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

Research output: Contribution to journalArticle

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

Fingerprint

Burkholderia pseudomallei
Operon
Melioidosis
Binding Sites
Anti-Bacterial Agents
Native Polyacrylamide Gel Electrophoresis
Intergenic DNA
Doxycycline
Nucleic Acid Regulatory Sequences
Sulfamethoxazole Drug Combination Trimethoprim
Multiple Drug Resistance
Electrophoretic Mobility Shift Assay
Esterases
Lipase
Genes
Carrier Proteins
Mutation
Proteins
Therapeutics

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

Molecular determinants of Burkholderia pseudomallei BpeEF-OprC efflux pump expression. / Rhodes, Katherine A.; Somprasong, Nawarat; Podnecky, Nicole L.; Mima, Takehiko; Chirakul, Sunisa; Schweizer, Herbert P.

In: Microbiology (United Kingdom), Vol. 164, No. 9, 000691, 01.09.2018, p. 1156-1167.

Research output: Contribution to journalArticle

Rhodes, KA, Somprasong, N, Podnecky, NL, Mima, T, Chirakul, S & Schweizer, HP 2018, 'Molecular determinants of Burkholderia pseudomallei BpeEF-OprC efflux pump expression', Microbiology (United Kingdom), vol. 164, no. 9, 000691, pp. 1156-1167. https://doi.org/10.1099/mic.0.000691
Rhodes, Katherine A. ; Somprasong, Nawarat ; Podnecky, Nicole L. ; Mima, Takehiko ; Chirakul, Sunisa ; Schweizer, Herbert P. / Molecular determinants of Burkholderia pseudomallei BpeEF-OprC efflux pump expression. In: Microbiology (United Kingdom). 2018 ; Vol. 164, No. 9. pp. 1156-1167.
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