scholarly journals The role of the Serratia marcescens SdeAB multidrug efflux pump and TolC homologue in fluoroquinolone resistance studied via gene-knockout mutagenesis

Microbiology ◽  
2008 ◽  
Vol 154 (2) ◽  
pp. 454-461 ◽  
Author(s):  
Sanela Begic ◽  
Elizabeth A. Worobec
Keyword(s):  
Serratia Marcescens ◽  
Efflux Pump ◽  
Gene Knockout ◽  
Fluoroquinolone Resistance ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump

Characterization of the Serratia marcescens SdeCDE multidrug efflux pump studied via gene knockout mutagenesis

2008 ◽  
Vol 54 (5) ◽  
pp. 411-416 ◽  
Author(s):  
Sanela Begic ◽  
Elizabeth A. Worobec
Keyword(s):  
Antibiotic Resistance ◽  
Substrate Specificity ◽  
Serratia Marcescens ◽  
Efflux Pump ◽  
Gene Knockout ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Active Efflux ◽  
Major Mechanism

Serratia marcescens is an important nosocomial agent having high antibiotic resistance. A major mechanism for S. marcescens antibiotic resistance is active efflux. To ascertain the substrate specificity of the S. marcescens SdeCDE efflux pump, we constructed pump gene deletion mutants. sdeCDE knockout strains showed no change in antibiotic susceptibility in comparison with the parental strains for any of the substrates, with the exception of novobiocin. In addition, novobiocin was the only antibiotic to be accumulated by sdeCDE-deficient strains. Based on the substrates used in our study, we conclude that SdeCDE is a Resistance–Nodulation–Cell Division family pump with limited substrate specificity.

scholarly journals Genetic Characterization of Highly Fluoroquinolone-Resistant Clinical Escherichia coli Strains from China: Role ofacrR Mutations

2001 ◽  
Vol 45 (5) ◽  
pp. 1515-1521 ◽  
Author(s):  
Hui Wang ◽  
Joann L. Dzink-Fox ◽  
Minjun Chen ◽  
Stuart B. Levy
Keyword(s):  
Escherichia Coli ◽  
Blot Analysis ◽  
Genetic Basis ◽  
Efflux Pump ◽  
Pcr Amplification ◽  
Fluoroquinolone Resistance ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
High Level

ABSTRACT The genetic basis for fluoroquinolone resistance was examined in 30 high-level fluoroquinolone-resistant Escherichia coliclinical isolates from Beijing, China. Each strain also demonstrated resistance to a variety of other antibiotics. PCR sequence analysis of the quinolone resistance-determining region of the topoisomerase genes (gyrA/B, parC) revealed three to five mutations known to be associated with fluoroquinolone resistance. Western blot analysis failed to demonstrate overexpression of MarA, and Northern blot analysis did not detect overexpression of soxS RNA in any of the clinical strains. The AcrA protein of the AcrAB multidrug efflux pump was overexpressed in 19 of 30 strains of E. colitested, and all 19 strains were tolerant to organic solvents. PCR amplification of the complete acrR (regulator/repressor) gene of eight isolates revealed amino acid changes in four isolates, a 9-bp deletion in another, and a 22-bp duplication in a sixth strain. Complementation with a plasmid-borne wild-type acrR gene reduced the level of AcrA in the mutants and partially restored antibiotic susceptibility 1.5- to 6-fold. This study shows that mutations in acrR are an additional genetic basis for fluoroquinolone resistance.

scholarly journals SmdAB, a Heterodimeric ABC-Type Multidrug Efflux Pump, in Serratia marcescens

2007 ◽  
Vol 190 (2) ◽  
pp. 648-654 ◽  
Author(s):  
Taira Matsuo ◽  
Jing Chen ◽  
Yusuke Minato ◽  
Wakano Ogawa ◽  
Tohru Mizushima ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Amino Acid Sequences ◽  
Multidrug Efflux ◽  
Chromosomal Dna ◽  
Atpase Inhibitor ◽  
Multidrug Efflux Pump ◽  
Hoechst 33342 ◽  
E Coli

ABSTRACT We cloned genes, designated smdAB, that encode a multidrug efflux pump from the chromosomal DNA of clinically isolated Serratia marcescens NUSM8906. For cells of the drug-hypersensitive strain Escherichia coli KAM32 harboring a recombinant plasmid carrying smdAB, structurally unrelated antimicrobial agents such as norfloxacin, tetracycline, 4′,6-diamidino-2-phenylindole (DAPI), and Hoechst 33342 showed elevated MICs. The deduced amino acid sequences of both SmdA and SmdB exhibited similarities to the sequences of ATP-binding cassette (ABC)-type multidrug efflux pumps. The efflux of DAPI and Hoechst 33342 from E. coli cells expressing SmdAB was observed, and the efflux activities were inhibited by sodium o-vanadate, which is a well-known ATPase inhibitor. The introduction of smdA or smdB alone into E. coli KAM32 did not elevate the MIC of DAPI; thus, both SmdA and SmdB were required for function. These results indicate that SmdAB is probably a heterodimeric multidrug efflux pump of the ABC family in S. marcescens.

scholarly journals Role of VltAB, an ABC Transporter Complex, in Viologen Tolerance inStreptococcus mutans

2011 ◽  
Vol 55 (4) ◽  
pp. 1460-1469 ◽  
Author(s):  
Saswati Biswas ◽  
Indranil Biswas
Keyword(s):  
Abc Transporter ◽  
Efflux Pump ◽  
Ammonium Compound ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Antibiotic Resistance Marker ◽  
Gram Positive ◽  
Wide Range ◽  
Insertion Mutants

ABSTRACTStreptococcus mutans, a Gram-positive organism, is the primary causative agent in the formation of dental caries in humans. To persist in the oral cavity,S. mutansmust be able to tolerate rapid environmental fluctuations and exposure to various toxic chemicals. However, the mechanisms underlying the ability of this cariogenic pathogen to survive and proliferate under harsh environmental conditions remain largely unknown. Here, we wanted to understand the mechanisms by whichS. mutanswithstands exposure to methyl viologen (MV), a quaternary ammonium compound (QAC) that generates superoxide radicals in the cell. To elucidate the essential genes for MV tolerance, screening of ∼3,500 mutants generated by ISS1mutagenesis, revealed 15 MV-sensitive mutants. Among them, five and four independent insertions had occurred in SMU.905 and SMU.906 genes, respectively. These two genes are appeared to be organized in an operon and encode a putative ABC transporter complex; we designated the genes asvltAandvltB, forviologentransporter. To verify our results,vltAwas deleted by using an antibiotic resistance marker; the mutant was just as sensitive to MV as the ISS1insertion mutants. Furthermore,vltAandvltBmutants were also sensitive to other viologen compounds such as benzyl and ethyl viologens. Complementation assays were also carried out to confirm the role of VltA and VltB in viologen tolerance. Sensitivity to various drugs, including a wide range of QACs, was evaluated. It appears that a functional VltA is also required for full resistance toward acriflavin, ethidium bromide, and safranin; all are well-known QACs. These results indicate that VltA/B constitute a heterodimeric multidrug efflux pump of the ABC family. BLAST-P analysis suggests that homologs of VltA/B are widely present in streptococci, enterococci, and other important Gram-positive pathogens.

scholarly journals Characterization of a Unique Outer Membrane Protein Required for Oxidative Stress Resistance and Virulence of Francisella tularensis

2018 ◽  
Vol 200 (8) ◽  
Author(s):  
Maha Alqahtani ◽  
Zhuo Ma ◽  
Harshada Ketkar ◽  
Ragavan Varadharajan Suresh ◽  
Meenakshi Malik ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Francisella Tularensis ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type

ABSTRACT Francisella tularensis , the causative agent of tularemia, lacks typical bacterial virulence factors and toxins but still exhibits extreme virulence. The bacterial multidrug efflux systems consist of an inner membrane, a transmembrane membrane fusion protein, and an outer membrane (OM) component that form a contiguous channel for the secretion of a multitude of bacterial products. Francisella contains three orthologs of the OM proteins; two of these, termed TolC and FtlC, are important for tularemia pathogenesis. The third OM protein, SilC, is homologous to the silver cation efflux protein of other bacterial pathogens. The silC gene ( FTL_0686 ) is located on an operon encoding an Emr-type multidrug efflux pump of F. tularensis . The role of SilC in tularemia pathogenesis is not known. In this study, we investigated the role of SilC in secretion and virulence of F. tularensis by generating a silC gene deletion (Δ silC ) mutant and its transcomplemented strain. Our results demonstrate that the Δ silC mutant exhibits increased sensitivity to antibiotics, oxidants, silver, diminished intramacrophage growth, and attenuated virulence in mice compared to wild-type F. tularensis . However, the secretion of antioxidant enzymes of F. tularensis is not impaired in the Δ silC mutant. The virulence of the Δ silC mutant is restored in NADPH oxidase-deficient mice, indicating that SilC resists oxidative stress in vivo . Collectively, this study demonstrates that the OM component SilC serves a specialized role in virulence of F. tularensis by conferring resistance against oxidative stress and silver. IMPORTANCE Francisella tularensis , the causative agent of a fatal human disease known as tularemia, is a category A select agent and a potential bioterror agent. The virulence mechanisms of Francisella are not completely understood. This study investigated the role of a unique outer membrane protein, SilC, of a multidrug efflux pump in the virulence of F. tularensis . This is the first report demonstrating that the OM component SilC plays an important role in efflux of silver and contributes to the virulence of F. tularensis primarily by providing resistance against oxidative stress. Characterization of these unique virulence mechanisms will provide an understanding of the pathogenesis of tularemia and identification of potential targets for the development of effective therapeutics and prophylactics for protection from this lethal disease.

scholarly journals Induction of Pseudomonas syringae pv. tomato DC3000 MexAB-OprM Multidrug Efflux Pump by Flavonoids Is Mediated by the Repressor PmeR

2011 ◽  
Vol 24 (10) ◽  
pp. 1207-1219 ◽  
Author(s):  
Paola Vargas ◽  
Antonia Felipe ◽  
Carmen Michán ◽  
María-Trinidad Gallegos
Keyword(s):  
Pseudomonas Syringae ◽  
Efflux Pump ◽  
Regulatory Gene ◽  
Multidrug Efflux ◽  
Tomato Dc3000 ◽  
Multidrug Efflux Pump ◽  
Tomato Plants ◽  
In Vivo Experiments

In this study, we have analyzed the expression of the Pseudomonas syringae pv. tomato DC3000 mexAB-oprM efflux pump operon and of the regulatory gene pmeR, and we have investigated the role of the PmeR protein on transcription from both promoters. We demonstrate that mexAB-oprM and pmeR are expressed in vivo at a relatively high and moderate basal level, respectively, which, in both cases, increases in the presence of different flavonoids and other compounds, such as butyl and methylparaben. We show that PmeR is the local repressor of the mexAB-oprM promoter and is able to regulate its own expression. The mechanism for this regulation includes binding to a pseudopalindromic operator site which overlaps both mexAB-oprM and pmeR promoters. We have also proven that flavonoids are able to interact with PmeR and induce a conformational change that interferes with the DNA binding ability of PmeR, thereby modulating mexAB-oprM and pmeR expression. Finally, we demonstrate by in vivo experiments that the PmeR/MexAB-OprM system contributes to the colonization of tomato plants. These results provide new insight into a transcriptional regulator and a transport system that play essential roles in the ability of P. syringae pv. tomato DC3000 to resist the action of flavonoids produced by the host.

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