Journal of Microbiology

Research Article

Functional importance of Ser323 in cysteine desulfhydrase and cystathionine gamma-lyase MccB of Staphylococcus aureus: Dukwon Lee, Hyojeong Lee, Kyumi Byun, Eun-Su Park, Nam-Chul Ha; J. Microbiol. 2025;63(2):e2411026. Published online February 27, 2025; DOI: https://doi.org/10.71150/jm.2411026

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Abstract PDF Supplementary Material: Pyridoxal 5'-phosphate (PLP)-dependent enzymes participate in various reactions involved in methionine and cysteine metabolism. The representative foodborne pathogen Staphylococcus aureus expresses the PLP-dependent enzyme MccB, which exhibits both cystathionine gamma-lyase (CGL) and cysteine desulfhydrase activities. In this study, we investigated the role of Ser323 in MccB, a conserved residue in many PLP-dependent enzymes in the transsulfuration pathway. Our findings reveal that Ser323 forms a hydrogen bond with the catalytic lysine in the absence of PLP, and upon internal aldimine formation, PLP-bound lysine is repositioned away from Ser323. Substituting Ser323 with alanine abolishes the enzymatic activity, similar to mutations at the catalytic lysine site. Spectroscopic analysis suggests that Ser323 is essential for the rapid formation of the internal aldimine with lysine in wild-type MccB. This study highlights the crucial role of Ser323 in catalysis, with broader implications for other PLP-dependent enzymes, and enhances our understanding of the molecular mechanisms involved in the selective control of foodborne pathogenic bacteria.

Journal Articles

Structural and Functional Analyses of the Flavoprotein Disulfide Reductase FN0820 of Fusobacterium nucleatum: Hyunwoo Shin , Yeongjin Baek , Dukwon Lee , Yongbin Xu , Yonghoon Kwon , Inseong Jo , Nam-Chul Ha; J. Microbiol. 2023;61(12):1033-1041. Published online December 20, 2023; DOI: https://doi.org/10.1007/s12275-023-00095-9

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Escherichia coli RclA and Staphylococcus aureus MerA are part of the Group I flavoprotein disulfide reductase (FDR) family and have been implicated in the contribution to bacterial pathogenesis by defending against the host immune response. Fusobacterium nucleatum is a pathogenic, anaerobic Gram-negative bacterial species commonly found in the human oral cavity and gastrointestinal tract. In this study, we discovered that the F. nucleatum protein FN0820, belonging to the Group I FDR family, exhibited a higher activity of a Cu2+- dependent NADH oxidase than E. coli RclA. Moreover, FN0820 decreased the dissolved oxygen level in the solution with higher NADH oxidase activity. We found that L-tryptophan and its analog 5-hydroxytryptophan inhibit the FN0820 activities of NADH oxidase and the concomitant reduction of oxygen. Our results have implications for developing new treatment strategies against pathogens that defend the host immune response with Group I FDRs.

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The role of metals in hypothiocyanite resistance in Escherichia coli
Michael J. Gray, Laurie E. Comstock
Journal of Bacteriology.2024;[Epub] CrossRef

Crystal structure of the phage-encoded N-acetyltransferase in complex with acetyl-CoA, revealing a novel dimeric arrangement: Nayeon Ki , Inseong Jo , Yongseong Hyun , Jinwook Lee , Nam-Chul Ha , Hyun-Myung Oh; J. Microbiol. 2022;60(7):746-755. Published online July 4, 2022; DOI: https://doi.org/10.1007/s12275-022-2030-2

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Abstract

Bacteriophages employ diverse mechanisms to facilitate the proliferation of bacteriophages. The Salmonella-infecting phage SPN3US contains a putative N-acetyltransferase, which is widely found in bacteriophages. However, due to low sequence similarity to the N-acetyltransferases from bacteria and eukaryotic cells, the structure and function of phage-encoded acetyltransferases are mainly unknown. This study determines the crystal structure of the putative N-acetyltransferase of SPN3US in complex with acetyl-CoA. The crystal structure showed a novel homodimeric arrangement stabilized by exchanging the C-terminal α-helix within the dimer. The following biochemical analyses suggested that the phageencoded acetyltransferase might have a very narrow substrate specificity. Further studies are required to reveal the biochemical activity, which would help elucidate the interaction between the phage and host bacteria in controlling pathogenic bacteria.

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Posttranslational modifications in bacteria during phage infection
Hannelore Longin, Nand Broeckaert, Vera van Noort, Rob Lavigne, Hanne Hendrix
Current Opinion in Microbiology.2024; 77: 102425. CrossRef

Crystal structure of the nuclease and capping domain of SbcD from Staphylococcus aureus: Jinwook Lee , Inseong Jo , Jinsook Ahn , Seokho Hong , Soyeon Jeong , Aeran Kwon , Nam-Chul Ha; J. Microbiol. 2021;59(6):584-589. Published online April 20, 2021; DOI: https://doi.org/10.1007/s12275-021-1012-0

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Abstract

The SbcCD complex is an essential component of the DNA double-strand break (DSB) repair system in bacteria. The bacterial SbcCD complex recognizes and cleaves the DNA ends in DSBs by ATP-dependent endo- and exonuclease activities as an early step of the DNA repair process. SbcD consists of nuclease, capping, and helix-loop-helix domains. Here, we present the crystal structure of a SbcD fragment from Staphylococcus aureus, which contained nuclease and capping domains, at a resolution of 2.9 Å. This structure shows a dimeric assembly similar to that of the corresponding domains of SbcD from Escherichia coli. The S. aureus SbcD fragment exhibited endonuclease activities on supercoiled DNA and exonuclease activity on linear and nicked DNA. This study contributes to the understanding of the molecular basis for how bacteria can resist sterilizing treatment, causing DNA damage.

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Staphylococcus aureus SOS response: Activation, impact, and drug targets
Kaiying Cheng, Yukang Sun, Huan Yu, Yingxuan Hu, Yini He, Yuanyuan Shen
mLife.2024; 3(3): 343. 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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Abstract

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.

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Molecular mechanisms of antibiotic resistance revisited
Elizabeth M. Darby, Eleftheria Trampari, Pauline Siasat, Maria Solsona Gaya, Ilyas Alav, Mark A. Webber, Jessica M. A. Blair
Nature Reviews Microbiology.2023; 21(5): 280. CrossRef
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
eLife.2022;[Epub] CrossRef
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
Biotechnological applications of type 1 secretion systems
Zohreh Pourhassan N., Sander H.J. Smits, Jung Hoon Ahn, Lutz Schmitt
Biotechnology Advances.2021; 53: 107864. CrossRef
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
Perspectives towards antibiotic resistance: from molecules to population
Joon-Hee Lee
Journal of Microbiology.2019; 57(3): 181. CrossRef

Journal Articles

Crystal structure of Streptomyces coelicolor RraAS2, an unusual member of the RNase E inhibitor RraA protein family: Nohra Park , Jihune Heo , Saemee Song , Inseong Jo , Kangseok Lee , Nam-Chul Ha; J. Microbiol. 2017;55(5):388-395. Published online April 29, 2017; DOI: https://doi.org/10.1007/s12275-017-7053-8

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Abstract

Bacterial ribonuclease E (RNase E) plays a crucial role in the processing and decay of RNAs. A small protein named RraA negatively regulates the activity of RNase E via protein-protein interaction in various bacteria. Recently, RraAS1 and RraAS2, which are functional homologs of RraA from Escherichia coli, were identified in the Gram-positive species Streptomyces coelicolor. RraAS1 and RraAS2 inhibit RNase ES ribonuclease activity in S. coelicolor. RraAS1 and RraAS2 have a C-termi-nal extension region unlike typical bacterial RraA proteins. In this study, we present the crystal structure of RraAS2, ex-hibiting a hexamer arranged in a dimer of trimers, consistent with size exclusion chromatographic results. Importantly, the C-terminal extension region formed a long α-helix at the junction of the neighboring subunit, which is similar to the trimeric RraA orthologs from Saccharomyces cerevisiae. Trun-cation of the C-terminal extension region resulted in loss of RNase ES inhibition, demonstrating its crucial role. Our find-ings present the first bacterial RraA that has a hexameric assembly with a C-terminal extension α-helical region, which plays an essential role in the regulation of RNase ES activity in S. coelicolor.

Citations

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Relaxed Cleavage Specificity of Hyperactive Variants of Escherichia coli RNase E on RNA I
Dayeong Bae, Hana Hyeon, Eunkyoung Shin, Ji-Hyun Yeom, Kangseok Lee
Journal of Microbiology.2023; 61(2): 211. CrossRef
An oxidative metabolic pathway of 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEHU) from alginate in an alginate-assimilating bacterium
Ryuji Nishiyama, Takao Ojima, Yuki Ohnishi, Yasuhiro Kumaki, Tomoyasu Aizawa, Akira Inoue
Communications Biology.2021;[Epub] CrossRef
The coordinated action of RNase III and RNase G controls enolase expression in response to oxygen availability in Escherichia coli
Minho Lee, Minju Joo, Minji Sim, Se-Hoon Sim, Hyun-Lee Kim, Jaejin Lee, Minkyung Ryu, Ji-Hyun Yeom, Yoonsoo Hahn, Nam-Chul Ha, Jang-Cheon Cho, Kangseok Lee
Scientific Reports.2019;[Epub] CrossRef
RNase G controls tpiA mRNA abundance in response to oxygen availability in Escherichia coli
Jaejin Lee, Dong-Ho Lee, Che Ok Jeon, Kangseok Lee
Journal of Microbiology.2019; 57(10): 910. CrossRef
Functional implications of hexameric assembly of RraA proteins from Vibrio vulnificus
Saemee Song, Seokho Hong, Jinyang Jang, Ji-Hyun Yeom, Nohra Park, Jaejin Lee, Yeri Lim, Jun-Yeong Jeon, Hyung-Kyoon Choi, Minho Lee, Nam-Chul Ha, Kangseok Lee, Eric Cascales
PLOS ONE.2017; 12(12): e0190064. CrossRef

RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity: Jihune Heo , Daeyoung Kim , Minju Joo , Boeun Lee , Sojin Seo , Jaejin Lee , Saemee Song , Ji-Hyun Yeom , Nam-Chul Ha , Kangseok Lee; J. Microbiol. 2016;54(10):660-666. Published online September 30, 2016; DOI: https://doi.org/10.1007/s12275-016-6417-9

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Abstract

RraA is a protein inhibitor of RNase E (Rne), which catalyzes the endoribonucleolytic cleavage of a large proportion of RNAs in Escherichia coli. The antibiotic‐producing bacterium Streptomyces coelicolor also contains homologs of RNase E and RraA, designated as RNase ES (Rns), RraAS1, and RraAS2, respectively. Here, we report that RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity. Analyses of the steady-state level of RNase E substrates indicated that coexpression of RraAS2 in E. coli cells overproducing Rns effectively inhibits the ribonucleolytic activity of full-length RNase ES, but its inhibitory effects were moderate or undetectable on other truncated forms of Rns, in which the N- or/and C-terminal scaffold domain was deleted. In addition, RraAS2 more efficiently inhibited the in vitro ribonucleolytic activity of RNase ES than that of a truncated form containing the catalytic domain only. Coimmunoprecipitation and in vivo cross-linking experiments further showed necessity of both scaffold domains of RNase ES for high-affinity binding of RraAS2 to the enzyme, resulting in decreased RNA-binding capacity of RNase ES. Our results indicate that RraAS2 is a protein inhibitor of RNase ES and provide clues to how this inhibitor affects the ribonucleolytic activity of RNase ES.

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Identification of the global regulatory roles of RraA via the integrative transcriptome and proteome in Vibrio alginolyticus
Huizhen Chen, Qian Gao, Bing Liu, Ying Zhang, Jianxiang Fang, Songbiao Wang, Youqi Chen, Chang Chen, Nicolas E. Buchler
mSphere.2024;[Epub] CrossRef
Streptomyces RNases – Function and impact on antibiotic synthesis
George H. Jones
Frontiers in Microbiology.2023;[Epub] CrossRef
Regulator of RNase E activity modulates the pathogenicity of Salmonella Typhimurium
Jaejin Lee, Eunkyoung Shin, Ji-Hyun Yeom, Jaeyoung Park, Sunwoo Kim, Minho Lee, Kangseok Lee
Microbial Pathogenesis.2022; 165: 105460. CrossRef
Regulator of ribonuclease activity modulates the pathogenicity of Vibrio vulnificus
Jaejin Lee, Eunkyoung Shin, Jaeyeong Park, Minho Lee, Kangseok Lee
Journal of Microbiology.2021; 59(12): 1133. CrossRef
Divergent rRNAs as regulators of gene expression at the ribosome level
Wooseok Song, Minju Joo, Ji-Hyun Yeom, Eunkyoung Shin, Minho Lee, Hyung-Kyoon Choi, Jihwan Hwang, Yong-In Kim, Ramin Seo, J. Eugene Lee, Christopher J. Moore, Yong-Hak Kim, Seong-il Eyun, Yoonsoo Hahn, Jeehyeon Bae, Kangseok Lee
Nature Microbiology.2019; 4(3): 515. CrossRef
RraAS1 inhibits the ribonucleolytic activity of RNase ES by interacting with its catalytic domain in Streptomyces coelicolor
Sojin Seo, Daeyoung Kim, Wooseok Song, Jihune Heo, Minju Joo, Yeri Lim, Ji-Hyun Yeom, Kangseok Lee
Journal of Microbiology.2017; 55(1): 37. CrossRef
Bdm-Mediated Regulation of Flagellar Biogenesis in Escherichia coli and Salmonella enterica Serovar Typhimurium
Jaejin Lee, Dae-Jun Kim, Ji-Hyun Yeom, Kangseok Lee
Current Microbiology.2017; 74(9): 1015. CrossRef
Functional implications of hexameric assembly of RraA proteins from Vibrio vulnificus
Saemee Song, Seokho Hong, Jinyang Jang, Ji-Hyun Yeom, Nohra Park, Jaejin Lee, Yeri Lim, Jun-Yeong Jeon, Hyung-Kyoon Choi, Minho Lee, Nam-Chul Ha, Kangseok Lee, Eric Cascales
PLOS ONE.2017; 12(12): e0190064. CrossRef
Crystal structure of Streptomyces coelicolor RraAS2, an unusual member of the RNase E inhibitor RraA protein family
Nohra Park, Jihune Heo, Saemee Song, Inseong Jo, Kangseok Lee, Nam-Chul Ha
Journal of Microbiology.2017; 55(5): 388. CrossRef

Review

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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Abstract

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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Structural Features and Energetics of the Periplasmic Entrance Opening of the Outer Membrane Channel TolC Revealed by Molecular Dynamics Simulation and Markov State Model Analysis
Jingwei Weng, Wenning Wang
Journal of Chemical Information and Modeling.2019; 59(5): 2359. 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
Antibiotic Hybrids: the Next Generation of Agents and Adjuvants against Gram-Negative Pathogens?
Ronald Domalaon, Temilolu Idowu, George G. Zhanel, Frank Schweizer
Clinical Microbiology Reviews.2018;[Epub] CrossRef
Genetic identification of factors for extracellular cellulose accumulation in the thermophilic cyanobacterium Thermosynechococcus vulcanus: proposal of a novel tripartite secretion system
Kaisei Maeda, Jyunya Tamura, Yukiko Okuda, Rei Narikawa, Takafumi Midorikawa, Masahiko Ikeuchi
Molecular Microbiology.2018; 109(1): 121. CrossRef
Switch Loop Flexibility Affects Substrate Transport of the AcrB Efflux Pump
Reinke T. Müller, Timothy Travers, Hi-jea Cha, Joshua L. Phillips, S. Gnanakaran, Klaas M. Pos
Journal of Molecular Biology.2017; 429(24): 3863. CrossRef
Molecular Rationale behind the Differential Substrate Specificity of Bacterial RND Multi-Drug Transporters
Venkata Krishnan Ramaswamy, Attilio V. Vargiu, Giuliano Malloci, Jürg Dreier, Paolo Ruggerone
Scientific Reports.2017;[Epub] CrossRef
Structure of the MacAB–TolC ABC-type tripartite multidrug efflux pump
Anthony W. P. Fitzpatrick, Salomé Llabrés, Arthur Neuberger, James N. Blaza, Xiao-Chen Bai, Ui Okada, Satoshi Murakami, Hendrik W. van Veen, Ulrich Zachariae, Sjors H. W. Scheres, Ben F. Luisi, Dijun Du
Nature Microbiology.2017;[Epub] CrossRef
Structural Basis for the Serratia marcescens Lipase Secretion System: Crystal Structures of the Membrane Fusion Protein and Nucleotide-Binding Domain
Daichi Murata, Hiroyuki Okano, Clement Angkawidjaja, Masato Akutsu, Shun-ichi Tanaka, Kenyu Kitahara, Takuya Yoshizawa, Hiroyoshi Matsumura, Yuji Kado, Eiichi Mizohata, Tsuyoshi Inoue, Satoshi Sano, Yuichi Koga, Shigenori Kanaya, Kazufumi Takano
Biochemistry.2017; 56(47): 6281. CrossRef
The Crystal Structure of the YknZ Extracellular Domain of ABC Transporter YknWXYZ from Bacillus amyloliquefaciens
Yongbin Xu, Jianyun Guo, Lulu Wang, Rui Jiang, Xiaoling Jin, Jing Liu, Shengdi Fan, Chun-Shan Quan, Nam-Chul Ha, Bostjan Kobe
PLOS ONE.2016; 11(5): e0155846. CrossRef

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

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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Abstract

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 α-Barrel Tip Region of Escherichia coli TolC Homologs of Vibrio vulnificus Interacts with the MacA Protein to Form the Functional Macrolide-Specific Efflux Pump MacAB-TolC: Minho Lee , Hyun-Lee Kim , Saemee Song , Minju Joo , Seunghwa Lee , Daeyoung Kim , Yoonsoo Hahn , Nam-Chul Ha , Kangseok Lee; J. Microbiol. 2013;51(2):154-159. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2699-3

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Abstract: TolC and its homologous family of proteins are outer membrane factors that are essential for exporting small molecules and toxins across the outer membrane in Gram-negative bacteria. Two open reading frames in the Vibrio vulnificus genome that encode proteins homologous to Escherichia coli TolC, designated TolCV1 and TolCV2, have 51.3% and 29.6% amino acid identity to TolC, respectively. In this study, we show that TolCV1 and TolCV2 functionally and physically interacted with the membrane fusion protein, MacA, a component of the macrolide-specific MacAB-TolC pump of E. coli. We further show that the conserved residues located at the aperture tip region of the α-hairpin of TolCV1 and TolCV2 played an essential role in the formation of the functional MacAB-TolC pump using site-directed mutational analyses. Our findings suggest that these outer membrane factors have conserved tip-to-tip interaction with the MacA membrane fusion protein for action of the drug efflux pump in Gramnegative bacteria.

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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Abstract: 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.

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