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Investigation of Bottleneck Enzyme Through Flux Balance Analysis to Improve Glycolic Acid Production in Escherichia coli
Jungyeon Kim, Ye-Bin Kim, Ju-Young Kim, Min-Ju Seo, Soo-Jin Yeom, Bong Hyun Sung
J. Microbiol. 2024;62(11):1023-1033.   Published online October 28, 2024
DOI: https://doi.org/10.1007/s12275-024-00175-4
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AbstractAbstract
Amid rising environmental concerns, attempts have been made to produce glycolic acid (GA) using microbial processes with renewable carbon resources instead of using chemicals. The Dahms pathway for GA production uses xylose as a substrate and consists of relatively simple enzymatic steps. However, employing it leads to a decrease in cell growth and GA productivity. Systematically identifying and addressing metabolic bottlenecks in the Dahms pathway are essential for efficient glycolic acid (GA) production have not yet been performed. Through metabolic flux balance analysis, we found that insufficient aldehyde dehydrogenase (AldA) activity lowers GA production and negatively affects cell growth due to reduced energy production. Thus, we discovered a novel AldA isolated from Buttiauxella agrestis (BaAldA) demonstrated a 1.69-fold lower KM and a 1.49-fold higher turnover rate (kcat/KM) than AldA from Escherichia coli (EcAldA). GA production in E. coli harboring BaAldA was 1.59 times higher than in the original strain. Fed-batch fermentation of E. coli harboring BaAldA produced 22.70 g/L GA with a yield of 0.497 g/gxylose (98.2% of the theoretical maximum yield in the Dahms pathway), showing a higher final yield for GA than previously reported in E. coli. Our novel BaAldA enzyme shows great potential for the production of GA using microorganisms or enzymes. Furthermore, our approach to identifying metabolic bottlenecks using flux balance analysis could be utilized to enhance the microbial production of various desirable products in future studies.
The putative polysaccharide synthase AfCps1 regulates Aspergillus fumigatus morphogenesis and conidia immune response in mouse bone marrow-derived macrophages
Sha Wang , Anjie Yuan , Liping Zeng , Sikai Hou , Meng Wang , Lei Li , Zhendong Cai , Guowei Zhong
J. Microbiol. 2021;59(1):64-75.   Published online November 17, 2020
DOI: https://doi.org/10.1007/s12275-021-0347-x
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  • 4 Web of Science
  • 3 Crossref
AbstractAbstract
Aspergillus fumigatus is a well-known opportunistic pathogen that causes invasive aspergillosis (IA) infections with high mortality in immunosuppressed individuals. Morphogenesis, including hyphal growth, conidiation, and cell wall biosynthesis is crucial in A. fumigatus pathogenesis. Based on a previous random insertional mutagenesis library, we identified the putative polysaccharide synthase gene Afcps1 and its paralog Afcps2. Homologs of the cps gene are commonly found in the genomes of most fungal and some bacterial pathogens. Afcps1/cpsA is important in sporulation, cell wall composition, and virulence. However, the precise regulation patterns of cell wall integrity by Afcps1/cpsA and further effects on the immune response are poorly understood. Specifically, our in-depth study revealed that Afcps1 affects cell-wall stability, showing an increased resistance of ΔAfcps1 to the chitinmicrofibril destabilizing compound calcofluor white (CFW) and susceptibility of ΔAfcps1 to the β-(1,3)-glucan synthase inhibitor echinocandin caspofungin (CS). Additionally, deletion of Afcps2 had a normal sporulation phenotype but caused hypersensitivity to Na+ stress, CFW, and Congo red (CR). Specifically, quantitative analysis of cell wall composition using high-performance anion exchange chromatography- pulsed amperometric detector (HPAEC-PAD) analysis revealed that depletion of Afcps1 reduced cell wall glucan and chitin contents, which was consistent with the downregulation of expression of the corresponding biosynthesis genes. Moreover, an elevated immune response stimulated by conidia of the ΔAfcps1 mutant in marrow-derived macrophages (BMMs) during phagocytosis was observed. Thus, our study provided new insights into the function of polysaccharide synthase Cps1, which is necessary for the maintenance of cell wall stability and the adaptation of conidia to the immune response of macrophages in A. fumigatus.

Citations

Citations to this article as recorded by  
  • Study on the metabolic changes and regulatory mechanism of Aspergillus flavus conidia germination
    Sifan Jia, Chong Li, Yu An, Desheng Qi, Erik F. Y. Hom
    Microbiology Spectrum.2024;[Epub]     CrossRef
  • Chitin Biosynthesis in Aspergillus Species
    Veronica S. Brauer, André M. Pessoni, Mateus S. Freitas, Marinaldo P. Cavalcanti-Neto, Laure N. A. Ries, Fausto Almeida
    Journal of Fungi.2023; 9(1): 89.     CrossRef
  • Evidencing New Roles for the Glycosyl-Transferase Cps1 in the Phytopathogenic Fungus Botrytis cinerea
    Matthieu Blandenet, Isabelle R. Gonçalves, Christine Rascle, Jean-William Dupuy, François-Xavier Gillet, Nathalie Poussereau, Mathias Choquer, Christophe Bruel
    Journal of Fungi.2022; 8(9): 899.     CrossRef
Review
REVIEW] Recent paradigm shift in the assembly of bacterial tripartite efflux pumps and the type I secretion system
Inseong Jo , Jin-Sik Kim , Yongbin Xu , Jaekyung Hyun , Kangseok Lee , Nam-Chul Ha
J. Microbiol. 2019;57(3):185-194.   Published online February 26, 2019
DOI: https://doi.org/10.1007/s12275-019-8520-1
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  • 9 Web of Science
  • 7 Crossref
AbstractAbstract
Tripartite efflux pumps and the type I secretion system of Gram-negative bacteria are large protein complexes that span the entire cell envelope. These complexes expel antibiotics and other toxic substances or transport protein toxins from bacterial cells. Elucidating the binary and ternary complex structures at an atomic resolution are crucial to understanding the assembly and working mechanism. Recent advances in cryoelectron microscopy along with the construction of chimeric proteins drastically shifted the assembly models. In this review, we describe the current assembly models from a historical perspective and emphasize the common assembly mechanism for the assembly of diverse tripartite pumps and type I secretion systems.

Citations

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    Elizabeth M. Darby, Eleftheria Trampari, Pauline Siasat, Maria Solsona Gaya, Ilyas Alav, Mark A. Webber, Jessica M. A. Blair
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  • Permeation of Fosfomycin through the Phosphate-Specific Channels OprP and OprO of Pseudomonas aeruginosa
    Vinaya Kumar Golla, Claudio Piselli, Ulrich Kleinekathöfer, Roland Benz
    The Journal of Physical Chemistry B.2022; 126(7): 1388.     CrossRef
  • Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability
    Eric Mandela, Christopher J Stubenrauch, David Ryoo, Hyea Hwang, Eli J Cohen, Von L Torres, Pankaj Deo, Chaille T Webb, Cheng Huang, Ralf B Schittenhelm, Morgan Beeby, JC Gumbart, Trevor Lithgow, Iain D Hay
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  • Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria
    Ilyas Alav, Jessica Kobylka, Miriam S. Kuth, Klaas M. Pos, Martin Picard, Jessica M. A. Blair, Vassiliy N. Bavro
    Chemical Reviews.2021; 121(9): 5479.     CrossRef
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  • Protein-Protein Interactions in the Cytoplasmic Membrane of Escherichia coli: Influence of the Overexpression of Diverse Transporter-Encoding Genes on the Activities of PTS Sugar Uptake Systems
    Mohammad Aboulwafa, Zhongge Zhang, Milton H. Saier Jr.
    Microbial Physiology.2020; 30(1-6): 36.     CrossRef
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Journal Article
Contribution of EmrAB efflux pumps to colistin resistance in Acinetobacter baumannii
Ming-Feng Lin , Yun-You Lin , Chung-Yu Lan
J. Microbiol. 2017;55(2):130-136.   Published online January 26, 2017
DOI: https://doi.org/10.1007/s12275-017-6408-5
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  • 68 Crossref
AbstractAbstract
Efflux pumps play an important role in antimicrobial resistance for Acinetobacter baumannii. However, the function of the Emr pump system and the relationship between Emr and drug resistance has not been characterized in A. baumannii. In this study, four possible groups of emr-like genes were found by searching a genome database. Among them, A1S_ 1772 (emrB) and A1S_1773 (emrA) were demonstrated to be co-transcribed as a single operon. Moreover, during osmotic stress, A1S_1772 showed the largest change in gene expression compared to the other emrB-like genes, and deletion of A1S_1772 (AB ΔemrB) significantly slowed cell growth in 20% sucrose. Using a phenotypic microarray analysis, the AB ΔemrB mutant was more susceptible to colistin and nafcillin, paromomycin, spiramycin, and D,L-serine hydroxmate than the wild type. The spot assay, time kill assay and minimal inhibition concentration determination also indicated that the wild type could tolerate colistin better than the AB ΔemrB mutant. Finally, the increased expression levels of all emrBlike genes, including A1S_0775, A1S_0909, A1S_1772, and A1S_1799, in colistin resistance-induced A. baumannii further supported the possible involvement of the emrB genes in A. baumannii colistin resistance. Together, the Emr pump systems in A. baumannii contribute to adaptation to osmotic stress and resistance to colistin.

Citations

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    Stefanie Gerson, Kai Lucaßen, Julia Wille, Carolina S. Nodari, Danuta Stefanik, Jennifer Nowak, Thorsten Wille, Jonathan W. Betts, Ignasi Roca, Jordi Vila, Jose M. Cisneros, Harald Seifert, Paul G. Higgins
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    Mohamed Abd El-Gawad El-Sayed Ahmed, Lan-Lan Zhong, Cong Shen, Yongqiang Yang, Yohei Doi, Guo-Bao Tian
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    Jing Liang, Xiucai Hu, Aijun Lü, Jingfeng Sun
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Research Support, Non-U.S. Gov't
Mutational inactivation of OprD in carbapenem-resistant Pseudomonas aeruginosa isolates from Korean hospitals
Chi Hyun Kim , Hee Young Kang , Bo Ra Kim , Hyejin Jeon , Yoo Chul Lee , Sang Hwa Lee , Je Chul Lee
J. Microbiol. 2016;54(1):44-49.   Published online January 5, 2016
DOI: https://doi.org/10.1007/s12275-016-5562-5
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AbstractAbstract
This study investigated the mechanisms underlying the carbapenem resistance of bloodstream isolates of Pseudomonas aeruginosa obtained from two Korean hospitals. Of the 79 P. aeruginosa isolates, 22 and 21 were resistant to imipenem and meropenem, respectively. The 22 imipenem-resistant P. aeruginosa isolates were classified into 7 sequence types (STs) and 13 pulsotypes. Twelve imipenem-resistant isolates from one hospital were found to belong to the international clone ST111. Two imipenem-resistant P. aeruginosa ST235 isolates carried the blaIMP-6 gene, but the remaining 20 isolates did not produce carbapenemases. Mutations in the oprD gene and a related decrease in gene expression were found in 21 and 5 isolates, respectively. However, all imipenemresistant P. aeruginosa isolates showed no significant expression of OprD in the outer membrane as compared with that of carbapenem-susceptible PAO1 strain. Overexpression of genes associated with efflux pumps, including mexB, mexD, mexF, and mexY, was not found in any imipenem-resistant isolate. One imipenem-resistant P. aeruginosa isolate overexpressed the ampC gene. Our results show that the low permeability of drugs due to the mutational inactivation of OprD is primarily responsible for carbapenem resistance in bloodstream isolates of P. aeruginosa from Korean hospitals.

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Reviews
MINIREVIEW] Multidrug efflux pumps in Staphylococcus aureus and their clinical implications
Soojin Jang
J. Microbiol. 2016;54(1):1-8.   Published online January 5, 2016
DOI: https://doi.org/10.1007/s12275-016-5159-z
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AbstractAbstract
Antibiotic resistance is rapidly spreading among bacteria such as Staphylococcus aureus, an opportunistic bacterial pathogen that causes a variety of diseases in humans. For the last two decades, bacterial multidrug efflux pumps have drawn attention due to their potential association with clinical multidrug resistance. Numerous researchers have demonstrated efflux-mediated resistance in vitro and in vivo and found novel multidrug transporters using advanced genomic information about bacteria. This article aims to provide a concise summary of multidrug efflux pumps and their important clinical implications, focusing on recent findings concerning S. aureus efflux pumps.

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MINIREVIEW] Molecular architecture of the bacterial tripartite multidrug efflux pump focusing on the adaptor bridging model
Saemee Song , Jin-Sik Kim , Kangseok Lee , Nam-Chul Ha
J. Microbiol. 2015;53(6):355-364.   Published online May 30, 2015
DOI: https://doi.org/10.1007/s12275-015-5248-4
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AbstractAbstract
Gram-negative bacteria expel a wide range of toxic substances through tripartite drug efflux pumps consisting of an inner membrane transporter, an outer membrane channel protein, and a periplasmic adaptor protein. These pumps form tripartite assemblies which can span the entire cell envelope, including the inner and outer membranes. There have been controversial findings regarding the assembly of the individual components in tripartite drug efflux pumps. Recent structural and functional studies have advanced our understanding of the assembly and working mechanisms of the pumps. Here, we re-evaluate the assembly models based on recent structural and functional studies. In particular, this study focuses on the ‘adaptor bridging model’, highlighting the intermeshing cogwheel-like interactions between the tip regions of the outer membrane channel protein and the periplasmic adaptor protein in the hexameric assembly.

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Research Support, Non-U.S. Gov'ts
Effect of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on killing Acinetobacter baumannii by colistin
Young Kyoung Park , Kwan Soo Ko
J. Microbiol. 2015;53(1):53-59.   Published online January 4, 2015
DOI: https://doi.org/10.1007/s12275-015-4498-5
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AbstractAbstract
We investigated the effect of cyanide 3-chlorophenylhydrazone (CCCP) and other efflux pump inhibitors (EPIs) on the colistin susceptibility in Acinetobacter baumannii. While minimum inhibitory concentrations (MICs) of colistin in all colistin-resistant strains decreased significantly with 25 μM of CCCP and 2,4-dinitrophenol (DNP), phenyl-arginine-β- naphthylamide (PAβN), and reserpine did not decrease the colistin MICs. However, CCCP and DNP as well as PAβN and reserpine did not have a significant effect on the MICs of the other agents. Efflux pump gene expressions in colistinresistant strains were not increased compared with those in colistin-susceptible strains. When only 5X MIC of colistin (5 mg/L) was provided to a colistin-susceptible A. baumannii strain, the bacterial cell number was reduced by 9 h after exposure to colistin, but regrowth was observed. When CCCP was added to colistin, bacterial cells were completely killed after 24 to 48 h of incubation, which was not due to the toxicity of CCCP itself. Colistin resistance in A. baumannii may not be due to efflux pumps. Our present study suggests that bacterial cells with reduced metabolic activity by CCCP are more susceptible to colistin in A. baumannii. It may show the possibility that combined therapy with colistin and other antimicrobial agents could effective against A. baumannii infections.

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    Ryo Nishikawa, Toshiyuki Murase, Hiroichi Ozaki
    Poultry Science.2019; 98(11): 5900.     CrossRef
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    Diana Machado, Jéssica Antunes, Ana Simões, João Perdigão, Isabel Couto, Matthew McCusker, Marta Martins, Isabel Portugal, Teresa Pacheco, Judite Batista, Cristina Toscano, Miguel Viveiros
    Journal of Medical Microbiology .2018; 67(6): 740.     CrossRef
  • Multiple Quorum Quenching Enzymes Are Active in the Nosocomial Pathogen Acinetobacter baumannii ATCC17978
    Celia Mayer, Andrea Muras, Manuel Romero, María López, María Tomás, Ana Otero
    Frontiers in Cellular and Infection Microbiology.2018;[Epub]     CrossRef
  • Genomic Insights Into Nitrofurantoin Resistance Mechanisms and Epidemiology in Clinical Enterobacteriaceae
    John Osei Sekyere
    Future Science OA.2018;[Epub]     CrossRef
  • Molecular characterization of fluoroquinolones, macrolides, and imipenem resistance in Haemophilus influenzae: analysis of the mutations in QRDRs and assessment of the extent of the AcrAB-TolC-mediated resistance
    Abdessalam Cherkaoui, Nadia Gaïa, Damien Baud, Stefano Leo, Adrien Fischer, Etienne Ruppe, Patrice François, Jacques Schrenzel
    European Journal of Clinical Microbiology & Infectious Diseases.2018; 37(11): 2201.     CrossRef
  • Overexpression of Stenotrophomonas maltophilia major facilitator superfamily protein MfsA increases resistance to fluoroquinolone antibiotics
    Paiboon Vattanaviboon, Punyawee Dulyayangkul, Skorn Mongkolsuk, Nisanart Charoenlap
    Journal of Antimicrobial Chemotherapy.2018; 73(5): 1263.     CrossRef
  • Mechanisms of Increased Resistance to Chlorhexidine and Cross-Resistance to Colistin following Exposure of Klebsiella pneumoniae Clinical Isolates to Chlorhexidine
    Matthew E. Wand, Lucy J. Bock, Laura C. Bonney, J. Mark Sutton
    Antimicrobial Agents and Chemotherapy.2017;[Epub]     CrossRef
  • Biology of Acinetobacter baumannii: Pathogenesis, Antibiotic Resistance Mechanisms, and Prospective Treatment Options
    Chang-Ro Lee, Jung Hun Lee, Moonhee Park, Kwang Seung Park, Il Kwon Bae, Young Bae Kim, Chang-Jun Cha, Byeong Chul Jeong, Sang Hee Lee
    Frontiers in Cellular and Infection Microbiology.2017;[Epub]     CrossRef
  • Carbonyl Cyanide m-Chlorophenylhydrazine (CCCP) Reverses Resistance to Colistin, but Not to Carbapenems and Tigecycline in Multidrug-Resistant Enterobacteriaceae
    John Osei Sekyere, Daniel G. Amoako
    Frontiers in Microbiology.2017;[Epub]     CrossRef
  • Old drug, new findings: colistin resistance and dependence of Acinetobacter baumannii
    Kwan Soo Ko, Yujin Choi, Ji-Young Lee
    Precision and Future Medicine.2017; 1(4): 159.     CrossRef
  • Effects of Efflux Pump Inhibitors on Colistin Resistance in Multidrug-Resistant Gram-Negative Bacteria
    Wentao Ni, Yanjun Li, Jie Guan, Jin Zhao, Junchang Cui, Rui Wang, Youning Liu
    Antimicrobial Agents and Chemotherapy.2016; 60(5): 3215.     CrossRef
Analysis of cepA Encoding an Efflux Pump-like Protein in Corynebacterium glutamicum
Soo-Yeon Sim , Eun-Ji Hong , Younhee Kim , Heung-Shick Lee
J. Microbiol. 2014;52(4):278-283.   Published online February 17, 2014
DOI: https://doi.org/10.1007/s12275-014-3461-1
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AbstractAbstract
A gene encoding a homolog of purine efflux proteins of Escherichia coli and Bacillus subtilis was identified in the genome of Corynebacterium glutamicum and designated as cepA. The gene encoded a putative protein product, containing 12 transmembrane helixes, which is a typical feature of integral membrane transport proteins. To elucidate the function of the gene, we constructed a cepA deletion mutant (ΔcepA) and a cepA-overexpressing strain and analyzed their physiological characteristics. The cepA gene could be deleted with no critical effect on cell growth. However, the cell yield of a ΔcepA strain was decreased by 10% as compared to that of a strain carrying a cepA-overexpression plasmid (P180-cepA). Further analysis identified increased resistance of the P180-cepA strain to the purine analogues 6-mercaptopurine and 6-mercaptoguanine, but not to 2-aminopurine and purine nucleoside analogues. Moreover, this strain showed increased resistance to the antibiotics nalidixic acid and ampicillin. Collectively, these data suggest that cepA is a novel multidrug resistance gene and probably functions in the efflux of toxic substances from the inside of cells to the environment, thus allowing cells to reach a higher cell yield.

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  • AutoBioTech─A Versatile Biofoundry for Automated Strain Engineering
    Tobias Michael Rosch, Julia Tenhaef, Tim Stoltmann, Till Redeker, Dominic Kösters, Niels Hollmann, Karin Krumbach, Wolfgang Wiechert, Michael Bott, Susana Matamouros, Jan Marienhagen, Stephan Noack
    ACS Synthetic Biology.2024; 13(7): 2227.     CrossRef
Interaction between the α-Barrel Tip of Vibrio vulnificus TolC Homologs and AcrA Implies the Adapter Bridging Model
Seunghwa Lee , Saemee Song , Minho Lee , Soonhye Hwang , Ji-Sun Kim , Nam-Chul Ha , Kangseok Lee
J. Microbiol. 2014;52(2):148-153.   Published online February 1, 2014
DOI: https://doi.org/10.1007/s12275-014-3578-2
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AbstractAbstract
The AcrAB-TolC multidrug efflux pump confers resistance to Escherichia coli against many antibiotics and toxic compounds. The TolC protein is an outer membrane factor that participates in the formation of type I secretion systems. The genome of Vibrio vulnificus encodes two proteins homologous to the E. coli TolC, designated TolCV1 and TolCV2. Here, we show that both TolCV1 and TolCV2 partially complement the E. coli TolC function and physically interact with the membrane fusion protein AcrA, a component of the E. coli AcrAB-TolC efflux pump. Using site-directed mutational analyses and an in vivo cross-linking assay, we demonstrated that the α-barrel tip region of TolC homologs plays a critical role in the formation of functional AcrAB-TolC efflux pumps. Our findings suggest the adapter bridging model as a general assembly mechanism for tripartite drug efflux pumps in Gram-negative bacteria.

Citations

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  • Progress of Antimicrobial Mechanisms of Stilbenoids
    Xiancai Li, Yongqing Li, Binghong Xiong, Shengxiang Qiu
    Pharmaceutics.2024; 16(5): 663.     CrossRef
  • Membrane Efflux Pumps of Pathogenic Vibrio Species: Role in Antimicrobial Resistance and Virulence
    Jerusha Stephen, Manjusha Lekshmi, Parvathi Ammini, Sanath H. Kumar, Manuel F. Varela
    Microorganisms.2022; 10(2): 382.     CrossRef
  • TolCV1 Has Multifaceted Roles During Vibrio vulnificus Infection
    Yue Gong, Rui Hong Guo, Joon Haeng Rhee, Young Ran Kim
    Frontiers in Cellular and Infection Microbiology.2021;[Epub]     CrossRef
  • Recent paradigm shift in the assembly of bacterial tripartite efflux pumps and the type I secretion system
    Inseong Jo, Jin-Sik Kim, Yongbin Xu, Jaekyung Hyun, Kangseok Lee, Nam-Chul Ha
    Journal of Microbiology.2019; 57(3): 185.     CrossRef
  • NaCl promotes antibiotic resistance by reducing redox states in Vibrio alginolyticus
    Jun Yang, Zao‐Hai Zeng, Man‐Jun Yang, Zhi‐Xue Cheng, Xuan‐Xian Peng, Hui Li
    Environmental Microbiology.2018; 20(11): 4022.     CrossRef
  • ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function
    Wen-Jung Lu, Hsuan-Ju Lin, Thamarai Janganan, Cheng-Yi Li, Wei-Chiang Chin, Vassiliy Bavro, Hong-Ting Lin
    International Journal of Molecular Sciences.2018; 19(4): 1000.     CrossRef
  • Functional analysis of Vibrio vulnificus RND efflux pumps homologous to Vibrio cholerae VexAB and VexCD, and to Escherichia coli AcrAB
    Seunghwa Lee, Ji-Hyun Yeom, Sojin Seo, Minho Lee, Sarang Kim, Jeehyeon Bae, Kangseok Lee, Jihwan Hwang
    Journal of Microbiology.2015; 53(4): 256.     CrossRef
  • Molecular architecture of the bacterial tripartite multidrug efflux pump focusing on the adaptor bridging model
    Saemee Song, Jin-Sik Kim, Kangseok Lee, Nam-Chul Ha
    Journal of Microbiology.2015; 53(6): 355.     CrossRef
  • Interaction Mediated by the Putative Tip Regions of MdsA and MdsC in the Formation of a Salmonella-Specific Tripartite Efflux Pump
    Saemee Song, Soonhye Hwang, Seunghwa Lee, Nam-Chul Ha, Kangseok Lee, Eric Cascales
    PLoS ONE.2014; 9(6): e100881.     CrossRef
  • Functional Analysis of TolC Homologs in Vibrio vulnificus
    Seunghwa Lee, Saemee Song, Kangseok Lee
    Current Microbiology.2014; 68(6): 729.     CrossRef
The PseEF Efflux System Is a Virulence Factor of Pseudomonas syringae pv. syringae
Hyosun Cho , Hyojeung Kang
J. Microbiol. 2012;50(1):79-90.   Published online February 27, 2012
DOI: https://doi.org/10.1007/s12275-012-1353-9
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AbstractAbstract
An ATP-binding cassette (ABC) transporter, called the PseEF efflux system, was identified at the left border of the syr-syp genomic island of Pseudomonas syringae pv. syringae strain B301D. The PseEF efflux system was located within a 3.3-kb operon that encodes a periplasmic membrane fusion protein (PseE), and an ABC-type cytoplasmic membrane protein (PseF). The PseEF efflux system exhibited amino acid homology to a putative ABC efflux system (MacAB) of E. coli W3104 with identities of 47.2% (i.e., PseE to MacA) and 57.6% (i.e., PseF to MacB). A nonpolar mutation within the pseF gene was generated by nptII insertional mutagenesis. The resultant mutant strain showed significant reduction in secretion of syringomycin (74%) and syringopeptin (71%), as compared to parental strain B301D. Quantitative real-time RT-PCR was used to determine transcript levels of the syringomycin (syrB1) and syringopeptin (sypA) synthetase genes in strain B301D-HK7 (a pseF mutant). Expression of the sypA gene by mutant strain B301D-HK7 was approximately 6.9% as compared to that of parental strain B301D, while the syrB1 gene expression by mutant strain B301D-HK7 was nearly 14.6%. In addition, mutant strain B301D-HK7 was less virulent by approximately 67% than parental strain B301D in immature cherry fruits. Mutant strain B301D-HK7 was not reduced in resistance to any antibiotics used in this study as compared to parental strain B301D. Expression (transcript levels) of the pseF gene was induced approximately six times by strain B301D grown on syringomycin minimum medium (SRM) supplemented with the plant signal molecules arbutin and D-fructose (SRMAF), as compared to that of strain B301D grown on SRM (in the absence of plant signal molecules). In addition, during infection of bean plants by P. syringae pv. syringae strain B728a, expression of the pseF gene increased at 3 days after inoculation (dai). More than 180-fold induction was observed in transcript levels of the pseF gene by parental strain B728a as compared to strain B728a-SL7 (a salA mutant). Thus, the PseEF efflux system, an ABC-type efflux system, has an important role in secretion of syringomycin and syringopeptin, and is required for full virulence in P. syringae pv. syringae.
Functional Definition of LuxS, an Autoinducer-2 (AI-2) Synthase and Its Role in Full Virulence of Streptococcus suis Serotype 2
Min Cao , Youjun Feng , Changjun Wang , Feng Zheng , Ming Li , Hui Liao , Yinghua Mao , Xiuzhen Pan , Jing Wang , Dan Hu , Fuquan Hu , Jiaqi Tang
J. Microbiol. 2011;49(6):1000-1011.   Published online December 28, 2011
DOI: https://doi.org/10.1007/s12275-011-1523-1
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AbstractAbstract
Quorum sensing is a widespread chemical communication in response to fluctuation of bacterial population density, and has been implicated into bacterial biofilm formation and regulation of expression of virulence factors. The luxS gene product, S-ribosylhomocysteinase, catalizes the last committed step in biosynthetic pathway of autoinducer 2 (AI-2), a signaling molecule for inter-species quorum sensing. We found a luxS homologue in 05ZYH33, an epidemic strain of Streptococcus suis serotype 2 (SS2) in China. A luxS null mutant (ΔluxS) of 05ZYH33 strain was obtained using an approach of homologous recombination. LuxS was determined to be required for AI-2 production in 05ZYH33 strain of S. suis 2. Inactivation of luxS gene led to a wide range of phenotypic changes including thinner capsular walls, increased tolerance to H2O2, reduced adherence capacity to epithelial cells, etc. In particular, loss of LuxS impaired dramatically its full virulence of SS2 in experimental model of piglets, and functional complementation restored it nearly to the level of parent strain. Genome-wide transcriptome analyses suggested that some known virulence factors such as CPS are down-regulated in the ΔluxS mutant, which might in part explain virulence attenuation by luxS deletion. Similarly, 29 of 71 genes with different expression level were proposed to be targets candidate regulated by LuxS/AI-2-dependent quorum sensing.
Periplasmic Domain of CusA in an Escherichia coli Cu+/Ag+ Transporter Has Metal Binding Sites
Bo-Young Yun , Yongbin Xu , Shunfu Piao , Nahee Kim , Jeong-Hyun Yoon , Hyun-Soo Cho , Kangseok Lee , Nam-Chul Ha
J. Microbiol. 2010;48(6):829-835.   Published online January 9, 2011
DOI: https://doi.org/10.1007/s12275-010-0339-8
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AbstractAbstract
The resistance nodulation division (RND)-type efflux systems are utilized in Gram-negative bacteria to export a variety of substrates. The CusCFBA system is the Cu+ and Ag+ efflux system in Escherichia coli, conferring resistance to lethal concentrations of Cu+ and Ag+. The periplasmic component, CusB, which is essential for the assembly of the protein complex, has Cu+ or Ag+ binding sites. The twelve-span membrane protein CusA is a homotrimeric transporter, and has a relatively large periplasmic domain. Here, we constructed the periplasmic domain of CusA by joining two DNA segments and then successfully expressed and purified the protein. Isothermal titration calorimetry experiments revealed Ag+ binding sites with Kds of 10-6-10-5 M. Our findings suggest that the metal binding in the periplasmic domain of CusA might play an important role in the function of the efflux pump.
Identification of Vibrio natriegens uvrA and uvrB Genes and Analysis of Gene Regulation Using Transcriptional Reporter Plasmids
Keryn L. Simons , Susan M. Thomas , Peter A. Anderson
J. Microbiol. 2010;48(5):644-656.   Published online November 3, 2010
DOI: https://doi.org/10.1007/s12275-010-9370-z
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AbstractAbstract
Nucleotide excision repair (NER) rectifies a variety of chemically and structurally distinct DNA lesions. The current model of NER is based upon the enteric bacterium Escherichia coli and there is scarce information about how other bacterial species respond to, and correct, DNA damage. Here we report the isolation and functional analysis of the uvrA and uvrB genes from Vibrio natriegens, a naturally occurring marine bacterium. Genetic studies were completed to assess the repair capabilities of V. natriegens uvrA and uvrB in E. coli uvrA and uvrB mutants. In addition to the genetic studies, transcriptional fusions between the luciferase gene and the 5′ regulatory regions of uvrA and uvrB gene of V. natriegens and E. coli were constructed. Luminescent measurements from E. coli transformed with these constructs showed that whilst the response to UV irradiation of either E. coli or V. natriegens uvrA regulatory sequences was similar, both the rate and induction of luminescence detected from the uvrB regulatory regions differed.
Journal Article
Molecular Characteristics and Resistant Mechanisms of Imipenem-Resistant Acinetobacter baumannii Isolates in Shenyang, China
Jing Ping Zhang , Wan Zhu , Su Fei Tian , Yun Zhuo Chu , Bai Yi Chen
J. Microbiol. 2010;48(5):689-694.   Published online November 3, 2010
DOI: https://doi.org/10.1007/s12275-010-0137-3
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AbstractAbstract
The investigation was carried out to elucidate the molecular characteristics and resistant mechanisms of imipenem-resistant Acinetobacter baumannii. Thirty-seven isolates were collected from January 2007 to December 2007. The homology of the isolates was analyzed by both pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The genes of β-lactamases, adeB, and class 1 integron were polymerase chain reaction amplified. Genotype analysis of the 37 A. baumannii isolates by PFGE revealed the circulation of four PFGE types (A-D) ; the A- and B-type accounted for 48.6% and 40.5%, respectively. MLST showed the existence of three allelic profiles. The agar dilution method was carried out to determine the MIC of imipenem, in the absence or presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 μg/ml). The MICs of the strains to imipenem were between 16 μg/ml and 128 μg/ml. When CCCP was added, a MIC decrease of at least four-fold was observed in 20 isolates, which belonged to the A- or C-type. AdeB and blaPER-1 genes were each detected in 35 isolates, blaOXA-23 gene in 34 isolates and blaOXA-58-like gene in 24 isolates. All isolates harbored blaOXA-51-like genes. No isolates carried the blaIMP-1 gene. Integron was detected in 25 isolates, which mediated the resistance to aminoglycosides and rifampin. The epidemiologic data suggested that the increasing infection of A. baumannii in our hospital was mainly caused by the inter-hospital spread of two epidemic clones. The AdeABC efflux system may be the important factor that leads to the high level of imipenem-resistance in PFGE A-type.

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