A Broad-Specificity Multidrug Efflux Pump Requiring a Pair of Homologous SMR-Type Proteins

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A Broad-Specificity Multidrug Efflux Pump
Requiring a Pair of Homologous SMR-Type Proteins

• Jack, Donald L., Michael L. Storms, Jason H.
  Tchieu, Ian T. Paulsen and Milton H. Saier Jr.
• Presented by Denique Boxhill

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Introduction

• Source URLhttp//fox.rollins.edu/egregory/B229ho
  me.htm...
• The genome of a gram ive bacteria, Bacillus
  subtilis, encodes 7 homologues. Homologues share
  the same morphology of genes.
• 6 of these 7 homologues are paired in 3 operons.
  An operon is a part of DNA that contains
  structural, operator and regulatory genes.

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• In Escherichia coli, co expression of one of
  these operons, namely ykkCD, results in a broad
  specificity multidrug-resistant phenotype. Which
  includes resistance to cationic, anionic and
  neutral drugs

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Background

• Some protein members belonging to 5 ubiquitous
  families of transport proteins can function in
  multidrug resistance (MDR)
• Families 1. Major facilitator superfamily (MFS)
  2. Resistance/ nodulation/cell division (RND)
  family 3. ATP-binding cassette (ABC) superfamily
  4. Drug/metabolite transporter family 5. Small
  multidrug resistance (SMR) family

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• SMR Family is unusual. Its proteins contain only
  100-120 amino acid residues and 4 transmembrane
  a-helical spanners.

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SMR family members found in B. subtilis and E.
coli
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• These proteins exist as oligomers, which are
  repeated sub units. They export cationic drugs
  from bacterial cytoplasm via a fairly hydrophobic
  transmembrane pathway.

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The Hydrophobic Transmembrane Pathway

• Multidrug transporters remove toxic reagents in
  exchange for hydrogen ions.
• Substrates are translocated through hydrophobic
  pathway

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Bacillus subtilis and Escherichia coli

• 6 of B. subtilis homologues and 2 of E. coli
  homologues are encoded from gene pairs in 4
  operons
• Gene pairs in B. subtilis ebrA and ebrB, yvdR
  and yvdS and ykkC and ykkD
• Gene pairs in E. coli b1599 and b1600
• One member of each B. subtilis protein pair is
  short, while the other is longer. This
  characteristic is also seen in the protein pairs
  of E. coli

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Bacillus subtilis and Escherichia coli

• Each of the 7 genes in B. subtilis were cloned
  and individually expressed in an E. coli strain,
  DH5a. No drug resistant phenotype was observed.
• Experimenters then hypothesized that perhaps both
  genes in any one operon needed to be coexpressed
  to observe drug resistant phenotype
• The procedure was redone in order to clone B.
  subtilis genes ykkC, ykkD and gene pair ykkCD
  into the expression vector pBAD24 and view the
  results.

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Effects of Coexpression of ykkC and ykkD genes on
Broad-spectrum MDR phenotype
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Experiment Results

• A broad-spectrum MDR phenotype observed when both
  the ykkC and ykkD genes were expressed together
  in E. coli strain.
• Resistance to broader range of toxic compounds
  than any previously studied pump observed.
• Toxic compounds cationic dyes, anionic and
  neutral antimicrobials.

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Conclusion

• SMR-Type proteins that exhibit an MDR phenotype
  export cationic drugs via a hydrophobic
  transmembrane pathway.
• Coexpression of the operon ykkCD in E. coli, not
  expression of either ykkC or ykkD alone, results
  in a broad specific multidrug-resistant
  phenotype.

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References

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• http//fox.rollins.edu/egregory/B229home.htm...