their activity.

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