scholarly journals Evolution of Aromatic β-Glucoside Utilization by Successive Mutational Steps in Escherichia coli

2014 ◽  
Vol 197 (4) ◽  
pp. 710-716 ◽  
Author(s):  
Parisa Zangoui ◽  
Kartika Vashishtha ◽  
Subramony Mahadevan
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Selection Pressure ◽  
Second Step ◽  
Content Type ◽  
Catabolic Activity ◽  
Nucleotide Insertion ◽  
Esculin Hydrolysis ◽  
Steady State Levels ◽  
Hydrolysis Of

ThebglAgene ofEscherichia coliencodes phospho-β-glucosidase A capable of hydrolyzing the plant-derived aromatic β-glucoside arbutin. We report that the sequential accumulation of mutations inbglAcan confer the ability to hydrolyze the related aromatic β-glucosides esculin and salicin in two steps. In the first step, esculin hydrolysis is achieved through the acquisition of a four-nucleotide insertion within the promoter of thebglAgene, resulting in enhanced steady-state levels of thebglAtranscript. In the second step, hydrolysis of salicin is achieved through the acquisition of a point mutation within thebglAstructural gene close to the active site without the loss of the original catabolic activity against arbutin. These studies underscore the ability of microorganisms to evolve additional metabolic capabilities by mutational modification of preexisting genetic systems under selection pressure, thereby expanding their repertoire of utilizable substrates.

scholarly journals Increased Hydrolysis of Oximino-β-Lactams by CMY-107, a Tyr199Cys Mutant Form of CMY-2 Produced by Escherichia coli

2015 ◽  
Vol 59 (12) ◽  
pp. 7894-7898 ◽  
Author(s):  
S. D. Kotsakis ◽  
V. Miriagou ◽  
E. E. Vetouli ◽  
E. Bozavoutoglou ◽  
E. Lebessi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Clinical Strain ◽  
Mutant Form ◽  
Content Type ◽  
Hydrolysis Of

ABSTRACTThe cephalosporinase CMY-107, a Tyr199Cys mutant form of CMY-2 encoded by an IncI self-transferable plasmid carried by anEscherichia coliclinical strain, was characterized. The enzyme hydrolyzed oximino-cephalosporins and aztreonam more efficiently than CMY-2 did.

scholarly journals Compartmentalization of the Carbaryl Degradation Pathway: Molecular Characterization of Inducible Periplasmic Carbaryl Hydrolase from Pseudomonas spp.

2017 ◽  
Vol 84 (2) ◽  
Author(s):  
Kamini ◽  
Dasvit Shetty ◽  
Vikas D. Trivedi ◽  
Madhushri Varunjikar ◽  
Prashant S. Phale
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
Metabolic Pathway ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Degradation Pathway ◽  
Signal Peptidase ◽  
Cellular Toxicity ◽  
Content Type ◽  
Hydrolysis Of

ABSTRACTPseudomonassp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis ofmcbA, encoding CH, in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4- to 6-fold higher in the periplasm than in the cytoplasm. The recombinant CH (rCH) showed 4-fold-higher activity in the periplasm ofEscherichia coli. The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of both Tmd and Sp (Tmd+Sp) resulted in expression of the inactive protein. Confocal microscopic analysis ofE. coliexpressing a (Tmd+Sp)-green fluorescent protein (GFP) fusion protein revealed the localization of GFP into the periplasm. Altogether, these results indicate that Tmd probably helps in anchoring of polypeptide to the inner membrane, while Sp assists folding and release of CH in the periplasm. The N-terminal sequence of the mature periplasmic CH confirms the absence of the Tmd+Sp region and confirms the signal peptidase cleavage site as Ala-Leu-Ala. CH purified from strains C5pp, C7, and rCHΔ(Tmd)a were found to be monomeric with molecular mass of ∼68 to 76 kDa and to catalyze hydrolysis of the ester bond with an apparentKmandVmaxin the range of 98 to 111 μM and 69 to 73 μmol · min−1· mg−1, respectively. The presence of low-affinity CH in the periplasm and 1-naphthol-metabolizing enzymes in the cytoplasm ofPseudomonasspp. suggests the compartmentalization of the metabolic pathway as a strategy for efficient degradation of carbaryl at higher concentrations without cellular toxicity of 1-naphthol.IMPORTANCEProteins in the periplasmic space of bacteria play an important role in various cellular processes, such as solute transport, nutrient binding, antibiotic resistance, substrate hydrolysis, and detoxification of xenobiotics. Carbaryl is one of the most widely used carbamate pesticides. Carbaryl hydrolase (CH), the first enzyme of the degradation pathway which converts carbaryl to 1-naphthol, was found to be localized in the periplasm ofPseudomonasspp. Predicted transmembrane domain and signal peptide sequences ofPseudomonaswere found to be functional inEscherichia coliand to translocate CH and GFP into the periplasm. The localization of low-affinity CH into the periplasm indicates controlled formation of toxic and recalcitrant 1-naphthol, thus minimizing its accumulation and interaction with various cellular components and thereby reducing the cellular toxicity. This study highlights the significance of compartmentalization of metabolic pathway enzymes for efficient removal of toxic compounds.

scholarly journals Interaction of L-threo and L-erythro isomers of 3-fluoroglutamate with glutamate decarboxylase from Escherichia coli

1985 ◽  
Vol 229 (3) ◽  
pp. 675-678 ◽  
Author(s):  
A Vidal-Cros ◽  
M Gaudry ◽  
A Marquet
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Glutamate Decarboxylase ◽  
Fluorine Atom ◽  
Pyridoxal Phosphate ◽  
Succinic Semialdehyde ◽  
E Coli ◽  
Rigid Conformation ◽  
The Difference ◽  
Hydrolysis Of

L-threo-3-Fluoroglutamate and L-erythro-3-fluoroglutamate were tested with glutamate decarboxylase from Escherichia coli. Both isomers were substrates: the threo isomer was decarboxylated into optically active 4-amino-3-fluorobutyrate, whereas the erythro isomer lost the fluorine atom during the reaction, yielding succinic semialdehyde after hydrolysis of the unstable intermediate enamine. The difference between the two isomers demonstrates that the glutamic acid-pyridoxal phosphate Schiff base is present at the active site under a rigid conformation. Furthermore, although the erythro isomer lost the fluorine atom, yielding a reactive aminoacrylic acid in the active site, no irreversible inactivation of E. coli glutamate decarboxylase was observed.

scholarly journals IncFII Conjugative Plasmid-Mediated Transmission of blaNDM-1 Elements among Animal-Borne Escherichia coli Strains

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
Dachuan Lin ◽  
Miaomiao Xie ◽  
Ruichao Li ◽  
Kaichao Chen ◽  
Edward Wai-Chi Chan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Selection Pressure ◽  
Structural Variation ◽  
Transmission Dynamics ◽  
Conjugative Plasmid ◽  
Gut Microflora ◽  
Content Type ◽  
E Coli

ABSTRACT This study aims to investigate the prevalence and transmission dynamics of the bla NDM-1 gene in animal Escherichia coli strains. Two IncFII bla NDM-1-encoding plasmids with only minor structural variation in the MDR region, pHNEC46-NDM and pHNEC55-NDM, were found to be responsible for the transmission of bla NDM-1 in these strains. The bla NDM-1 gene can be incorporated into plasmids and stably inherited in animal-borne E. coli strains that can be maintained in animal gut microflora even without carbapenem selection pressure.

The ATPase complex of Escherichia coli

1984 ◽  
Vol 62 (11) ◽  
pp. 1190-1197 ◽  
Author(s):  
Philip D. Bragg
Keyword(s):  
Escherichia Coli ◽  
Chemical Modification ◽  
Active Site ◽  
Active Sites ◽  
Transmembrane Protein ◽  
Proton Translocation ◽  
Amino Terminal ◽  
Loop Region ◽  
Cytoplasmic Surface ◽  
Hydrolysis Of

The ATPase (ATP synthase) complex of Escherichia coli is composed of an extrinsic membrane protein (ECF1), which contains the active site for ATP formation and hydrolysis, and is attached to ECF0, a transmembrane protein through which protons move to or from the active site on ECF1. ECF1 is composed of five subunits (α–ε) with a stoichiometry of α3β3γδε. The stoichiometry of the three subunits (a–c) of ECF0 is probably a1b2c10–15. In addition to 3 mol tightly bound adenine nucleotide/mol ECF1, three other "exchangeable" nucleotide binding sites can be detected. These sites are still present in the α and β subunit defective ECF1 of uncA401 and uncD412 mutants, although some changes in the tightness of binding are evident. The active sites of ECF1 require normal a and p subunits and may be present at αβ subunit interfaces. Hydrolysis of ATP requires cooperative interactions between α and β subunits. At low concentrations of ATP, in the absence of added divalent cations, hydrolysis of this substrate can occur at a single site without release of the product. This is consistent with alternating or sequential site mechanisms for ATP hydrolysis or synthesis. Predictions of secondary and tertiary structures from the known primary amino acid sequences of polypeptides a, b, and c have led to the following conclusions. Polypeptide a forms six or seven transmembrane a helices. The amino-terminal sequence of polypeptide b spans the membrane, but most of the protein is exposed on the cytoplasmic surface of the membrane where it can be cleaved by proteases in vitro. Polypeptide c consists of two nonpolar membrane-spanning α helices linked by a polar segment at the cytoplasmic surface of the membrane. This loop region interacts with ECF1 or is close to the ECF1-binding site. This is shown by competition between ECF1 and antibody for binding to polypeptide c. Chemical modification of arginyl residues in the loop region of polypeptide c inhibits ECF1 binding. Protease cleavage of polypeptide b affects, but does not abolish, binding of ECF1 to ECF0. Presumably, polypeptide b interacts with ECF1 also. The individual roles of the ECF0 polypeptides in proton translocation are not clear. Mutants in any of the three polypeptides may be defective in proton translocation. However, mutant and chemical modification studies support a role for the polypeptide c oligomer in the transmembrane proton pathway.

scholarly journals Newly Identified Thermostable Esterase from Sulfobacillus acidophilus: Properties and Performance in Phthalate Ester Degradation

2014 ◽  
Vol 80 (22) ◽  
pp. 6870-6878 ◽  
Author(s):  
Xiao-Yan Zhang ◽  
Xiang Fan ◽  
Yong-Jun Qiu ◽  
Cheng-Yuan Li ◽  
Shuai Xing ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Phthalate Esters ◽  
Phthalate Ester ◽  
Content Type ◽  
Potential Value ◽  
Specificity Constant ◽  
And Performance ◽  
Time Required ◽  
Hydrolysis Of ◽  
Optimal Ph

ABSTRACTEstS1, a newly identified thermostable esterase fromSulfobacillus acidophilusDSM10332, was heterologously expressed inEscherichia coliand shown to enzymatically degrade phthalate esters (PAEs) to their corresponding monoalkyl PAEs. The optimal pH and temperature of the esterase were found to be 8.0 and 70°C, respectively. The half-life of EstS1 at 60°C was 15 h, indicating that the enzyme had good thermostability. The specificity constant (kcat/Km) of the enzyme forp-nitrophenyl butyrate was as high as 6,770 mM−1s−1. The potential value of EstS1 was demonstrated by its ability to effectively hydrolyze 35 to 82% of PAEs (10 mM) within 2 min at 37°C, with all substrates being completely degraded within 24 h. At 60°C, the time required for complete hydrolysis of most PAEs was reduced by half. To our knowledge, this enzyme is a new esterase identified from thermophiles that is able to degrade various PAEs at high temperatures.

scholarly journals Functional Analysis of Family GH36 α-Galactosidases from Ruminococcus gnavus E1: Insights into the Metabolism of a Plant Oligosaccharide by a Human Gut Symbiont

2012 ◽  
Vol 78 (21) ◽  
pp. 7720-7732 ◽  
Author(s):  
M. Cervera-Tison ◽  
L. E. Tailford ◽  
C. Fuell ◽  
L. Bruel ◽  
G. Sulzenbacher ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Glycoside Hydrolase ◽  
Analytical Ultracentrifugation ◽  
Common Species ◽  
Phosphotransferase System ◽  
Human Gut ◽  
Content Type ◽  
Encoding Gene ◽  
Strict Specificity

ABSTRACTRuminococcus gnavusbelongs to the 57 most common species present in 90% of individuals. Previously, we identified an α-galactosidase (Aga1) belonging to glycoside hydrolase (GH) family 36 fromR. gnavusE1 (M. Aguilera, H. Rakotoarivonina, A. Brutus, T. Giardina, G. Simon, and M. Fons, Res. Microbiol. 163:14–21, 2012). Here, we identified a novel GH36-encoding gene from the same strain and termed itaga2. Althoughaga1showed a very simple genetic organization,aga2is part of an operon of unique structure, including genes putatively encoding a regulator, a GH13, two phosphotransferase system (PTS) sequences, and a GH32, probably involved in extracellular and intracellular sucrose assimilation. The 727-amino-acid (aa) deduced Aga2 protein shares approximately 45% identity with Aga1. Both Aga1 and Aga2 expressed inEscherichia colishowed strict specificity for α-linked galactose. Both enzymes were active on natural substrates such as melibiose, raffinose, and stachyose. Aga1 and Aga2 occurred as homotetramers in solution, as shown by analytical ultracentrifugation. Modeling of Aga1 and Aga2 identified key amino acids which may be involved in substrate specificity and stabilization of the α-linked galactoside substrates within the active site. Furthermore, Aga1 and Aga2 were both able to perform transglycosylation reactions with α-(1,6) regioselectivity, leading to the formation of product structures up to [Hex]12and [Hex]8, respectively. We suggest that Aga1 and Aga2 play essential roles in the metabolism of dietary oligosaccharides and could be used for the design of galacto-oligosaccharide (GOS) prebiotics, known to selectively modulate the beneficial gut microbiota.

The C-Terminal Active Site Cysteine of Escherichia coli Glutaredoxin 1 Determines the Glutathione Specificity of the Second Step of Peptide Deglutathionylation

2009 ◽  
Vol 11 (8) ◽  
pp. 1819-1828 ◽  
Author(s):  
Mirva J. Saaranen ◽  
Kirsi E. H. Salo ◽  
Maria K. Latva-Ranta ◽  
Vuokko L. Kinnula ◽  
Lloyd W. Ruddock
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Second Step ◽  
Active Site Cysteine

scholarly journals Target-Based Resistance in Pseudomonas aeruginosa and Escherichia coli to NBTI 5463, a Novel Bacterial Type II Topoisomerase Inhibitor

2014 ◽  
Vol 59 (1) ◽  
pp. 331-337 ◽  
Author(s):  
Asha S. Nayar ◽  
Thomas J. Dougherty ◽  
Folkert Reck ◽  
Jason Thresher ◽  
Ning Gao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Second Step ◽  
Type Ii ◽  
Resistance Mutations ◽  
Topoisomerase Iv ◽  
Content Type ◽  
E Coli ◽  
Topoisomerase Inhibitor ◽  
Bacterial Type

ABSTRACTIn a previous report (T. J. Dougherty, A. Nayar, J. V. Newman, S. Hopkins, G. G. Stone, M. Johnstone, A. B. Shapiro, M. Cronin, F. Reck, and D. E. Ehmann, Antimicrob Agents Chemother 58:2657–2664, 2014), a novel bacterial type II topoisomerase inhibitor, NBTI 5463, with activity against Gram-negative pathogens was described. First-step resistance mutations inPseudomonas aeruginosaarose exclusively in thenfxBgene, a regulator of the MexCD-OprJ efflux pump system. The present report describes further resistance studies with NBTI 5463 in bothPseudomonas aeruginosaandEscherichia coli. Second-step mutations inP. aeruginosaarose at aspartate 82 of the gyrase A subunit and led to 4- to 8-fold increases in the MIC over those seen in the parental strain with a first-stepnfxBefflux mutation. A third-step mutant showed additional GyrA changes, with no changes in topoisomerase IV. Despite repeated efforts, resistance mutations could not be selected inE. coli. Genetic introduction of the Asp82 mutations observed inP. aeruginosadid not significantly increase the NBTI MIC inE. coli. However, with the aspartate 82 mutation present, it was possible to select second-step mutations in topoisomerase IV that did lead to MIC increases of 16- and 128-fold. As with the gyrase aspartate 82 mutation, the mutations in topoisomerase IV did not by themselves raise the NBTI MIC inE. coli. Only the presence of mutations in both targets ofE. coliled to an increase in NBTI MIC values. This represents a demonstration of the value of balanced dual-target activity in mitigating resistance development.

scholarly journals Stability of the Osmoregulated Promoter-DerivedproPmRNA Is Posttranscriptionally Regulated by RNase III in Escherichia coli

2015 ◽  
Vol 197 (7) ◽  
pp. 1297-1305 ◽  
Author(s):  
Boram Lim ◽  
Kangseok Lee
Keyword(s):  
Escherichia Coli ◽  
Osmotic Stress ◽  
Cellular Response ◽  
Rna Binding ◽  
Binding Capacity ◽  
Rnase Iii ◽  
Content Type ◽  
Steady State Levels ◽  
The Stability

ABSTRACTThe enzymatic activity ofEscherichia coliendo-RNase III determines the stability of a subgroup of mRNA species, includingbdm,betT, andproU, whose protein products are associated with the cellular response to osmotic stress. Here, we report that the stability ofproPmRNA, which encodes a transporter of osmoprotectants, is controlled by RNase III in response to osmotic stress. We observed that steady-state levels ofproPmRNA and ProP protein are inversely correlated with cellular RNase III activity and, in turn, affect the proline uptake capacity of the cell.In vitroandin vivoanalyses ofproPmRNA revealed RNase III cleavage sites in a stem-loop within the 5′ untranslated region present only inproPmRNA species synthesized from the osmoregulated P1 promoter. Introduction of nucleotide substitutions in the cleavage site identified inhibited the ribonucleolytic activity of RNase III onproPmRNA, increasing the steady-state levels and half-life of the mRNA. In addition, decreased RNase III activity coincided with a significant increase in both the half-life and abundance ofproPmRNA under hyperosmotic stress conditions. Analysis of the RNA bound to RNase III viain vivocross-linking and immunoprecipitation indicated that this phenomenon is related to the decreased RNA binding capacity of RNase III. Our findings suggest the existence of an RNase III-mediated osmoregulatory network that rapidly balances the expression levels of factors associated with the cellular response to osmotic stress inE. coli.IMPORTANCEOur results demonstrate that RNase III activity onproPmRNA degradation is downregulated inEscherichia colicells under osmotic stress. In addition, we show that the downregulation of RNase III activity is associated with decreased RNA binding capacity of RNase III under hyperosmotic conditions. In particular, our findings demonstrate a link between osmotic stress and RNase III activity, underscoring the growing importance of posttranscriptional regulation in modulating rapid physiological adjustment to environmental changes.

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