scholarly journals High-Level Chromosomally Mediated Tetracycline Resistance in Neisseria gonorrhoeae Results from a Point Mutation in the rpsJ Gene Encoding Ribosomal Protein S10 in Combination with the mtrR and penB Resistance Determinants

2005 ◽  
Vol 49 (10) ◽  
pp. 4327-4334 ◽  
Author(s):  
Mei Hu ◽  
Sobhan Nandi ◽  
Christopher Davies ◽  
Robert A. Nicholas
Keyword(s):  
Amino Acids ◽  
Neisseria Gonorrhoeae ◽  
Ribosomal Protein ◽  
Point Mutation ◽  
Single Point ◽  
Ribosomal Subunit ◽  
Tetracycline Resistance ◽  
Saturation Mutagenesis ◽  
Single Point Mutation ◽  
High Level

ABSTRACT Neisseria gonorrhoeae becomes resistant to tetracycline by two major mechanisms: expression of a plasmid-encoded TetM protein and mutations in endogenous genes (chromosomally mediated resistance). Early studies by Sparling and colleagues (P. F. Sparling F. A. J. Sarubbi, and E. Blackman, J. Bacteriol. 124:740-749, 1975) demonstrated that three genes were involved in high-level chromosomally mediated tetracycline resistance (MIC of tetracycline ≥ 2 μg/ml): ery-2 (now referred to as mtrR), penB, and tet-2. While the identities of the first two genes are known, the tet-2 gene has not been identified. We cloned the tet-2 gene, which confers tetracycline resistance, from tetracycline-resistant clinical isolate N. gonorrhoeae FA6140 and show that resistance is due to a single point mutation (Val-57 to Met) in the rpsJ gene (rpsJ1) encoding ribosomal protein S10. Moreover, the identical mutation was found in six distinct tetracycline-resistant clinical isolates in which the MIC of tetracycline was ≥2 μg/ml. Site-saturation mutagenesis of the codon for Val-57 identified two other amino acids (Leu and Gln) that conferred identical levels of resistance as the Met-57 mutation. The mutation maps to the vertex of a loop in S10 that is near the aminoacyl-tRNA site in the structure of the 30S ribosomal subunit from Thermus thermophilus, and the residue equivalent to Val-57 in T. thermophilus S10, Lys-55, is within 8 to 9 Å of bound tetracycline. These data suggest that large noncharged amino acids alter the rRNA structure near the tetracycline-binding site, leading to a lower affinity of the antibiotic.

scholarly journals High-Level Azithromycin Resistance Occurs in Neisseria gonorrhoeae as a Result of a Single Point Mutation in the 23S rRNA Genes

2010 ◽  
Vol 54 (9) ◽  
pp. 3812-3816 ◽  
Author(s):  
Stephanie A. Chisholm ◽  
Jayshree Dave ◽  
Catherine A. Ison
Keyword(s):  
United Kingdom ◽  
Neisseria Gonorrhoeae ◽  
Point Mutation ◽  
Single Point ◽  
23S Rrna ◽  
Single Point Mutation ◽  
The United Kingdom ◽  
High Level ◽  
Domain V ◽  
Azithromycin Resistance

ABSTRACT High-level azithromycin resistance (AZM-HR), defined as a MIC of ≥256 mg/liter, emerged in Neisseria gonorrhoeae in the United Kingdom in 2004. To determine the mechanism of this novel phenotype, isolates from the United Kingdom that were AZM-HR (n, 19), moderately AZM resistant (MICs, 2 to 8 mg/liter) (n, 26), or sensitive (MICs, 0.12 to 0.25 mg/liter) (n, 4) were screened for methylase (erm) genes and for mutations in the mtrR promoter region, associated with efflux pump upregulation. All AZM-resistant isolates and 12 sensitive isolates were screened for mutations in domain V of each 23S rRNA allele. All AZM-HR isolates contained the A2059G mutation (Escherichia coli numbering) in three (3 isolates) or four (16 isolates) 23S rRNA alleles. Most (22/26) moderately AZM resistant isolates contained the C2611T mutation in at least 3/4 alleles. The remainder contained four wild-type alleles, as did 8/12 sensitive isolates, while one allele was mutated in the remaining four sensitive isolates. Serial passage of AZM-sensitive colonies on an erythromycin-containing medium selected AZM-HR if the parent strain already contained mutation A2059G in one 23S rRNA allele. The resultant AZM-HR strains contained four mutated alleles. Eight isolates (five moderately AZM resistant and three AZM-HR) contained mutations in the mtrR promoter. No methylase genes were detected. This is the first evidence that AZM-HR in gonococci may result from a single point mutation (A2059G) in the peptidyltransferase loop in domain V of the 23S rRNA gene. Mutation of a single allele is insufficient to confer AZM-HR, but AZM-HR can develop under selection pressure. The description of a novel resistance mechanism will aid in screening for the AZM-HR phenotype.

Abstract 52: The Role of Kindlin-2 in Integrin Activation Depends on a Short C-Terminal Region

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Jamila Hirbawi ◽  
Kamila Bledzka ◽  
Yan Qing Ma ◽  
Jun Qin ◽  
Edward F Plow
Keyword(s):  
Amino Acids ◽  
Point Mutation ◽  
Single Point ◽  
Terminal Region ◽  
Membrane Receptors ◽  
Single Point Mutation ◽  
Loss Of Function ◽  
Integrin Activation ◽  
C Terminus

Integrins are heterodimeric cell membrane receptors that regulate cell adhesion, migration, and survival. The kindlins are known to be key regulators of integrin activation, the transition from a low affinity, default state to a high affinity state for ligand. This function depends on their binding, together with talin, to the cytoplasmic tails (CT) of the β subunit of integrins. Kindlins are FERM domain containing proteins, and it is its F3 (PTB) subdomain of the FERM that is the primary binding site for integrin β CT. At its very C-terminus, beyond the F3, is a short extension of 21 amino acids, K2 660-680, and we have focused on the role of this region in the co-activator function of kindlin-2 (K2). For this analysis, we performed PAC-1 (antibody to detect activated αIIbβ3 integrin) binding assays in CHO cells stably expressing integrin α IIb β 3 that were transiently transfected with talin head domain and K2 mutants. Expression levels of all proteins were verified to be similar by western blotting and FACS. Truncation of K2 at residue 660 essentially eliminated the co-activator function of K2. Deletion of smaller segments also reduced co-activator activity by 50% to 100%. Deletion of just the last two amino acids in the sequence, W 679 V 680 , resulted in a 50% reduction in co-activator activity and a single point mutation of Y 673 A also led to a 50% loss of function. A combination mutant consisting of the W 679 V 680 deletion and the Y 673 point mutation resulted in 100% loss of kindlin-2 co-activator activity. Pull-down experiments performed using GST tagged β 3 CT and CHO lysates transfected with GFP-kindlin-2 forms suggested that the C-terminal deletion did not disrupt binding to β 3 CT. This observation was corroborated by surface plasmon resonance studies in which the binding of full-length K2 and K2Δ666C (Δ666) was compared, and their K D values for immobilized β3 CT were found to be essentially the same. Overall, these data establish an important and unanticipated role of the carboxy-terminal region of kindlin-2 in its integrin co-activator function that is not dependent of its binding to integrin.

scholarly journals Assessing the capability ofin silicomutation protocols for predicting the finite temperature conformation of amino acids

2018 ◽  
Vol 20 (40) ◽  
pp. 25901-25909 ◽  
Author(s):  
Rodrigo Ochoa ◽  
Miguel A. Soler ◽  
Alessandro Laio ◽  
Pilar Cossio
Keyword(s):  
Molecular Dynamics ◽  
Amino Acids ◽  
Molecular Dynamics Simulations ◽  
Point Mutation ◽  
Finite Temperature ◽  
Single Point ◽  
Single Point Mutation ◽  
Equilibrium Configurations ◽  
Dynamics Simulations

Single-point mutation protocols based on backbone-dependent rotamer libraries show the best performance in predicting equilibrium configurations from molecular dynamics simulations.

scholarly journals Biochemical and molecular characterization of Alternaria alternata isolates highly resistant to procymidone from broccoli and cabbage

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Bingran Wang ◽  
Tiancheng Lou ◽  
Lingling Wei ◽  
Wenchan Chen ◽  
Longbing Huang ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Single Point ◽  
Sodium Dodecyl ◽  
Cross Resistance ◽  
Molecular Characteristics ◽  
Single Point Mutation ◽  
Wide Range ◽  
Significant Difference ◽  
Leaf Blights ◽  
High Level

AbstractAlternaria alternata, a causal agent of leaf blights and spots on a wide range of hosts, has a high risk of developing resistance to fungicides. Procymidone, a dicarboximide fungicide (DCF), has been widely used in controlling Alternaria leaf blights in China for decades. However, the resistance of A. alternata against DCFs has rarely been reported from crucifer plants. A total of 198 A. alternata isolates were collected from commercial fields of broccoli and cabbage during 2018–2019, and their sensitivities to procymidone were determined. Biochemical and molecular characteristics were subsequently compared between the high-level procymidone-resistant (ProHR) and procymidone-sensitive (ProS) isolates, and also between ProHR isolates from broccoli and cabbage. Compared with ProS isolates, the mycelial growth rate, sporulation capacity and virulence of most ProHR isolates were reduced; ProHR isolates displayed an increased sensitivity to osmotic stresses and a reduced sensitivity to sodium dodecyl sulfate (SDS); all ProHR isolates showed a reduced sensitivity to hydrogen peroxide (H2O2) except for the isolate B102. Correlation analysis revealed a positive cross-resistance between procymidone and iprodione, or fludioxonil. When treated with 10 μg/mL of procymidone, both mycelial intracellular glycerol accumulations (MIGAs) and relative expression of AaHK1 in ProS isolates were higher than those in ProHR isolates. Sequence alignment of AaHK1 from ten ProHR isolates demonstrated that five of them possessed a single-point mutation (P94A, V612L, E708K or Q924STOP), and four isolates had an insertion or a deletion in their coding regions. No significant difference in biochemical characteristics was observed among ProHR isolates from two different hosts, though mutations in AaHK1 of the cabbage-originated ProHR isolates were distinct from those of the broccoli-originated ProHR isolates.

scholarly journals A single point mutation converts a glutaryl-7-aminocephalosporanic acid acylase into an N-acyl-homoserine lactone acylase

2021 ◽  
Author(s):  
Shereen A. Murugayah ◽  
Gary B. Evans ◽  
Joel D. A. Tyndall ◽  
Monica L. Gerth
Keyword(s):  
Single Point ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Acyl Homoserine Lactone ◽  
Signalling Molecules ◽  
Single Amino Acid Residue

Abstract Objective To change the specificity of a glutaryl-7-aminocephalosporanic acid acylase (GCA) towards N-acyl homoserine lactones (AHLs; quorum sensing signalling molecules) by site-directed mutagenesis. Results Seven residues were identified by analysis of existing crystal structures as potential determinants of substrate specificity. Site-saturation mutagenesis libraries were created for each of the seven selected positions. High-throughput activity screening of each library identified two variants—Arg255Ala, Arg255Gly—with new activities towards N-acyl homoserine lactone substrates. Structural modelling of the Arg255Gly mutation suggests that the smaller side-chain of glycine (as compared to arginine in the wild-type enzyme) avoids a key clash with the acyl group of the N-acyl homoserine lactone substrate. Conclusions Mutation of a single amino acid residue successfully converted a GCA (with no detectable activity against AHLs) into an AHL acylase. This approach may be useful for further engineering of ‘quorum quenching’ enzymes.

scholarly journals A single point mutation converts a proton-pumping rhodopsin into a red-shifted, turn-on fluorescent sensor for chloride

2021 ◽  
Author(s):  
Jasmine N. Tutol ◽  
Jessica Lee ◽  
Hsichuan Chi ◽  
Farah N. Faizuddin ◽  
Sameera S. Abeyrathna ◽  
...  
Keyword(s):  
Point Mutation ◽  
Fluorescent Sensor ◽  
Single Point ◽  
Proton Pumping ◽  
Single Point Mutation ◽  
Turn On ◽  
Gloeobacter Violaceus

By utilizing laboratory-guided evolution, we have converted the fluorescent proton-pumping rhodopsin GR from Gloeobacter violaceus into GR1, a red-shifted, turn-on fluorescent sensor for chloride.

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