Quorum Sensing

Chapter 15

Identification of AHL- and BDSF-Controlled Proteins

inBurkholderia cenocepaciaby Proteomics

Yilei Liu, Gabriella Pessi, Katharina Riedel, and Leo Eberl

Abstract

We used comparative proteome analysis to determine the target genes of the two quorum sensing (QS)
circuits in the opportunistic pathogenBurkholderia cenocepacia: theN-acyl homoserine lactone (AHL)-
based CepIR system and the BDSF (Burkholderiadiffusiblesignalfactor,cis-2-dodecenoic acid)-based
RpfFR system. In this book chapter, we focus on the description of the practical procedure we currently use
in the laboratory to perform a sensitive GeLC-MS/MS shotgun proteomics experiment; we also briefly
describe the downstream bioinformatic data analysis.

Key wordsQuorum sensing, Cell fractionation, SDS-PAGE, In-gel tryptic digestion, Mass

spectrometry

1 Introduction

B. cenocepaciais an opportunistic pathogen that, together with other

19 closely related bacterial species, belongs to theBurkholderia

cepaciacomplex (Bcc) [1, 2]. All Bcc strains contain a highly con-

served AHL-dependent LuxIR type QS system, known as CepIR

[3, 4], with CepI encoding the AHL synthase, responsible for the

production of theN-octanoyl homoserine lactone (C8-HSL) and, in

minor amounts of theN-hexanoyl homoserine lactone (C6-HSL),

and CepR acting as a transcriptional regulator. Certain Bcc members

harbor additional AHL-based QS systems, such as BviIR inB. viet-

namiensis[5]andCciIRinB. cenocepaciastrains that both belong to

the epidemic ET12 lineage [6]. The presence of yet another QS

system inB. cenocepacia, which uses the fatty acidcis-2-dodecenoic

acid (also known asBurkholderiadiffusible signal factor or BDSF) as

signal molecule, was reported in 2008 [7]. The key enzyme for

BDSF biosynthesis is RpfF, an enzyme that has both dehydratase

and thioesterase activities [8]. The BDSF receptor protein RpfR has

recently been identified and was shown to modulate the intracellular

c-di-GMP level [9]. The BDSF-dependent RpfFR system is highly

Livia Leoni and Giordano Rampioni (eds.),Quorum Sensing: Methods and Protocols, Methods in Molecular Biology,
vol. 1673,https://doi.org/10.1007/978-1-4939-7309-5_15,©Springer Science+Business Media LLC 2018

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