Journal of Microbiology

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Pycnogenol reduces the expression of P. aeruginosa T3SS and inflammatory response in NCI-H292 cells: Seung-Ho Kim, Da Yun Seo, Sang-Bae Han, Un-Hwan Ha, Ji-Won Park, Kyung-Seop Ahn; J. Microbiol. 2025;63(10):2503004. Published online September 19, 2025; DOI: https://doi.org/10.71150/jm.2503004

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Abstract PDF Supplementary Material: Nosocomial infections caused by Pseudomonas aeruginosa (P. aeruginosa) have become increasingly common, particularly among immunocompromised individuals, who experience high mortality rates and prolonged treatment durations due to the limited availability of effective therapies. In this study, we screened for anti-ExoS compounds targeting P. aeruginosa and identified pycnogenol (PYC) as a potent inhibitor of the type III secretion system (T3SS), a major virulence mechanism responsible for the translocation of effectors such as ExoS. Using ELISA, western blotting, and real-time PCR analyses in both P. aeruginosa and infected H292 cells, we found that PYC significantly reduced T3SS activity. Mechanistically, PYC suppressed the transcription of T3SS-related genes by downregulating exsA expression in P. aeruginosa. Furthermore, pretreatment with PYC attenuated the cytotoxic effects and reduced the expression of proinflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-18 (IL-18), in P. aeruginosa-infected H292 cells. These effects were associated with the inhibition of NF-κB signaling and inflammasome activation. Taken together, our findings suggest that PYC may serve as a promising therapeutic candidate against P. aeruginosa infections by targeting T3SS-mediated virulence and modulating host inflammatory responses.

Alizarin, which reduces ExoS, attenuates inflammation by P. aeruginosa in H292 cells: Seung-Ho Kim, Hye In Ahn, Jae-Hoon Oh, Da Yun Seo, Jung-Hee Kim, Ok-kyoung Kwon, Ji-Won Park, Kyung-Seop Ahn; J. Microbiol. 2025;63(5):e2411012. Published online May 27, 2025; DOI: https://doi.org/10.71150/jm.2411012

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Pseudomonas aeruginosa (P. aeruginosa) is resistant to several drugs as well as antibiotics and is thus classified as multidrug resistant and extensively drug resistant. These bacteria have a secretion system called the "type 3 secretion system (T3SS)", which facilitates infection by delivering an effector protein. ExoenzymeS (ExoS) is known to induce cell death and activate caspase-1. In particular, patients infected with P. aeruginosa develop diseases associated with high mortality, such as pneumonia, because no drug targets an ExoS or T3SS. We selected natural compounds to treat T3SS-mediated pneumonia and chose alizarin, a red dye. We confirmed the effects of alizarin on T3SS by bacterial PCR and ELISA. It was confirmed that alizarin regulates ExoS by inhibiting exsA but also popD and pscF. Furthermore, in infected H292 cells, it not only attenuates inflammation by inhibiting lipopolysaccharide (LPS)-induced phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 but also interferes with the level of ExoS delivered into the host and modulates caspase-1. We confirmed this result and determined that it led to decreases in proinflammatory cytokines such as Interleukin-1beta (IL-1β), Interleukin-6 (IL-6), and Interleukin-18 (IL-18). Therefore, we suggest that alizarin is a suitable drug for treating pneumonia caused by P. aeruginosa because it helps to attenuate inflammation by regulating T3SS and NF-κB signaling.

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Beyond pathogenicity: applications of the type III secretion system (T3SS) of Pseudomonas aeruginosa
Tianqi Su, Lin Zhang, Jie Shen, Danyu Qian, Yulei Guo, Zhenpeng Li
Frontiers in Microbiology.2025;[Epub] CrossRef

Research Article

Enoxacin adversely affects Salmonella enterica virulence and host pathogenesis through interference with type III secretion system type II (T3SS-II) and disruption of translocation of Salmonella Pathogenicity Island-2 (SPI2) effectors: El-Sayed Khafagy, Gamal A. Soliman, Maged S. Abdel-Kader, Mahmoud M. Bendary, Wael A. H. Hegazy, Momen Askoura; J. Microbiol. 2025;63(2):e2410015. Published online February 27, 2025; DOI: https://doi.org/10.71150/jm.2410015

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Abstract PDF: Salmonella enterica is a clinically significant oro-fecal pathogen that causes a wide variety of illnesses and can lead to epidemics. S. enterica expresses a lot of virulence factors that enhance its pathogenesis in host. For instance, S. enterica employs a type three secretion system (T3SS) to translocate a wide array of effector proteins that could change the surrounding niche ensuring suitable conditions for the thrive of Salmonella infection. Many antimicrobials have been recently introduced to overcome the annoying bacterial resistance to antibiotics. Enoxacin is member of the second-generation quinolones that possesses a considerable activity against S. enterica. The present study aimed to evaluate the effect of enoxacin at sub-minimum inhibitory concentration (sub-MIC) on S. enterica virulence capability and pathogenesis in host. Enoxacin at sub-MIC significantly diminished both Salmonella invasion and intracellular replication within the host cells. The observed inhibitory effect of enoxacin on S. enterica internalization could be attributed to its ability to interfere with translocation of the T3SS effector proteins. These results were further confirmed by the finding that enoxacin at sub-MIC down-regulated the expression of the genes encoding for T3SS-type II (T3SS-II). Moreover, enoxacin at sub-MIC lessened bacterial adhesion to abiotic surface and biofilm formation which indicates a potential anti-virulence activity. Importantly, in vivo results showed a significant ability of enoxacin to protect mice against S. enterica infection and decreased bacterial colonization within animal tissues. In nutshell, current findings shed light on an additional mechanism of enoxacin at sub-MIC by interfering with Salmonella intracellular replication. The outcomes presented herein could be further invested in conquering bacterial resistance and open the door for additional effective clinical applications.

Journal Articles

Comparative Secretory Efficiency of Two Chitosanase Signal Peptides from Bacillus subtilis in Escherichia coli: Tae-Yang Eom, Yehui Gang, Youngdeuk Lee, Yoon-Hyeok Kang, Eunyoung Jo, Svini Dileepa Marasinghe, Heung Sik Park, Gun-Hoo Park, Chulhong Oh; J. Microbiol. 2024;62(12):1155-1164. Published online November 25, 2024; DOI: https://doi.org/10.1007/s12275-024-00186-1

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The production of recombinant proteins in Escherichia coli is often challenged by cytoplasmic expression due to proteolytic degradation and inclusion body formation. Extracellular expression can overcome these problems by simplifying downstream processing and improving protein yields. This study aims to compare the efficiency of two Bacillus subtilis chitosanase signal peptides in mediating extracellular secretion in E. coli. We identified a naturally occurring mutant signal peptide (mCsn2-SP) from B. subtilis CH2 chitosanase (CH2CSN), which is characterized by a deletion of six amino acids in the N-region relative to the signal peptide (Csn1-SP) from B. subtilis CH1 chitosanase (CH1CSN). The CH1CSN and CH2CSN genes were cloned into the pET-11a vector and protein secretion was evaluated in E. coli BL21(DE3) host cells. Expression was induced with 0.1 mM and 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30 °C for one and three days. CH2CSN showed higher secretion levels compared to CH1CSN under all experimental conditions, especially with 0.1 mM IPTG induction for 3 days, which resulted in a 2.37-fold increase in secretion. Furthermore, it was demonstrated that mCsn2-SP is capable of secreting human Cu,Zn-superoxide dismutase (hSOD) in E. coli BL21(DE3) and successfully translocating it to the periplasmic region. This study represents the inaugural investigation into the utilisation of a naturally modified signal peptide, thereby corroborating the assertion that signal peptide deletion variants can influence protein secretion efficiency. Furthermore, the findings substantiate the proposition that such variants can serve as a viable alternative for the secretion of heterologous proteins in E. coli.

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Signal Peptides: From Molecular Mechanisms to Applications in Protein and Vaccine Engineering
Shuai Zhang, Zhihui He, Hui Wang, Jingbo Zhai
Biomolecules.2025; 15(6): 897. CrossRef

Relaxed Cleavage Specificity of Hyperactive Variants of Escherichia coli RNase E on RNA I: Dayeong Bae , Hana Hyeon , Eunkyoung Shin , Ji&# , Kangseok Lee; J. Microbiol. 2023;61(2):211-220. Published online February 22, 2023; DOI: https://doi.org/10.1007/s12275-023-00013-z

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RNase E is an essential enzyme in Escherichia coli. The cleavage site of this single-stranded specific endoribonuclease is well-characterized in many RNA substrates. Here, we report that the upregulation of RNase E cleavage activity by a mutation that affects either RNA binding (Q36R) or enzyme multimerization (E429G) was accompanied by relaxed cleavage specificity. Both mutations led to enhanced RNase E cleavage in RNA I, an antisense RNA of ColE1-type plasmid replication, at a major site and other cryptic sites. Expression of a truncated RNA I with a major RNase E cleavage site deletion at the 5′-end (RNA I- 5) resulted in an approximately twofold increase in the steady-state levels of RNA I- 5 and the copy number of ColE1-type plasmid in E. coli cells expressing wild-type or variant RNase E compared to those expressing RNA I. These
results
indicate that RNA I- 5 does not efficiently function as an antisense RNA despite having a triphosphate group at the 5′-end, which protects the RNA from ribonuclease attack. Our study suggests that increased cleavage rates of RNase E lead to relaxed cleavage specificity on RNA I and the inability of the cleavage product of RNA I as an antisense regulator in vivo does not stem from its instability by having 5′-monophosphorylated end.

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Engineering an Escherichia coli based in vivo mRNA manufacturing platform
Edward Curry, George Muir, Jixin Qu, Zoltán Kis, Martyn Hulley, Adam Brown
Biotechnology and Bioengineering.2024; 121(6): 1912. CrossRef

The novel antifungal agent AB-22 displays in vitro activity against hyphal growth and biofilm formation in Candida albicans and potency for treating systemic candidiasis: Kyung-Tae Lee , Dong-Gi Lee , Ji Won Choi , Jong-Hyun Park , Ki Duk Park , Jong-Seung Lee , Yong-Sun Bahn; J. Microbiol. 2022;60(4):438-443. Published online March 14, 2022; DOI: https://doi.org/10.1007/s12275-022-2016-0

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Systemic candidiasis, which is mainly caused by Candida albicans, is a serious acute fungal infection in the clinical setting. In a previous study, we reported that compound 22h (designated as AB-22 in this study), a vinyl sulfate compound, is a fast-acting fungicidal agent against a broad spectrum of fungal pathogens. In this study, we aimed to further analyze the in vitro and in vivo efficacy of AB-22 against filamentation, biofilm formation, and virulence of C. albicans. Under in vitro hyphal growth-inducing condition, AB-22 effectively inhibited germ tube formation and hyphal growth, which are required for the initiation of biofilm formation. Indeed, AB-22 significantly suppressed C. albicans biofilm formation in a dose-dependent manner. Moreover, AB-22 treatment inhibited the normal induction of ALS3, HWP1, and ECE1, which are all required for hyphal transition in C. albicans. Furthermore, AB-22 treatment increased the survival of mice systemically infected with C. albicans. In conclusion, in addition to its fungicidal activity, AB-22 inhibits filamentation and biofilm formation in C. albicans, which could collectively contribute to its potent in vivo efficacy against systemic candidiasis.

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Preparation and analysis of quinoa active protein (QAP) and its mechanism of inhibiting Candida albicans from a transcriptome perspective
Xufei Zhang, Chunmei Zheng, Wenxuan Ge, Xueying Li, Xiuzhang Wang, Yanxia Sun, Xiaoyong Wu
PeerJ.2025; 13: e18961. CrossRef
Inhibition of candidalysin production by methoxy-apo-enterobactin from Streptomyces ambofaciens CJD34 as a novel antifungal strategy against Candida albicans
Eui-Seong Kim, Hyeongju Jeong, Mustansir Abbas, Soohyun Um, Juntack Oh, Kyuho Moon, Kyung-Tae Lee
Journal of Microbiology.2025; 63(6): e2504019. CrossRef

Novosphingobium sp. PP1Y as a novel source of outer membrane vesicles: Federica De Lise , Francesca Mensitieri , Giulia Rusciano , Fabrizio Dal Piaz , Giovanni Forte , Flaviana Di Lorenzo , Antonio Molinaro , Armando Zarrelli , Valeria Romanucci , Valeria Cafaro , Antonio Sasso , Amelia Filippelli , Alberto Di Donato , Viviana Izzo; J. Microbiol. 2019;57(6):498-508. Published online May 27, 2019; DOI: https://doi.org/10.1007/s12275-019-8483-2

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Outer membrane vesicles (OMVs) are nanostructures of 20– 200 nm diameter deriving from the surface of several Gramnegative bacteria. OMVs are emerging as shuttles involved in several mechanisms of communication and environmental adaptation. In this work, OMVs were isolated and characterized from Novosphingobium sp. PP1Y, a Gram-negative non-pathogenic microorganism lacking LPS on the outer membrane surface and whose genome was sequenced and annotated. Scanning electron microscopy performed on samples obtained from a culture in minimal medium highlighted the presence of PP1Y cells embedded in an extracellular matrix rich in vesicular structures. OMVs were collected from the exhausted growth medium during the mid-exponential phase, and purified by ultracentrifugation on a sucrose gradient. Atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis showed that purified PP1Y OMVs had a spherical morphology with a diameter of ca. 150 nm and were homogenous in size and shape. Moreover, proteomic and fatty acid analysis of purified OMVs revealed a specific biochemical “fingerprint”, suggesting interesting details concerning their biogenesis and physiological role. Moreover, these extracellular nanostructures do not appear to be cytotoxic on HaCaT cell line, thus paving the way to their future use as novel drug delivery systems.

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Outer membrane vesicles from Escherichia coli as a presentation platform for AR-23 antiviral peptide
Francesca Mensitieri, Federica Dell’Annunziata, Giulia Gaudino, Veronica Folliero, Gianluigi Franci, Fabrizio Dal Piaz, Viviana Izzo
Frontiers in Molecular Biosciences.2025;[Epub] CrossRef
Proteomic analysis of meropenem-induced outer membrane vesicles released by carbapenem-resistant Klebsiella pneumoniae
Fangfang Fan, Guangzhang Chen, Siqian Deng, Li Wei, Mariola J. Ferraro
Microbiology Spectrum.2024;[Epub] CrossRef
LuxR402 of Novosphingobium sp. HR1a regulates the correct configuration of cell envelopes
Ana Segura, Lázaro Molina
Frontiers in Microbiology.2023;[Epub] CrossRef
Genomic and physiological characterization of Novosphingobium terrae sp. nov., an alphaproteobacterium isolated from Cerrado soil containing a mega-sized chromid
Aline Belmok, Felipe Marques de Almeida, Rodrigo Theodoro Rocha, Carla Simone Vizzotto, Marcos Rogério Tótola, Marcelo Henrique Soller Ramada, Ricardo Henrique Krüger, Cynthia Maria Kyaw, Georgios J. Pappas
Brazilian Journal of Microbiology.2023; 54(1): 239. CrossRef
Outer Membrane Vesicles Derived from Klebsiella pneumoniae Are a Driving Force for Horizontal Gene Transfer
Federica Dell’Annunziata, Carmela Dell’Aversana, Nunzianna Doti, Giuliana Donadio, Fabrizio Dal Piaz, Viviana Izzo, Anna De Filippis, Marilena Galdiero, Lucia Altucci, Giovanni Boccia, Massimiliano Galdiero, Veronica Folliero, Gianluigi Franci
International Journal of Molecular Sciences.2021; 22(16): 8732. CrossRef

ZntR positively regulates T6SS4 expression in Yersinia pseudotuberculosis: Tietao Wang , Keqi Chen , Fen Gao , Yiwen Kang , Muhammad Tausif Chaudhry , Zhuo Wang , Yao Wang , Xihui Shen; J. Microbiol. 2017;55(6):448-456. Published online March 10, 2017; DOI: https://doi.org/10.1007/s12275-017-6540-2

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The type VI secretion system (T6SS) is a widespread and versatile protein secretion system found in most Gram- negative bacteria. Studies of T6SS have mainly focused on its role in virulence toward host cells and inter-bacterial inter-actions, but studies have also shown that T6SS4 in Yersinia pseudotuberculosis participates in the acquisition of zinc ions to alleviate the accumulation of hydroxyl radicals induced by multiple stressors. Here, by comparing the gene expression patterns of wild-type and zntR mutant Y. pseudotubercu-losis cells using RNA-seq analysis, T6SS4 and 17 other bio-logical processes were found to be regulated by ZntR. T6SS4 was positively regulated by ZntR in Y. pseudotuberculosis, and further investigation demonstrated that ZntR regulates T6SS4 by directly binding to its promoter region. T6SS4 ex-pression is regulated by zinc via ZntR, which maintains in-tracellular zinc homeostasis and controls the concentration of reactive oxygen species to prevent bacterial death under oxidative stress. This study provides new insights into the regulation of T6SS4 by a zinc-dependent transcriptional regu-lator, and it provides a foundation for further investigation of the mechanism of zinc transport by T6SS.

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Genome-wide phenotypic profiling of transcription factors and identification of novel targets to control the virulence of Vibrio vulnificus
Dayoung Sung, Garam Choi, Minji Ahn, Hokyung Byun, Tae Young Kim, Hojun Lee, Zee-Won Lee, Ji Yong Park, Young Hyun Jung, Ho Jae Han, Sang Ho Choi
Nucleic Acids Research.2025;[Epub] CrossRef
Regulation of the H1 Type VI Secretion System by the Transcriptional Regulator NfxB in Pseudomonas aeruginosa
Shuhui Liu, Ziyuan Wu, Wenbo Yan, Qian Liu, Yuanli Zhao, Tingting Gao, Yiming Yang, Linke Cao, Ruixue Tao, Meng Li, Lijun Liu, Yani Zhang, Tietao Wang
International Journal of Molecular Sciences.2025; 26(4): 1472. CrossRef
ZntR is a critical regulator for zinc homeostasis and involved in pathogenicity in Riemerella anatipestifer
Hongmeng Ma, Mengying Wang, Yizhou Yao, Shutong Zhang, Mingshu Wang, Dekang Zhu, Renyong Jia, Shun Chen, Xinxin Zhao, Qiao Yang, Ying Wu, Shaqiu Zhang, Juan Huang, Bin Tian, Xumin Ou, Di Sun, Yu He, Zhen Wu, Ling Zhang, Yanling Yu, Anchun Cheng, Mafeng Li
Microbiology Spectrum.2025;[Epub] CrossRef
Two-Component Signaling System RegAB Represses Pseudomonas syringae pv. actinidiae T3SS by Directly Binding to the promoter of hrpRS1
Mengsi Zhang, Mingming Yang, Xiaoxue Zhang, Shuying Li, Shuaiwu Wang, Alex Muremi Fulano, Yongting Meng, Xihui Shen, Li-li Huang, Yao Wang
Journal of Integrative Agriculture.2024;[Epub] CrossRef
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Jorge Dias Carlier, Gustavo Magno dos Reis Ferreira, Rosane Freitas Schwan, Cristina Ferreira da Silva, Maria Clara Costa
Environmental Advances.2024; 15: 100479. CrossRef
OxyR-regulated T6SS functions in coordination with siderophore to resist oxidative stress
Changfu Li, Zhiyan Wei, Xinquan He, Haiyang He, Yuqi Liu, Yuxin Zuo, He Xiao, Yao Wang, Xihui Shen, Lingfang Zhu, Olaya Rendueles
Microbiology Spectrum.2024;[Epub] CrossRef
A σE-mediated temperature gauge orchestrates type VI secretion system, biofilm formation and cell invasion in pathogen Pseudomonas plecoglossicida
Yibei Zhang, Yuping Huang, Haoyuan Ding, Jiabao Ma, Xinyu Tong, Yuanxing Zhang, Zhen Tao, Qiyao Wang
Microbiological Research.2023; 266: 127220. CrossRef
Impact of lead (Pb2+) on the growth and biological activity of Serratia marcescens selected for wastewater treatment and identification of its zntR gene—a metal efflux regulator
Gustavo Magno dos Reis Ferreira, Josiane Ferreira Pires, Luciana Silva Ribeiro, Jorge Dias Carlier, Maria Clara Costa, Rosane Freitas Schwan, Cristina Ferreira Silva
World Journal of Microbiology and Biotechnology.2023;[Epub] CrossRef
MlrA, a MerR family regulator in Vibrio cholerae , senses the anaerobic signal in the small intestine of the host to promote bacterial intestinal colonization
Jialin Wu, Yutao Liu, Wendi Li, Fan Li, Ruiying Liu, Hao Sun, Jingliang Qin, Xiaohui Feng, Di Huang, Bin Liu
Gut Microbes.2022;[Epub] CrossRef
Nutritional immunity: the battle for nutrient metals at the host–pathogen interface
Caitlin C. Murdoch, Eric P. Skaar
Nature Reviews Microbiology.2022; 20(11): 657. CrossRef
The transcriptional regulator Zur regulates the expression of ZnuABC and T6SS4 in response to stresses in Yersinia pseudotuberculosis
Ran Cai, Fen Gao, Junfeng Pan, Xinwei Hao, Zonglan Yu, Yichen Qu, Jialin Li, Dandan Wang, Yao Wang, Xihui Shen, Xingyu Liu, Yantao Yang
Microbiological Research.2021; 249: 126787. CrossRef
T6SS Mediated Stress Responses for Bacterial Environmental Survival and Host Adaptation
Kai-Wei Yu, Peng Xue, Yang Fu, Liang Yang
International Journal of Molecular Sciences.2021; 22(2): 478. CrossRef
Yersiniabactin contributes to overcoming zinc restriction during Yersinia pestis infection of mammalian and insect hosts
Sarah L. Price, Viveka Vadyvaloo, Jennifer K. DeMarco, Amanda Brady, Phoenix A. Gray, Thomas E. Kehl-Fie, Sylvie Garneau-Tsodikova, Robert D. Perry, Matthew B. Lawrenz
Proceedings of the National Academy of Sciences.2021;[Epub] CrossRef
Roles of Type VI Secretion System in Transport of Metal Ions
Xiaobing Yang, Hai Liu, Yanxiong Zhang, Xihui Shen
Frontiers in Microbiology.2021;[Epub] CrossRef
Beyond dueling: roles of the type VI secretion system in microbiome modulation, pathogenesis and stress resistance
Jinshui Lin, Lei Xu, Jianshe Yang, Zhuo Wang, Xihui Shen
Stress Biology.2021;[Epub] CrossRef
Coordinated regulation of anthranilate metabolism and bacterial virulence by the GntR family regulator MpaR in Pseudomonas aeruginosa
Tietao Wang, Yihang Qi, Zhihan Wang, Jingru Zhao, Linxuan Ji, Jun Li, Zhao Cai, Liang Yang, Min Wu, Haihua Liang
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Vanessa Knittel, Pooja Sadana, Stephanie Seekircher, Anne-Sophie Stolle, Britta Körner, Marcel Volk, Cy M. Jeffries, Dmitri I. Svergun, Ann Kathrin Heroven, Andrea Scrima, Petra Dersch, Joan Mecsas
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The type VI secretion system protein AsaA in Acinetobacter baumannii is a periplasmic protein physically interacting with TssM and required for T6SS assembly
Lei Li, Yi-Nuo Wang, Hong-Bing Jia, Ping Wang, Jun-Fang Dong, Juan Deng, Feng-Min Lu, Qing-Hua Zou
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Can Chen, Xiaobing Yang, Xihui Shen
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Xiaobing Yang, Yunhong Song, Qingyun Dai, Hongyun Zhang, Li Song, Zhuo Wang, Junfeng Pan, Yao Wang
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Identification of essential genes of Pseudomonas aeruginosa for its growth in airway mucus: Mohammed Abd Alrahman , Sang Sun Yoon; J. Microbiol. 2017;55(1):68-74. Published online December 30, 2016; DOI: https://doi.org/10.1007/s12275-017-6515-3

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Pseudomonas aeruginosa has been identified as an important causative agent of airway infection, mainly in cystic fibrosis. This disease is characterized by defective mucociliary clearance induced in part by mucus hyper-production. Mucin is a major component of airway mucus and is heavily O-glycosylated, with a protein backbone. Airway infection is known to be established with bacterial adhesion to mucin. However, the genes involved in mucin degradation or utilization remain elusive. In this study, we sought to provide a genetic basis of P. aeruginosa airway growth by identifying those genes. First, using RNASeq analyses, we compared genome-wide expression profiles of PAO1, a prototype P. aeruginosa laboratory strain, grown in M9-mucin (M9M) and M9-glucose (M9G) media. Additionally, a PAO1 transposon (Tn) insertion mutants library was screened for mutants defective in growth in M9M medium. One mutant with a Tn insertion in the xcpU gene (PA3100) was determined to exhibit faulty growth in M9M medium. This gene contributes to the type II secretion system, suggesting that P. aeruginosa uses this secretion system to produce a number of proteins to break down and assimilate the mucin molecule. Furthermore, we screened the PAO1 genome for genes with protease activity. Of 13 mutants, one with mutation in PA3247 gene exhibited defective growth in M9M, suggesting that the PA3247-encoded protease plays a role in mucin utilization. Further mechanistic dissection of this particular process will reveal new drug targets, the inhibition of which could control recalcitrant P. aeruginosa infections.

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Citrobacter rodentium possesses a functional type II secretion system necessary for successful host infection
Z Krekhno, SE Woodward, A Serapio-Palacios, J Peña-Díaz, KM Moon, LJ Foster, BB Finlay
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Cross-talk between cancer and Pseudomonas aeruginosa mediates tumor suppression
Juliana K. Choi, Samer A. Naffouje, Masahide Goto, Jing Wang, Konstantin Christov, David J. Rademacher, Albert Green, Arlene A. Stecenko, Ananda M. Chakrabarty, Tapas K. Das Gupta, Tohru Yamada
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Pseudomonas aeruginosa type IV pili actively induce mucus contraction to form biofilms in tissue-engineered human airways
Tamara Rossy, Tania Distler, Lucas A. Meirelles, Joern Pezoldt, Jaemin Kim, Lorenzo Talà, Nikolaos Bouklas, Bart Deplancke, Alexandre Persat, Victor Sourjik
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Impact of diet and the bacterial microbiome on the mucous barrier and immune disorders
Charlotte A. Alemao, Kurtis F. Budden, Henry M. Gomez, Saima F. Rehman, Jacqueline E. Marshall, Shakti D. Shukla, Chantal Donovan, Samuel C. Forster, Ian A. Yang, Simon Keely, Elizabeth R. Mann, Emad M. El Omar, Gabrielle T. Belz, Philip M. Hansbro
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Frontiers in Microbiology.2021;[Epub] CrossRef
Structural and functional analysis of the carotenoid biosynthesis genes of aPseudomonasstrain isolated from the excrement of Autumn Darter
Yuki Fukaya, Miho Takemura, Takashi Koyanagi, Takashi Maoka, Kazutoshi Shindo, Norihiko Misawa
Bioscience, Biotechnology, and Biochemistry.2018; 82(6): 1043. CrossRef
Evolutionary conservation of the antimicrobial function of mucus: a first defence against infection
Cassie R Bakshani, Ana L Morales-Garcia, Mike Althaus, Matthew D Wilcox, Jeffrey P Pearson, John C Bythell, J Grant Burgess
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Infection and Immunity.2017;[Epub] CrossRef

Review

Against friend and foe: Type 6 effectors in plant-associated bacteria: Choong-Min Ryu; J. Microbiol. 2015;53(3):201-208. Published online March 3, 2015; DOI: https://doi.org/10.1007/s12275-015-5055-y

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Abstract PDF

Bacterial secretion systems play critical roles in communication with neighboring bacteria and in the modulation of host immune responses via the secretion of small proteins called effectors. Several secretion systems have been identified and these are denoted types I-II. Of these, the type VI secretion system (T6SS) and its effectors were only recently elucidated. Most studies on the role and significance of the T6SS and its effectors have focused on human pathogens. In this review, type 6 effectors from plant-associated beneficial and pathogenic bacteria are discussed, including effectors from Agrobacterium tumefaciens, Dickeya dadanti, Rhizobium leguminosarum, Pectobacterium atroseptium, Ralstonia solanacearum, Pseudomonas syringae, Pseudomonas fluorescens, and Pseudomonas protegens. Type 6 effectors act in symbiosis, biofilm formation, virulence, and interbacterial competition. Understanding the impact of type 6 effectors on pathogenesis will contribute to the management of bacterial pathogens in crop plants by allowing the manipulation of intra and inter-specific interactions.

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VimR is a conserved master transcription regulator controlling virulence and metabolism in Pseudomonas
Fang Chen, Yue Sun, Jiadai Huang, Tianmin Li, Beifang Lu, Zhe He, Jingwei Li, Tianqi Wang, Haiyan Hu, Xin Deng
Nucleic Acids Research.2025;[Epub] CrossRef
Adapting the inoculation methods of kiwifruit canker disease to identify efficient biocontrol bacteria from branch microbiome
Xiaolong Shao, Qianhua Wu, Li Li, Weimei He, Xueting He, Dongjin Cheng, Aprodisia Murero, Long Lin, Limin Wang, Caihong Zhong, Lili Huang, Guoliang Qian
Molecular Plant Pathology.2024;[Epub] CrossRef
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Research Support, Non-U.S. Gov'ts

Crystal structure of the bacterial type VI secretion system component TssL from Vibrio cholerae: Jeong Ho Chang , Yeon-Gil Kim; J. Microbiol. 2015;53(1):32-37. Published online December 4, 2014; DOI: https://doi.org/10.1007/s12275-015-4539-0

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The type VI secretion system (T6SS), commonly found in Gram-negative bacteria, is responsible for exporting effector proteins. The T6SS has been reported to be cytotoxic to host cells. While the components and assembly of the T6SS complex have been largely assessed, structural data on T6SS components from virulent bacteria is remarkably insufficient. Here, we report the crystal structure of Vibrio cholerae TssL (VcTssL), a core component of T6SS. In spite of a relatively low sequence identity, the overall structure of VcTssL is largely similar to those from other bacterial homologs except for several differences found in local structural elements. A unique feature attributed to the C-terminal fragment of Vc- TssL is a crystallographic artifact. This incidental feature of VcTssL may provide insights into screening of molecular partners for the cytoplasmic domain of TssL. Additionally, our results may help in the design of molecular probes for a detailed understanding of the functional relationship between TssL and other T6SS components.

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Structural Characterization of TssL from Acinetobacter baumannii: a Key Component of the Type VI Secretion System
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NOTE] Comparative Assessment of the Intracellular Survival of the Burkholderia pseudomallei bopC Mutant: Varintip Srinon , Sunsiree Muangman , Nithima Imyaem , Veerachat Muangsombut , Natalie R. Lazar Adler , Edouard E. Galyov , Sunee Korbsrisate; J. Microbiol. 2013;51(4):522-526. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-2557-3

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Burkholderia pseudomallei, the causative agent of melioidosis, is a Gram-negative saprophytic bacterium capable of surviving within phagocytic cells. To assess the role of BopC (a type III secreted effector protein) in the pathogenesis of B. pseudomallei, a B. pseudomallei bopC mutant was used to infect J774A.1 macrophage-like cells. The bopC mutant showed significantly reduced intracellular survival in infected macrophages compared to wild-type B. pseudomallei. In addition, the bopC mutant displayed delayed escape from endocytic vesicles compared with the wild-type strain. This indicates that BopC is important, and at least in part, needed for intracellular survival of B. pseudomallei.

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Transitioning from Soil to Host: Comparative Transcriptome Analysis Reveals the Burkholderia pseudomallei Response to Different Niches
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Varintip Srinon, Patoo Withatanung, Somjit Chaiwattanarungruengpaisan, Metawee Thongdee, Chatruthai Meethai, Joanne M. Stevens, Richard W. Titball, Sunee Korbsrisate
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Role of Toll-Like Receptor 5 (TLR5) in Experimental Melioidosis
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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 PDF

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.

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Secretion of Truncated Recombinant Rabies Virus Glycoprotein with Preserved Antigenic Properties Using a Co-Expression System in Hansenula polymorpha: Weidong Qian , Frank Aguilar , Ting Wang , Bingsheng Qiu; J. Microbiol. 2013;51(2):234-240. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2337-0

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Rabies virus infection remains a serious public health threat in the developing world, where cost-concerns make widescale public health interventions impractical. The development of novel and inexpensive ELISA diagnostic antigens is critical in early detection and prevention of complications. The transmembrane glycoprotein (G) of rabies virus (RV) contains an external domain capable of inducing the synthesis of anti-rabies, virus-neutralizing antibodies, in infected or immunized hosts. In our study, the external G domain was synthesized and fused in-frame with a polyhistidine-tag coding sequence present in the expression plasmid. Soluble truncated recombinant G was secreted in Hansenula polymorpha (H. polymorpha) using H. polymorpha-derived calnexin (HpCNE1) overproduction and found to be correctly N-glycosylated. The truncated recombinant G was purified from cell culture supernatant by Ni-agarose affinity chromatography and when compared with the full-length glycoprotein, found to be similarly immunogenic in vaccinated rabbits. These results subsequently led us to explore the potential of truncated recombinant G as a diagnostic antigen in ELISA. Our results show that the truncated recombinant G can detect antibodies directed to both whole virion and native glycoprotein. More sophisticated applications of truncated recombinant G would profit from the correctly N-glycosylated and soluble monomer.

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Review

REVIEW] The Role of Type III Secretion System 2 in Vibrio parahaemolyticus Pathogenicity: Hyeilin Ham , Kim Orth; J. Microbiol. 2012;50(5):719-725. Published online November 4, 2012; DOI: https://doi.org/10.1007/s12275-012-2550-2

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Abstract

Vibrio parahaemolyticus, a Gram-negative marine bacterial pathogen, is emerging as a major cause of food-borne illnesses worldwide due to the consumption of raw seafood leading to diseases including gastroenteritis, wound infection, and septicemia. The bacteria utilize toxins and type III secretion system (T3SS) to trigger virulence. T3SS is a multi-subunit needle-like apparatus used to deliver bacterial proteins, termed effectors, into the host cytoplasm which then target various eukaryotic signaling pathways. V. parahaemolyticus carries two T3SSs in each of its two chromosomes, named T3SS1 and T3SS2, both of which play crucial yet distinct roles during infection: T3SS1 causes cytotoxicity whereas T3SS2 is mainly associated with enterotoxicity. Each T3SS secretes a unique set of effectors that contribute to virulence by acting on different host targets and serving different functions. Emerging studies on T3SS2 of V. parahaemolyticus, reveal its regulation, translocation, discovery, characterization of its effectors, and development of animal models to understand the enterotoxicity. This review on recent findings for T3SS2 of V. parahaemolyticus highlights a novel mechanism of invasion that appears to be conserved by other marine bacteria.

Citations

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Journal Article

Disruption of SCO5461 Gene Coding for a Mono-ADP-Ribosyltransferase Enzyme Produces a Conditional Pleiotropic Phenotype Affecting Morphological Differentiation and Antibiotic Production in Streptomyces coelicolor: Krisztina Szirák , Judit Keser&# , Sándor Biró , Iván Schmelczer , György Barabás , András Penyige; J. Microbiol. 2012;50(3):409-418. Published online June 30, 2012; DOI: https://doi.org/10.1007/s12275-012-1440-y

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Abstract PDF: The SCO5461 gene of Streptomyces coelicolor A3(2) codes for an ADP-ribosyltransferase enzyme that is predicted to be a transmembrane protein with an extracellular catalytic domain. PCR-targeted disruption of the gene resulted in a mutant that differentiated normally on complex SFM medium; however, morphological differentiation in minimal medium was significantly delayed and this phenotype was even more pronounced on osmotically enhanced minimal medium. The mutant did not sporulate when it was grown on R5 medium, however the normal morphological differentiation was restored when the strain was cultivated beside the wild-type S. coelicolor M145 strain. Comparison of the pattern of ADP-ribosylated proteins showed a difference between the mutant and the wild type, fewer modified proteins were present in the cellular crude extract of the mutant strain. These results support our previous suggestions that protein ADP-ribosylation is involved in the regulation of differentiation and antibiotic production and secretion in Streptomyces.

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

Effect of Acidic pH on the Invasion Efficiency and the Type III Secretion System of Burkholderia thailandensis: Siroj Jitprasutwit , Wisansanee Thaewpia , Veerachat Muangsombut , Aroonlug Lulitanond , Chanvit Leelayuwat , Ganjana Lertmemongkolchai , Sunee Korbsrisate; J. Microbiol. 2010;48(4):526-532. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-0078-x

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Abstract PDF: Burkholderia thailandensis is a close relative of Burkholderia pseudomallei. These organisms are very similar, but B. thailandensis is far less virulent than B. pseudomallei. Nucleotide sequencing and analysis of 14 B. thailandensis isolates revealed variation in the regions coding for the type III secreted BipD protein. The degree of B. thailandensis BipD sequence variation was greater than that found in B. pseudomallei. Western blot analysis indicated that, unlike B. pseudomallei, B. thailandensis type III secreted proteins including BipD and BopE could not be detected in the supernatant of culture medium unless induced by acidic conditions. In addition, culturing B. thailandensis under acidic growth conditions (pH 4.5) can induce the ability of this bacterium to invade human respiratory epithelial cells A549. The identification of an environmental stimulus that increases the invasion capability of B. thailandensis invasion is of value for those who would like to use this bacterium as a model to study B. pseudomallei virulence.

Stable Expression and Secretion of Polyhydroxybutyrate Depolymerase of Paucimonas lemoignei in Escherichia coli: Se Whan Park , Moon Gyu Chung , Hwa Young Lee , Jeong Yoon Kim , Young Ha Rhee; J. Microbiol. 2008;46(6):662-669. Published online December 24, 2008; DOI: https://doi.org/10.1007/s12275-008-0283-z

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Abstract PDF: An efficient strategy for the expression and secretion of extracellular polyhydroxybutyrate depolymerase (PhaZ1) of Paucimonas lemoignei in Escherichia coli was developed by employing the signal peptide of PhaZ1 and a truncated ice nucleation protein anchoring motif (INPNC). Directly synthesized mature form of PhaZ1 was present in the cytoplasm of host cells as inclusion bodies, while a construct containing PhaZ1 and its own N-terminal signal peptide (PrePhaZ1) enabled the secretion of active PhaZ1 into the extracellular medium. However, the PrePhaZ1 construct was harmful to the host cell and resulted in atypical growth and instability of the plasmid during the cultivation. In contrast, INPNC-PhaZ1 and INPNCPrePhaZ1 fusion constructs did not affect growth of host cells. INPNC-PhaZ1 was successfully displayed on the cell surface with its fusion form, but did not retain PhaZ1 activity. In the case of INPNC-PrePhaZ1, the initially synthesized fusion form was separated by precise cleavage of the signal peptide, and active PhaZ1 was consequently released into the culture medium. The amount of PhaZ1 derived from E. coli (INPNC-PrePhaZ1) was almost twice as great as that directly expressed from E. coli (PrePhaZ1), and was predominantly (approximately 85%) located in the periplasm when cultivated at 22°C but was efficiently secreted into the extracellular medium when cultivated at 37°C.

Two Forms of Vibrio vulnificus Metalloprotease VvpE are Secreted via the Type II General Secretion System: Jong Park , So-Yeon Ryu , Choon-Mee Kim , Sung-Heui Shin; J. Microbiol. 2008;46(3):338-343. Published online July 5, 2008; DOI: https://doi.org/10.1007/s12275-008-0058-6

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Abstract PDF

Vibrio vulnificus has been known to secrete one form of metalloprotease VvpE (45 kDa) that is cleaved to 34 kDa-VvpE and 11 kDa-C-terminal propeptide via extracellular autoproteolysis. However, we found that extracellular secretion of both the 34 and 45 kDa forms of VvpE began in the early growth phase; moreover, 34 kDa-VvpE existed as the major form in V. vulnificus cell lysates and culture supernatants. In addition, extracellular secretion of both 34 and 45 kDa-VvpE was blocked by mutation of the pilD gene, which encodes for the type IV leader peptidase/N-methyltransferase of the type II general secretion system, and the blocked VvpE secretion was recovered by in trans-complementation of the wild-type pilD gene. These results indicate that 34 kDa-VvpE is the major form secreted along with 45 kDa-VvpE from the early growth phase via the PilD-mediated type II general secretion system.

Citations

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Structure and assembly of pilotin-dependent and -independent secretins of the type II secretion system
S. Peter Howard, Leandro F. Estrozi, Quentin Bertrand, Carlos Contreras-Martel, Timothy Strozen, Viviana Job, Alexandre Martins, Daphna Fenel, Guy Schoehn, Andréa Dessen, Tomoko Kubori
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SmcR, the Quorum-sensing Master Regulator, Is Partially Involved in Temperature/Salinity-mediated Changes in MetalloproteasevvpEExpression inVibrio vulnificus
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Functional Characterization of EpsC, a Component of the Type II Secretion System, in the Pathogenicity of Vibrio vulnificus
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Genomic and proteomic analyses of the coral pathogen Vibrio coralliilyticus reveal a diverse virulence repertoire
Eidy de O Santos, Nelson Alves, Graciela M Dias, Ana Maria Mazotto, Alane Vermelho, Gary J Vora, Bryan Wilson, Victor H Beltran, David G Bourne, Frédérique Le Roux, Fabiano L Thompson
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Enhanced Secretion of Cell Wall Bound Enolase into Culture Medium by the soo1-1 Mutation of Saccharomyces cerevisiae: Ki-Hyun Kim , Hee-Moon Park; J. Microbiol. 2004;42(3):248-252.; DOI: https://doi.org/2080 [pii]

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Abstract PDF: In order to identify the protein(s) secreted into culture medium by the soo1-1/ret1-1 mutation of Saccharomyces cerevisiae, proteins from the culture medium of cells grown at permissive (28^oC) and non-permissive temperatures (37^oC), were analyzed. Comparison of protein bands separated by SDS-PAGE identified a prominent band of 47-kDa band from a mutant grown at 37^oC. N-terminal amino acid sequencing of this 47-kDa protein showed high identity with enolases 1 and 2. Western blot analysis revealed that most of the cell wall-bound enolase was released into the culture medium of the mutant grown at 37^oC, some of which were separated as those with lower molecular weights. Our results, presented here, indicate the impairment of cell wall enolase biogenesis and assembly by the soo1-1/ret1-1 mutation of S. cerevisiae.

Construction of secretion vectors using the α-amylase signal sequence of bacillus subtilis NA64: Kim, Sung Il , Lee, Se Yong; J. Microbiol. 1996;34(1):74-81.

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Abstract PDF: Two secretion vectors, pUBA240 and pUB340 were constructed by using the promoter and secretory signal region of the α-amylase gene from an α-amylase hyperproducing strain, Bacillus subtilis NA64. In this secretion vector system, various restriction enzyme sites are located immediately after the proregion of the α-amylase gene for easy replacement of various foreign structural genes. To evaluate this secretion vectors, the β-lactamase gene of pBR322 was used as a reporter gene. The expressed and biologically active β-lactamase was secreted into the culture broth from B. subtilis LKS86 transformants harboring each β-lactamase secreting plasmid, pUBAbla and pUBSble. In both cases, more than 92% of expressed β- lactamases were located idn the culture medium. The amount of the secreted β-lactamase was about 80% of the total secreted proteins in the culture medium.

A possible mechanism responsible for translocation and secretion an alkaliphilic bacillus sp. S-1 pullulanase: Shim, Jae Kyoung , Kim, Kyoung Sook , Kim, Cheorl Ho; J. Microbiol. 1997;35(3):213-221.

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Abstract PDF: The secretion of the alkaliphilic Bacillus sp. S-1 extracellular pullulanase involves translocation across the cytoplasmic membrane of the Gram-positive bacterial cell envelope. Translocation of the intracellular pullulanase PUL-I, was traced to elucidate the mechanism and pathway of protein secretion from an alkaliphilic Bacillus sp. S-1. Pullulanase could be slowly but quantitatively released into the medium during growth of the cells in medium containing proteinase K. The released pullulanase lacked the N-terminal domain. The N-terminus is the sole membrane anchor in the pullulanase protein and was not affected by proteases, confirming that it is not exposed on the cell surface. Processing of a 180,000M_r pullulanase to a 140,000M_r polypeptide has been demonstrated in cell extracts using antibodies raised against 140,000M_r extracellular form. Processing of the 180,000 M_r protein occured during the preparation of extracts in an alkaline pH condition. A modified rapid extraction procedure suggested that the processing event also occured in vivo. Processing apparently increased the activity of pullulanase. The western blotting analysis with mouse anti-serum against 140-kDa extracellular pullulanase PUL-E showed that PUL-I is processed into PUL-X via intermediate form of PUL-E. Possible explanationa for the translocation are discussed.

Controlled Expression and Secretion of Aspergillus oryzae Alkaline Protease in Aspergillus nidulans: Eun Ah Kim , Jeong Goo Lee , Mi Kyung Whang , Hee Moon Park , Jeong Yoon Kim , Suhn Kee Chae , Pil Jae Maeng; J. Microbiol. 2001;39(2):95-101.

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Abstract PDF: In an effort to develop an efficient expression and secretion system for heterologous proteins in Aspergillus nidulans, the PCR-amplified coding sequence for alkaline protease (AlpA) of A. oryzae was cloned into a fungal expression vector downstream of A. nidulans alcA (alcohol dehydrogenase) promoter to yield pRAAlp. Transformation of A. nidulans with pRAAlp gave stable transformants harboring various copy numbers (3 to 10) of integrated alpA gene, from among which 6 representatives were selected. On a medium containing 0.8% ammonium sulfate that represses the expression of the hosts own protease, the alcA promoter-controlled AlpA expression was strongly induced by threonine but repressed by glucose. The level of AlpA secretion was highest (approximately 666 mU/ml) in transformant ALP6 containing the largest copy number integrated alpA. However, the level of AlpA secretion was not necessarily proportional to the copy numbers of the integrated alpA genes. The N-terminal sequence of the secreted mature AlpA was determined to be Gly-Leu-Thr-Thr-Gln-Lys-Ser and its molecular mass to be approximately 34 kDa, indicating that AlpA is properly processed by the removal of 121 N-terminal amino acids.

Genetic and Environmental Control of Salmonella Invasion: Craig Altier; J. Microbiol. 2005;43(1):85-92.

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Abstract PDF: An early step in the pathogenesis of non-typhoidal Salmonella species is the ability to penetrate the intestinal epithelial monolayer. This process of cell invasion requires the production and transport of secreted effector proteins by a type III secretion apparatus encoded in Salmonella pathogenicity island I (SPI-1). The control of invasion involves a number of genetic regulators and environmental stimuli in complex relationships. SPI-1 itself encodes several transcriptional regulators (HilA, HilD, HilC, and InvF) with overlapping sets of target genes. These regulators are, in turn, controlled by both positive and regulators outside SPI-1, including the two-component regulators BarA/SirA and PhoP/Q, and the csr post-transcriptional control system. Additionally, several environmental conditions are known to regulate invasion, including pH, osmolarity, oxygen tension, bile, Mg^2+ concentration, and short chain fatty acids. This review will discuss the current understanding of invasion control, with emphasis on the interaction of environmental factors with genetic regulators that leads to productive infection.

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