scholarly journals In Vivo Selection of Campylobacter Isolates with High Levels of Fluoroquinolone Resistance Associated with gyrA Mutations and the Function of the CmeABC Efflux Pump

2003 ◽  
Vol 47 (1) ◽  
pp. 390-394 ◽  
Author(s):  
Naidan Luo ◽  
Orhan Sahin ◽  
Jun Lin ◽  
Linda O. Michel ◽  
Qijing Zhang
Keyword(s):  
Intestinal Tract ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Single Point ◽  
Point Mutations ◽  
Fluoroquinolone Resistance ◽  
Quinolone Antibiotics ◽  
Single Point Mutations ◽  
Selection Of

ABSTRACT Enrofloxacin treatment of chickens infected with fluoroquinolone(FQ)-sensitive Campylobacter promoted the emergence of FQ-resistant Campylobacter mutants which propagated in the intestinal tract and recolonized the chickens. The recovered isolates were highly resistant to quinolone antibiotics but remained susceptible to non-FQ antimicrobial agents. Specific single-point mutations in the gyrA gene and the function of the CmeABC efflux pump were linked to the acquired FQ resistance. These results reveal that Campylobacter is hypermutable in vivo under the selection pressure of FQ and highlight the need for the prudent use of FQ antibiotics.

scholarly journals Stabilizing the Closed SARS-CoV-2 Spike Trimer

2020 ◽  
Author(s):  
Jarek Juraszek ◽  
Lucy Rutten ◽  
Sven Blokland ◽  
Pascale Bouchier ◽  
Richard Voorzaat ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Single Point ◽  
Point Mutations ◽  
Heptad Repeat ◽  
S Protein ◽  
Single Point Mutations ◽  
Primary Focus ◽  
Immunogenic Properties ◽  
Serological Diagnostics ◽  
Selection Of

AbstractThe trimeric spike (S) protein of SARS-CoV-2 is the primary focus of most vaccine design and development efforts. Due to intrinsic instability typical of class I fusion proteins, S tends to prematurely refold to the post-fusion conformation, compromising immunogenic properties and prefusion trimer yields. To support ongoing vaccine development efforts, we report the structure-based design of soluble S trimers, with increased yields and stabilities, based on introduction of single point mutations and disulfide-bridges. We identify two regions in the S-protein critical for the protein’s stability: the heptad repeat region 1 of the S2 subunit and subunit domain 1 at the interface with S2. We combined a minimal selection of mostly interprotomeric mutations to create a stable S-closed variant with a 6.4-fold higher expression than the parental construct while no longer containing a heterologous trimerization domain. The cryo-EM structure reveals a correctly folded, predominantly closed pre-fusion conformation. Highly stable and well producing S protein and the increased understanding of S protein structure will support vaccine development and serological diagnostics.

scholarly journals First Characterization of Fluoroquinolone Resistance in Streptococcus suis

2006 ◽  
Vol 51 (2) ◽  
pp. 777-782 ◽  
Author(s):  
Jose Antonio Escudero ◽  
Alvaro San Millan ◽  
Ana Catalan ◽  
Adela G. de la Campa ◽  
Estefania Rivero ◽  
...  
Keyword(s):  
Horizontal Gene Transfer ◽  
Single Point ◽  
Point Mutations ◽  
Streptococcus Suis ◽  
Fluoroquinolone Resistance ◽  
Genes Encoding ◽  
Resistant Strains ◽  
Single Point Mutations ◽  
Clonal Spread

ABSTRACT We have identified and sequenced the genes encoding the quinolone-resistance determining region (QRDR) of ParC and GyrA in fluoroquinolone-susceptible and -resistant Streptococcus suis clinical isolates. Resistance is the consequence of single point mutations in the QRDRs of ParC and GyrA and is not due to clonal spread of resistant strains or horizontal gene transfer with other bacteria.

scholarly journals Molecular Basis of Resistance to Selected Antimicrobial Agents in the Emerging Zoonotic Pathogen Streptococcus suis

2015 ◽  
Vol 53 (7) ◽  
pp. 2332-2336 ◽  
Author(s):  
Mamata Gurung ◽  
Migma Dorji Tamang ◽  
Dong Chan Moon ◽  
Su-Ran Kim ◽  
Jin-Ha Jeong ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Antimicrobial Agents ◽  
Single Point ◽  
Point Mutations ◽  
Streptococcus Suis ◽  
Quinolone Resistance ◽  
Content Type ◽  
Zoonotic Pathogen ◽  
Single Point Mutations

Characterization of 227Streptococcus suisstrains isolated from pigs during 2010 to 2013 showed high levels of resistance to clindamycin (95.6%), tilmicosin (94.7%), tylosin (93.8%), oxytetracycline (89.4%), chlortetracycline (86.8%), tiamulin (72.7%), neomycin (70.0%), enrofloxacin (56.4%), penicillin (56.4%), ceftiofur (55.9%), and gentamicin (55.1%). Resistance to tetracyclines, macrolides, aminoglycosides, and fluoroquinolone was attributed to thetetgene,erm(B),erm(C),mph(C), andmef(A) and/ormef(E) genes,aph(3′)-IIIaandaac(6′)-Ie-aph(2″)-Iagenes, and single point mutations in the quinolone resistance-determining region of ParC and GyrA, respectively.

Detection of grlA and gyrA Mutations in 344 Staphylococcus aureus Strains

1998 ◽  
Vol 42 (2) ◽  
pp. 236-240 ◽  
Author(s):  
Tong Wang ◽  
Mayumi Tanaka ◽  
Kenichi Sato
Keyword(s):  
Staphylococcus Aureus ◽  
Direct Sequencing ◽  
Single Point ◽  
Point Mutations ◽  
Single Strand Conformation Polymorphism ◽  
Fluoroquinolone Resistance ◽  
Polymorphism Analysis ◽  
Ciprofloxacin Resistance ◽  
Single Point Mutations ◽  
Length Analysis

ABSTRACT Mutations in the grlA and gyrA genes of 344 clinical strains of Staphylococcus aureus isolated in 1994 in Japan were identified by combinations of single-strand conformation polymorphism analysis, restriction fragment length analysis, and direct sequencing to identify possible relationships to fluoroquinolone resistance. Five types of single-point mutations and four types of double mutations were observed in the grlA genes of 204 strains (59.3%). Four types of single-point mutations and four types of double mutations were found in the gyrA genes of 188 strains (54.7%). Among them, the grlA mutation of TCC→TTC or TAC (Ser-80→Phe or Tyr) and the gyrAmutation of TCA→TTA (Ser-84→Leu) were principal, being detected in 137 (39.8%) and 121 (35.9%) isolates, respectively. ThegrlA point mutations of CAT→CAC (His-77 [silent]), TCA→CCA (Ser-81→Pro), and ATA→ATT (Ile-100 [silent]) were novel, as was the GAC→GGC (Asp-73→Gly) change in gyrA. A total of 15 types of mutation combinations within both genes were related to ciprofloxacin resistance (MIC ≥ 3.13 μg/ml) and were present in 193 mutants (56.1%). Strains containing mutations in both genes were highly resistant to ciprofloxacin (MIC at which 50% of the isolates are inhibited [MIC50] = 50 μg/ml). Those with the Ser-80→Phe or Tyr alteration in grlA but wild-typegyrA showed a lower level of ciprofloxacin resistance (MIC50 ≤ 12.5 μg/ml). Levofloxacin was active against 68 of 193 isolates (35.2%) with mutations at codon 80 of grlAin the presence or absence of a concomitant mutation at codon 73, 84, or 88 in gyrA (MIC ≤ 6.25 μg/ml). The new fluoroquinolone DU-6859a showed good activity with 186 of 193 isolates (96.4%) for which the MIC was ≤6.25 μg/ml.

Cupin Variants as Macromolecular Ligand Library for Stereoselective Michael Addition of Nitroalkanes

2019 ◽  
Author(s):  
Nobutaka Fujieda ◽  
Miho Yuasa ◽  
Yosuke Nishikawa ◽  
Genji Kurisu ◽  
Shinobu Itoh ◽  
...  
Keyword(s):  
Michael Addition ◽  
In Silico ◽  
Addition Reaction ◽  
Single Point ◽  
Point Mutations ◽  
Unsaturated Ketone ◽  
Michael Addition Reaction ◽  
Beta Barrel ◽  
Cupin Superfamily ◽  
Single Point Mutations

Cupin superfamily proteins (TM1459) work as a macromolecular ligand framework with a double-stranded beta-barrel structure ligating to a Cu ion through histidine side chains. Variegating the first coordination sphere of TM1459 revealed that H52A and H54A/H58A mutants effectively catalyzed the diastereo- and enantio-selective Michael addition reaction of nitroalkanes to an α,β-unsaturated ketone. Moreover, in silico substrate docking signified C106N and F104W single-point mutations, which inverted the diastereoselectivity of H52A and further improved the stereoselectivity of H54A/H58A, respectively.

Single-Point Mutations in Qβ Virus-like Particles Change Binding to Cells

2021 ◽  
Author(s):  
Marisa L. Martino ◽  
Stephen N. Crooke ◽  
Marianne Manchester ◽  
M.G. Finn
Keyword(s):  
Single Point ◽  
Point Mutations ◽  
Virus Like Particles ◽  
Single Point Mutations

scholarly journals In Vivo Evidence for TonB Dimerization

2003 ◽  
Vol 185 (19) ◽  
pp. 5747-5754 ◽  
Author(s):  
Annette Sauter ◽  
S. Peter Howard ◽  
Volkmar Braun
Keyword(s):  
Single Point ◽  
Point Mutations ◽  
Ferric Citrate ◽  
Site Directed Mutagenesis ◽  
Toxin Gene ◽  
Transmembrane Region ◽  
Dimer Formation ◽  
Citrate Transport ◽  
Hybrid Proteins

ABSTRACT TonB, in complex with ExbB and ExbD, is required for the energy-dependent transport of ferric siderophores across the outer membrane of Escherichia coli, the killing of cells by group B colicins, and infection by phages T1 and φ80. To gain insights into the protein complex, TonB dimerization was studied by constructing hybrid proteins from complete TonB (containing amino acids 1 to 239) [TonB(1-239)] and the cytoplasmic fragment of ToxR which, when dimerized, activates the transcription of the cholera toxin gene ctx. ToxR(1-182)-TonB(1-239) activated the transcription of lacZ under the control of the ctx promoter (P ctx ::lacZ). Replacement of the TonB transmembrane region by the ToxR transmembrane region resulted in the hybrid proteins ToxR(1-210)-TonB(33-239) and ToxR(1-210)-TonB(164-239), of which only the latter activated P ctx ::lacZ transcription. Dimer formation was reduced but not abolished in a mutant lacking ExbB and ExbD, suggesting that these complex components may influence dimerization but are not strictly required and that the N-terminal cytoplasmic membrane anchor and the C-terminal region are important for dimer formation. The periplasmic TonB fragment, TonB(33-239), inhibits ferrichrome and ferric citrate transport and induction of the ferric citrate transport system. This competition provided a means to positively screen for TonB(33-239) mutants which displayed no inhibition. Single point mutations of inactive fragments selected in this manner were introduced into complete TonB, and the phenotypes of the TonB mutant strains were determined. The mutations located in the C-terminal half of TonB, three of which (Y163C, V188E, and R204C) were obtained separately by site-directed mutagenesis, as was the isolated F230V mutation, were studied in more detail. They displayed different activity levels for various TonB-dependent functions, suggesting function-related specificities which reflect differences in the interactions of TonB with various transporters and receptors.

MinD directly interacting with FtsZ at the H10 helix suggests a model for robust activation of MinC to destabilize FtsZ polymers

2017 ◽  
Vol 474 (18) ◽  
pp. 3189-3205 ◽  
Author(s):  
Ashoka Chary Taviti ◽  
Tushar Kant Beuria
Keyword(s):  
Cell Division ◽  
Single Point ◽  
Point Mutations ◽  
Direct Interaction ◽  
Ring Formation ◽  
Z Ring ◽  
Single Point Mutations ◽  
Biochemical Analyses ◽  
Ftsz Assembly ◽  
The Mind

Cell division in bacteria is a highly controlled and regulated process. FtsZ, a bacterial cytoskeletal protein, forms a ring-like structure known as the Z-ring and recruits more than a dozen other cell division proteins. The Min system oscillates between the poles and inhibits the Z-ring formation at the poles by perturbing FtsZ assembly. This leads to an increase in the FtsZ concentration at the mid-cell and helps in Z-ring positioning. MinC, the effector protein, interferes with Z-ring formation through two different mechanisms mediated by its two domains with the help of MinD. However, the mechanism by which MinD triggers MinC activity is not yet known. We showed that MinD directly interacts with FtsZ with an affinity stronger than the reported MinC–FtsZ interaction. We determined the MinD-binding site of FtsZ using computational, mutational and biochemical analyses. Our study showed that MinD binds to the H10 helix of FtsZ. Single-point mutations at the charged residues in the H10 helix resulted in a decrease in the FtsZ affinity towards MinD. Based on our findings, we propose a novel model for MinCD–FtsZ interaction, where MinD through its direct interaction with FtsZ would trigger MinC activity to inhibit FtsZ functions.

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