Functional Heterologous Expression of Mature Lipase LipA from Pseudomonas aeruginosa PSA01 in Escherichia coli SHuffle and BL21 (DE3): Effect of the Expression Host on Thermal Stability and Solvent Tolerance of the Enzyme Produced

This study aimed to express heterologously the lipase LipA from Pseudomonas aeruginosa PSA01 obtained from palm fruit residues. In previous approaches, LipA was expressed in Escherichia coli fused with its signal peptide and without its disulfide bond, displaying low activity. We cloned the mature LipA with its truncated chaperone Lif in a dual plasmid and overexpressed the enzyme in two E. coli strains: the traditional BL21 (DE3) and the SHuffle® strain, engineered to produce stable cytoplasmic disulfide bonds. We evaluated the effect of the disulfide bond on LipA stability using molecular dynamics. We expressed LipA successfully under isopropyl β-d-1-thio-galactopyranoside (IPTG) and slow autoinducing conditions. The SHuffle LipA showed higher residual activity at 45 °C and a greater hyperactivation after incubation with ethanol than the enzyme produced by E. coli BL21 (DE3). Conversely, the latter was slightly more stable in methanol 50% and 60% (t½: 49.5 min and 9 min) than the SHuffle LipA (t½: 31.5 min and 7.4 min). The molecular dynamics simulations showed that removing the disulfide bond caused some regions of LipA to become less flexible and some others to become more flexible, significantly affecting the closing lid and partially exposing the active site at all times.

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

International Journal of Antibiotics and Probiotics ◽

10.31405/ijap.1-2.17.03 ◽

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.

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

FEMS Microbiology Letters ◽

10.1093/femsle/fnaa185 ◽

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.

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

Nanomaterials ◽

10.3390/nano10081541 ◽

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.

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Molecular Dynamics Simulations of the Predicted E. Coli FtsB-FtsL Tetrameric Complex

Biophysical Journal ◽

10.1016/j.bpj.2016.11.2712 ◽

2017 ◽

Vol 112 (3) ◽

pp. 501a

Author(s):

Gladys Diaz Vazquez ◽

Samson Condon ◽

Qiang Cui ◽

Alessandro Senes

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

E Coli ◽

Dynamics Simulations ◽

Tetrameric Complex

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Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI

Applied and Environmental Microbiology ◽

10.1128/aem.01704-15 ◽

2015 ◽

Vol 81 (20) ◽

pp. 6953-6963 ◽

Cited By ~ 5

Author(s):

Zhe Zhao ◽

Lauren J. Eberhart ◽

Lisa H. Orfe ◽

Shao-Yeh Lu ◽

Thomas E. Besser ◽

...

Keyword(s):

Escherichia Coli ◽

Atp Synthase ◽

Disulfide Bonds ◽

Gene Knockout ◽

Single Gene ◽

Site Directed Mutagenesis ◽

Content Type ◽

E Coli ◽

Synthase Inhibitor ◽

Loss Of Susceptibility

ABSTRACTThe microcin PDI inhibits a diverse group of pathogenicEscherichia colistrains. Coculture of a single-gene knockout library (BW25113;n= 3,985 mutants) against a microcin PDI-producing strain (E. coli25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts inE. coliO157:H7 Sakai. Heterologous expression ofE. coliompFconferred susceptibility toSalmonella entericaandYersinia enterocoliticastrains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49region within the first extracellular loop ofE. coliOmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator forompF, and consequently loss of susceptibility by the ΔompRstrain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. Intransexpression ofompFin the ΔdsbAand ΔdsbBstrains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

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Characterization of Soluble, Disulfide Bond-Stabilized, Prokaryotically Expressed Human Thyrotropin Receptor Ectodomain*

Endocrinology ◽

10.1210/endo.138.2.4920 ◽

1997 ◽

Vol 138 (2) ◽

pp. 588-593 ◽

Cited By ~ 8

Author(s):

Y. Bobovnikova ◽

P. N. Graves ◽

H. Vlase ◽

T. F. Davies

Keyword(s):

Amino Acids ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Thioredoxin Reductase ◽

Biologically Active ◽

Receptor Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Fusion Partner ◽

E Coli ◽

Recombinant Receptor

Abstract To study the interaction of TSH receptor (TSHR) autoantibodies with receptor protein, it is necessary first to express the receptor in the proper conformation including the formation of correct disulfide bridges. However, the reducing environment of the Escherichia coli (E. coli) cytoplasm prevents the generation of protein disulfide bonds and limits the solubility and immunoreactivity of recombinant human TSHR (hTSHR) products. To circumvent these limitations, hTSHR complementary DNA encoding the extracellular domain (hTSHR-ecd; amino acids 21–415) was inserted into the vector pGEX-2TK by directional cloning and used to transform the thioredoxin reductase mutant strain of E. coli (Ad494), which allowed formation of disulfide bonds in the cytoplasm. After induction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTSHR amino acids 21–35. This showed that over 50% of the expressed hTSHR-ecd was soluble in contrast to expression in a wild-type E. coli (strain αF′), where the majority of the recombinant receptor was insoluble. The soluble recombinant receptor was affinity purified and characterized. Under nonreducing SDS-PAGE conditions, the soluble hTSHR-ecd migrated as refolded, disulfide bond-stabilized, multimeric species, whose formation was independent of fusion partner protein. This product was found to be biologically active as evidenced by the inhibition of the binding of 125I-TSH to the full-length hTSHR expressed in transfected CHO cells and was used to develop a competitive capture enzyme-linked immunosorbent assay for mapping of hTSHR antibody epitopes. Hence, hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation was found to be highly soluble and biologically relevant.

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Structure and Dynamics of FosA-Mediated Fosfomycin Resistance in Klebsiella pneumoniae and Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01572-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 15

Author(s):

Erik H. Klontz ◽

Adam D. Tomich ◽

Sebastian Günther ◽

Justin A. Lemkul ◽

Daniel Deredge ◽

...

Keyword(s):

Escherichia Coli ◽

Molecular Dynamics ◽

Klebsiella Pneumoniae ◽

Crystal Structures ◽

Active Sites ◽

Content Type ◽

Dimer Interface ◽

Fosfomycin Resistance ◽

Gram Negative Organisms ◽

Dynamics Simulations

ABSTRACT Fosfomycin exhibits broad-spectrum antibacterial activity and is being reevaluated for the treatment of extensively drug-resistant pathogens. Its activity in Gram-negative organisms, however, can be compromised by expression of FosA, a metal-dependent transferase that catalyzes the conjugation of glutathione to fosfomycin, rendering the antibiotic inactive. In this study, we solved the crystal structures of two of the most clinically relevant FosA enzymes: plasmid-encoded FosA3 from Escherichia coli and chromosomally encoded FosA from Klebsiella pneumoniae (FosAKP). The structure, molecular dynamics, catalytic activity, and fosfomycin resistance of FosA3 and FosAKP were also compared to those of FosA from Pseudomonas aeruginosa (FosAPA), for which prior crystal structures exist. E. coli TOP10 transformants expressing FosA3 and FosAKP conferred significantly greater fosfomycin resistance (MIC, >1,024 μg/ml) than those expressing FosAPA (MIC, 16 μg/ml), which could be explained in part by the higher catalytic efficiencies of the FosA3 and FosAKP enzymes. Interestingly, these differences in enzyme activity could not be attributed to structural differences at their active sites. Instead, molecular dynamics simulations and hydrogen-deuterium exchange experiments with FosAKP revealed dynamic interconnectivity between its active sites and a loop structure that extends from the active site of each monomer and traverses the dimer interface. This dimer interface loop is longer and more extended in FosAKP and FosA3 than in FosAPA, and kinetic analyses of FosAKP and FosAPA loop-swapped chimeric enzymes highlighted its importance in FosA activity. Collectively, these data yield novel insights into fosfomycin resistance that could be leveraged to develop new strategies to inhibit FosA and potentiate fosfomycin activity.

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