scholarly journals HORMA domain proteins and a Pch2-like ATPase regulate bacterial cGAS-like enzymes to mediate bacteriophage immunity

2019 ◽  
Author(s):  
Qiaozhen Ye ◽  
Rebecca K. Lau ◽  
Ian T. Mathews ◽  
Jeramie D. Watrous ◽  
Camillia S. Azimi ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mechanisms ◽  
Protein Detection ◽  
Second Messenger ◽  
Negative Regulation ◽  
Bacteriophage Λ ◽  
E Coli ◽  
Aaa Atpase ◽  
Broad Array ◽  
Biochemical Mechanisms

AbstractBacteria are continually challenged by foreign invaders including bacteriophages, and have evolved a variety of defenses against these invaders. Here, we describe the structural and biochemical mechanisms of a bacteriophage immunity pathway found in a broad array of bacteria, including pathogenic E. coli and Pseudomonas aeruginosa. This pathway employs eukaryotic-like HORMA domain proteins that recognize specific peptides, then bind and activate a cGAS/DncV-like nucleotidyltransferase (CD-NTase) to generate a cyclic tri-AMP (cAAA) second messenger; cAAA in turn activates an endonuclease effector, NucC. Signaling is attenuated by a homolog of the AAA+ ATPase Pch2/TRIP13, which binds and likely disassembles the active HORMA-CD-NTase complex. When expressed in non-pathogenic E. coli, this pathway confers immunity against bacteriophage λ infection. Our findings reveal the molecular mechanisms of a bacterial defense pathway integrating a cGAS-like nucleotidyltransferase with HORMA domain proteins for threat sensing through protein detection, and negative regulation by a Pch2-like ATPase.

scholarly journals Mechanistic insights into c-di-GMP–dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa

2015 ◽  
Vol 113 (2) ◽  
pp. E209-E218 ◽  
Author(s):  
Bruno Y. Matsuyama ◽  
Petya V. Krasteva ◽  
Claudine Baraquet ◽  
Caroline S. Harwood ◽  
Holger Sondermann ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Active Site ◽  
Quaternary Structure ◽  
Second Messenger ◽  
Bacterial Biofilm ◽  
Binding Motif ◽  
Atpase Domain ◽  
Chronic Infections ◽  
Interaction Domain ◽  
Aaa Atpase

Bacterial biofilm formation during chronic infections confers increased fitness, antibiotic tolerance, and cytotoxicity. In many pathogens, the transition from a planktonic lifestyle to collaborative, sessile biofilms represents a regulated process orchestrated by the intracellular second-messenger c-di-GMP. A main effector for c-di-GMP signaling in the opportunistic pathogen Pseudomonas aeruginosa is the transcription regulator FleQ. FleQ is a bacterial enhancer-binding protein (bEBP) with a central AAA+ ATPase σ54-interaction domain, flanked by a C-terminal helix-turn-helix DNA-binding motif and a divergent N-terminal receiver domain. Together with a second ATPase, FleN, FleQ regulates the expression of flagellar and exopolysaccharide biosynthesis genes in response to cellular c-di-GMP. Here we report structural and functional data that reveal an unexpected mode of c-di-GMP recognition that is associated with major conformational rearrangements in FleQ. Crystal structures of FleQ’s AAA+ ATPase domain in its apo-state or bound to ADP or ATP-γ-S show conformations reminiscent of the activated ring-shaped assemblies of other bEBPs. As revealed by the structure of c-di-GMP–complexed FleQ, the second messenger interacts with the AAA+ ATPase domain at a site distinct from the ATP binding pocket. c-di-GMP interaction leads to active site obstruction, hexameric ring destabilization, and discrete quaternary structure transitions. Solution and cell-based studies confirm coupling of the ATPase active site and c-di-GMP binding, as well as the functional significance of crystallographic interprotomer interfaces. Taken together, our data offer unprecedented insight into conserved regulatory mechanisms of gene expression under direct c-di-GMP control via FleQ and FleQ-like bEBPs.

Антимікробна резистентність провідних збудників інфекцій сечових шляхів у Києві (Україна)

2017 ◽  
Vol 1 (2) ◽  
pp. 48-60
Author(s):  
A.G. Salmanov ◽  
A.V. Rudenko
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Klebsiella Pneumoniae ◽  
Staphylococcus Epidermidis ◽  
Klebsiella Oxytoca ◽  
Enterobacter Aerogenes ◽  
Enterobacter Cloacae ◽  
Proteus Vulgaris ◽  
Providencia Rettgeri ◽  
E Coli

Мета роботи — вивчити резистентність до антибіотиків бактеріальних збудників інфекцій сечових шляхів (ІСШ), виділених у пацієнтів урологічного стаціонару в м. Києві. Матеріали і методи. Досліджено 1612 штамів бактерій, виділених із сечі хворих з ІСШ (цистит, уретрит, пієлонефрит), госпіталізованих в урологічне відділення ДУ «Інститут урології НАМН України» у м. Києві протягом 2016 р. Серед пацієнтів переважали жінки — 1201 (74,5 %). Вік хворих становив від 17 до 74 років. Для збору даних використано медичну документацію лікарні. Мікробіологічні дослідження виконано у лабораторії мікробіології ДУ «Інститут урології НАМН України». Аналізували результати культурального дослідження зразків сечі, зібраних за наявності клінічних ознак ІСШ. Дослідження клінічного матеріалу та інтерпретацію отриманих результатів проводили загальноприйнятими методами. Вивчено чутливість уропатогенів до 31 антибіотика дискодифузійним методом відповідно до рекомендацій Інституту клінічних та лабораторних стандартів США (Clinical and Laboratory Standards Institute (CLSI)). Результати та обговорення. Аналіз мікробного спектра сечі виявив домінування серед уропатогенів штамів Escherichia coli (32,0 %), Enterococcus faecalis (19,5 %), Klebsiella pneumoniae (10,9 %), Staphylococcus epidermidis (8,9 %), S. haemolyticus (6,5 %) та Pseudomonas aeruginosa (6,4 %). Частка Enterococcus faecium, Enterobacter aerogenes і Streptococcus viridans становила відповідно 2,5, 2,2 і 1,6 %, Enterobacter cloacae, Klebsiella oxytoca, Acinetobacter baumannii, Proteus vulgaris та Providencia rettgeri — менше 1,0 %. У більшості випадків (69,7 %) мікроорганізми виділено у монокультурі, у решті випадків — у мікробних асоціа- ціях. Високу резистентність до тестованих антибіотиків виявили штами E. aerogenes (45,1 %), E. cloacae (45,7 %), E. faecium (40,9 %), E. faecalis (40,7 %), E. coli (39,9 %), P. aeruginosa (34,0 %), K. pneumoniae (28,6 %). Найбільш активними до уропатогенів були іміпенем (E. coli — 87,6 %, P. aeruginosa — 75,7 %, E. cloacae — 67,3 %, E. aerogenes — 72,6 %, K. pneumoniae — 93,2 %), меропенем (E. coli — 89,1 %, P. aeruginosa — 76,7 %, K. pneumoniae — 82,6 %), лефлоцин (E. coli — 74,5 %, ентерококи — 78,7 %, P. aeruginosa — 76,7 %, E. cloacae — 73,9 %, E. aerogenes — 80,4 %, K. pneumoniae — 83,5 %), амоксицилін/клавуланат (ентерококи — 84,6 %), фурагін (ентерококи — 82,6 %), цефоперазон (K. pneumoniae — 89,2 %, P. aeruginosa — 73,8 %), цефтріаксон (K. pneumoniae — 80,1 %). Висновки. Антибіотикорезистентність збудників ІСШ — важлива терапевтична проблема. Найбільшою активністю до уропатогенів характеризуються іміпенем, меропенем, лефлоцин, амоксицилін/ клавуланат, фурагін, цефоперазон, цефтріаксон, які можна розглядати як препарат вибору для призначення стартової терапії ІСШ. Необхідно здійснювати постійний моніторинг за резистентністю до дії антибіотиків. Політику використання антибіотиків у кожному стаціонарі слід визначати залежно від локальних даних щодо резистентності до протимікробних препаратів.

The Characterization of Antibiotic Resistance of Bacterial Isolates from Intensive Care Unit Patient Samples in a University Affiliated Hospital in Romania

2019 ◽  
Vol 70 (5) ◽  
pp. 1778-1783
Author(s):  
Andreea-Loredana Golli ◽  
Floarea Mimi Nitu ◽  
Maria Balasoiu ◽  
Marina Alina Lungu ◽  
Cristiana Cerasella Dragomirescu ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Bacterial Pathogens ◽  
Resistance Rate ◽  
Resistance Pattern ◽  
Bacterial Isolates ◽  
E Coli ◽  
Acinetobacter Spp ◽  
Almost All ◽  
Klebsiella Spp

To determine the resistance pattern of bacterial pathogens involved in infections of the patients aged between 18-64 years, admitted in a ICU from a 1518-bed university-affiliated hospital. A retrospective study of bacterial pathogens was carried out on 351 patients aged between 18-64 years admitted to the ICU, from January to December 2017. In this study there were analysed 469 samples from 351 patients (18-64 years). A total of 566 bacterial isolates were obtained, of which 120 strains of Klebsiella spp. (35.39%%), followed by Nonfermenting Gram negative bacilli, other than Pseudomonas and Acinetobacter (NFB) (75- 22.12%), Acinetobacter spp. (53 - 15.63%), Pseudomonas aeruginosa and Proteus (51 - 15.04%), and Escherichia coli (49 - 14.45%). The most common isolates were from respiratory tract (394 isolates � 69.61%). High rates of MDR were found for Pseudomonas aeruginosa (64.70%), MRSA (62.65%) and Klebsiella spp. (53.33%), while almost all of the isolated NFB strains were MDR (97.33%). There was statistic difference between the drug resistance rate of Klebsiella and E. coli strains to ceftazidime and ceftriaxone (p[0.001), cefuroxime (p[0.01) and to cefepime (p[0.01). The study revealed an alarming pattern of antibiotic resistance in the majority of ICU isolates.

scholarly journals Production, characterization, and cross-reactivity of a polyclonal antibody against Arabidopsis TARGET OF RAPAMYCIN

2019 ◽  
Vol 62 (1) ◽  
Author(s):  
Gyeong-Im Shin ◽  
Sun Young Moon ◽  
Song Yi Jeong ◽  
Myung Geun Ji ◽  
Joon-Yung Cha ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cross Reactivity ◽  
Target Of Rapamycin ◽  
Loss Of Function ◽  
Anticancer Activities ◽  
Purification Method ◽  
E Coli ◽  
Related Family ◽  
Large Gene ◽  
Truncated Variants

AbstractTARGET OF RAPAMYCIN (TOR), a member of the phosphatidylinositol 3-kinase-related family of protein kinases, is encoded by a single, large gene and is evolutionarily conserved in all eukaryotes. TOR plays a role as a master regulator that integrates nutrient, energy, and stress signaling to orchestrate development. TOR was first identified in yeast mutant screens, as its mutants conferred resistance to rapamycin, an antibiotic with immunosuppressive and anticancer activities. In Arabidopsis thaliana, the loss-of-function tor mutant displays embryo lethality, but the precise mechanisms of TOR function are still unknown. Moreover, a lack of reliable molecular and biochemical assay tools limits our ability to explore TOR functions in plants. Here, we produced a polyclonal α-TOR antibody using two truncated variants of TOR (1–200 and 1113–1304 amino acids) as antigens because recombinant full-length TOR is challenging to express in Escherichia coli. Recombinant His-TOR1−200 and His-TOR1113−1304 proteins were individually expressed in E. coli, and a mixture of proteins (at a 1:1 ratio) was used for immunizing rabbits. Antiserum was purified by an antigen-specific purification method, and the purified polyclonal α-TOR antibody successfully detected endogenous TOR proteins in wild-type Arabidopsis and TOR orthologous in major crop plants, including tomato, maize, and alfalfa. Moreover, our α-TOR antibody is useful for coimmunoprecipitation assays. In summary, we generated a polyclonal α-TOR antibody that detects endogenous TOR in various plant species. Our antibody could be used in future studies to determine the precise molecular mechanisms of TOR, which has largely unknown multifunctional roles in plants.

scholarly journals High-density transposon libraries utilising outward-oriented promoters identify mechanisms of action and resistance to antimicrobials

2020 ◽  
Vol 367 (22) ◽  
Author(s):  
Chris Coward ◽  
Gopujara Dharmalingham ◽  
Omar Abdulle ◽  
Tim Avis ◽  
Stephan Beisken ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Selective Advantage ◽  
Mechanisms Of Action ◽  
Over Expression ◽  
E Coli ◽  
Synthase Inhibitor ◽  
Established Technique ◽  
Bacterial Transposon

ABSTRACT The use of bacterial transposon mutant libraries in phenotypic screens is a well-established technique for determining which genes are essential or advantageous for growth in conditions of interest. Standard, inactivating, transposon libraries cannot give direct information about genes whose over-expression gives a selective advantage. We report the development of a system wherein outward-oriented promoters are included in mini-transposons, generation of transposon mutant libraries in Escherichia coli and Pseudomonas aeruginosa and their use to probe genes important for growth under selection with the antimicrobial fosfomycin, and a recently-developed leucyl-tRNA synthase inhibitor. In addition to the identification of known mechanisms of action and resistance, we identify the carbon–phosphorous lyase complex as a potential resistance liability for fosfomycin in E. coli and P. aeruginosa. The use of this technology can facilitate the development of novel mechanism-of-action antimicrobials that are urgently required to combat the increasing threat worldwide from antimicrobial-resistant pathogenic bacteria.

scholarly journals In Escherichia coli Ammonia Inhibits Cytochrome bo3 But Activates Cytochrome bd-I

Antioxidants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 13
Author(s):  
Elena Forte ◽  
Sergey A. Siletsky ◽  
Vitaliy B. Borisov
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Dissociation Constants ◽  
Reductase Activity ◽  
Ammonia Concentration ◽  
High Concentration ◽  
E Coli ◽  
Cytochrome Bd ◽  
Cytochrome Bo3 ◽  
Oxygen Reductase

Interaction of two redox enzymes of Escherichia coli, cytochrome bo3 and cytochrome bd-I, with ammonium sulfate/ammonia at pH 7.0 and 8.3 was studied using high-resolution respirometry and absorption spectroscopy. At pH 7.0, the oxygen reductase activity of none of the enzymes is affected by the ligand. At pH 8.3, cytochrome bo3 is inhibited by the ligand, with 40% maximum inhibition at 100 mM (NH4)2SO4. In contrast, the activity of cytochrome bd-I at pH 8.3 increases with increasing the ligand concentration, the largest increase (140%) is observed at 100 mM (NH4)2SO4. In both cases, the effector molecule is apparently not NH4+ but NH3. The ligand induces changes in absorption spectra of both oxidized cytochromes at pH 8.3. The magnitude of these changes increases as ammonia concentration is increased, yielding apparent dissociation constants Kdapp of 24.3 ± 2.7 mM (NH4)2SO4 (4.9 ± 0.5 mM NH3) for the Soret region in cytochrome bo3, and 35.9 ± 7.1 and 24.6 ± 12.4 mM (NH4)2SO4 (7.2 ± 1.4 and 4.9 ± 2.5 mM NH3) for the Soret and visible regions, respectively, in cytochrome bd-I. Consistently, addition of (NH4)2SO4 to cells of the E. coli mutant containing cytochrome bd-I as the only terminal oxidase at pH 8.3 accelerates the O2 consumption rate, the highest one (140%) being at 27 mM (NH4)2SO4. We discuss possible molecular mechanisms and physiological significance of modulation of the enzymatic activities by ammonia present at high concentration in the intestines, a niche occupied by E. coli.

scholarly journals Structure and dynamics of the E. coli chemotaxis core signaling complex by cryo-electron tomography and molecular simulations

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
C. Keith Cassidy ◽  
Benjamin A. Himes ◽  
Dapeng Sun ◽  
Jun Ma ◽  
Gongpu Zhao ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Electron Tomography ◽  
Conformational Dynamics ◽  
Molecular Simulations ◽  
Adaptor Protein ◽  
Signaling Mechanism ◽  
E Coli ◽  
Signaling Complex ◽  
Chemical Gradients ◽  
Cryo Electron Tomography

AbstractTo enable the processing of chemical gradients, chemotactic bacteria possess large arrays of transmembrane chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW, organized as coupled core-signaling units (CSU). Despite decades of study, important questions surrounding the molecular mechanisms of sensory signal transduction remain unresolved, owing especially to the lack of a high-resolution CSU structure. Here, we use cryo-electron tomography and sub-tomogram averaging to determine a structure of the Escherichia coli CSU at sub-nanometer resolution. Based on our experimental data, we use molecular simulations to construct an atomistic model of the CSU, enabling a detailed characterization of CheA conformational dynamics in its native structural context. We identify multiple, distinct conformations of the critical P4 domain as well as asymmetries in the localization of the P3 bundle, offering several novel insights into the CheA signaling mechanism.

scholarly journals Fabrication of a Novel AgBr/Ag2MoO4@InVO4 Composite with Excellent Visible Light Photocatalytic Property for Antibacterial Use

Nanomaterials ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1541
Author(s):  
Jie Zhang ◽  
Jia Wang ◽  
Qingjun Zhu ◽  
Binbin Zhang ◽  
Huihui Xu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Degradation Rate ◽  
Photocatalytic Property ◽  
Photocatalytic Reaction ◽  
Composite Photocatalyst ◽  
E Coli ◽  
Radical Trapping ◽  
Visible Light Photocatalytic

A novel AgBr/Ag2MoO4@InVO4 composite photocatalyst with different heterojunction structures was successfully constructed by compounding InVO4 with Ag2MoO4 and AgBr. According to the degradation, antibacterial and free radical trapping data, the photocatalytic antibacterial and antifouling activities of AgBr/Ag2MoO4@InVO4 composite were evaluated, and the corresponding photocatalytic reaction mechanism was proposed. Adding AgBr/Ag2MoO4@InVO4 composite, the degradation rate of ciprofloxacin (CIP) achieved 95.5% within 120 min. At the same time, the antibacterial rates of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) achieved 99.99%. The AgBr/Ag2MoO4@InVO4 composite photocatalyst showed promising usage in photocatalytic antibacterial and purification areas.

scholarly journals Molecular mechanisms of master regulator VqsM mediating quorum-sensing and antibiotic resistance in Pseudomonas aeruginosa

2014 ◽  
Vol 42 (16) ◽  
pp. 10307-10320 ◽  
Author(s):  
Haihua Liang ◽  
Xin Deng ◽  
Xuefeng Li ◽  
Yan Ye ◽  
Min Wu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Quorum Sensing ◽  
Molecular Mechanisms ◽  
Master Regulator

Cloning the gene for the heat shock response positiwe regulator (sigma 32 homolog) from Pseudomonas aeruginosa

1995 ◽  
Vol 41 (1) ◽  
pp. 75-87 ◽  
Author(s):  
Zerlina M. Naczynski ◽  
Andrew M. Kropinski ◽  
Chris Mueller
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Heat Shock ◽  
Sigma Factor ◽  
Oligonucleotide Probe ◽  
In Vitro Transcription ◽  
Translation System ◽  
Amino Acid Homology ◽  
E Coli ◽  
Transcriptional Start Sites

A 31 base pair synthetic oligonucleotide based on the genes for the Escherichia coli heat shock sigma factor (rpoH) and the Pseudomonas aeruginosa housekeeping sigma factor (rpoD) was employed in conjunction with the Tanaka et al. (K. Tanaka, T. Shiina, and H. Takahashi, 1988. Science (Washington, D.C.), 242: 1040–1042) RpoD box probe to identify the location of the rpoH gene in P. aeruginosa genomic digests. This gene was cloned into plasmid pGEM3Z(f+), sequenced, and found to share 67% nucleotide identity and 77% amino acid homology with the rpoH gene and its product (σ32) of E. coli. The plasmid containing the rpoH gene complemented the function of σ32 in an E. coli rpoH deletion mutant. Furthermore, this plasmid directed the synthesis of a 32-kDa protein in an E. coli S-30 in vitro transcription–translation system. Primer extension studies were used to identify the transcriptional start sites under control and heat-stressed (45 and 50 °C) conditions. Two promoter sites were identified having sequence homology to the E. coli σ70 and σ24 consensus sequences.Key words: heat shock, Pseudomonas aeruginosa, sigma factor, transcription, oligonucleotide probe.

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