Molecular determinants of Burkholderia pseudomallei BpeEF-OprC efflux pump expression

Katherine A. Rhodes, Nawarat Somprasong, Nicole L. Podnecky, Takehiko Mima, Sunisa Chirakul, Herbert P. Schweizer

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Abstract

Burkholderia pseudomallei, the cause of melioidosis, is intrinsically resistant to many antibiotics. Acquired multidrug resistance, including resistance to doxycycline and co-trimoxazole used for melioidosis eradication phase therapy, is mainly attributed to constitutive expression of the BpeEF-OprC efflux pump. Constitutive expression of this pump is caused by mutations affecting two highly similar LysR-type transcriptional regulators (LTTR), BpeT and BpeS, but their interaction with the regulatory region governing BpeEF-OprC expression has not yet been studied. The bpeE-bpeF-oprC genes are distally located in the llpE-bpeEbpeF-oprC operon. The llpE gene encodes a putative lipase/esterase of unknown function. We show that in a bpeT mutant llpE is constitutively co-transcribed with bpeE-bpeF-oprC. As expected from previous studies with B. cenocepacia, deletion of llpE does not affect antibiotic efflux. Using transcriptional bpeE′-lacZ fusions, we demonstrate that the 188 bp bpeT-llpE intergenic region located between bpeT and the llpE-bpeE-bpeF-oprC operon contains regulatory elements needed for control of bpeT and llpEbpeE-bpeF-oprC operon expression. By native polyacrylamide gel electrophoresis and electrophoretic mobility shift assays with purified recombinant BpeT and BpeS proteins, we show BpeT and BpeS form oligomers that share a 14 bp binding site overlapping the essential region required for llpE-bpeE-bpeF-oprC expression. The binding site contains the conserved T-N11-A LTTR box motif involved in binding of LysR proteins, which in concert with two other possible LTTR boxes may mediate BpeT and BpeS regulation of BpeEF-OprC expression. These studies form the basis for further investigation of BpeEF-OprC expression and regulation at the molecular level by yet unknown external stimuli.

Original languageEnglish
Article number000691
Pages (from-to)1156-1167
Number of pages12
JournalMicrobiology (United Kingdom)
Volume164
Issue number9
DOIs
Publication statusPublished - Sep 1 2018

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Keywords

  • Antibiotic resistance
  • Burkholderia pseudomallei
  • Efflux
  • Melioidosis
  • Regulation

ASJC Scopus subject areas

  • Microbiology

Cite this

Rhodes, K. A., Somprasong, N., Podnecky, N. L., Mima, T., Chirakul, S., & Schweizer, H. P. (2018). Molecular determinants of Burkholderia pseudomallei BpeEF-OprC efflux pump expression. Microbiology (United Kingdom), 164(9), 1156-1167. [000691]. https://doi.org/10.1099/mic.0.000691