scholarly journals The channel-tunnel HI1462 of Haemophilus influenzae reveals differences to Escherichia coli TolC

Microbiology ◽  
2006 ◽  
Vol 152 (6) ◽  
pp. 1639-1647 ◽  
Author(s):  
Georg Polleichtner ◽  
Christian Andersen
Keyword(s):  
Escherichia Coli ◽  
Single Channel ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
Tunnel Entrance

Efflux pumps play a major role in multidrug resistance of pathogenic bacteria. The TolC homologue HI1462 was identified as the single channel-tunnel in Haemophilus influenzae required to form a functional multidrug efflux pump. The outer-membrane protein was expressed in Escherichia coli, purified and reconstituted in black lipid membranes. It exhibited a comparatively small single-channel conductance of 43 pS in 1 M KCl and is the first known TolC homologue which is anion-selective. The HI1462 structure was modelled and an arginine residue lining the tunnel entrance was identified. The channel-tunnel of a mutant with the arginine substituted by an alanine residue was cation-selective and had a sevenfold higher single-channel conductance compared to wild-type. These results confirm that the arginine is responsible for anion selectivity and forms a salt bridge with a glutamate residue of the adjacent monomer, establishing a circular network, which keeps the tunnel entrance in a tightly closed conformation. In in vivo experiments, both the wild-type HI1462 and the mutant were able to substitute for E. coli TolC in the haemolysin secretion system, but not in the AcrAB/TolC multidrug efflux pump. The structure–function relationship of HI1462 is discussed in the context of the well-studied TolC channel-tunnel of E. coli.

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 NorA plasmid resistance to fluoroquinolones: role of copy number and norA frameshift mutations.

1996 ◽  
Vol 40 (7) ◽  
pp. 1665-1669 ◽  
Author(s):  
L Sun ◽  
S Sreedharan ◽  
K Plummer ◽  
L M Fisher
Keyword(s):  
Copy Number ◽  
Efflux Pump ◽  
Gene Promoter ◽  
Fluoroquinolone Resistance ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
Copy Numbers ◽  
Mutational Activation

Staphylococcus aureus NorA protein is a transmembrane multidrug efflux pump that confers low-level resistance to hydrophilic fluoroquinolones. The norA gene promoter is active in Escherichia coli HB101. We have examined the genetic basis of norA-mediated resistance in E. coli by introducing a wild-type norA gene into HB101 in plasmid pCL1921, pBR322, or pUC18 exhibiting copy numbers that spanned a 22-fold range. Increased ciprofloxacin resistance correlated with norA transcript levels seen by Northern (RNA) analysis. Thus, contrary to some reports, a wild-type norA gene confers fluoroquinolone resistance in E. coli in a copy-number-dependent fashion and does not require mutational activation. Interestingly, a multicopy pUC19norA derivative gave transformants exhibiting a range of resistance phenotypes. The norA gene of one transformant carried a single base deletion (ATACAAT to AACAAT; the deleted base is underlined) in the putative--10 Pribnow box resulting in a promoter down-regulatory mutation; a second plasmid had acquired a frameshift producing a null mutation at codon 112. These mutations override the dual resistance-growth-inhibitory phenotype of high-copy-number norA plasmids. The results have implications for using the standard E. coli HB101 system to assess NorA function and potentially for plasmid-borne transmission of norA-mediated drug resistance.

scholarly journals Expression in Escherichia coli of a New Multidrug Efflux Pump, MexXY, from Pseudomonas aeruginosa

1999 ◽  
Vol 43 (2) ◽  
pp. 415-417 ◽  
Author(s):  
Tomoyuki Mine ◽  
Yuji Morita ◽  
Atsuko Kataoka ◽  
Tohru Mizushima ◽  
Tomofusa Tsuchiya
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Multidrug Resistance ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
Expression In Escherichia Coli ◽  
New Genes

ABSTRACT Two new genes (mexXY) similar to mexAB,mexCD, and mexEF and mediating multidrug resistance were cloned from the chromosome of Pseudomonas aeruginosa. Elevated ethidium extrusion was observed withEscherichia coli cells harboring the plasmid carryingmexXY. This MexXY system confers higher resistance to fluoroquinolones than the MexAB and MexCD systems, and E. coli TolC or P. aeruginosa OprM is necessary for the function of the MexXY system.

scholarly journals MarA-Like Regulator of Multidrug Resistance in Yersinia pestis

2006 ◽  
Vol 50 (9) ◽  
pp. 2971-2975 ◽  
Author(s):  
Rupa A. Udani ◽  
Stuart B. Levy
Keyword(s):  
Escherichia Coli ◽  
Multidrug Resistance ◽  
Yersinia Pestis ◽  
Efflux Pump ◽  
Multidrug Resistant ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
N Terminus ◽  
Resistant Mutants

ABSTRACT MarA47Yp from Yersinia pestis, showing 47% identity to Escherichia coli MarA in its N terminus, caused resistance to antibiotics and to organic solvents when expressed in both E. coli and Y. pestis. Resistance was linked to increased expression of the AcrAB multidrug efflux pump. In four of five spontaneous multidrug-resistant mutants of Y. pestis independently selected by growth on tetracycline, the marA47 Yp gene was overexpressed. The findings suggest that marA47 Yp is a marA ortholog in Y. pestis.

scholarly journals The eefABC Multidrug Efflux Pump Operon Is Repressed by H-NS in Enterobacter aerogenes

2005 ◽  
Vol 187 (11) ◽  
pp. 3894-3897 ◽  
Author(s):  
Muriel Masi ◽  
Jean-Marie Pagès ◽  
Claude Villard ◽  
Elizabeth Pradel
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Enterobacter Aerogenes ◽  
Dominant Negative ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
Laboratory Conditions ◽  
Wide Range

ABSTRACT The Enterobacter aerogenes eefABC locus, which encodes a tripartite efflux pump, was cloned by complementation of an Escherichia coli tolC mutant. E. aerogenes ΔacrA expressing EefABC became less susceptible to a wide range of antibiotics. Data from eef::lacZ fusions showed that eefABC was not transcribed in the various laboratory conditions tested. However, increased transcription from Peef was observed in an E. coli hns mutant. In addition, EefA was detected in E. aerogenes expressing a dominant negative E. coli hns allele.

scholarly journals Selection of multiple-antibiotic-resistant (mar) mutants of Escherichia coli by using the disinfectant pine oil: roles of the mar and acrAB loci.

1997 ◽  
Vol 41 (12) ◽  
pp. 2770-2772 ◽  
Author(s):  
M C Moken ◽  
L M McMurry ◽  
S B Levy
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Antibiotic Resistant ◽  
Pine Oil ◽  
Mutant Strains ◽  
Selection Of ◽  
Multiple Antibiotics

Mutants of Escherichia coli selected for resistance to the disinfectant pine oil or to a household product containing pine oil also showed resistance to multiple antibiotics (tetracycline, ampicillin, chloramphenicol, and nalidixic acid) and overexpressed the marA gene. Likewise, antibiotic-selected Mar mutants, which also overexpress marA, were resistant to pine oil. Deletion of the mar or acrAB locus, the latter encoding a multidrug efflux pump positively regulated in part by MarA, increased the susceptibility of wild-type and mutant strains to pine oil.

scholarly journals Plasmid-Encoded Multidrug Efflux Pump Conferring Resistance to Olaquindox in Escherichia coli

2004 ◽  
Vol 48 (9) ◽  
pp. 3332-3337 ◽  
Author(s):  
Lars Hestbjerg Hansen ◽  
Elsebetta Johannesen ◽  
Mette Burmølle ◽  
Anders Hay Sørensen ◽  
Søren J. Sørensen
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Resistance Mechanism ◽  
Open Reading Frames ◽  
Conjugative Plasmid ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
Control Plasmid ◽  
Reading Frames

ABSTRACT We report here the first gene-encoded resistance mechanism to the swine growth enhancer olaquindox. The genetic elements involved in resistance to olaquindox were subcloned and sequenced from a conjugative plasmid isolated from Escherichia coli. The subcloned fragment contained two open reading frames, oqxA and oqxB, that are homologous to several resistance-nodulation-cell-division family efflux systems from different species. The putative protein sequences were aligned to both experimentally verified and putative efflux pumps. We show that oqxA and oqxB are expressed in E. coli. Plasmids containing the oqxAB genes yielded high (>128 μg/ml) resistance to olaquindox in E. coli, whereas strains containing the control plasmid showed low resistance to the drug (8 μg/ml). The oqxAB-encoded pump also conferred high (>64 μg/ml) resistance to chloramphenicol. We demonstrate that the subcloned fragment conferred H+-dependent ethidium efflux abilities to E. coli strain N43. In addition, we show that the efflux system is dependent on the host TolC outer membrane protein when expressed in E. coli.

The assembly and disassembly of the AcrAB-TolC three-component multidrug efflux pump

2015 ◽  
Vol 396 (9-10) ◽  
pp. 1083-1089 ◽  
Author(s):  
Reinke Tobias Müller ◽  
Klaas Martinus Pos
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Gram Negative Bacteria ◽  
Electrochemical Gradient ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Gram Negative ◽  
E Coli ◽  
Pump Assembly ◽  
Multiple Antibiotics

Abstract In Gram-negative bacteria, tripartite efflux pumps, like AcrAB-TolC from Escherichia coli, play a prominent role in the resistance against multiple antibiotics. Transport of the drugs across the outer membrane and its coupling to the electrochemical gradient is dependent on the presence of all three components. As the activity of the E. coli AcrAB-TolC efflux pump is dependent on both the concentration of substrates and the extent of the electrochemical gradient across the inner membrane, the dynamics of tripartite pump assembly and disassembly might be crucial for effective net transport of drugs towards the outside of the cell.

scholarly journals AcrAB Multidrug Efflux Pump Is Associated with Reduced Levels of Susceptibility to Tigecycline (GAR-936) in Proteus mirabilis

2003 ◽  
Vol 47 (2) ◽  
pp. 665-669 ◽  
Author(s):  
Melissa A. Visalli ◽  
Ellen Murphy ◽  
Steven J. Projan ◽  
Patricia A. Bradford
Keyword(s):  
Proteus Mirabilis ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Transposon Insertion ◽  
Spectrum Activity ◽  
Insertion Mutants ◽  
Broad Spectrum Activity ◽  
Increased Susceptibility

ABSTRACT Tigecycline has good broad-spectrum activity against many gram-positive and gram-negative pathogens with the notable exception of the Proteeae. A study was performed to identify the mechanism responsible for the reduced susceptibility to tigecycline in Proteus mirabilis. Two independent transposon insertion mutants of P. mirabilis that had 16-fold-increased susceptibility to tigecycline were mapped to the acrB gene homolog of the Escherichia coli AcrRAB efflux system. Wild-type levels of decreased susceptibility to tigecycline were restored to the insertion mutants by complementation with a clone containing a PCR-derived fragment from the parental wild-type acrRAB efflux gene cluster. The AcrAB transport system appears to be associated with the intrinsic reduced susceptibility to tigecycline in P. mirabilis.

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