scholarly journals A Fluorescent Microplate Assay Quantifies Bacterial Efflux and Demonstrates Two Distinct Compound Binding Sites in AcrB

2015 ◽  
Vol 59 (4) ◽  
pp. 2388-2397 ◽  
Author(s):  
Ramkumar Iyer ◽  
Annette Ferrari ◽  
R. Rijnbrand ◽  
Alice L. Erwin
Keyword(s):  
Binding Sites ◽  
Nile Red ◽  
Content Type ◽  
E Coli ◽  
Chemical Structures ◽  
Low Mass ◽  
Substrate Binding Sites ◽  
Related Compounds ◽  
Distal Site ◽  
Modest Correlation

ABSTRACTA direct assay of efflux byEscherichia coliAcrAB-TolC and related multidrug pumps would have great value in discovery of new Gram-negative antibiotics. The current understanding of how efflux is affected by the chemical structure and physical properties of molecules is extremely limited, derived from antibacterial data for compounds that inhibit growth of wild-typeE. coli. We adapted a previously described fluorescent efflux assay to a 96-well microplate format that measured the ability of test compounds to compete for efflux with Nile Red (an environment-sensitive fluor), independent of antibacterial activity. We show that Nile Red and the lipid-sensitive probe DiBAC4-(3) [bis-(1,3-dibutylbarbituric acid)-trimethine oxonol] can quantify efflux competition inE. coli. We extend the previous findings that the tetracyclines compete with Nile Red and show that DiBAC4-(3) competes with macrolides. The extent of the competition shows a modest correlation with the effect of theacrBdeletion on MICs within the compound sets for both dyes. Crystallographic studies identified at least two substrate binding sites in AcrB, the proximal and distal pockets. High-molecular-mass substrates bound the proximal pocket, while low-mass substrates occupied the distal pocket. As DiBAC4-(3) competes with macrolides but not with Nile Red, we propose that DiBAC4-(3) binds the proximal pocket and Nile Red likely binds the distal site. In conclusion, competition with fluorescent probes can be used to study the efflux process for diverse chemical structures and may provide information as to the site of binding and, in some cases, enable rank-ordering a series of related compounds by efflux.

scholarly journals One Intact Transmembrane Substrate Binding Site Is Sufficient for the Function of the Homodimeric Type I ATP-Binding Cassette Importer for Positively Charged Amino Acids Art(MP) 2 of Geobacillus stearothermophilus

2018 ◽  
Vol 200 (12) ◽  
Author(s):  
Johanna Heuveling ◽  
Heidi Landmesser ◽  
Erwin Schneider
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Atp Binding ◽  
Content Type ◽  
Charged Amino Acids ◽  
Substrate Binding Sites ◽  
Positively Charged ◽  
Atp Binding Cassette

ABSTRACT ATP-binding cassette (ABC) transport systems comprise two transmembrane domains/subunits that form a translocation path and two nucleotide-binding domains/subunits that bind and hydrolyze ATP. Prokaryotic canonical ABC import systems require an extracellular substrate-binding protein for function. Knowledge of substrate-binding sites within the transmembrane subunits is scarce. Recent crystal structures of the ABC importer Art(QN) 2 for positively charged amino acids of Thermoanerobacter tengcongensis revealed the presence of one substrate molecule in a defined binding pocket in each of the transmembrane subunits, ArtQ (J. Yu, J. Ge, J. Heuveling, E. Schneider, and M. Yang, Proc Natl Acad Sci U S A 112:5243–5248, 2015, https://doi.org/10.1073/pnas.1415037112 ). This finding raised the question of whether both sites must be loaded with substrate prior to initiation of the transport cycle. To address this matter, we first explored the role of key residues that form the binding pocket in the closely related Art(MP) 2 transporter of Geobacillus stearothermophilus , by monitoring consequences of mutations in ArtM on ATPase and transport activity at the level of purified proteins embedded in liposomes. Our results emphasize that two negatively charged residues (E153 and D160) are crucial for wild-type function. Furthermore, the variant Art[M(L67D)P] 2 exhibited strongly impaired activities, which is why it was considered for construction of a hybrid complex containing one intact and one impaired substrate-binding site. Activity assays clearly revealed that one intact binding site was sufficient for function. To our knowledge, our study provides the first biochemical evidence on transmembrane substrate-binding sites of an ABC importer. IMPORTANCE Canonical prokaryotic ATP-binding cassette importers mediate the uptake of a large variety of chemicals, including nutrients, osmoprotectants, growth factors, and trace elements. Some also play a role in bacterial pathogenesis, which is why full understanding of their mode of action is of the utmost importance. One of the unsolved problems refers to the chemical nature and number of substrate binding sites formed by the transmembrane subunits. Here, we report that a hybrid amino acid transporter of G. stearothermophilus , encompassing one intact and one impaired transmembrane binding site, is fully competent in transport, suggesting that the binding of one substrate molecule is sufficient to trigger the translocation process.

DNA photolyase of enterococci: possible explanation for its low sunlight inactivation rate

Biologia ◽  
2009 ◽  
Vol 64 (5) ◽  
Author(s):  
Mushtaq Hussain ◽  
Syeda Qamarunnissa ◽  
Saboohi Raza ◽  
Javed Qureshi ◽  
Abdul Wajid ◽  
...  
Keyword(s):  
Binding Sites ◽  
Light Harvesting ◽  
Inactivation Rate ◽  
Dna Photolyase ◽  
Multiple Sequence ◽  
Efficient Energy Transfer ◽  
Tertiary Structures ◽  
E Coli ◽  
The Difference ◽  
Substrate Binding Sites

AbstractDNA photolyase is perhaps the most ancient and direct arsenal in curing the UV-induced dimers formed in the microbial genome. Out of two cofactors of the enzyme, catalytic and light harvesting, differences in the latter have provided basis for categorizing photolyases of prokaryotes as folate and deazaflavin types. In the present study, the homology modeling of DNA photolyase of Enterococcus faecalis was undertaken. The predicted models were structurally compared with the crystal structure coordinates of photolyases from Escherichia coli (folate type) and Anacystis nidulans (deazaflavin type). Discrepancies present in the multiple sequence alignment and tertiary structures, particularly at the light harvesting cofactor (methenyltetrahydrofolic acid, MTHF; 8-hydroxy-5-deazaflavin, 8-HDF) binding sites indicated the mechanistic nature of enterococcal photolyase. Concisely, despite the greater holistic homology with folate-type photolyase, enterococcal photolyase was characterized as deazaflavin-type. The presence of 8-HDF binding sites and groove architecture of substrate binding sites were also found supportive in this regard. The inter cofactor distance and/or orientation also implied to the efficient energy transfer in photolyase of Enterococcus in comparison with E. coli. In addition, we observed relatively high protein deformability in the enterococcal genome, which may favors the repair action of photolyase. The findings are expected to provide molecular insights into the difference in sunlight inactivation rate of two important fecal contamination indicators, namely Enterococcus and E. coli.

scholarly journals Pimozide Inhibits the AcrAB-TolC Efflux Pump in Escherichia coli

2013 ◽  
Vol 7 (1) ◽  
pp. 83-86 ◽  
Author(s):  
Jürgen A Bohnert ◽  
Sabine Schuster ◽  
Winfried V Kern
Keyword(s):  
Escherichia Coli ◽  
Ethidium Bromide ◽  
Binding Sites ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Nile Red ◽  
Neuroleptic Drug ◽  
Antimicrobial Compounds ◽  
Efflux Pump Inhibitors ◽  
Substrate Binding Sites

Efflux pump inhibitors (EPIs) are attractive compounds to reverse multidrug-resistance in clinically relevant bacterial pathogens. In this study we tested the ability of the neuroleptic drug pimozide to inhibit the Escherichia coli AcrAB-TolC efflux pump, whose overproduction confers resistance to various antimicrobial agents. A real-time Nile red efflux assay in the AcrAB – overproducing strain 3-AG100 revealed that pimozide was capable of full inhibition of this pump at a concentration of 100 µM, which is far below its intrinsic MIC (>1mM). However, MIC assay demonstrated very little effect of pimozide with regard to reduction in MICs of various antimicrobial compounds. Only oxacillin MICs were reduced twofold in the presence of pimozide at 100 and 200 µM. Since pimozide did considerably enhance accumulation of ethidium bromide in a fluorescence assay, ethidium bromide MIC assays in the presence and absence of this putative EPI were performed. They revealed that pimozide was able to reduce the MICs of ethidium bromide by 4-fold. In line with previous reports we suggest that the capability of EPIs to restore the susceptibility to antimicrobial agents can be highly substrate-specific due to different substrate binding sites.

scholarly journals CsrA-Mediated Translational Activation of ymdA Expression in Escherichia coli

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Andrew Renda ◽  
Stephanie Poly ◽  
Ying-Jung Lai ◽  
Archana Pannuri ◽  
Helen Yakhnin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Binding Sites ◽  
Biofilm Formation ◽  
Central Component ◽  
Content Type ◽  
E Coli ◽  
Ribosome Binding ◽  
Activation Mechanisms ◽  
Translational Activation

ABSTRACT The sequence-specific RNA-binding protein CsrA is the central component of the conserved global regulatory Csr system. In Escherichia coli, CsrA regulates many cellular processes, including biofilm formation, motility, carbon metabolism, iron homeostasis, and stress responses. Such regulation often involves translational repression by CsrA binding to an mRNA target, thereby inhibiting ribosome binding. While CsrA also extensively activates gene expression, no detailed mechanism for CsrA-mediated translational activation has been demonstrated. An integrated transcriptomic study identified ymdA as having the strongest CsrA-mediated activation across the E. coli transcriptome. Here, we determined that CsrA activates ymdA expression posttranscriptionally. Gel mobility shift, footprint, toeprint, and in vitro coupled transcription-translation assays identified two CsrA binding sites in the leader region of the ymdA transcript that are critical for translational activation. Reporter fusion assays confirmed that CsrA activates ymdA expression at the posttranscriptional level in vivo. Furthermore, loss of binding at either of the two CsrA binding sites abolished CsrA-dependent activation. mRNA half-life studies revealed that CsrA also contributes to stabilization of ymdA mRNA. RNA structure prediction revealed an RNA hairpin upstream of the ymdA start codon that sequesters the Shine-Dalgarno (SD) sequence, which would inhibit ribosome binding. This hairpin also contains one of the two critical CsrA binding sites, with the other site located just upstream. Our results demonstrate that bound CsrA destabilizes the SD-sequestering hairpin such that the ribosome can bind and initiate translation. Since YmdA represses biofilm formation, CsrA-mediated activation of ymdA expression may repress biofilm formation under certain conditions. IMPORTANCE The Csr system of E. coli controls gene expression and physiology on a global scale. CsrA protein, the central component of this system, represses translation initiation of numerous genes by binding to target transcripts, thereby competing with ribosome binding. Variations of this mechanism are so common that CsrA is sometimes called a translational repressor. Although CsrA-mediated activation mechanisms have been elucidated in which bound CsrA inhibits RNA degradation, no translation activation mechanism has been defined. Here, we demonstrate that CsrA binding to two sites in the 5′ untranslated leader of ymdA mRNA activates translation by destabilizing a structure that otherwise prevents ribosome binding. The extensive role of CsrA in activating gene expression suggests the common occurrence of similar activation mechanisms.

scholarly journals Widespread Strain-Specific Distinctions in Chromosomal Binding Dynamics of a Highly Conserved Escherichia coli Transcription Factor

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
James P. R. Connolly ◽  
Nicky O’Boyle ◽  
Andrew J. Roe
Keyword(s):  
Escherichia Coli ◽  
Gene Regulation ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Binding Sites ◽  
Regulatory Networks ◽  
Surprising Result ◽  
Bacterial Gene ◽  
Content Type ◽  
E Coli

ABSTRACT Bacterial gene regulation is governed by often hundreds of transcription factors (TFs) that bind directly to targets on the chromosome. Global studies of TFs usually make assumptions that regulatory targets within model strains will be conserved between members of the same species harboring common genetic targets. We recently discovered that YhaJ of Escherichia coli is crucial for virulence in two different pathotypes but binds to distinct regions of their genomes and regulates no common genes. This surprising result leads to strain-specific mechanisms of virulence regulation, but the implications for other E. coli pathotypes or commensals were unclear. Here, we report that heterogenous binding of YhaJ is widespread within the E. coli species. We analyzed the global YhaJ binding dynamics of four evolutionarily distinct E. coli isolates under two conditions, revealing 78 significant sites on the core genome as well as horizontally acquired loci. Condition-dependent dosage of YhaJ correlated with the number of occupied sites in vivo but did not significantly alter its enrichment at regions bound in both conditions, explaining the availability of this TF to occupy accessory sites in response to the environment. Strikingly, only ∼15% of YhaJ binding sites were common to all strains. Furthermore, differences in enrichment of uncommon sites were observed largely in chromosomal regions found in all strains and not explained exclusively by binding to strain-specific horizontally acquired elements or mutations in the DNA binding sequence. This observation suggests that intraspecies distinctions in TF binding dynamics are a widespread phenomenon and represent strain-specific gene regulatory potential. IMPORTANCE In bacterial cells, hundreds of transcription factors coordinate gene regulation and thus are a major driver of cellular processes. However, the immense diversity in bacterial genome structure and content makes deciphering regulatory networks challenging. This is particularly apparent for the model organism Escherichia coli as evolution has driven the emergence of species members with highly distinct genomes, which occupy extremely different niches in nature. While it is well-known that transcription factors must integrate horizontally acquired DNA into the regulatory network of the cell, the extent of regulatory diversity beyond single model strains is unclear. We have explored this concept in four evolutionarily distinct E. coli strains and show that a highly conserved transcription factor displays unprecedented diversity in chromosomal binding sites. Importantly, this diversity is not restricted to strain-specific DNA or mutation in binding sites. This observation suggests that strain-specific regulatory networks are potentially widespread within individual bacterial species.

Biotransformation of Coumarins by Filamentous Fungi: An Alternative Way for Achievement of Bioactive Analogs

2019 ◽  
Vol 16 (6) ◽  
pp. 568-577 ◽  
Author(s):  
Jainara Santos do Nascimento ◽  
João Carlos Silva Conceição ◽  
Eliane de Oliveira Silva
Keyword(s):  
Filamentous Fungi ◽  
Chemical Reactions ◽  
Biological Activities ◽  
Chemical Transformations ◽  
Chemical Structures ◽  
Pattern Structures ◽  
Pharmacological Interest ◽  
Aspergillus Sp ◽  
The Impact ◽  
Related Compounds

Coumarins are natural 1,2-benzopyrones, present in remarkable amounts as secondary metabolites in edible and medicinal plants. The low yield in the coumarins isolation from natural sources, along with the difficulties faced by the total synthesis, make them attractive for biotechnological studies. The current literature contains several reports on the biotransformation of coumarins by fungi, which can generate chemical analogs with high selectivity, using mild and eco-friendly conditions. Prompted by the enormous pharmacological interest in the coumarin-related compounds, their alimentary and chemical applications, this review covers the biotransformation of coumarins by filamentous fungi. The chemical structures of the analogs were presented and compared with those from the pattern structures. The main chemical reactions catalyzed the insertion of functional groups, and the impact on the biological activities caused by the chemical transformations were discussed. Several chemical reactions can be catalyzed by filamentous fungi in the coumarin scores, mainly lactone ring opening, C3-C4 reduction and hydroxylation. Chunninghamella sp. and Aspergillus sp. are the most common fungi used in these transformations. Concerning the substrates, the biotransformation of pyranocoumarins is a rarer process. Sometimes, the bioactivities were improved by the chemical modifications and coincidences with the mammalian metabolism were pointed out.

scholarly journals Tailoring a Global Iron Regulon to a Uropathogen

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Rajdeep Banerjee ◽  
Erin Weisenhorn ◽  
Kevin J. Schwartz ◽  
Kevin S. Myers ◽  
Jeremy D. Glasner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Urinary Tract ◽  
Amino Acid ◽  
Regulatory Network ◽  
Iron Homeostasis ◽  
Iron Limitation ◽  
Content Type ◽  
E Coli ◽  
General Stress ◽  
K 12

ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

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