scholarly journals Hydrolysis Spectrum Extension of CMY-2-Like β-Lactamases Resulting from Structural Alteration in the Y-X-N Loop

2012 ◽  
Vol 56 (3) ◽  
pp. 1151-1156 ◽  
Author(s):  
Sandrine Dahyot ◽  
Hedi Mammeri
Keyword(s):  
Hot Spot ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Hydrolysis Rate ◽  
Directed Mutagenesis ◽  
Content Type ◽  
E Coli ◽  
Class A ◽  
Extended Spectrum ◽  
Cloning Experiment

ABSTRACTTheCitrobacter freundiiisolate CHA, which was responsible for postoperative peritonitis after 10 days of cefepime therapy, displayed a phenotype of resistance consistent with extended-spectrum AmpC (ESAC) β-lactamase. The chromosome-borneblaAmpC-CHAgene was amplified and sequenced, revealing five amino acid substitutions, I125V, R148H, Q196H, V305A, and V348A, in the product compared to the sequence of native AmpC. A cloning experiment yielded theEscherichia coliTOP10(pAmpC-CHA) strain, which was resistant to all extended-spectrum cephalosporins (ESCs), including cefepime. To ascertain whether the R148H substitution accounted for the hydrolysis spectrum extension, it was reverted by site-directed mutagenesis. The resultingE. coliTOP10(pAmpC-CHA-H148R) strain was fully susceptible to cefepime, thus confirming that the Arg-148 replacement was mandatory for substrate profile enlargement. To further characterize the phenotypical and biochemical effects induced by the R148H change, it was introduced by site-directed mutagenesis into the CMY-2 β-lactamase, which is structurally related to the chromosome-borne cephalosporinase ofC. freundii. The CMY-2-R148H variant conferred increased MICs of ESCs, whereas those of carbapenems were unchanged even in a porin-deficientE. colistrain. Moreover, it exhibited increased catalytic efficiency (kcat/Km) toward ceftazidime (100-fold) due to an enhanced hydrolysis rate (kcat), whereas the enzymatic parameters toward imipenem were unchanged. The structural analysis of the AmpC variant showed that the R148H replacement occurred in the loop containing the Y-X-N motif, which is the counterpart of the SDN loop in class A β-lactamases. This study shows that the Y-X-N loop is a novel hot spot for mutations accounting for hydrolysis spectrum extension in CMY-2-type enzymes.

scholarly journals Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

2012 ◽  
Vol 78 (11) ◽  
pp. 3880-3884 ◽  
Author(s):  
Yu-Ri Lim ◽  
Soo-Jin Yeom ◽  
Deok-Kun Oh
Keyword(s):  
Specific Activity ◽  
Thermus Thermophilus ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Geobacillus Thermodenitrificans ◽  
Wild Type Enzyme ◽  
Content Type ◽  
Mannose 6 Phosphate

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

scholarly journals Structural Insights into the TLA-3 Extended-Spectrum β-Lactamase and Its Inhibition by Avibactam and OP0595

2017 ◽  
Vol 61 (10) ◽  
Author(s):  
Wanchun Jin ◽  
Jun-ichi Wachino ◽  
Yoshihiro Yamaguchi ◽  
Kouji Kimura ◽  
Anupriya Kumar ◽  
...  
Keyword(s):  
Rate Constant ◽  
Catalytic Efficiency ◽  
Amino Acid Residues ◽  
Inhibitory Effects ◽  
Content Type ◽  
Class A ◽  
Extended Spectrum ◽  
Structural Template ◽  
Structural Insights ◽  
Positively Charged

ABSTRACT The development of effective inhibitors that block extended-spectrum β-lactamases (ESBLs) and restore the action of β-lactams represents an effective strategy against ESBL-producing Enterobacteriaceae. We evaluated the inhibitory effects of the diazabicyclooctanes avibactam and OP0595 against TLA-3, an ESBL that we identified previously. Avibactam and OP0595 inhibited TLA-3 with apparent inhibitor constants (Ki app) of 1.71 ± 0.10 and 1.49 ± 0.05 μM, respectively, and could restore susceptibility to cephalosporins in the TLA-3-producing Escherichia coli strain. The value of the second-order acylation rate constant (k 2/K, where k 2 is the acylation rate constant and K is the equilibrium constant) of avibactam [(3.25 ± 0.03) × 103 M−1 · s−1] was closer to that of class C and D β-lactamases (k 2/K, <104 M−1 · s−1) than that of class A β-lactamases (k 2/K, >104 M−1 · s−1). In addition, we determined the structure of TLA-3 and that of TLA-3 complexed with avibactam or OP0595 at resolutions of 1.6, 1.6, and 2.0 Å, respectively. TLA-3 contains an inverted Ω loop and an extended loop between the β5 and β6 strands (insertion after Ser237), which appear only in PER-type class A β-lactamases. These structures might favor the accommodation of cephalosporins harboring bulky R1 side chains. TLA-3 presented a high catalytic efficiency (k cat/Km ) against cephalosporins, including cephalothin, cefuroxime, and cefotaxime. Avibactam and OP0595 bound covalently to TLA-3 via the Ser70 residue and made contacts with residues Ser130, Thr235, and Ser237, which are conserved in ESBLs. Additionally, the sulfate group of the inhibitors formed polar contacts with amino acid residues in a positively charged pocket of TLA-3. Our findings provide a structural template for designing improved diazabicyclooctane-based inhibitors that are effective against ESBL-producing Enterobacteriaceae.

scholarly journals Tinkering with Osmotically Controlled Transcription Allows Enhanced Production and Excretion of Ectoine and Hydroxyectoine from a Microbial Cell Factory

2017 ◽  
Vol 84 (2) ◽  
Author(s):  
Laura Czech ◽  
Sebastian Poehl ◽  
Philipp Hub ◽  
Nadine Stöveken ◽  
Erhard Bremer
Keyword(s):  
Osmotic Stress ◽  
Growth Medium ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Cell Factory ◽  
Biosynthetic Gene ◽  
Content Type ◽  
E Coli ◽  
Synthetic Cell ◽  
Sigma 70

ABSTRACTEctoine and hydroxyectoine are widely synthesized by members of theBacteriaand a few members of theArchaeaas potent osmostress protectants. We have studied the salient features of the osmostress-responsive promoter directing the transcription of the ectoine/hydroxyectoine biosynthetic gene cluster from the plant-root-associated bacteriumPseudomonas stutzeriby transferring it intoEscherichia coli, an enterobacterium that does not produce ectoines naturally. Usingect-lacZreporter fusions, we found that the heterologousectpromoter reacted with exquisite sensitivity in its transcriptional profile to graded increases in sustained high salinity, responded to a true osmotic signal, and required the buildup of an osmotically effective gradient across the cytoplasmic membrane for its induction. The involvement of the −10, −35, and spacer regions of the sigma-70-typeectpromoter in setting promoter strength and response to osmotic stress was assessed through site-directed mutagenesis. Moderate changes in theectpromoter sequence that increase its resemblance to housekeeping sigma-70-type promoters ofE. coliafforded substantially enhanced expression, both in the absence and in the presence of osmotic stress. Building on this set ofectpromoter mutants, we engineered anE. colichassis strain for the heterologous production of ectoines. This synthetic cell factory lacks the genes for the osmostress-responsive synthesis of trehalose and the compatible solute importers ProP and ProU, and it continuously excretes ectoines into the growth medium. By combining appropriate host strains and different plasmid variants, excretion of ectoine, hydroxyectoine, or a mixture of both compounds was achieved under mild osmotic stress conditions.IMPORTANCEEctoines are compatible solutes, organic osmolytes that are used by microorganisms to fend off the negative consequences of high environmental osmolarity on cellular physiology. An understanding of the salient features of osmostress-responsive promoters directing the expression of the ectoine/hydroxyectoine biosynthetic gene clusters is lacking. We exploited theectpromoter from an ectoine/hydroxyectoine-producing soil bacterium for such a study by transferring it into a surrogate bacterial host. Despite the fact thatE. colidoes not synthesize ectoines naturally, theectpromoter retained its exquisitely sensitive osmotic control, indicating that osmoregulation ofecttranscription is an inherent feature of the promoter and its flanking sequences. These sequences were narrowed to a 116-bp DNA fragment. Ectoines have interesting commercial applications. Building on data from a site-directed mutagenesis study of theectpromoter, we designed a synthetic cell factory that secretes ectoine, hydroxyectoine, or a mixture of both compounds into the growth medium.

scholarly journals Characterization of CTX-M-140, a Variant of CTX-M-14 Extended-Spectrum β-Lactamase with Decreased Cephalosporin Hydrolytic Activity, from Cephalosporin-Resistant Proteus mirabilis

2016 ◽  
Vol 60 (10) ◽  
pp. 6121-6126 ◽  
Author(s):  
Guo-Bao Tian ◽  
Yi-Qi Jiang ◽  
Ying-Min Huang ◽  
Yun Qin ◽  
Lian-Qiang Feng ◽  
...  
Keyword(s):  
Proteus Mirabilis ◽  
Gc Content ◽  
Hydrolytic Activity ◽  
Site Directed Mutagenesis ◽  
Kinetic Properties ◽  
Content Type ◽  
E Coli ◽  
Extended Spectrum ◽  
A Genome ◽  
M 14

ABSTRACTCTX-M-140, a novel CTX-M-type extended-spectrum β-lactamase (ESBL), was identified in cephalosporin-resistant clinical isolates ofProteus mirabilis. CTX-M-140 contained an alanine-to-threonine substitution at position 109 compared to its putative progenitor, CTX-M-14. When it was expressed in anEscherichia coliisogenic background, CTX-M-140 conferred 4- to 32-fold lower MICs of cephalosporins than those with CTX-M-14, indicating that the phenotype was attributable to this single substitution. For four mutants of CTX-M-14 that were constructed by site-directed mutagenesis (A109E, A109D, A109K, and A109R mutants), MICs of cephalosporins were similar to those for theE. colihost strain, which suggested that the alanine at position 109 was essential for cephalosporin hydrolysis. The kinetic properties of native CTX-M-14 and CTX-M-140 were consistent with the MICs for theE. coliclones. Compared with that of CTX-M-14, a lower hydrolytic activity against cephalosporins was observed for CTX-M-140.blaCTX-M-140is located on the chromosome as determined by I-CeuI pulsed-field gel electrophoresis (I-CeuI-PFGE) and Southern hybridization. The genetic environment surroundingblaCTX-M-140is identical to the sequence found in different plasmids withblaCTX-M-9-groupgenes among theEnterobacteriaceae. Genome sequencing and analysis showed thatP. mirabilisstrains withblaCTX-M-140have a genome size of ∼4 Mbp, with a GC content of 38.7% and 23 putative antibiotic resistance genes. Our results indicate that alanine at position 109 is critical for the hydrolytic activity of CTX-M-14 against oxyimino-cephalosporins.

scholarly journals Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

2013 ◽  
Vol 79 (13) ◽  
pp. 4072-4077 ◽  
Author(s):  
Xuguo Duan ◽  
Jian Chen ◽  
Jing Wu
Keyword(s):  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Ph Optimum ◽  
Widespread Application ◽  
Content Type ◽  
Major Factors

ABSTRACTPullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Its instability and low catalytic efficiency are the major factors preventing its widespread application. To address these issues, Asp437 and Asp503 of the pullulanase fromBacillus deramificanswere selected in this study as targets for site-directed mutagenesis based on a structure-guided consensus approach. Four mutants (carrying the mutations D503F, D437H, D503Y, and D437H/D503Y) were generated and characterized in detail. The results showed that the D503F, D437H, and D503Y mutants had an optimum temperature of 55°C and a pH optimum of 4.5, similar to that of the wild-type enzyme. However, the half-lives of the mutants at 60°C were twice as long as that of the wild-type enzyme. In addition, the D437H/D503Y double mutant displayed a larger shift in thermostability, with an optimal temperature of 60°C and a half-life at 60°C of more than 4.3-fold that of the wild-type enzyme. Kinetic studies showed that theKmvalues for the D503F, D437H, D503Y, and D437H/D503Y mutants decreased by 7.1%, 11.4%, 41.4%, and 45.7% and theKcat/Kmvalues increased by 10%, 20%, 140%, and 100%, respectively, compared to those of the wild-type enzyme. Mechanisms that could account for these enhancements were explored. Moreover, in conjunction with the enzyme glucoamylase, the D503Y and D437H/D503Y mutants exhibited an improved reaction rate and glucose yield during starch hydrolysis compared to those of the wild-type enzyme, confirming the enhanced properties of the mutants. The mutants generated in this study have potential applications in the starch industry.

scholarly journals Mutations in blaKPC-3 That Confer Ceftazidime-Avibactam Resistance Encode Novel KPC-3 Variants That Function as Extended-Spectrum β-Lactamases

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Ghady Haidar ◽  
Cornelius J. Clancy ◽  
Ryan K. Shields ◽  
Binghua Hao ◽  
Shaoji Cheng ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Extended Spectrum

ABSTRACT We identified four bla KPC-3 mutations in ceftazidime-avibactam-resistant clinical Klebsiella pneumoniae isolates, corresponding to D179Y, T243M, D179Y/T243M, and EL165-166 KPC-3 variants. Using site-directed mutagenesis and transforming vectors into Escherichia coli, we conclusively demonstrated that mutant bla KPC-3 encoded enzymes that functioned as extended-spectrum β-lactamases; mutations directly conferred higher MICs of ceftazidime-avibactam and decreased the MICs of carbapenems and other β-lactams. Impact was strongest for the D179Y mutant, highlighting the importance of the KPC Ω-loop.

scholarly journals Activity of NXL104 in Combination with β-Lactams against Genetically Characterized Escherichia coli and Klebsiella pneumoniae Isolates Producing Class A Extended-Spectrum β-Lactamases and Class C β-Lactamases

2011 ◽  
Vol 55 (5) ◽  
pp. 2434-2437 ◽  
Author(s):  
P. R. S. Lagacé-Wiens ◽  
F. Tailor ◽  
P. Simner ◽  
M. DeCorby ◽  
J. A. Karlowsky ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
The Novel ◽  
Content Type ◽  
E Coli ◽  
Class A ◽  
Extended Spectrum ◽  
Class C ◽  
Lactamase Inhibitor

ABSTRACTThe novel non-β-lactam β-lactamase inhibitor NXL104, in combination with cefepime, ceftazidime, ceftriaxone, amdinocillin, and meropenem, was tested against 190 extended-spectrum β-lactamase (ESBL)-producingEscherichia coliandKlebsiella pneumoniaeisolates, 94 AmpC-hyperproducingE. coliisolates, and 8 AmpC/ESBL-coexpressingE. coliisolates. NXL104 restored 100% susceptibility to the partner cephalosporins for all isolates tested. Amdinocillin and meropenem MICs were modestly improved (2 to 32 times lower) by NXL104. These results suggest that NXL104 may be useful in combination with β-lactams for the treatment of infections caused by ESBL- and AmpC-producingEnterobacteriaceae.

scholarly journals A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit.

1994 ◽  
Vol 180 (6) ◽  
pp. 2147-2153 ◽  
Author(s):  
M Pizza ◽  
M R Fontana ◽  
M M Giuliani ◽  
M Domenighini ◽  
C Magagnoli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Neutralizing Antibodies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
E Coli ◽  
Heat Labile ◽  
A Subunit ◽  
Adp Ribosyltransferase ◽  
Derivatives Of

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.

scholarly journals A One Health Comparative Assessment of Antimicrobial Resistance in Generic and Extended-Spectrum Cephalosporin-Resistant Escherichia coli from Beef Production, Sewage and Clinical Settings

2020 ◽  
Vol 8 (6) ◽  
pp. 885 ◽  
Author(s):  
Emelia H. Adator ◽  
Claudia Narvaez-Bravo ◽  
Rahat Zaheer ◽  
Shaun R. Cook ◽  
Lisa Tymensen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
One Health ◽  
Hot Spot ◽  
Municipal Sewage ◽  
Multi Drug Resistance ◽  
Beef Production ◽  
E Coli ◽  
Beef Processing ◽  
Extended Spectrum

This study aimed to compare antimicrobial resistance (AMR) in extended-spectrum cephalosporin-resistant and generic Escherichia coli from a One Health continuum of the beef production system in Alberta, Canada. A total of 705 extended-spectrum cephalosporin-resistant E. coli (ESCr) were obtained from: cattle feces (CFeces, n = 382), catch basins (CBasins, n = 137), surrounding streams (SStreams, n = 59), beef processing plants (BProcessing, n = 4), municipal sewage (MSewage; n = 98) and human clinical specimens (CHumans, n = 25). Generic isolates (663) included: CFeces (n = 142), CBasins (n = 185), SStreams (n = 81), BProcessing (n = 159) and MSewage (n = 96). All isolates were screened for antimicrobial susceptibility to 9 antimicrobials and two clavulanic acid combinations. In ESCr, oxytetracycline (87.7%), ampicillin (84.4%) and streptomycin (73.8%) resistance phenotypes were the most common, with source influencing AMR prevalence (p < 0.001). In generic E. coli, oxytetracycline (51.1%), streptomycin (22.6%), ampicillin (22.5%) and sulfisoxazole (14.3%) resistance were most common. Overall, 88.8% of ESCr, and 26.7% of generic isolates exhibited multi-drug resistance (MDR). MDR in ESCr was high from all sources: CFeces (97.1%), MSewage (96.9%), CHumans (96%), BProcessing (100%), CBasins (70.5%) and SStreams (61.4%). MDR in generic E. coli was lower with CFeces (45.1%), CBasins (34.6%), SStreams (23.5%), MSewage (13.6%) and BProcessing (10.7%). ESBL phenotypes were confirmed in 24.7% (n = 174) ESCr and 0.6% of generic E. coli. Prevalence of bla genes in ESCr were blaCTXM (30.1%), blaCTXM-1 (21.6%), blaTEM (20%), blaCTXM-9 (7.9%), blaOXA (3.0%), blaCTXM-2 (6.4%), blaSHV (1.4%) and AmpC β-lactamase blaCMY (81.3%). The lower AMR in ESCr from SStreams and BProcessing and higher AMR in CHumans and CFeces likely reflects antimicrobial use in these environments. Although MDR levels were higher in ESCr as compared to generic E. coli, AMR to the same antimicrobials ranked high in both ESCr and generic E. coli sub-populations. This suggests that both sub-populations reflect similar AMR trends and are equally useful for AMR surveillance. Considering that MDR ESCr MSewage isolates were obtained without enrichment, while those from CFeces were obtained with enrichment, MSewage may serve as a hot spot for MDR emergence and dissemination.

scholarly journals Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

2014 ◽  
Vol 80 (20) ◽  
pp. 6549-6559 ◽  
Author(s):  
Sabrina Wemhoff ◽  
Roland Klassen ◽  
Friedhelm Meinhardt
Keyword(s):  
Kluyveromyces Lactis ◽  
Killer Toxin ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Target Cells ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Protein Toxin ◽  
Assisted Delivery

ABSTRACTZymocin is aKluyveromyces lactisprotein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activityin vitrothat might be important during γ import.Saccharomyces cerevisiaestrains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase fromPichia acaciae(P. acaciaeOrf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.

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