scholarly journals Resistance to fusidic acid in Escherichia coli mediated by the type I variant of chloramphenicol acetyltransferase. A plasmid-encoded mechanism involving antibiotic binding

1983 ◽  
Vol 215 (1) ◽  
pp. 29-38 ◽  
Author(s):  
A D Bennett ◽  
W V Shaw
Keyword(s):  
Escherichia Coli ◽  
Fusidic Acid ◽  
Acid Resistance ◽  
Chloramphenicol Acetyltransferase ◽  
Chain Elongation ◽  
Type I ◽  
Structural Constraints ◽  
Binding Studies ◽  
Chloramphenicol Resistance

Plasmid-encoded fusidic acid resistance in Escherichia coli is mediated by a common variant of chloramphenicol acetyltransferase (EC 2.3.1.28), an enzyme which is an effector of chloramphenicol resistance. Resistance to chloramphenicol is a consequence of acetylation of the antibiotic catalysed by the enzyme and the failure of the 3-acetoxy product to bind to bacterial ribosomes. Cell-free coupled transcription and translation studies are in agreement with genetic studies which indicated that the entire structural gene for the type I chloramphenicol acetyltransferase is necessary for the fusidic acid resistance phenotype. The mechanism of resistance does not involve covalent modification of the antibiotic. The other naturally occurring enterobacterial chloramphenicol acetyltransferase variants (types II and III) do not cause fusidic acid resistance. Steady-state kinetic studies with the type I enzyme have shown that the binding of fusidic acid is competitive with respect to chloramphenicol. The inhibition of polypeptide chain elongation in vitro which is observed in the presence of fusidic acid is relieved by addition of purified chloramphenicol acetyltransferase, and equilibrium dialysis experiments with [3H]fusidate and the type I enzyme have defined the stoichiometry and apparent affinity of fusidate for the type I enzyme. Further binding studies with fusidate analogues, including bile salts, have shown some of the structural constraints on the steroidal skeleton of the ligand which are necessary for binding to the enzyme. Determinations of antibiotic resistance levels and estimates of intracellular chloramphenicol acetyltransferase concentrations in vivo support the data from experiments in vitro to give a coherent mechanism for fusidic acid resistance based on reversible binding of the antibiotic to the enzyme.

scholarly journals The use of naturally occurring hybrid variants of chloramphenicol acetyltransferase to investigate subunit contacts

1981 ◽  
Vol 193 (2) ◽  
pp. 541-552 ◽  
Author(s):  
L C Packman ◽  
W V Shaw
Keyword(s):  
Chloramphenicol Acetyltransferase ◽  
Alpha Subunit ◽  
Type I ◽  
Chloramphenicol Resistance ◽  
Type Iii ◽  
Naturally Occurring ◽  
Alpha Beta ◽  
Beta 2

1. Hybrids of the tetrameric enzyme chloramphenicol acetyltransferase (EC 2.3.1.28) were formed in vivo in a strain of Escherichia coli which harbours two different plasmids, each of which normally confers chloramphenicol resistance and specifies an easily distinguished enzyme variant (type I or type III) which is composed of identical subunits. Cell-free extracts of the dual-plasmid strain were found to contain five species of active enzyme, two of which were the homomeric enzymes corresponding to the naturally occurring tetramers of the type-I (beta 4) and type-III (alpha 4) enzymes. The other three variants were judged to be the heteromeric hybrid variants (alpha 3 beta, alpha 2 beta 2, alpha beta 3). 2. The alpha 3 beta and alpha 2 beta 2 hybrids of chloramphenicol acetyltransferase were purified to homogeneity by combining the techniques of affinity and ion-exchange chromatography. The alpha beta 3 variant was not recovered and may be unstable in vitro. 3. The unique lysine residues that could not be modified with methyl acetimidate in each of the native homomeric enzymes were also investigated in the heteromeric tetramers. 4. Lysine-136 remains buried in each beta subunit of the parental (type I) enzyme and in each of the hybrid tetramers. Lysine-38 of each alpha subunit is similarly unreactive in the native type-III chloramphenicol acetyltransferase (alpha 4), but in the alpha 2 beta 2 hybird lysine-38 of each alpha subunit is fully exposed to solvent. Another lysine residue, fully reactive in the alpha 4 enzyme, was observed to be inaccessible to modification in the symmetrical hybrid. The results obtained for the alpha 3 beta enzyme suggest that lysine-38 in two subunits and a different lysine group (that identified in the alpha 2 beta 2 enzyme) in the third alpha subunit are buried. 5. A tentative model for the subunit interactions of chloramphenicol acetyltransferase is proposed on the basis of the results described.

scholarly journals Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

2021 ◽  
Author(s):  
Kazuto Yoshimi ◽  
Kohei TAKESHITA ◽  
Noriyuki Kodera ◽  
Satomi Shibumura ◽  
Yuko Yamauchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Speed ◽  
Dna Helicase ◽  
Type I ◽  
Loop Formation ◽  
Force Microscopy ◽  
Atomic Force ◽  
Target Strand ◽  
Target Dna

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific ssDNA cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target dsDNA substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.

scholarly journals Esterases in Serum-Containing Growth Media Counteract Chloramphenicol Acetyltransferase Activity In Vitro

1999 ◽  
Vol 43 (3) ◽  
pp. 655-660 ◽  
Author(s):  
Charles D. Sohaskey ◽  
Alan G. Barbour
Keyword(s):  
Human Serum ◽  
Horse Serum ◽  
Chloramphenicol Acetyltransferase ◽  
Rabbit Serum ◽  
Growth Media ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Cell Extracts

ABSTRACT The spirochete Borrelia burgdorferi was unexpectedly found to be as susceptible to diacetyl chloramphenicol, the product of the enzyme chloramphenicol acetyltransferase, as it was to chloramphenicol itself. The susceptibilities of Escherichia coli and Bacillus subtilis, as well as that ofB. burgdorferi, to diacetyl chloramphenicol were then assayed in different media. All three species were susceptible to diacetyl chloramphenicol when growth media were supplemented with rabbit serum or, to a lesser extent, human serum. Susceptibility ofE. coli and B. subtilis to diacetyl chloramphenicol was not observed in the absence of serum, when horse serum was used, or when the rabbit or human serum was heated first. In the presence of 10% rabbit serum, a strain of E. colibearing the chloramphenicol acetyltransferase (cat) gene had a fourfold-lower resistance to chloramphenicol than in the absence of serum. A plate bioassay for chloramphenicol activity showed the conversion by rabbit, mouse, and human sera but not bacterial cell extracts or heated serum of diacetyl chloramphenicol to an inhibitory compound. Deacetylation of acetyl chloramphenicol by serum components was demonstrated by using fluorescent substrates and thin-layer chromatography. These studies indicate that esterases of serum can convert diacetyl chloramphenicol back to an active antibiotic, and thus, in vitro findings may not accurately reflect the level of chloramphenicol resistance by cat-bearing bacteria in vivo.

scholarly journals Platelet-derived microparticles associate with fibrin during thrombosis

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4651-4663 ◽  
Author(s):  
P Siljander ◽  
O Carpen ◽  
R Lassila
Keyword(s):  
Type I Collagen ◽  
Thrombus Formation ◽  
Flow Chamber ◽  
Type I ◽  
Binding Studies ◽  
Vitro Binding ◽  
Large Platelet ◽  
Platelet Aggregates ◽  
Thrombin Antithrombin Iii Complex

Platelet-derived microparticles (MP) are reported to express both pro- and anticoagulant activities. Nevertheless, their functional significance has remained unresolved. The present study monitored the generation and fate of MP in an experimental model of thrombosis with costimulation of platelets by collagen and thrombin. When minimally anticoagulated (0.5 micromol/L PPACK) blood was perfused over immobilized fibrillar type I collagen in a flow chamber at a low shear rate (300 s(-1)), endogenous thrombin was generated, as evidenced by thrombin-antithrombin III complex. In contrast to full anticoagulation 150 micromol/L PPACK) and the absence of collagen, large platelet aggregates and fibrin ensued during perfusions over collagen in the presence of thrombin. In these thrombi, MP, defined as GPIIbIIIa- and P- selectin-positive vesicles (<1 micron), were found to align fibrin in immunofluorescence and scanning immunoelectron microscopy. Moreover, in sections of embolectomized thromboemboli from patients GPIIbIIIa- and P- selectin-positive material compatible with MP was detected in a fibrin strand-like pattern. In vitro binding studies showed that MP bound to fibrin and acted there as procoagulants. In summary, we show that MP generated during thrombus formation associate with local fibrin. This adhesive function fibrin could imply a sustained modulatory role for MP in evolving thrombi.

scholarly journals Compensatory Adaptation to the Loss of Biological Fitness Associated with Acquisition of Fusidic Acid Resistance in Staphylococcus aureus

2005 ◽  
Vol 49 (4) ◽  
pp. 1426-1431 ◽  
Author(s):  
Silke Besier ◽  
Albrecht Ludwig ◽  
Volker Brade ◽  
Thomas A. Wichelhaus
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Fusidic Acid ◽  
Wild Type Strain ◽  
Elongation Factor ◽  
Acid Resistance ◽  
Resistant Bacteria ◽  
Wild Type ◽  
Compensatory Adaptation

ABSTRACT Recent studies have shown that individual amino acid exchanges within elongation factor G (EF-G) cause fusidic acid resistance in Staphylococcus aureus. The data from the present study illustrate that the fusidic acid resistance-mediating amino acid substitutions P406L and H457Y are associated with a marked impairment of the biological fitness of S. aureus. In particular, strains producing EF-G derivatives with these mutations showed reduced growth, decreased plasma coagulase activity, and an impaired capability to compete with the isogenic wild-type strain. Second-site mutations within EF-G, such as A67T and S416F, that have been encountered in clinical fusidic acid-resistant isolates containing the amino acid exchanges P406L and H457Y, respectively, were shown not to contribute to resistance. Furthermore, the substitution A67T had no impact on the biological fitness in vitro. The exchange S416F, however, was found to function as a fitness-compensating mutation in S. aureus carrying the substitution H457Y in EF-G. In conclusion, the data presented in this report provide evidence at the molecular level that the deleterious effects of fusidic acid resistance-mediating exchanges within EF-G of S. aureus can be reduced considerably by specific compensating mutations in this target protein. This compensatory adaptation most likely plays a significant role in the stabilization of resistant bacteria within a given population.

scholarly journals Fur Represses Adhesion to, Invasion of, and Intracellular Bacterial Community Formation within Bladder Epithelial Cells and Motility in Uropathogenic Escherichia coli

2016 ◽  
Vol 84 (11) ◽  
pp. 3220-3231 ◽  
Author(s):  
Kumiko Kurabayashi ◽  
Tomohiro Agata ◽  
Hirofumi Asano ◽  
Haruyoshi Tomita ◽  
Hidetada Hirakawa
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Antimicrobial Agents ◽  
Host Cells ◽  
Type I ◽  
Wild Type ◽  
Content Type ◽  
Type I Fimbriae ◽  
Tract Infections

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infections (UTIs). This bacterium adheres to and invades the host cells in the bladder, where it forms biofilm-like polymicrobial structures termed intracellular bacterial communities (IBCs) that protect UPEC from antimicrobial agents and the host immune systems. Using genetic screening, we found that deletion of the fur gene, which encodes an iron-binding transcriptional repressor for iron uptake systems, elevated the expression of type I fimbriae and motility when UPEC was grown under iron-rich conditions, and it led to an increased number of UPEC cells adhering to and internalized in bladder epithelial cells. Consequently, the IBC colonies that the fur mutant formed in host cells were denser and larger than those formed by the wild-type parent strain. Fur is inactivated under iron-restricted conditions. When iron was depleted from the bacterial cultures, wild-type UPEC adhesion, invasion, and motility increased, similar to the case with the fur mutant. The purified Fur protein bound to regions upstream of fimA and flhD , which encode type I fimbriae and an activator of flagellar expression that contributes to motility, respectively. These results suggest that Fur is a repressor of fimA and flhD and that its repression is abolished under iron-depleted conditions. Based on our in vitro experiments, we conclude that UPEC adhesion, invasion, IBC formation, and motility are suppressed by Fur under iron-rich conditions but derepressed under iron-restricted conditions, such as in patients with UTIs.

Relative occupation of type-I and type-II corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications

1987 ◽  
Vol 115 (3) ◽  
pp. 459-467 ◽  
Author(s):  
J. M. H. M. Reul ◽  
F. R. van den Bosch ◽  
E. R. de Kloet
Keyword(s):  
Rat Brain ◽  
Plasma Concentrations ◽  
Striking Difference ◽  
Classical Type ◽  
Type I ◽  
Type Ii ◽  
Binding Studies ◽  
Receptor Sites ◽  
Basal Plasma

ABSTRACT The rat brain contains two receptor systems for corticosterone: the type-I corticosterone-preferring receptor and the classical type-II glucocorticoid receptor. The two receptor populations can be distinguished in binding studies with the 'pure' synthetic glucocorticoid 11β,17β-dihydroxy-6-methyl-17α (1-propynyl)-androsta-1,4,6-trione-3-one (RU 28362). In-vitro autoradiography and quantitative image analysis showed that the type-I receptor was localized almost exclusively in the hippocampus, whereas the type-II receptor extended throughout the brain, with the highest levels in the nucleus paraventricularis, nucleus supraopticus and in the thalamic, amygdaloid, hippocampal and septal regions. Unoccupied type-I and type-II receptor sites, as measured in vitro by cytosol binding of 3H-labelled steroids, displayed a large difference in the rate of appearance after adrenalectomy. The availability of type-I receptors exhibited a marked increase, reaching maximal levels within 4–7 h, and then remained constant until 2 weeks after adrenalectomy. The availability of type-II receptors did not change considerably during the first 24 h after adrenalectomy, but displayed a large increase in capacity during the subsequent 2 weeks. After adrenocortical activation as a consequence of exposure to a novel environment, plasma concentrations of corticosterone increased to reach a peak of 811 nmol/l after 30 min and attained the basal concentration (43 nmol/l) after 240 min. During this time, occupation of type-I receptors increased from 77·8% at 0 min to 97% at 30–60 min and then declined to 84·8% after 240 min. Occupation of the type-II receptors was 28·1% at 0 min, 74·5% after 30 min and 32·8% after 240 min. Injection of dexamethasone (25 μg/100 g body wt) at 08.00 h resulted in suppression of basal plasma concentrations of corticosterone and prevented the circadian-driven rise in circulating corticosterone. Occupation of type-I receptors did not change considerably as a result of injection of dexamethasone, but occupation of type-II receptors was markedly increased till 16.00 h compared with that after injection of vehicle. It was concluded that the type-I and type-II receptors are not only localized differently in the rat brain, but also exhibit a striking difference in occupation after manipulation of the pituitary-adrenocortical system. The data further support the concept of a type-I receptor-mediated tonic activating influence and a type-II receptor-mediated feedback action of corticosterone on brain function. J. Endocr. (1987) 115, 459–467

scholarly journals Glutamate Decarboxylase-Dependent Acid Resistance in Brucella spp.: Distribution and Contribution to Fitness under Extremely Acidic Conditions

2014 ◽  
Vol 81 (2) ◽  
pp. 578-586 ◽  
Author(s):  
Maria Alessandra Damiano ◽  
Daniela Bastianelli ◽  
Sascha Al Dahouk ◽  
Stephan Köhler ◽  
Axel Cloeckaert ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Glutamate Decarboxylase ◽  
Acid Resistance ◽  
Oral Route ◽  
Genome Sequences ◽  
Content Type ◽  
Resistant Phenotype ◽  
Wide Range ◽  
Acidic Conditions

ABSTRACTBrucellais an expanding genus of major zoonotic pathogens, including at least 10 genetically very close species occupying a wide range of niches from soil to wildlife, livestock, and humans. Recently, we have shown that in the new speciesBrucella microti, the glutamate decarboxylase (Gad)-dependent system (GAD system) contributes to survival at a pH of 2.5 and also to infection in mice by the oral route. In order to study the functionality of the GAD system in the genusBrucella, 47 isolates, representative of all known species and strains of this genus, and 16 strains of the closest neighbor genus,Ochrobactrum, were studied using microbiological, biochemical, and genetic approaches. In agreement with the genome sequences, the GAD system of classical species was not functional, unlike that of most strains ofBrucella ceti,Brucella pinnipedialis, and newly described species (B. microti,Brucella inopinataBO1,B. inopinata-like BO2, andBrucellasp. isolated from bullfrogs). In the presence of glutamate, these species were more acid resistantin vitrothan classical terrestrial brucellae. Expression intransof thegadlocus from representativeBrucellaspecies in theEscherichia coliMG1655 mutant strain lacking the GAD system restored the acid-resistant phenotype. The highly conserved GAD system of the newly described or atypicalBrucellaspecies may play an important role in their adaptation to acidic external and host environments. Furthermore, the GAD phenotype was shown to be a useful diagnostic tool to distinguish these latterBrucellastrains fromOchrobactrumand from classical terrestrial pathogenicBrucellaspecies, which are GAD negative.

scholarly journals Subcellular localization of transglutaminase. Effect of collagen

1988 ◽  
Vol 250 (2) ◽  
pp. 421-427 ◽  
Author(s):  
M Juprelle-Soret ◽  
S Wattiaux-De Coninck ◽  
R Wattiaux
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Collagen Type I ◽  
Type I ◽  
Nuclear Fraction ◽  
Binding Studies ◽  
Isopycnic Centrifugation

1. The subcellular distribution of transglutaminase was investigated by using the analytical approach of differential and isopycnic centrifugation as applied to three organs of the rat: liver, kidney and lung. After differential centrifugation by the method of de Duve, Pressman, Gianetto, Wattiaux & Appelmans [(1955) Biochem. J. 63, 604-617], transglutaminase is mostly recovered in the unsedimentable fraction S and the nuclear fraction N. After isopycnic centrifugation of the N fraction in a sucrose density gradient, a high proportion of the enzyme remains at the top of the gradient; a second but minor peak of activity is present in high-density regions, where a small proportion of 5′-nucleotidase, a plasma-membrane marker, is present together with a large proportion of collagen recovered in that fraction. 2. Fractions where a peak of transglutaminase was apparent in the sucrose gradient were examined by electron microscopy. The main components are large membrane sheets with extracellular matrix and free collagen fibers. 3. As these results seem to indicate that some correlation exists between particulate transglutaminase distribution and those of collagen and plasma membranes, the possible binding of transglutaminase by collagen (type I) and by purified rat liver plasma membrane was investigated. 4. The binding studies indicated that collagen is able to bind transglutaminase and to make complexes with plasma-membrane fragments whose density is higher than that of plasma-membrane fragments alone. Transglutaminase cannot be removed from such complexes by 1% Triton X-100, but can be to a relatively large extent by 0.5 M-KCl and by 50% (w/v) glycerol. 5. Such results suggest that the apparent association of transglutaminase with plasma membrane originates from binding in vitro of the cytosolic enzyme to plasma membrane bound to collagen, which takes place during homogenization of the tissue, when the soluble enzyme and extracellular components are brought together.

scholarly journals The Acyl-Homoserine Lactone Synthase YenI from Yersinia enterocolitica Modulates Virulence Gene Expression in Enterohemorrhagic Escherichia coli O157:H7

2013 ◽  
Vol 81 (11) ◽  
pp. 4192-4199 ◽  
Author(s):  
Y N. Nguyen ◽  
Haiqing Sheng ◽  
Rambabu Dakarapu ◽  
John R. Falck ◽  
Carolyn J. Hovde ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Yersinia Enterocolitica ◽  
Virulence Gene ◽  
Acid Resistance ◽  
Homoserine Lactone ◽  
Enterohemorrhagic Escherichia Coli ◽  
Content Type ◽  
Fecal Shedding ◽  
Virulence Gene Expression

ABSTRACTThe human pathogen enterohemorrhagicEscherichia coli(EHEC) O157:H7 colonizes the rectoanal junction (RAJ) in cattle, its natural reservoir. Colonization at the RAJ poses a serious risk for fecal shedding and contamination of the environment. We previously demonstrated that EHEC senses acyl-homoserine lactones (AHLs) produced by the microbiota in the rumen to activate thegadacid resistance genes necessary for survival through the acidic stomachs in cattle and to repress the locus of enterocyte effacement (LEE) genes important for colonization of the RAJ, but unnecessary in the rumen. Devoid of AHLs, the RAJ is the prominent site of colonization of EHEC in cattle. To determine if the presence of AHLs in the RAJ could repress colonization at this site, we engineered EHEC to express theYersinia enterocoliticaAHL synthase geneyenI, which constitutively produces AHLs, to mimic a constant exposure of AHLs in the environment. TheyenI+EHEC produces oxo-C6-homoserine lactone (oxo-C6-HSL) and had a significant reduction in LEE expression, effector protein secretion, and attaching and effacing (A/E) lesion formationin vitrocompared to the wild type (WT). TheyenI+EHEC also activated expression of thegadgenes. To assess whether AHL production, which decreases LEE expression, would decrease RAJ colonization by EHEC, cattle were challenged at the RAJ with WT oryenI+EHEC. Although theyenI+EHEC colonized the RAJ with efficiency equal to that of the WT, there was a trend for the cattle to shed the WT strain longer than theyenI+EHEC.

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