scholarly journals Acyl depsipeptide (ADEP) resistance in Streptomyces

Microbiology ◽  
2011 ◽  
Vol 157 (8) ◽  
pp. 2226-2234 ◽  
Author(s):  
Myriam Gominet ◽  
Nicolas Seghezzi ◽  
Philippe Mazodier
Keyword(s):  
Multidrug Resistance ◽  
Abc Transporter ◽  
Mode Of Action ◽  
Protease Activity ◽  
Streptomyces Coelicolor ◽  
Antibiotic Activity ◽  
Wild Type ◽  
Clp Protease ◽  
Clp Proteases ◽  
Proteolytic Chamber

ADEP, a molecule of the acyl depsipeptide family, has an antibiotic activity with a unique mode of action. ADEP binding to the ubiquitous protease ClpP alters the structure of the enzyme. Access of protein to the ClpP proteolytic chamber is therefore facilitated and its cohort regulatory ATPases (ClpA, ClpC, ClpX) are not required. The consequent uncontrolled protein degradation in the cell appears to kill the ADEP-treated bacteria. ADEP is produced by Streptomyces hawaiiensis. Most sequenced genomes of Streptomyces have five clpP genes, organized as two distinct bicistronic operons, clpP1clpP2 and clpP3clpP4, and a single clpP5 gene. We investigated whether the different Clp proteases are all sensitive to ADEP. We report that ClpP1 is a target of ADEP whereas ClpP3 is largely insensitive. In wild-type Streptomyces lividans, clpP3clpP4 expression is constitutively repressed and the reason for the maintenance of this operon in Streptomyces has been elusive. ClpP activity is indispensable for survival of actinomycetes; we therefore tested whether the clpP3clpP4 operon, encoding an ADEP-insensitive Clp protease, contributes to a mechanism of ADEP resistance by target substitution. We report that in S. lividans, inactivation of ClpP1ClpP2 production or protease activity is indeed a mode of resistance to ADEP although it is neither the only nor the most frequent mode of resistance. The ABC transporter SclAB (orthologous to the Streptomyces coelicolor multidrug resistance pump SCO4959–SCO4960) is also able to confer ADEP resistance, and analysis of strains with sclAB deletions indicates that there are also other mechanisms of ADEP resistance.

scholarly journals Mutational Disruption of Plasma Membrane Trafficking of Saccharomyces cerevisiae Yor1p, a Homologue of Mammalian Multidrug Resistance Protein

1999 ◽  
Vol 19 (4) ◽  
pp. 2998-3009 ◽  
Author(s):  
David J. Katzmann ◽  
Eric A. Epping ◽  
W. Scott Moye-Rowley
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Multidrug Resistance ◽  
Abc Transporter ◽  
Membrane Trafficking ◽  
Predicted Amino Acid Sequence ◽  
Multidrug Resistance Protein ◽  
Wild Type ◽  
Amino Terminal ◽  
Resistance Protein

ABSTRACT The ATP binding cassette (ABC) transporter protein Yor1p was identified on the basis of its ability to elevate oligomycin resistance when it was overproduced from a high-copy-number plasmid. Analysis of the predicted amino acid sequence of Yor1p indicated that this protein was a new member of a subfamily of ABC transporter proteins defined by the multidrug resistance protein (MRP). In this work, Yor1p is demonstrated to localize to the Saccharomyces cerevisiaeplasma membrane by both indirect immunofluorescence and biochemical fractionation studies. Several mutations were generated in the amino-terminal nucleotide binding domain (NBD1) of Yor1p to test if the high degree of sequence conservation in this region of the protein was important for function. Deletion of a phenylalanine residue at Yor1p position 670 led to a mutant protein that appeared to be retained in the endoplasmic reticulum (ER) and that was unstable. As shown by others, deletion of the analogous residue from a second mammalian MRP family member, the cystic fibrosis transmembrane conductance regulator (CFTR), also led to retention of this normally plasma membrane-localized protein in the ER. Changes in the spacing between or the sequences flanking functional motifs of Yor1p NBD1 led to defective trafficking or decreased activity of the mutant proteins. Analyses of the degradation of wild-type and ΔF670 Yor1p indicated that the half-life of ΔF670 Yor1p was dramatically shortened. While the vacuole was the primary site for turnover of wild-type Yor1p, degradation of ΔF670 Yor1p was found to be more complex with both proteasomal and vacuolar contributions.

scholarly journals Conserved amino acids in the region connecting membrane spanning domain 1 to nucleotide binding domain 1 are essential for expression of the MRP1 (ABCC1) transporter

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246727
Author(s):  
Emma E. Smith ◽  
Gwenaëlle Conseil ◽  
Susan P. C. Cole
Keyword(s):  
Plasma Membrane ◽  
Multidrug Resistance ◽  
Abc Transporter ◽  
Organic Anion ◽  
Nucleotide Binding ◽  
Wild Type ◽  
Membrane Expression ◽  
Validation Data ◽  
Active Efflux ◽  
Membrane Spanning

Multidrug resistance protein 1 (MRP1) (gene symbol ABCC1) is an ATP-binding cassette (ABC) transporter which effluxes xeno- and endobiotic organic anions including estradiol glucuronide and the pro-inflammatory leukotriene C4. MRP1 also confers multidrug resistance by reducing intracellular drug accumulation through active efflux. MRP1 has three membrane spanning domains (MSD), and two nucleotide binding domains (NBD). MSD1 and MSD2 are linked to NBD1 and NBD2 by connecting regions (CR) 1 and CR2, respectively. Here we targeted four residues in CR1 (Ser612, Arg615, His622, Glu624) for alanine substitution and unexpectedly, found that cellular levels of three mutants (S612A, R615A, E624A) in transfected HEK cells were substantially lower than wild-type MRP1. Whereas CR1-H622A properly trafficked to the plasma membrane and exhibited organic anion transport activity comparable to wild-type MRP1, the poorly expressing R615A and E624A (and to a lesser extent S612A) mutant proteins were retained intracellularly. Analyses of cryogenic electron microscopic and atomic homology models of MRP1 indicated that Arg615 and Glu624 might participate in bonding interactions with nearby residues to stabilize expression of the transporter. However, this was not supported by double exchange mutations E624K/K406E, R615D/D430R and R615F/F619R which failed to improve MRP1 levels. Nevertheless, these experiments revealed that the highly conserved CR1-Phe619 and distal Lys406 in the first cytoplasmic loop of MSD1 are also essential for expression of MRP1 protein. This study is the first to demonstrate that CR1 contains several highly conserved residues critical for plasma membrane expression of MRP1 but thus far, currently available structures and models do not provide any insights into the underlying mechanism(s). Additional structures with rigorous biochemical validation data are needed to fully understand the bonding interactions critical to stable expression of this clinically important ABC transporter.

ABC Transporter Inhibitors in Reversing Multidrug Resistance to Chemotherapy

2015 ◽  
Vol 16 (12) ◽  
pp. 1356-1371 ◽  
Author(s):  
Haigang Cui ◽  
Anna Zhang ◽  
Mingwei Chen ◽  
Johnson Liu
Keyword(s):  
Multidrug Resistance ◽  
Abc Transporter ◽  
Resistance To Chemotherapy

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate

2001 ◽  
Vol 281 (4) ◽  
pp. G1034-G1043 ◽  
Author(s):  
Kousei Ito ◽  
Hiroshi Suzuki ◽  
Yuichi Sugiyama
Keyword(s):  
Amino Acids ◽  
Multidrug Resistance ◽  
Amino Acid ◽  
Membrane Vesicles ◽  
Single Amino Acid ◽  
Sf9 Cells ◽  
Transport Activity ◽  
Wild Type ◽  
Cationic Amino Acids ◽  
The Difference

Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC). The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains. For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined. Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates. Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates. These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.

scholarly journals New Sporulation Loci in Streptomyces coelicolor A3(2)

1999 ◽  
Vol 181 (17) ◽  
pp. 5419-5425 ◽  
Author(s):  
N. Jamie Ryding ◽  
Maureen J. Bibb ◽  
Virginie Molle ◽  
Kim C. Findlay ◽  
Keith F. Chater ◽  
...  
Keyword(s):  
Phase Contrast ◽  
Streptomyces Coelicolor ◽  
Cosmid Library ◽  
Phase Contrast Microscopy ◽  
Wild Type ◽  
Chromosomal Dna ◽  
East Anglia ◽  
Wild Type Phenotype ◽  
Contrast Microscopy ◽  
Spore Pigment

ABSTRACT Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the grey polyketide spore pigment, and such white (whi) mutants had been used to define eight sporulation loci, whiA,whiB, whiD, whiE, whiG,whiH, whiI, and whiJ (K. F. Chater, J. Gen. Microbiol. 72:9–28, 1972; N. J. Ryding, Ph.D. thesis, University of East Anglia, 1995). In an attempt to identify new whi loci, we mutagenized S. coelicolor M145 spores with nitrosoguanidine and identified 770 mutants with colonies ranging from white to medium grey. After excluding unstable strains, we examined the isolates by phase-contrast microscopy and chose 115 whi mutants with clear morphological phenotypes for further study. To exclude mutants representing cloned whi genes, self-transmissible SCP2*-derived plasmids carrying whiA, whiB,whiG, whiH, or whiJ (but notwhiD, whiE, or whiI) were introduced into each mutant by conjugation, and strains in which the wild-type phenotype was restored either partially or completely by any of these plasmids were excluded from further analysis. In an attempt to complement some of the remaining 31 whi mutants, an SCP2* library of wild-type S. coelicolor chromosomal DNA was introduced into 19 of the mutants by conjugation. Clones restoring the wild-type phenotype to 12 of the 19 strains were isolated and found to represent five distinct loci, designated whiK,whiL, whiM, whiN, andwhiO. Each of the five loci was located on the ordered cosmid library: whiL, whiM, whiN, and whiO occupied positions distinct from previously clonedwhi genes; whiK was located on the same cosmid overlap as whiD, but the two loci were shown by complementation to be distinct. The phenotypes resulting from mutations at each of these new loci are described.

scholarly journals RNase III Is Required for Actinomycin Production in Streptomyces antibioticus

2013 ◽  
Vol 79 (20) ◽  
pp. 6447-6451 ◽  
Author(s):  
Jung-Hoon Lee ◽  
Marcha L. Gatewood ◽  
George H. Jones
Keyword(s):  
Parental Strain ◽  
Insertional Mutagenesis ◽  
Southern Blotting ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Production Medium ◽  
Wild Type ◽  
Rnase Iii ◽  
Content Type ◽  
Streptomyces Antibioticus

ABSTRACTUsing insertional mutagenesis, we have disrupted the RNase III gene,rnc, of the actinomycin-producing streptomycete,Streptomyces antibioticus. Disruption was verified by Southern blotting. The resulting strain grows more vigorously than its parent on actinomycin production medium but produces significantly lower levels of actinomycin. Complementation of therncdisruption with the wild-typerncgene fromS. antibioticusrestored actinomycin production to nearly wild-type levels. Western blotting experiments demonstrated that the disruptant did not produce full-length or truncated forms of RNase III. Thus, as is the case inStreptomyces coelicolor, RNase III is required for antibiotic production inS. antibioticus. No differences in the chemical half-lives of bulk mRNA were observed in a comparison of theS. antibioticus rncmutant and its parental strain.

scholarly journals ThemtfATranscription Factor Gene Controls Morphogenesis, Gliotoxin Production, and Virulence in the Opportunistic Human Pathogen Aspergillus fumigatus

2014 ◽  
Vol 13 (6) ◽  
pp. 766-775 ◽  
Author(s):  
Timothy D. Smith ◽  
Ana M. Calvo
Keyword(s):  
Aspergillus Fumigatus ◽  
Protease Activity ◽  
Galleria Mellonella ◽  
Molecular Genetic ◽  
Colony Growth ◽  
Infection Model ◽  
Slight Reduction ◽  
Wild Type ◽  
Content Type ◽  
Factor Gene

ABSTRACTAspergillus fumigatusis the leading causative agent of invasive aspergillosis (IA). The number of cases is on the rise, with mortality rates as high as 90% among immunocompromised patients. Molecular genetic studies inA. fumigatuscould provide novel targets to potentially set the basis for antifungal therapies. In the current study, we investigated the role of the transcription factor genemtfAinA. fumigatus. Our results revealed thatmtfAplays a role in the growth and development of the fungus. Deletion or overexpression ofmtfAleads to a slight reduction in colony growth, as well as a reduction in conidiation levels, in the overexpression strain compared to the wild-type strain. Furthermore, production of the secondary metabolite gliotoxin increased whenmtfAwas overexpressed, coinciding with an increase in the transcription levels of the gliotoxin genesgliZandgliPwith respect to the wild type. In addition, our study showed thatmtfAis also necessary for normal protease activity inA. fumigatus; deletion ofmtfAresulted in a reduction of protease activity compared to wild-type levels. Importantly, the absence ofmtfAcaused a decrease in virulence in theGalleria mellonellainfection model, indicating thatmtfAis necessary forA. fumigatuswild-type pathogenesis.

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