scholarly journals Guanidine Riboswitch-Regulated Efflux Transporters Protect Bacteria against Ionic Liquid Toxicity

2019 ◽  
Vol 201 (13) ◽  
Author(s):  
Douglas A. Higgins ◽  
John M. Gladden ◽  
Jeff A. Kimbrel ◽  
Blake A. Simmons ◽  
Steven W. Singer ◽  
...  
Keyword(s):  
Ionic Liquid ◽  
Multidrug Resistance ◽  
Critical Role ◽  
Efflux Transporters ◽  
Imidazolium Ionic Liquid ◽  
Content Type ◽  
Small Multidrug Resistance ◽  
Cellulosic Sugars ◽  
Industrial Strains ◽  
Biochemical Production

ABSTRACT Plant cell walls contain a renewable, nearly limitless supply of sugar that could be used to support microbial production of commodity chemicals and biofuels. Imidazolium ionic liquid (IIL) solvents are among the best reagents for gaining access to the sugars in this otherwise recalcitrant biomass. However, the sugars from IIL-treated biomass are inevitably contaminated with residual IILs that inhibit growth in bacteria and yeast, blocking biochemical production by these organisms. IIL toxicity is, therefore, a critical roadblock in many industrial biosynthetic pathways. Although several IIL-tolerant (IILT) bacterial and yeast isolates have been identified in nature, few genetic mechanisms have been identified. In this study, we identified two IILT Bacillus isolates as well as a spontaneous IILT Escherichia coli lab strain that are tolerant to high levels of two widely used IILs. We demonstrate that all three IILT strains contain one or more pumps of the small multidrug resistance (SMR) family, and two of these strains contain mutations that affect an adjacent regulatory guanidine riboswitch. Furthermore, we show that the regulation of E. coli sugE by the guanidine II riboswitch can be exploited to promote IIL tolerance by the simple addition of guanidine to the medium. Our results demonstrate the critical role that transporter genes play in IIL tolerance in their native bacterial hosts. The study presented here is another step in engineering IIL tolerance into industrial strains toward overcoming this key gap in biofuels and industrial biochemical production processes. IMPORTANCE This study identifies bacteria that are tolerant to ionic liquid solvents used in the production of biofuels and industrial biochemicals. For industrial microbiology, it is essential to find less-harmful reagents and microbes that are resistant to their cytotoxic effects. We identified a family of small multidrug resistance efflux transporters, which are responsible for the tolerance of these strains. We also found that this resistance can be caused by mutations in the sequences of guanidine-specific riboswitches that regulate these efflux pumps. Extending this knowledge, we demonstrated that guanidine itself can promote ionic liquid tolerance. Our findings will inform genetic engineering strategies that improve conversion of cellulosic sugars into biofuels and biochemicals in processes where low concentrations of ionic liquids surpass bacterial tolerance.

scholarly journals Peptide-Based Efflux Pump Inhibitors of the Small Multidrug Resistance Protein from Pseudomonas aeruginosa

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Chloe J Mitchell ◽  
Tracy A. Stone ◽  
Charles M. Deber
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistance ◽  
Efflux Pump ◽  
Membrane Integrity ◽  
Resistant Bacteria ◽  
Binding Motif ◽  
Sequence Motif ◽  
Content Type ◽  
Binding Interface ◽  
Small Multidrug Resistance

ABSTRACT Bacteria have acquired multiple mechanisms to evade the lethal effects of current therapeutics, hindering treatment of bacterial infections, such as those caused by the pathogen Pseudomonas aeruginosa, which is responsible for nosocomial and cystic fibrosis lung infections. One resistance mechanism involves membrane-embedded multidrug efflux pumps that can effectively extrude an array of substrates, including common antibiotics, dyes, and biocides. Among these is a small multidrug resistance (SMR) efflux protein, consisting of four transmembrane (TM) helices, that functions as an antiparallel dimer. TM helices 1 to 3 (TM1 to TM3) comprise the substrate binding pocket, while TM4 contains a GG7 heptad sequence motif that mediates the SMR TM4-TM4 dimerization. In the present work, we synthesized a series of peptides containing the residues centered on the TM4-TM4 binding interface found in the P. aeruginosa SMR (PAsmr), typified by Ac-Ala-(Sar)3-LLGIGLIIAGVLV-KKK-NH2 (helix-helix interaction residues are underlined). Here, the acetylated N-terminal sarcosine (N-methyl-Gly) tag [Ac-Ala-(Sar)3] promotes membrane penetration, while the C-terminal Lys tag promotes selectivity for the negatively charged bacterial membranes. This peptide was observed to competitively disrupt PAsmr-mediated efflux, as measured by efflux inhibition of the fluorescent dye ethidium bromide, while having no effect on cell membrane integrity. Alternatively, a corresponding peptide in which the TM4 binding motif is scrambled was inactive in this assay. In addition, when Escherichia coli cells expressing PAsmr were combined with sublethal concentrations of several biocides, growth was significantly inhibited when peptide was added, notably, by up to 95% with the disinfectant benzylalkonium chloride. These results demonstrate promise for an efflux pump inhibitor to address the increasing threat of antibiotic-resistant bacteria.

scholarly journals Drug Efflux by a Small Multidrug Resistance Protein Is Inhibited by a Transmembrane Peptide

2012 ◽  
Vol 56 (7) ◽  
pp. 3911-3916 ◽  
Author(s):  
Bradley E. Poulsen ◽  
Charles M. Deber
Keyword(s):  
Multidrug Resistance ◽  
Rate Constant ◽  
Halobacterium Salinarum ◽  
Resistant Bacteria ◽  
Bacterial Cells ◽  
Multidrug Resistance Proteins ◽  
Content Type ◽  
Hydrophobic Peptides ◽  
Small Multidrug Resistance ◽  
Efflux Rate Constant

ABSTRACTDrug-resistant bacteria use several families of membrane-embedded transporters to remove antibiotics from the cell. One such family is the small multidrug resistance proteins (SMRs) that, because of their relatively small size (ca. 110 residues with four transmembrane [TM] helices), must form (at least) dimers to efflux drugs. Here, we use a Lys-tagged synthetic peptide with exactly the same sequence as TM4 of the full-length SMR Hsmr fromHalobacterium salinarum[TM4 sequence: AcA(Sar)3-VAGVVGLALIVAGVVVLNVAS-KKK (Sar =N-methylglycine)] to compete with and disrupt the native TM4-TM4 interactions believed to constitute the locus of Hsmr dimerization. Using a cellular efflux assay of the fluorescent SMR substrate ethidium bromide, we determined that bacterial cells containing Hsmr are able to remove cellular ethidium via first-order exponential decay with a rate constant (k) of 10.1 × 10−3± 0.7 × 10−3s−1. Upon treatment of the cells with the TM4 peptide, we observed a saturable ∼60% decrease in the efflux rate constant to 3.7 × 10−3± 0.2 × 10−3s−1. In corresponding experiments with control peptides, including scrambled sequences and a sequence withd-chirality, a decrease in ethidium efflux either was not observed or was marginal, likely from nonspecific effects. The designed peptides did not evoke bacterial lysis, indicating that they act via the α-helicity and membrane insertion propensities of the native TM4 helix. Our overall results suggest that this approach could conceivably be used to design hydrophobic peptides for disruption of key TM-TM interactions of membrane proteins and represent a valuable route to the discovery of new therapeutics.

scholarly journals Riboswitch-Associated Guanidinium-Selective Efflux Pumps Frequently Transmitted on Proteobacterial Plasmids Increase Escherichia coli Biofilm Tolerance to Disinfectants

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Carmine J. Slipski ◽  
Taylor R. Jamieson ◽  
George G. Zhanel ◽  
Denice C. Bay
Keyword(s):  
Escherichia Coli ◽  
Multidrug Resistance ◽  
Quaternary Ammonium ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Cloning And Expression ◽  
Upstream Region ◽  
Ammonium Compound ◽  
Content Type ◽  
Small Multidrug Resistance

ABSTRACT Members of the small multidrug resistance (SMR) efflux pump family known as SugE (recently renamed Gdx) are known for their narrow substrate selectivity to small guanidinium (Gdm+) compounds and disinfectant quaternary ammonium compounds (QACs). Gdx members have been identified on multidrug resistance plasmids in Gram-negative bacilli, but their functional role remains unclear, as few have been characterized. Here, we conducted a survey of sequenced proteobacterial plasmids that encoded one or more SugE/Gdx sequences in an effort to (i) identify the most frequently represented Gdx member(s) on these plasmids and their sequence diversity, (ii) verify if Gdx sequences possess a Gdm+ riboswitch that regulates their translation similarly to chromosomally encoded Gdx members, and (iii) determine the antimicrobial susceptibility profile of the most predominate Gdx member to various QACs and antibiotics in Escherichia coli strains BW25113 and KAM32. The results of this study determined 14 unique SugE sequences, but only one Gdx sequence, annotated as “SugE(p),” predominated among the >140 plasmids we surveyed. Enterobacterales plasmids carrying sugE(p) possessed a guanidine II riboswitch similar to the upstream region of E. coli gdx. Cloning and expression of sugE(p), gdx, and emrE sequences into a low-copy-number expression vector (pMS119EH) revealed significant increases in QAC resistance to a limited range of detergent-like QACs only when gdx and sugE(p) transformants were grown as biofilms. These findings suggest that sugE(p) presence on proteobacterial plasmids may be driven by species that frequently encounter Gdm+ and QAC exposure. IMPORTANCE This study characterized the function of antimicrobial-resistant phenotypes attributed to plasmid-encoded guanidinium-selective small multidrug resistance (Gdm/SugE) efflux pumps. These sequences are frequently monitored as biocide resistance markers in antimicrobial resistance surveillance studies. Our findings reveal that enterobacterial gdm sequences transmitted on plasmids possess a guanidine II riboswitch, which restricts transcript translation in the presence of guanidinium. Cloning and overexpression of this gdm sequence revealed that it confers higher resistance to quaternary ammonium compound (QAC) disinfectants (which possess guanidium moieties) when grown as biofilms. Since biofilms are commonly eradicated with QAC-containing compounds, the presence of this gene on plasmids and its biofilm-specific resistance are a growing concern for clinical and food safety prevention measures.

scholarly journals Few Conserved Amino Acids in the Small Multidrug Resistance Transporter EmrE Influence Drug Polyselectivity

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Marwah Saleh ◽  
Denice C. Bay ◽  
Raymond J. Turner
Keyword(s):  
Amino Acids ◽  
Multidrug Resistance ◽  
Drug Binding ◽  
Conserved Residues ◽  
Content Type ◽  
Small Multidrug Resistance ◽  
Transporter Family ◽  
Wide Range ◽  
Multidrug Resistance Transporter ◽  
Conserved Amino Acids

ABSTRACT EmrE is the archetypical member of the small multidrug resistance transporter family and confers resistance to a wide range of disinfectants and dyes known as quaternary cation compounds (QCCs). The aim of this study was to examine which conserved amino acids play an important role in substrate selectivity. On the basis of a previous analysis of EmrE homologues, a total of 33 conserved residues were targeted for cysteine or alanine replacement within E. coli EmrE. The antimicrobial resistance of each EmrE variant expressed in Escherichia coli strain JW0451 (lacking dominant pump acrB) to a collection of 16 different QCCs was tested using agar spot dilution plating to determine MIC values. The results determined that only a few conserved residues were drug polyselective, based on ≥4-fold decreases in MIC values: the active-site residue E14 (E14D and E14A) and 4 additional conserved residues (A10C, F44C, L47C, W63A). EmrE variants I11C, V15C, P32C, I62C, L93C, and S105C enhanced resistance to polyaromatic QCCs, while the remaining EmrE variants reduced resistance to one or more QCCs with shared chemical features: acylation, tri- and tetraphenylation, aromaticity, and dicationic charge. Mapping of EmrE variants onto transmembrane helical wheel projections using the highest resolved EmrE structure suggests that polyselective EmrE variants were located closest to the helical faces surrounding the predicted drug binding pocket, while EmrE variants with greater drug specificity mapped onto distal helical faces. This study reveals that few conserved residues are essential for drug polyselectivity and indicates that aromatic QCC selection involves a greater portion of conserved residues than that in other QCCs.

scholarly journals Fructose Induces Fluconazole Resistance in Candida albicans through Activation of Mdr1 and Cdr1 Transporters

2021 ◽  
Vol 22 (4) ◽  
pp. 2127
Author(s):  
Jakub Suchodolski ◽  
Anna Krasowska
Keyword(s):  
Candida Albicans ◽  
Multidrug Resistance ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
De Novo ◽  
Pathogenic Fungus ◽  
Serum Levels ◽  
Efflux Transporters ◽  
Fluconazole Resistance ◽  
Green Fluorescent

Candida albicans is a pathogenic fungus that is increasingly developing multidrug resistance (MDR), including resistance to azole drugs such as fluconazole (FLC). This is partially a result of the increased synthesis of membrane efflux transporters Cdr1p, Cdr2p, and Mdr1p. Although all these proteins can export FLC, only Cdr1p is expressed constitutively. In this study, the effect of elevated fructose, as a carbon source, on the MDR was evaluated. It was shown that fructose, elevated in the serum of diabetics, promotes FLC resistance. Using C. albicans strains with green fluorescent protein (GFP) tagged MDR transporters, it was determined that the FLC-resistance phenotype occurs as a result of Mdr1p activation and via the increased induction of higher Cdr1p levels. It was observed that fructose-grown C. albicans cells displayed a high efflux activity of both transporters as opposed to glucose-grown cells, which synthesize Cdr1p but not Mdr1p. Additionally, it was concluded that elevated fructose serum levels induce the de novo production of Mdr1p after 60 min. In combination with glucose, however, fructose induces Mdr1p production as soon as after 30 min. It is proposed that fructose may be one of the biochemical factors responsible for Mdr1p production in C. albicans cells.

Microwave-Assisted Preparation of Molecularly Imprinted Monolith Combining Imidazolium Ionic Liquid and POSS for Enhanced Extraction of Baicalin-Like Compounds in Scutellaria Baicalensis by means of In-Capillary SPME Followed by on-line LC and off-line LC-MS/MS

2021 ◽  
Author(s):  
Xiaohan Zhang ◽  
Le Gao ◽  
Liying Niu ◽  
Xiaodong Bi
Keyword(s):  
Ionic Liquid ◽  
Molecularly Imprinted Polymers ◽  
Scutellaria Baicalensis ◽  
Synthetic Methods ◽  
Imidazolium Ionic Liquid ◽  
Molecularly Imprinted ◽  
Imprinted Polymers ◽  
Microwave Assisted ◽  
On Line ◽  
Molecularly Imprinted Monolith

Molecularly imprinted polymers (MIPs) possess target-customized and range-adjustable selectivity, and hence have been attracting increasing efforts to develop new synthetic methods and new forms of applications. By wisely choosing functional...

Accessing resources for service innovation – the critical role of network relationships

2014 ◽  
Vol 25 (1) ◽  
pp. 2-29 ◽  
Author(s):  
Helena Rusanen ◽  
Aino Halinen ◽  
Elina Jaakkola
Keyword(s):  
Critical Role ◽  
Service Innovation ◽  
Innovation Process ◽  
Research Strategy ◽  
Future Research ◽  
Theoretical Understanding ◽  
Resource Access ◽  
Content Type ◽  
External Resources ◽  
Different Types

Purpose – This paper aims to explore how companies access resources through network relationships when developing service innovations. The paper identifies the types of resource that companies seek from other actors and examines the nature of relationships and resource access strategies that can be applied to access each type of resource. Design/methodology/approach – A longitudinal, multi-case study is conducted in the field of technical business-to-business (b-to-b) services. An abductive research strategy is applied to create a new theoretical understanding of resource access. Findings – Companies seek a range of resources through different types of network relationships for service innovation. Four types of resource access strategies were identified: absorption, acquisition, sharing, and co-creation. The findings show how easily transferable resources can be accessed through weak relationships and low-intensity collaboration. Access to resources that are difficult to transfer, instead, necessitates strong relationships and high-intensity collaboration. Research limitations/implications – The findings are valid for technical b-to-b services, but should also be tested for other kinds of innovations. Future research should also study how actors integrate the resources gained through networks in the innovation process. Practical implications – Managers should note that key resources for service innovation may be accessible through a variety of actors and relationships ranging from formal arrangements to miscellaneous social contacts. To make use of tacit resources such as knowledge, firms need to engage in intensive collaboration. Originality/value – Despite attention paid to network relationships, innovation collaboration, and external resources, previous research has neither linked these issues nor studied their mutual contingencies. This paper provides a theoretical model that characterizes the service innovation resources accessible through different types of relationships and access strategies.

scholarly journals Characterization of RarA, a Novel AraC Family Multidrug Resistance Regulator in Klebsiella pneumoniae

2012 ◽  
Vol 56 (8) ◽  
pp. 4450-4458 ◽  
Author(s):  
Mark Veleba ◽  
Paul G. Higgins ◽  
Gerardo Gonzalez ◽  
Harald Seifert ◽  
Thamarai Schneiders
Keyword(s):  
Multidrug Resistance ◽  
Klebsiella Pneumoniae ◽  
Efflux Pump ◽  
Content Type ◽  
Resistance Phenotype ◽  
Serratia Proteamaculans ◽  
Virulence Potential ◽  
Article Type ◽  
Acrab Efflux Pump

ABSTRACTTranscriptional regulators, such as SoxS, RamA, MarA, and Rob, which upregulate the AcrAB efflux pump, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria. In addition to the multidrug resistance phenotype, these regulators have also been shown to play a role in the cellular metabolism and possibly the virulence potential of microbial cells. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes.Klebsiella pneumoniaeis a major nosocomial pathogen which can express the SoxS, MarA, Rob, and RamA proteins, and the accompanying paper shows that the increased transcription oframAis associated with tigecycline resistance (M. Veleba and T. Schneiders, Antimicrob. Agents Chemother. 56:4466–4467, 2012). Bioinformatic analyses of the availableKlebsiellagenome sequences show that an additional AraC-type regulator is encoded chromosomally. In this work, we characterize this novel AraC-type regulator, hereby called RarA (Regulator of antibiotic resistance A), which is encoded inK. pneumoniae,Enterobactersp. 638,Serratia proteamaculans568, andEnterobacter cloacae. We show that the overexpression ofrarAresults in a multidrug resistance phenotype which requires a functional AcrAB efflux pump but is independent of the other AraC regulators. Quantitative real-time PCR experiments show thatrarA(MGH 78578 KPN_02968) and its neighboring efflux pump operonoqxAB(KPN_02969_02970) are consistently upregulated in clinical isolates collected from various geographical locations (Chile, Turkey, and Germany). Our results suggest thatrarAoverexpression upregulates theoqxABefflux pump. Additionally, it appears thatoqxR, encoding a GntR-type regulator adjacent to theoqxABoperon, is able to downregulate the expression of theoqxABefflux pump, where OqxR complementation resulted in reductions to olaquindox MICs.

scholarly journals Overlapping Substrate Specificities in the Small Multidrug Resistance (SMR) Family of Transporters

2020 ◽  
Vol 118 (3) ◽  
pp. 130a
Author(s):  
Christian B. Macdonald ◽  
Ali A. Kermani ◽  
Randy B. Stockbridge
Keyword(s):  
Multidrug Resistance ◽  
Substrate Specificities ◽  
Small Multidrug Resistance
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