Quorum sensing regulates electric current generation of Pseudomonas aeruginosa PA14 in bioelectrochemical systems Arvind Venkataraman a , Miriam Rosenbaum a , Jan B.A. Arends a,1 , Rayko Halitschke b , Largus T. Angenent a, * a Department of Biological and Environmental Engineering, Cornell University, 214 Riley-Robb Hall, Ithaca, NY 14853, USA b Ecology and Evolutionary Biology, Cornell University, 425 Corson Hall, Ithaca, NY 14853, USA article info Article history: Received 11 December 2009 Received in revised form 12 January 2010 Accepted 13 January 2010 Available online 18 January 2010 Keywords: Pseudomonas aeruginosa Quorum sensing Bioelectrochemical systems Phenazine retS abstract Here, we show that quorum sensing (QS) modulates the current generation of the anode-respiring bac- terium Pseudomonas aeruginosa because it controls the production of phenazines, which mediate the electron transfer to the anode. The current generation by a wildtype (WT) strain P. aeruginosa PA14 and the GacS/GacA protein-regulatory mutant retS was investigated under different environmental con- ditions. The retS mutant generated significantly higher current (45-fold) than the WT under anaerobic conditions. Anaerobic current generation by the WT was 28-fold higher with extraneously supplied lac- tones (a QS-signaling molecule). Compared to anaerobic conditions, the WT with some oxygen (microaer- obic conditions) exhibited enhanced phenazine production (39-fold) and current levels (48-fold). Iron- rich medium and microaerobic conditions had a negative impact on current generation by retS. All these results were directly linked to QS activity in P. aeruginosa, thus, demonstrating the importance of this bac- terial communication system for current generation in BESs. We also show that BESs represent a new tool for real-time investigation of phenazine-related QS activity. Ó 2010 Elsevier B.V. All rights reserved. 1. Introduction Bioelectrochemical systems (BESs) are gaining importance as innovative biotechnological devices for the renewable generation of electricity from wastewater in microbial fuel cells (MFCs), gen- eration of chemical products in microbial electrolysis cells, and sequestration of CO 2 . In previous studies, Pseudomonas sp. in the anodic microbial community of an MFC was related to current gen- eration [1,2]. Phenazines produced by Pseudomonas aeruginosa (pyocyanin, 1-hydroxyphenazine, phenazine-1-carboxamide, and phenazine-1-carboxylate) act as redox-shuttles to facilitate respi- ration of P. aeruginosa with the electrode [2]. It has been recently shown that these endogeneous phenazines are responsible for sur- vival of P. aeruginosa PA14 under anaerobic conditions [3]. Besides its importance in BESs, P. aeruginosa is also a model organism for understanding quorum sensing (QS). QS is the bacte- rial mode of communication via secreted signaling factors [4]. The core components of the QS system in P. aeruginosa are the las and rhl systems, respectively, consisting of transcriptional regulatory proteins (LasR and RhlR) and autoinducer synthases (LasI and RhlI) (Fig. 1). LasI controls the production of 3-oxo-dodecanoyl homo- serine lactone (3O-C12-HSL) and RhlI controls the production of N-butyryl homoserine lactone (C4-HSL), which initiate the QS cas- cade (Fig. 1) [5]. In addition to controlling rhlI, the transcriptional regulator LasR also positively regulates the pqsABCDE operon, which generates the Pseudomonas quinolone signal (PQS), a third QS signal for P. aeruginosa (Fig. 1). The pqs operon in turn controls the phzABCDEFG operon, which is required for metabolizing choris- mate to phenazine-1-carboxylate. The genes phzS, phzM, and phzH in turn metabolize phenazine-1-carboxylate to 1-hydroxyphen- azine, pyocyanin, and phenazine-1-carboxamide, respectively [6]. QS in P. aeruginosa is also controlled by several two-component regulatory systems, such as GacS/GacA [7] (Fig. 1). GacS/GacA is negatively regulated by RetS and in the absence of RetS (retS mu- tant) a GacS/GacA signaling cascade results in the activation of the rhl system (we, therefore, hypothesize an upregulation of QS – see discussion). We screened seven mutants and the wildtype (WT) strain of P. aeruginosa PA14 for their electrochemical behavior. Six of these mutants have transposon insertions in genes involved in: GacS/ GacA regulation (retS); in type IV pili and flagellum formation (pilB and fliC, respectively); and in phenazine production (phzS, phzM, and phzH). The seventh mutant (Dphz) lacks both gene operons phzA1-G1 and phzA2-G2, which are required for phenazine synthe- sis [8]. Here, we investigated the protein-regulatory mutant retS 1388-2481/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2010.01.019 * Corresponding author. Tel.: +1 607 255 2480; fax: +1 607 255 4080. E-mail address: la249@cornell.edu (L.T. Angenent). 1 Present address: Ghent University, Belgium. Electrochemistry Communications 12 (2010) 459–462 Contents lists available at ScienceDirect Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom