Bioelectrochemistry 119 (2018) 150–160

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Deciphering the electric code of Geobacter sulfurreducens in cocultures with Pseudomonas aeruginosa via SWATH-MS proteomics Lucie Semenec a, Andrew E. Laloo b, Benjamin L. Schulz c, Ismael A. Vergara d, Philip L. Bond b, Ashley E. Franks a,⁎ a

Department of Physiology, Anatomy and Microbiology, La Trobe University, Melbourne, Victoria, Australia Advanced Water Management Centre, The University of Queensland, Brisbane, Queensland, Australia School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia d Bioinformatics and Cancer Genomics, Research Division, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia b c

a r t i c l e

i n f o

Article history: Received 8 April 2017 Received in revised form 27 September 2017 Accepted 28 September 2017 Available online 03 October 2017 Keywords: Direct interspecies electron transfer (DIET) Adaptive evolution Hydrogen interspecies electron transfer (HIT) SWATH-MS proteomics Syntrophic coculture

a b s t r a c t Interspecies electron transfer (IET) occurs in many microbial communities, enabling extracellular electron exchange for syntrophic utilization of mixed resources. Various mechanisms of IET have been characterized including direct IET (DIET) and hydrogen IET (HIT) but their evolution throughout syntrophic adaptation has not been investigated through an omics approach. A syntrophic coculture of Geobacter sulfurreducens and Pseudomonas aeruginosa was established and evolved in restricted medium. The medium required cooperative metabolism due to preferential utilization of formate and fumarate by P. aeruginosa and G. sulfurreducens respectively. Pure cultures did not yield significant growth while substantial growth was observed in cocultures. The syntrophy was not reliant on phenazine, since Δphz mutant strain cocultures grew, however appeared to rely on cytochromes as evidenced from the stunted growth G. sulfurreducens ΔomcZ and ΔomcS mutant cocultures. SWATH (sequential window acquisition of all theoretical spectra) MS (mass spectrometry) proteomic analysis of initial cocultures revealed upregulation in DIET-associated cytochromes, whereas adapted cocultures revealed upregulation in HybA, a G. sulfurreducens uptake hydrogenase critical to HIT. This suggests DIET plays a critical role in the establishment of syntrophy between G. sulfurreducens and P. aeruginosa but is later consolidated with HIT as the cocultures adapt. This is the first instance to show a temporal distribution of DIET and HIT within the same coculture. © 2017 Published by Elsevier B.V.

1. Introduction Extracellular electron transfer (EET) is the ability to receive or donate electrons extracellularly from external electron donors or acceptors to facilitate metabolism and respiration [1,2]. This ability plays a critical role in biogeochemical cycling of metals and is useful to bioremediation, biofuel production and biomining applications [1–4]. Despite the vast diversity within the prokaryotic domain, the capability of extracellular electron transfer (EET) occurs most significantly in Desulfobulbaceae and Desulfuromonadales [5]. Bacteria capable of EET are commonly referred to as electrogens and have been reported to utilize a range of electron donors and acceptors including metabolic intermediates, other bacteria, electrodes, metals and metalloids [6]. Therefore, various mechanisms of EET have been identified including direct EET (DEET) [7–12], shuttle-mediated EET (SEET) [13–17], pilinmediated EET (PEET) [11,18–21], mineral mediated DEET [22–25], quinone-mediated interspecies EET (QUIET) [26] as well as hydrogen/ ⁎ Corresponding author at: School of Life Sciences, Biological Sciences 1, La Trobe University, Bundoora, VIC 3086, Australia. E-mail address: [email protected] (A.E. Franks).

https://doi.org/10.1016/j.bioelechem.2017.09.013 1567-5394/© 2017 Published by Elsevier B.V.

formate interspecies EET (HIT/FIT) [27–30] and direct interspecies EET (DIET) [31–35], the latter of which incorporates many of the former mechanisms. Geobacter spp. are model electrogens, with a developed genetic system [36] and well characterized for their ability to undergo DIET and HIT/FIT with a diverse range of microorganisms. This includes DIET with members of the Archaea domain Methanosarcina barkeri [32] and Methanosaeta harundinacea [37] as well as within their own genus between G. sulfurreducens and G. metallireducens [34]. Geobacter spp. are also known for their utilization of uptake hydrogenases like HybA for the syntrophic consumption of hydrogen via HIT with methanogens [30,38]. They can also utilize exogenous electron shuttles such as formate and H2 [28,30,39] in HIT/FIT which are modes of electron uptake in ocean sediments [40,41]. Both DIET [42,43] and HIT [29,38,44] appear to be common modes of IET in methanogenic environments. Previous metabolic modelling has suggested that DIET is more energetically favorable than HIT or FIT [45], however this was based on intracellular metabolism only. More recently, a study that included extracellular limitations in their model determined that FIT is the most thermodynamically favorable mode of IET, followed by DIET and then HIT [46]. Despite these energetic preferences,

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organisms capable of both DIET and HIT like G. sulfurreducens may use either of these respiratory pathways depending on their microbial partner. For instance, G. sulfurreducens was shown to undergo DIET with G. metallireducens [24,28,34,47] but HIT with Pelobacter carbinolicus [28, 34,47] in the same medium, where ethanol and fumarate were provided as the sole electron donor and acceptor respectively. The choice between either DIET or HIT seems to depend on three factors. First, the ability of the electron donating microbe to produce hydrogen; in the case of G. metallireducens it could not produce H2 whereas P. carbinolicus could. Second, the ability of the electron donating microorganism to reduce insoluble electron acceptors. G. metallireducens is capable of reduction of Fe(III) oxides or electrodes whereas P. carbinolicus is not. Lastly, the ability of the electron receiving microbe to utilize H2 or an electrode as an electron donor. G. sulfurreducens can do both. Thus, G. sulfurreducens performs HIT with P. carbinolicus which produces H2 but cannot reduce insoluble electron acceptors, and DIET with G. metallireducens which cannot produce H2 but can reduce insoluble electron acceptors. It is not yet clear however, which mechanism of EET is preferred when the electron donating microbe can both produce hydrogen and utilize insoluble electron acceptors. Geobacter spp. are ubiquitous in subsurface environments such as marine sediments, anaerobic soils and wastewater. Pseudomonas spp. are also highly adaptable and inhabit many of the same niches including soil, aquatic and marine environments but are also found in plants and animals [48–50]. Pseudomonas spp. are normally adapted to respiring soluble electron acceptors like oxygen and nitrate but also have the unique capacity for SEET via redox mediators in restrictive environments. Such endogenously produced mediators, known as phenazines, allow for shuttling of electrons between themselves and other bacteria including Brevibacillus sp. [51]. What has not yet been demonstrated however is the potential for DIET between electrogens like Geobacter that undergo DEET and PEET and those like Pseudomonas that produce redox shuttles to aid in SEET. Since these two clades of bacteria often co-habit many of the same environments, it is important to understand their capacity for interaction with one another. Geobacter is known to have over a hundred different cytochromes many of whose function is yet uncharacterized [52]. Evidence suggests that a variety of cytochromes act together to aid in EET [10,53–55]. The cytochromes employed for reduction of an electron acceptor depend on its reduction potential and thus a different set of cytochromes are associated with each different electron acceptor it respires [6,56, 57]. Given this complexity, we applied a next-generation proteomics approach, SWATH-MS [58], to explore which components may be involved temporally in the development of a syntrophic interaction between Geobacter and Pseudomonas. Furthermore, since the cocultures exhibited improved growth over successive generations, this approach would allow for investigation of which EET components may be optimizing this syntrophic cocultures adaptive evolution. By characterizing their proteomes when in coculture, the array of EET components involved in their syntrophic interaction could be studied as they evolved. Since bacterial monocultures evolve polymorphisms when grown in nutrient-limited environments [59,60], it is important to understand whether EET mechanisms also changed over time with adaptive evolution. Thus, a temporal investigation of EET mechanism changes that occur in cultures evolved via serial-transfers was performed, where adapted proteomes were compared to original non-adapted cultures. 2. Materials and methods 2.1. Bacterial strains and growth conditions G. sulfurreducens DL-1 (PCA strain, ATCC51573), Δ omcZ [11] and Δ omcS [10] mutants were kindly provided for this study by Professor Derek Lovley. Strains were grown under strictly anaerobic conditions in NBAF as previously described [36], except no resazurin was added. For growth of the pure culture, 20 mM acetate and 40 mM fumarate

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were used as the electron donor and acceptor respectively. P. aeruginosa PAO1, PA14 and PA14 Δphz1/2 [61] (herein referred to as PA14 Δphz) strains were kindly provided for this study by Professor Lars Dietrich and grown under strictly anaerobic conditions in NB medium containing 20 mM formate and 20 mM nitrate as the electron donor and acceptor. For growth of the cocultures, 20 mM formate and 40 mM fumarate were provided as the electron donor and acceptor. Adaptation of the cocultures involved transferring 1% of culture to fresh NB(formate/fumarate) medium once they reached stationary phase, the growth stage when P. aeruginosa begin producing phenazines [61], as determined by optical density at 600 (OD600). The cultures were adapted to up to thirteen transfers. The initial culture is referred herein as subculture 0 (s0) and the thirteenth transfer culture is called subculture 13 (s13). In total, thirteen transfers were made and stocks of cultures were flash frozen in liquid nitrogen and stored at −80°C. 2.2. Harvesting cultures for proteomics Each of the initial s0 cocultures, PAO1 + DL-1, PA14 + DL-1 and PA14 Δphz + DL-1, were grown in triplicate. Since these triplicate cocultures were individually transferred thirteen times for adaptive evolution (until s13); they were considered biological replicates. To obtain sufficient quantities of protein for SWATH-MS, a high culture volume was required. Eight technical replicates of each coculture (including all biological replicates) grown in 10 ml of medium for a total volume of 80 ml. These replicates were pooled per sample to ensure enough culture was harvested for obtaining a sufficient amount of protein during extraction. Harvesting for SWATH-MS was conducted when cultures reached stationary phase. Technical replicate cultures were pooled into 50 ml Sorval tubes in the anaerobic chamber to avoid any protein changes upon exposure to oxygen. The samples were centrifuged at 14,000 rcf for 15 min and the collected pellets were snap frozen in liquid nitrogen and stored at −80°C until protein extractions were done. 2.3. Protein extraction Cell pellets stored at −80 °C were thawed on ice and lysed in extraction buffer containing B-Per II (Thermo Fisher Scientific), 50 mM DTT (Sigma-Aldrich) and Complete mini EDTA-free Protease inhibitor cocktail (Roche). Three freeze thaw cycles in liquid nitrogen were performed after which samples were incubated at room temperature for 30 min. Samples were then centrifuged at 18,000 rcf for 15 min at 4 °C. Supernatants were mixed with 0.04% sodium deoxycholate and incubated on ice for 15 min. Subsequent protein precipitation with 10% TCA was done at 4 °C overnight. The following day, samples were centrifuged at 18,000 rcf for 15 min and the supernatant was discarded. To purify the proteins, pellets were resuspended in 80% acetone and sonicated at 4 °C until complete resuspension of pellet was obtained. This was followed by 20 min incubation at −20 °C. The protein pellet was obtained by centrifugation at 18,000 rcf for 15 min and then allowed to dry for 5 min. Protein pellets were resuspended in buffer containing 2 M thiourea, 7 M urea and 100 mM ammonium bicarbonate. The 2-D Quant Kit (GE Healthcare) was used to measure protein concentrations and all samples were normalized to 10 μg/μl. Reduction of proteins with 5 mM DTT for 30 min at 56 °C was followed by a 30 min alkylation with 25 mM iodoacetamide (Sigma-Aldrich) at room temperature in the dark. An additional 5 mM DTT was added to quench excess iodoacetamide. Digestion with 1:600 enzyme:protein ratio of rLys-C (Promega) was performed at 37 °C for 7 h. Samples were then diluted with 50 mM ammonium bicarbonate buffer to reduce the urea concentration to 2 M. A second digestion with 1:100 enzyme:protein ratio of trypsin was performed at 37 °C for 14 h. Proteins were desalted and purified using ZipTip (Millipore) clean-up with washes in 5% acetonitrile(ACN)/0.1% trifluoroacetic acid (TFA) and eluted with 80% ACN/0.1% TFA into autosampler vials for mass spectrometry (Agilent

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Technologies). Eluted peptides were dried by vacuum centrifugation to remove ACN and resuspended in 0.1% formic acid. 2.4. Proteomics SWATH-MS was done by analyzing peptides via LC-ESI-MS/MS with a Prominence nanoLC system (Shimadzu) and Triple ToF 5600 mass spectrometer with a Nanospray III interface (ABSciex). The obtained mass-spectrometry (MS) data was input into ProteinPilot software and IDA searches were performed against the downloaded G. sulfurreducens (PCA) and P. aeruginosa (PAO1 and PA14) proteomes obtained from Uniprot Swiss-Prot database (August 2016 release), at a global FDR of 1%. The PeakView program was used to generate SWATH files containing quantified proteomes with settings set to 5 peptides per protein and 3 transitions per peptide. The “exclude shared peptides” option was selected to avoid the issue of shared peptides in conserved proteins of the two bacteria present in each coculture. The MSstats statistics package [62] for R was used for all statistical analyses. To increase the stringency of analysis, only proteins with intensity values existing for ≥50% of the peptides detected were included in the analysis. Furthermore, normalization setting was set to false to avoid shifting median intensity values by a constant. Due to the nature of the cocultures being a mix of two bacterial species, MSStats analyses were performed twice for each coculture sample, once for only G. sulfurreducens proteins and the second analysis was done specifically on P. aeruginosa proteins. Protein abundance changes between G. sulfurreducens DL-1 in coculture with P. aeruginosa relative to pure culture of DL-1 were measured as the log2 Fold Change (log2FC) difference. Similarly, the protein abundance changes between each P. aeruginosa strain PAO1, PA14 and PA14 Δphz relative to its pure culture equivalent were reported as log2FC differences (Table S4). Pathways, molecular function, biological processes and cellular components for each protein were obtained from the Kyoto Encyclopedia of Genes and Genomes [63] (KEGG: www.genome.jp/kegg) and Gene Ontology (GO) [64] resources. 3. Results and discussion 3.1. Syntrophy between Geobacter and Pseudomonas cocultures in restricted medium The ability of G. sulfurreducens and P. aeruginosa to utilize a range of electron donors and acceptors was tested (Tables S1 and S2). P. aeruginosa was confirmed to preferentially utilize formate as an electron donor compared to G. sulfurreducens as evident by growth in formate/nitrate compared to formate/fumarate (Fig. 1A). The low growth of G. sulfurreducens with formate as the electron donor (Fig. 1A) is

consistent with previous reports on its inability to utilize formate as the sole electron donor [34,65]. However, G. sulfurreducens does contain a formate dehydrogenase which enables it to undergo formate interspecies electron transfer (FIT) [28], but given the limited growth of DL-1 pure cultures in formate/fumarate, it is likely this enzyme is more active in cocultures rather than in pure cultures. Growth of P. aeruginosa in formate/nitrate is consistent with previous characterization of P. aeruginosa formate dehydrogenase [66]. Fumarate could only be utilized by G. sulfurreducens as an electron acceptor, with no growth observed in pure cultures of both P. aeruginosa strains PAO1 and PA14 in formate and fumarate medium (Fig. 1A). To determine whether G. sulfurreducens and P. aeruginosa could establish syntrophic interaction, cocultures were established in restricted medium. Formate was provided as a potential electron donor for utilization by P. aeruginosa and fumarate as a potential electron acceptor by G. sulfurreducens. This media was considered syntrophic because P. aeruginosa could not utilize fumarate as an electron acceptor and G. sulfurreducens was unable to produce significant growth with formate as an electron donor. Such a syntrophic medium, where electron donor and acceptor are limited to either pair of bacteria in a coculture has previously been demonstrated to enhance and evolve syntrophic interactions [34]. Pure cultures of either PAO1 or PA14 strain did not show any signs of growth in the syntrophic coculture medium and only limited growth of the DL-1 pure culture was observed (Fig. 1B). Priming cultures of P. aeruginosa were grown in formate and nitrate, which do not result in significant growth of G. sulfurreducens, to acclimate P. aeruginosa toward formate utilization. Similarly, G. sulfurreducens was primed for growth in acetate and fumarate, neither of which can be utilized by P. aeruginosa, before being transferred into syntrophic medium with PAO1 and PA14 strains of P. aeruginosa. Both PAO1 and PA14 strains differ in their virulence [67], but both PA14 [68] and PAO1 produce phenazine redox shuttles. Phenazines participate in EET to enable survival in anaerobic conditions and are also involved in IET with syntrophic partners [51,69,70]. However, their capacity for phenazine production differs depending on their environment. PA14 produces 10-fold more phenazine (in particular pyocyanin) than PAO1 when grown in Luria-Bertani (LB) [61]. Conversely, pyocyanin production is nearly 4-fold higher in PAO1 than PA14 when grown in medium that mimics the cystic fibrosis lung [71]. It is therefore important to compare the potential for syntrophy of both these two strains of P. aeruginosa with G. sulfurreducens. A significant improvement in growth occurred for G. sulfurreducens and P. aeruginosa cocultures in this syntrophic medium (Fig. 1B) relative to G. sulfurreducens pure cultures, which still had a low level of growth. Enhanced growth in coculture medium suggests a mechanism of syntrophic interaction exists where G. sulfurreducens and P. aeruginosa

Fig. 1. Growth of P. aeruginosa and G. sulfurreducens pure cultures in priming medium and syntrophic medium. A) Growth of priming cultures; P. aeruginosa in NB(formate/nitrate) and G. sulfurreducens in NB(acetate/fumarate) and PAO1, PA14 and DL-1 pure cultures in the syntrophic NB(formate/fumarate) medium. B) Growth in NB(formate/fumarate) medium shows enhanced growth of cocultures versus pure cultures. Symbols A): PAO1 (formate/nitrate); PA14 (formate/nitrate); PAO1(formate/nitrate); PA14 (formate/fumarate); DL-1 (formate/fumarate); DL-1 (acetate/fumarate). Symbols B): PAO1 (formate/fumarate); PA14 (formate/fumarate); DL-1 (formate/fumarate); PAO1 + DL-1 (formate/ fumarate); PA14 + DL-1 (formate/fumarate).

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combine their metabolism to more efficiently utilize both formate and fumarate. Possible syntrophic mechanisms include either DIET or HIT to allow for growth. If HIT is occurring in the cocultures, P. aeruginosa oxidation of formate (Eq. (1)) would be followed by subsequent production of hydrogen (Eq. (2)) as seen in Escherichia coli and Thermococcus litoralis [72–75]. Syntrophic consumption of the resulting hydrogen by G. sulfurreducens would be then be used for reduction of fumarate to succinate (Eq. (3)) resulting in the overall reaction (Eq. (4)). HCO2 – →CO2 þ Hþ þ 2e−

ð1Þ

2Hþ þ 2e− →H2

ð2Þ

H2 þ C4 H4 O4 →C4 H6 O4

ð3Þ

HCO2 – þ Hþ þ C4 H4 O4 →C4 H6 O4 þ CO2

ð4Þ

Although to the best of our knowledge, hydrogen production via formate oxidation has not yet been reported in P. aeruginosa, it does contain formate dehydrogenase and hydrogenase proteins that make this reaction possible. Furthermore, since G. sulfurreducens has previously been shown to consume H2 in syntrophic coculture with P. carbinolicus [28], the proposed syntrophy between P. aeruginosa and G. sulfurreducens via HIT is a strong possibility. If instead DIET occurs in the cocultures, formate oxidation by P. aeruginosa (Eq. (1)) would generate electrons that may then be directly transferred to G. sulfurreducens via DIET and utilized for fumarate reduction (Eq. (5)). This would yield the same net reaction for DIET as for HIT (Eq. (4)). C4 H4 O4 þ 2Hþ þ 2e− →C4 H6 O4

ð5Þ

To test which of these mechanisms was most likely to explain the syntrophic growth, further analysis was conducted. 3.2. Potential for DIET between Geobacter and Pseudomonas species during syntrophic interaction Previous studies have reported that phenazines can enhance interspecies EET [76] between Pseudomonas and other microbial species. To test whether P. aeruginosa produced phenazines were a major mechanism by which electrons are shuttled extracellularly to G. sulfurreducens during syntrophic growth, a kindly provided phenazine knockout strain PA14 Δphz1/2 [61] (herein referred to as PA14 Δphz) was grown in coculture with G. sulfurreducens. As with the wild-type PAO1 and PA14

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strains in cocultures, the phenazine mutant coculture with G. sulfurreducens exhibited the same level of syntrophic growth compared to the wildtype cocultures (Fig. 2). The growth of PA14 Δphz coculture indicates that phenazines are not required for the syntrophic growth observed between the Geobacter and Pseudomonas species. To test if previously reported G. sulfurreducens EET components [1, 77] such as OmcZ and OmcS c-type cytochromes were involved in their syntrophic interaction with P. aeruginosa, we also established cocultures with single mutant strains of G. sulfurreducens Δ omcS and Δ omcZ. Neither the pure cultures of G. sulfurreducens Δ omcS and ΔomcZ cytochrome mutants nor the cocultures of these mutants with P. aeruginosa formed viable cultures in the syntrophic coculture medium (Fig. 2). Since previous cocultures of G. sulfurreducens and G. metallireducens where omcS is deleted fail to perform DIET [34], the lack of growth in the G. sulfurreducens ΔomcZ and ΔomcS mutant cocultures with P. aeruginosa suggests their potential role in syntrophic DIET with P. aeruginosa. However, it must be noted that although growth was significantly stunted in the ΔomcZ (Fig. 2B) and ΔomcS (Fig. 2A) mutant cocultures, the appearance of a few tiny light pink aggregates occurred for all cocultures with these mutants over the duration of monitoring growth (data not shown). Cultures were propagated over several generations to allow for potential evolution of enhanced syntrophic interactions as previously observed with G. sulfurreducens and G. metallireducens cocultures [34]. By the thirteenth transfer, the doubling time decreased on average of over five-fold and a substantial shortening of the lag-phase was observed (Fig. 3A–C). Growth between eight technical replicates differed more in the original non-adapted (herein referred to as subculture 0– s0) cocultures than in the adapted (herein referred to as subculture 13–s13) cocultures (Fig. 3). Therefore, as the cocultures adapted, their growth dynamics became more consistent between technical replicates. Phenotypic changes were also observed between the s0 and s13 cocultures. Initially PAO1/DL-1 cocultures displayed biofilms on the bottom of the culture tubes as well as pink and red aggregates reminiscent to those seen in cocultures previously shown to undergo DIET [34,37,42]. This phenotype changed to a more homogenous looking biofilm in the s13 cocultures (Table S3, Fig. S3) indicating a potential switch in the IET mechanism being used throughout their adaptive evolution. The growth of the PA14 and PA14 Δ phz cocultures with DL-1 also became more consistent between replicates by the thirteenth transfer (Fig. 3B–C). There was also a change in culture phenotype throughout adaptation where initially the presence of red aggregates occurs in both cocultures, whereas by the adapted s13 cultures the PA14/DL-1 appeared like initial cocultures except its biofilm formation became more prolific (Table S3, Fig. S3). For the PA14 Δphz/DL-1 cocultures, the red

Fig. 2. Growth of initial G. sulfurreducens OmcZ and OmcS deficient mutants with P. aeruginosa Phz deficient mutants in syntrophic coculture medium NB(formate/fumarate). A) G. sulfurreducens ΔomcZ mutant did not grow in pure culture or in cocultures with P. aeruginosa in the syntrophic NB(formate/fumarate) medium. B) G. sulfurreducens ΔomcS mutants did not grow either in pure culture or in cocultures with P. aeruginosa in the syntrophic NB(formate/fumarate) medium. Phenazine-deficient PA14 Δphz mutant of P. aeruginosa did not grow in pure culture but grew in coculture with wild-type G. sulfurreducens DL-1 in the syntrophic medium. Symbols A): DL-1 ΔomcS; PA14 Δphz; PA14 Δphz + DL-1; PA14 + DL-1 ΔomcS; PAO1 + DL-1 ΔomcS; PA14 Δphz + DL-1 ΔomcS. Symbols B) DL-1 ΔomcZ; PA14 Δphz; PA14 Δphz + DL-1; PA14 + DL-1 ΔomcZ; PAO1 + DL-1 ΔomcZ; PA14 Δphz + DL-1 ΔomcZ.

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Fig. 3. Growth comparison of initial versus adapted G. sulfurreducens and P. aeruginosa cocultures. A) PAO1 + DL-1 cocultures, B) PA14 + DL-1 cocultures, C) PA14 phz + DL-1 cocultures. Orange curves depict the mean growth of all eight technical replicates of adapted (s13) cocultures with standard error bars indicated. The small error bars corresponding to the standard error between the average of these eight replicates indicate the high level of homogeneity that was seen between the replicates. Blue curves depict individual growth of each of the eight technical replicates of non-adapted (s0) cocultures. The spread of the s0 growth curves indicate the heterogeneity observed in these initial cocultures. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

aggregates disappeared but loose pink aggregates remained in the adapted s13 cultures. Although growth curves became more consistent between replicates in the adapted PA14/DL-1 and PA14 Δphz/DL-1 cocultures, their culture phenotype maintained heterogeneity throughout evolution of the eight technical replicates (Table S3). This suggests multiple mechanisms of respiration exist throughout their adaptive evolution including a potential for DIET particularly in initial cultures where red aggregates were abundant. 3.3. G. sulfurreducens appears to dominate in co-cultures with P. aeruginosa To understand the functional adaptive changes that allowed enhanced growth of cocultures with successive generations, proteomes were obtained by SWATH-MS of initial (s0) and adapted (s13) cocultures. The G. sulfurreducens portions of the coculture proteomes were compared to that of G. sulfurreducens pure culture in media with acetate and fumarate as the electron donor and acceptor. Similarly, the P. aeruginosa portion of the proteomes was compared to P. aeruginosa pure cultures in medium with formate and nitrate as the electron donor and acceptor respectively. G. sulfurreducens appeared to have increased activity when P. aeruginosa was provided as a potential electron

donor compared to acetate due to the overall significantly increased protein abundances of G. sulfurreducens proteins in initial s0 cocultures (Fig. 4A–B, E). In contrast, P. aeruginosa proteins were overall significantly decreased in abundance when provided with G. sulfurreducens was provided as an electron acceptor compared to nitrate (Fig. 4C–D, G). This suggests that G. sulfurreducens activity may be more dominant, particularly within the initial s0 cocultures. This dynamic, where the syntrophic partner receiving electrons is more dominant, is similar to that previously observed in G. sulfurreducens and Pelobacter carbinolicus cocultures, where Geobacter is more highly present [28]. This may be due to G. sulfurreducens not only utilizing hydrogen as an electron donor via HIT but also low level utilization of formate as an electron donor via FIT. In our study, the higher activity of G. sulfurreducens over P. aeruginosa could potentially also be attributed to the fact that unlike P. aeruginosa, which is solely reliant on G. sulfurreducens to act as an electron acceptor, G. sulfurreducens can either undergo DIET with P. aeruginosa to receive electrons or to a low level utilize formate. With successive transfers, the abundance for both G. sulfurreducens and P. aeruginosa proteins is lower than in their initial interaction (Fig. 4B,D). Perhaps through adaptive evolution, key proteins essential for growth are selected for in earlier generations, allowing for subsequent downregulation of unnecessary proteins in later generations. Since the

Fig. 4. Changes in protein abundances from initial to adapted G. sulfurreducens and P. aeruginosa PAO1 cocultures in NB(formate/fumarate). A–D) Venn diagrams depicting proportion of shared significantly upregulated-increased abundance (pink) and downregulated-decreased abundance (blue) proteins between initial cocultures (s0) and adapted cocultures s(13). A–B) PA + DL-1 versus DL-1 includes proteins with significant changes to abundance in all DL-1 cocultures (PAO1/DL-1, PA14/DL-1 and PA14 Δphz/DL-1) relative to DL-1 pure culture. C–D) PA + DL-1 versus PA values includes proteins with significant changes to abundance in all P. aeruginosa cocultures (PAO1/DL-1, PA14/DL-1 and PA14 Δphz/DL-1) relative to all P. aeruginosa pure cultures. E–H) Volcano plots with significantly upregulated (pink) and downregulated (blue) proteins in the PAO1 + DL-1 cocultures in NB(formate/fumarate); E–F) as compared to DL-1 pure culture in NB(acetate/fumarate); G–H) PAO1 proteins from cocultures in NB(formate/fumarate) versus PAO1 pure culture in NB(formate/nitrate). Green dots are proteins whose abundance was not significantly different from pure cultures. E–F; G. sulfurreducens DL-1 proteins and G–H; P. aeruginosa PAO1 proteins from the cocultures. Significance is defined as Log2FC values ≥ |1.0| and adjusted p-value ≤ 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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decrease in P. aeruginosa proteins is still greater than that for G. sulfurreducens in adapted s13 cocultures (panel F vs. H), the dominance of G. sulfurreducens appears to remain consistent throughout their evolution. Many of the P. aeruginosa proteins significantly decreased in abundance were involved in metabolism, transcription and translation (Table S4) in both original and adapted cocultures. Although there was also a marked decrease in abundance of G. sulfurreducens proteins involved in tricarboxylic acid cycle metabolism and translation as it adapted, the abundance of biosynthesis proteins remained to be increased in the adapted cocultures (Table S5). These results also suggest that Pseudomonas is not as metabolically active as Geobacter within these cocultures. Furthermore, inspection of the mean peptide spectral intensity readings, indicate that G. sulfurreducens proteins were more abundant than P. aeruginosa proteins in all coculture proteomes across all three biological replicates (Table 1). This further confirms the predominance of G. sulfurreducens in the PAO1/DL-1, PA14/DL-1 and PA14 Δphz/DL-1 cocultures. Despite this, the abundance of P. aeruginosa formate dehydrogenase (FdnG) is higher than G. sulfurreducens FdnG and thus P. aeruginosa is still actively undergoing formate oxidation and providing G. sulfurreducens with an electron donor as a result (Fig. S5). In previous studies where G. sulfurreducens was found to predominate in coculture with G. metallireducens, it was attributed to its ability to utilize both electrons via DIET through ethanol oxidation by G. metallireducens as well as the acetate produced as a byproduct of this oxidation [34,78]. The fact that G. sulfurreducens is more abundant than P. aeruginosa in this study may also indicate that G. sulfurreducens is obtaining energy by more than one respiratory mechanism. 3.4. Mechanisms of interspecies electron transfer between Geobacter and Pseudomonas A list of G. sulfurreducens genes potentially involved in DIET has been previously described [47]. We searched our proteomics data for the proteins encoded by these DIET associated genes, and additionally PilY1-2, PilW and all proteins annotated as cytochromes were investigated. Furthermore, G. sulfurreducens proteins encoded by genes involved in HIT/ FIT [47] were also inspected. A strong upregulation of G. sulfurreducens OmcS, OmcC, MacA and PgcA cytochromes was observed, particularly in the initial s0 cocultures (Fig. 5) when comparing pure culture G. sulfurreducens proteins to those in cocultures. OmcS, OmcC and MacA cytochromes are involved the reduction of insoluble electron acceptors Table 1 Significant difference between mean spectral peptide intensities from G. sulfurreducens and P. aeruginosa coculture proteomes. Coculture

PAO1 + DL-1 s0

PAO1 + DL-1 s13

PA14 + DL-1 s0

PA14 + DL-1 s13

PA14 Δphz + DL-1 s0

PA14 Δphz + DL-1 s13

Strain

PAO1 DL-1 ⁎p-Value PAO1 DL-1 ⁎p-Value PA14 DL-1 p-value PA14 DL-1 ⁎p-Value PA14 Δphz DL-1 ⁎p-Value PA14 Δphz DL-1 ⁎p-Value

Mean peptide intensity Replicate 1

Replicate 2

Replicate 3

117,400 123,800 1.48E-06 67,270 71,520 1.04E-08 24,620 68,430 2.23E-96 31,330 90,700 1.60E-116 30,600 107,300 4.45E-123 29,890 102,200 2.64E-129

87,990 93,740 1.80E-08 87,380 92,870 2.53E-07 34,180 107,000 1.24E-127 31,950 87,060 1.22E-106 37,280 123,000 9.28E-153 22,510 65,470 1.48E-100

73,140 78,360 3.49E-09 93,160 98,500 8.58E-06 32,380 101,200 7.40E-121 27,710 72,770 4.61E-99 39,410 124,600 5.09E-123 26,280 86,250 5.70E-122

⁎ Unpaired Wilcoxon Rank-Sum test p-values.

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via EET [34,53,79] however their role in receiving extracellular electrons is not well characterized. A recent theory proposes that cytochromes form supramolecular “respiratory complexes” that function as a relay network in conjunction with pili structures to allow for superexchange of electrons between electroactive biofilm members [54]. It was seen that, OmcC, OmcS, OmcE and unidentified 50 kDa and 30 kDa proteins formed the proposed supramolecular respiratory structures [54]. MacA is a 36 kDa protein [53] and was found upregulated in a similar pattern to OmcC and OmcS in G. sulfurreducens and P. aeruginosa cocultures. Therefore, it is possible that a supramolecular respiratory complex may exist between OmcC, MacA and OmcS, particularly in initial cocultures of G. sulfurreducens and P. aeruginosa, however, elucidating the localization of these proteins in the cocultures is necessary to test this hypothesis. PgcA is also involved in EET to insoluble electron acceptors such as Fe(III) oxides [80]. Additionally, it is predicted to function as a redox shuttle cytochrome [81]. Given its strong upregulation in cocultures where P. aeruginosa is donating electrons to G. sulfurreducens, its role in receiving rather than donating extracellular electrons warrants further investigation. The significantly increased abundance of OmcS in the cocultures corresponded well with the poor growth seen for G. sulfurreducens ΔomcS mutants with P. aeruginosa (Fig. 2A). The ΔomcZ mutants were also deficient in growth with P. aeruginosa (Fig. 2B), but surprisingly there was no significant change in OmcZ abundance in the cocultures. This is likely due to OmcZ being localized to the extracellular matrix, particularly when being utilized for EET [82] resulting in OmcZ not being sufficiently captured during protein extraction. The localization of OmcZ in these cocultures will need to be investigated in future studies. As the cocultures evolved, the abundance of OmcC, MacA, OmcS and PgcA, c-type cytochromes decreased while HybA became significantly upregulated in the PAO1 and PA14 cocultures (Schematic 1). HybA is an uptake hydrogenase subunit that is utilized for coupling the oxidation of hydrogen to fumarate reduction [83]. The upregulation of G. sulfurreducens HybA in PAO1 and PA14 cocultures with DL-1 is consistent with its high transcript abundance seen in cocultures of P. carbinolicus and G. sulfurreducens that undergo hydrogen interspecies electron transfer (HIT) [28]. These results suggest DIET between G. sulfurreducens and P. aeruginosa, likely involving OmcC, MacA, OmcS and PgcA, is initially the major mechanism of IET but as they adapt over successive generations, HIT also becomes apparent for PAO1 and PA14 cocultures (Schematic 1). Since the overall abundance of the cytochromes was still higher than that for HybA, DIET still appears to be involved in the syntrophic interactions (Fig. S4), however it is no longer the only mechanism of syntrophy. HIT does not appear to occur in either (s0 or s13) of the PA14 phenazine deficient mutant cocultures as indicated by a lack of HybA. Since phenazine redox shuttles did not appear to be important for EET between G. sulfurreducens and P. aeruginosa (Fig. 2), perhaps they play an antimicrobial role [84–86] rather than a redox shuttling role. The relieved strain from antimicrobial attacks by Pseudomonas phenazines in the phenazine deficient mutant cocultures would then allow G. sulfurreducens to continue relying on close interactions via DIET rather than switching to a more long-distance interaction via HIT, however this warrants further investigation. Interestingly, there was also a significant upregulation of G. sulfurreducens formate dehydrogenase, FdnG, which is usually indicative of formate interspecies electron transfer [28], in the PA14 and PA14 Δ phz cocultures. Since formate is provided as the sole electron donor and hence not a by-product of P. aeruginosa metabolism, formate utilization by G. sulfurreducens rather than FIT is likely occurring in addition to DIET (Schematic 1). This low level of formate utilization was already evident when DL-1 pure cultures had slight growth in syntrophic NB(formate/fumarate) medium (Fig. 1). FdnG abundance increased as the cocultures evolved (Fig. S4), decreasing G. sulfurreducens need for syntrophic interaction with P. aeruginosa. However, P. aeruginosa formate dehydrogenase activity remained present but in lower abundance as well in the adapted co-cultures so it was still responsible for some

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Fig. 5. Changes to G. sulfurreducens DIET, HIT and formate utilization protein abundances from cocultures of G. sulfurreducens and P. aeruginosa in NB(formate/fumarate) relative to wildtype G. sulfurreducens DL-1 strain in NB(acetate/fumarate). Heatmap shows the Log2 Fold Change (log2FC) between protein abundance in G. sulfurreducens cocultures with P. aeruginosa relative to pure cultures of G. sulfurreducens. Proteins above the black line include DIET-associated proteins and those below represent HIT/FIT associated proteins. Grey areas indicate proteins missing from the corresponding proteomes. Log2(FC) values obtained from MSStats of SWATH-MS proteomics data. **adjusted p-value ≤ 0.01, * adjusted p-value ≤ 0.05, no asterisk indicates adjusted p-value N 0.05.

formate oxidation (Fig. S5). This finding supports the earlier observation that G. sulfurreducens appears to be more metabolically active and abundant than P. aeruginosa. 4. Conclusions Through the exploratory investigation of EET mechanisms within syntrophic cocultures of G. sulfurreducens and P. aeruginosa via

SWATH-MS proteomics, a catalogue of proteins involved in DIET within the cocultures was established. Additionally, by allowing for their adaptation through serial transfers, it was possible to investigate the evolution of their EET mechanisms by comparing un-adapted to adapted whole proteomes. This is the first study to provide an omics comparison of electrogenic cocultures throughout their syntrophic evolution. Another important finding was the suggestion of a heterogeneous combination of syntrophic respiratory mechanisms, DIET and HIT, being used

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Schematic 1. Proposed model of IET mechanisms in syntrophic cocultures of G. sulfurreducens DL-1 strain (red cells) and P. aeruginosa PAO1, PA14 and PA14 phz strains (yellow cells) throughout evolution. A) proposed model of EET between initial PAO1 and DL-1 cocultures. B) proposed model of EET between adapted PAO1 and DL-1 cocultures. C) proposed model of EET between initial PA14 and DL-1 cocultures. D) proposed model of EET between adapted PA14 and DL-1 cocultures. E) proposed model of EET between initial PA14 Δphz and DL1 cocultures. D) proposed model of EET between adapted PA14 Δphz and DL-1 cocultures. All proteins indicated in the models are G. sulfurreducens DL-1 proteins from the cocultures with log2FC ≥ 0.7 and adjusted p-value ≤ 0.05 relative to DL-1 pure cultures. Formate is written as HCO− 2 . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

within the same coculture at the same time. Initial establishment of the coculture resulted in a significant upregulation in DIET associated proteins. As the two bacteria adapted to living in this strict medium, HIT appeared to also take place as seen by the significant upregulation of uptake hydrogenase, HybA. There was also a significant upregulation of G. sulfurreducens formate dehydrogenase in PA14 and PA14 Δphz cocultures with DL-1, suggesting an adaptation to formate utilization. Thus, DIET appears to be a critical initial stress response to allow for respiration in nutrient limited environments whereas HIT forms of syntrophy take longer to establish but contribute to improved growth in conjunction with DIET. Furthermore, particularly for the PA14 cocultures with DL-1, the ability of G. sulfurreducens to rely less on P. aeruginosa and more on its own formate dehydrogenase for formate oxidation indicates a shift from DIET to formate utilization. This research has laid the groundwork for future investigations into the combined role of DIET with HIT and formate utilization given strict environmental constraints. This examination of proteomic changes in Geobacter in restrictive conditions with Pseudomonas also provides a solid initial step toward deciphering the electron transfer mechanisms between these electrogens. Additionally, insight is gained toward Geobacter's adaptation to restrictive growth conditions that mimic many of the stringent environments it thrives in. Although many insights into this syntrophy have been gained, many more questions arise from the use of this powerful omics tool. How does their unique ability to transfer electrons between one another and those of other species influence their adaptation to nutrient limited environments? Also, is it more beneficial for them to perform redox reactions on their own as non-electric microbes do or does sharing of electrons via DIET or HIT give them an evolutionary advantage over non-electric microbes sharing their environment?

The use of proteomics to study bacteria under restrictive conditions gives insight into the preferred respiratory and metabolic mechanisms selected for and may help uncover synthetic biology targets for enhancing favorable microbial traits in various biotechnology applications. This research could also be useful to the study of bacterial growth in space, where carbon and electron donor/acceptor sources may be scarcely abundant, thus necessitating the need for microbial syntrophy to allow survival. Most importantly, knowledge of how bacteria adapt to restricted conditions will help to further elucidate their mechanisms of electron transfer and contribute to their utilization in MFC applications such as bioremediation, wastewater treatment, current generation and biofuel production. Supplementary data to this article can be found online at https://doi. org/10.1016/j.bioelechem.2017.09.013.

Declaration of interest The authors declare no conflict of interest.

Contributions L.S., P.L.B. and A.E.F were involved in the conception and design of the study, L.S., A.E.L. were involved in the acquisition of the data. L.S., B.L.S., A.E.L., I.A.V., P.L.B. and A.E.F. were involved in the analysis and interpretation of data. All authors were involved in the writing of the manuscript and have revised critically for important intellectual content as well as approving the version to be submitted.

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Acknowledgements We would like to thank Dr. Derek Lovley and Dr. Lars Dietrich for their generous gift of the Geobacter strains and mutants and PA14 and PA14 Δ phz1/2 strains of P. aeruginosa respectively. This research has been partially financially supported by the Human Protection and Performance Program of the Defence Science Institute (Award no. DSI001). The Applied and Environmental Microbiology Laboratory receives support from the Defence Science and Technology Group (DSTG), Office of Naval Research Global (Award no. N626909-13-1-N259), Asian Office of Aerospace Research and Development (AOARD; Award no. FA2386-14-1-4032) and the Australian Research Council (ARC Award no. LP140100459). References [1] D.R. Lovley, Electromicrobiology, Annu. Rev. Microbiol. 66 (2012) 391–409. [2] L. Shi, H.L. Dong, G. Reguera, H. Beyenal, A.H. Lu, J. Liu, H.Q. Yu, J.K. Fredrickson, Extracellular electron transfer mechanisms between microorganisms and minerals, Nat. Rev. Microbiol. 14 (2016) 651–662. [3] B.E. Logan, D. Call, S. Cheng, H.V. Hamelers, T.H. Sleutels, A.W. Jeremiasse, R.A. Rozendal, Microbial electrolysis cells for high yield hydrogen gas production from organic matter, Environ. Sci. Technol. 42 (2008) 8630–8640. [4] B.E. Logan, B. Hamelers, R.A. Rozendal, U. Schröder, J. Keller, S. Freguia, P. Aelterman, W. Verstraete, K. Rabaey, Microbial fuel cells: methodology and technology, Environ. Sci. Technol. 40 (2006) 5181–5192. [5] S. Ishii, S. Suzuki, T.M. Norden-Krichmar, A. Tenney, P.S.G. Chain, M.B. Scholz, K.H. Nealson, O. Bretschger, A novel metatranscriptomic approach to identify gene expression dynamics during extracellular electron transfer, Nat. Commun. 4 (2013). [6] L. Semenec, A.E. Franks, The microbiology of microbial electrolysis cells, Microbiol. Aust. 35 (2014) 201–206. [7] D.R. Bond, D.E. Holmes, L.M. Tender, D.R. Lovley, Electrode-reducing microorganisms that harvest energy from marine sediments, Science (New York, N.Y.) 295 (2002) 483–485. [8] C. Bücking, A. Piepenbrock, A. Kappler, J. Gescher, Outer-membrane cytochromeindependent reduction of extracellular electron acceptors in Shewanella oneidensis, Microbiology 158 (2012) 2144–2157. [9] K.B. Gregory, D.R. Bond, D.R. Lovley, Graphite electrodes as electron donors for anaerobic respiration, Environ. Microbiol. 6 (2004) 596–604. [10] T. Mehta, M.V. Coppi, S.E. Childers, D.R. Lovley, Outer membrane c -type cytochromes required for Fe (III) and Mn (IV) oxide reduction in Geobacter sulfurreducens, Appl. Environ. Microbiol. 71 (2005) 8634–8641. [11] K.P. Nevin, B.-C. Kim, R.H. Glaven, J.P. Johnson, T.L. Woodard, B.A. Methé, R.J. DiDonato Jr., S.F. Covalla, A.E. Franks, A. Liu, Anode biofilm transcriptomics reveals outer surface components essential for high density current production in Geobacter sulfurreducens fuel cells, PLoS One 4 (2009), e5628. [12] S. Xu, Y. Jangir, M.Y. El-Naggar, Disentangling the roles of free and cytochromebound flavins in extracellular electron transport from Shewanella oneidensis MR-1, Electrochim. Acta 198 (2016) 49–55. [13] D. Baron, E. LaBelle, D. Coursolle, J.a. Gralnick, D.R. Bond, Electrochemical measurement of electron transfer kinetics by Shewanella oneidensis MR-1, J. Biol. Chem. 284 (2009) 28865–28873. [14] M.E. Hernandez, A. Kappler, K. Dianne, M.E. Hernandez, A. Kappler, D.K. Newman, Phenazines and other redox-active antibiotics promote microbial mineral reduction, Appl. Environ. Microbiol. 70 (2004) 921–928. [15] E. Marsili, D.B. Baron, I.D. Shikhare, D. Coursolle, J.a. Gralnick, D.R. Bond, Shewanella secretes flavins that mediate extracellular electron transfer, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 3968–3973. [16] K. Rabaey, N. Boon, M. Höfte, W. Verstraete, Microbial phenazine production enhances electron transfer in biofuel cells, Environ. Sci. Technol. 39 (2005) 3401–3408. [17] H. von Canstein, J. Ogawa, S. Shimizu, J.R. Lloyd, Secretion of flavins by Shewanella species and their role in extracellular electron transfer, Appl. Environ. Microbiol. 74 (2008) 615–623. [18] S.E. Childers, S. Ciufo, D.R. Lovley, Geobacter metallireducens accesses insoluble Fe(III) oxide by chemotaxis, Nature 416 (2002) 767–769. [19] D.E.D. Holmes, Y., D.J.F. Walker, D.R. Lovley, The electrically conductive pili of Geobacter species are a recently evolved feature for extracellular electron transfer, Microbial Genom. 2 (2016) 1–20. [20] G. Reguera, K.D. McCarthy, T. Mehta, J.S. Nicoll, M.T. Tuominen, D.R. Lovley, Extracellular electron transfer via microbial nanowires, Nature 435 (2005) 1098–1101. [21] G. Reguera, K.P. Nevin, J.S. Nicoll, S.F. Covalla, T.L. Woodard, D.R. Lovley, Biofilm and nanowire production leads to increased current in Geobacter sulfurreducens fuel cells, Appl. Environ. Microbiol. 72 (2006) 7345–7348. [22] S. Chen, A.-E. Rotaru, P.M. Shrestha, N.S. Malvankar, F. Liu, W. Fan, K.P. Nevin, D.R. Lovley, Promoting interspecies electron transfer with biochar, Sci Rep 4 (2014) 5019. [23] S. Kato, K. Hashimoto, K. Watanabe, Microbial interspecies electron transfer via electric currents through conductive minerals, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 10042–10046.

[24] F.H. Liu, A.E. Rotaru, P.M. Shrestha, N.S. Malvankar, K.P. Nevin, D.R. Lovley, Promoting direct interspecies electron transfer with activated carbon, Energy Environ. Sci. 5 (2012) 8982–8989. [25] Z. Zhao, Y. Zhang, D.E. Holmes, Y. Dang, T.L. Woodard, K.P. Nevin, D.R. Lovley, Potential enhancement of direct interspecies electron transfer for syntrophic metabolism of propionate and butyrate with biochar in up-flow anaerobic sludge blanket reactors, Bioresour. Technol. 209 (2016) 148–156. [26] J.A. Smith, K.P. Nevin, D.R. Lovley, Syntrophic growth via quinone-mediated interspecies electron transfer, Front. Microbiol. 6 (2015) 121. [27] M.J. McInerney, J.R. Sieber, R.P. Gunsalus, Syntrophy in anaerobic global carbon cycles, Curr. Opin. Biotechnol. 20 (2009) 623–632. [28] A.-E. Rotaru, P.M. Shrestha, F. Liu, T. Ueki, K. Nevin, Z.M. Summers, D.R. Lovley, Interspecies electron transfer via hydrogen and formate rather than direct electrical connections in cocultures of Pelobacter carbinolicus and Geobacter sulfurreducens, Appl. Environ. Microbiol. 78 (2012) 7645–7651. [29] J.R. Sieber, M.J. McInerney, R.P. Gunsalus, Genomic insights into syntrophy: the paradigm for anaerobic metabolic cooperation, Annu. Rev. Microbiol. 66 (2012) 429–452. [30] J.H. Thiele, J.G. Zeikus, Control of interspecies electron flow during anaerobic digestion: significance of formate transfer versus hydrogen transfer during syntrophic methanogenesis in flocs, Appl. Environ. Microbiol. 54 (1988) 20–29. [31] M. Morita, N.S. Malvankar, A.E. Franks, Z.M. Summers, L. Giloteaux, A.E. Rotaru, C. Rotaru, D.R. Lovley, Potential for direct interspecies electron transfer in methanogenic wastewater digester aggregates, MBio 2 (2011). [32] A.-E. Rotaru, P.M. Shrestha, F. Liu, B. Markovaite, S. Chen, K. Nevin, D. Lovley, Direct interspecies electron transfer between Geobacter metallireducens and Methanosarcina barkeri, Appl. Environ. Microbiol. 80 (2014) 4599–4605. [33] A.-E. Rotaru, P.M. Shrestha, F. Liu, M. Shrestha, D. Shrestha, M. Embree, K. Zengler, C. Wardman, K.P. Nevin, D.R. Lovley, A new model for electron flow during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane, Energy Environ. Sci. 7 (2014) 408–415. [34] Z.M. Summers, H.E. Fogarty, C. Leang, A.E. Franks, N.S. Malvankar, D.R. Lovley, Direct exchange of electrons within aggregates of an evolved syntrophic coculture of anaerobic bacteria, Science (New York, N.Y.) 330 (2010) 1413–1415. [35] L.Y. Wang, K.P. Nevin, T.L. Woodard, B.Z. Mu, D.R. Lovley, Expanding the Diet for DIET: electron donors supporting direct interspecies electron transfer (DIET) in defined co-cultures, Front. Microbiol. 7 (2016). [36] M.V. Coppi, C. Leang, S.J. Sandler, D.R. Lovley, Development of a genetic system for Geobacter sulfurreducens, Appl. Environ. Microbiol. 67 (2001) 3180–3187. [37] A.-E.S.P.M.L. Rotaru, Fanghua, Minita Shrestha, Devesh Shrestha, Mallory Embree, Karsten Zengler, Colin Wardman, Kelly P. Nevin, Derek R. Lovley, A new model for electron flow during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane, Energy Environ. Sci. 7 (2014) 408–415. [38] A.J.M. Stams, C.M. Plugge, Electron transfer in syntrophic communities of anaerobic bacteria and archaea, Nat. Rev. Microbiol. 7 (2009) 568–577. [39] A.J.M. Stams, F.A.M. De Bok, C.M. Plugge, V. Eekert, H.A. Miriam, J. Dolfing, G. Schraa, Exocellular electron transfer in anaerobic microbial communities, Appl. Environ. Microbiol. 8 (2006) 371–382. [40] M.V. Coppi, The hydrogenases of Geobacter sulfurreducens: a comparative genomic perspective, Microbiology 151 (2005) 1239–1254. [41] B.A. Methe, J. Webster, K. Nevin, J. Butler, D.R. Lovley, DNA microarray analysis of nitrogen fixation and Fe(III) reduction in Geobacter sulfurreducens, Appl. Environ. Microbiol. 71 (2005) 2530–2538. [42] A.E. Rotaru, P.M. Shrestha, F. Liu, B. Markovaite, S. Chen, K.P. Nevin, D.R. Lovley, Direct interspecies electron transfer between Geobacter metallireducens and Methanosarcina barkeri, Appl. Environ. Microbiol. 80 (2014) 4599–4605. [43] A.E. Rotaru, P.M. Shrestha, F.H. Liu, M. Shrestha, D. Shrestha, M. Embree, K. Zengler, C. Wardman, K.P. Nevin, D.R. Lovley, A new model for electron flow during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane, Energy Environ. Sci. 7 (2014) 408–415. [44] P.M. Shrestha, A.-E. Rotaru, Plugging in or going wireless: strategies for interspecies electron transfer, Front. Microbiol. 5 (2014) 237. [45] H. Nagarajan, M. Embree, A.E. Rotaru, P.M. Shrestha, A.M. Feist, B.O. Palsson, D.R. Lovley, K. Zengler, Characterization and modelling of interspecies electron transfer mechanisms and microbial community dynamics of a syntrophic association, Nat. Commun. 4 (2013). [46] T. Storck, B. Virdis, D.J. Batstone, Modelling extracellular limitations for mediated versus direct interspecies electron transfer, ISME J. 10 (2016) 621–631. [47] P.M. Shrestha, A.E. Rotaru, Z.M. Summers, M. Shrestha, F. Liu, D.R. Lovley, Transcriptomic and genetic analysis of direct interspecies electron transfer, Appl. Environ. Microbiol. 79 (2013) 2397–2404. [48] N.H. Khan, M. Ahsan, W.D. Taylor, K. Kogure, Culturability and survival of marine, freshwater and clinical Pseudomonas aeruginosa, Microbes Environ. 25 (2010) 266–274. [49] N. Kimata, T. Nishino, S. Suzuki, K. Kogure, Pseudomonas aeruginosa isolated from marine environments in Tokyo Bay, Microb. Ecol. 47 (2004) 41–47. [50] C.K. Stover, X.Q. Pham, A.L. Erwin, S.D. Mizoguchi, P. Warrener, M.J. Hickey, F.S.L. Brinkman, W.O. Hufnagle, D.J. Kowalik, M. Lagrou, R.L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L.L. Brody, S.N. Coulter, K.R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G.K.S. Wong, Z. Wu, I.T. Paulsen, J. Reizer, M.H. Saier, R.E.W. Hancock, S. Lory, M.V. Olson, Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen, Nature 406 (2000) 959–964.

L. Semenec et al. / Bioelectrochemistry 119 (2018) 150–160 [51] T.H. Pham, N. Boon, P. Aelterman, P. Clauwaert, L. De Schamphelaire, L. Vanhaecke, K. De Maeyer, M. Hofte, W. Verstraete, K. Rabaey, Metabolites produced by Pseudomonas sp. enable a Gram-positive bacterium to achieve extracellular electron transfer, Appl. Microbiol. Biotechnol. 77 (2008) 1119–1129. [52] B.A. Methe, K.E. Nelson, J.A. Eisen, I.T. Paulsen, W. Nelson, J.F. Heidelberg, D. Wu, M. Wu, N. Ward, M.J. Beanan, R.J. Dodson, R. Madupu, L.M. Brinkac, S.C. Daugherty, R.T. DeBoy, A.S. Durkin, M. Gwinn, J.F. Kolonay, S.A. Sullivan, D.H. Haft, J. Selengut, T.M. Davidsen, N. Zafar, O. White, B. Tran, C. Romero, H.A. Forberger, J. Weidman, H. Khouri, T.V. Feldblyum, T.R. Utterback, S.E. Van Aken, D.R. Lovley, C.M. Fraser, Genome of Geobacter sulfurreducens: metal reduction in subsurface environments, Science 302 (2003) 1967–1969. [53] J.E. Butler, F. Kaufmann, M.V. Coppi, C. Nunez, D.R. Lovley, MacA a diheme c-type cytochrome involved in Fe(III) reduction by Geobacter sulfurreducens, J. Bacteriol. 186 (2004) 4042–4045. [54] M.V. Ordonez, G.D. Schrott, D.A. Massazza, J.P. Busalmen, The relay network of Geobacter biofilms, Energy Environ. Sci. 9 (2016) 2677–2681. [55] J.W. Voordeckers, B.C. Kim, M. Izallalen, D.R. Lovley, Role of Geobacter sulfurreducens outer surface c-type cytochromes in reduction of soil humic acid and anthraquinone-2,6-disulfonate, Appl. Environ. Microbiol. 76 (2010) 2371–2375. [56] C.E. Levar, C.H. Chan, M.G. Mehta-Kolte, D.R. Bond, An inner membrane cytochrome required only for reduction of high redox potential extracellular electron acceptors, MBio 5 (2014). [57] L. Zacharoff, C.H. Chan, D.R. Bond, Reduction of low potential electron acceptors requires the CbcL inner membrane cytochrome of Geobacter sulfurreducens, Bioelectrochemistry 107 (2016) 7–13. [58] L.C. Gillet, P. Navarro, S. Tate, H. Rost, N. Selevsek, L. Reiter, R. Bonner, R. Aebersold, Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis, Mol. Cell. Proteomics 11 (2012) (O111 016717). [59] R.F. Rosenzweig, R.R. Sharp, D.S. Treves, J. Adams, Microbial evolution in a simple unstructured environment - genetic differentiation in Escherichia coli, Genetics 137 (1994) 903–917. [60] D.S. Treves, S. Manning, J. Adams, Repeated evolution of an acetate-crossfeeding polymorphism in long-term populations of Escherichia coli, Mol. Biol. Evol. 15 (1998) 789–797. [61] L.E.P. Dietrich, A. Price-Whelan, A. Petersen, M. Whiteley, D.K. Newman, The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa, Mol. Microbiol. 61 (2006) 1308–1321. [62] M. Choi, C.Y. Chang, T. Clough, D. Broudy, T. Killeen, B. MacLean, O. Vitek, MSstats: an R package for statistical analysis of quantitative mass spectrometry-based proteomic experiments, Bioinformatics 30 (2014) 2524–2526. [63] M. Kanehisa, Y. Sato, M. Kawashima, M. Furumichi, M. Tanabe, KEGG as a reference resource for gene and protein annotation, Nucleic Acids Res. 44 (2016) D457–D462. [64] C. Gene Ontology, Gene ontology consortium: going forward, Nucleic Acids Res. 43 (2015) D1049–1056. [65] F. Caccavo, D.J. Lonergan, D.R. Lovley, M. Davis, J.F. Stolz, M.J. Mcinerney, Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metalreducing microorganism, Appl. Environ. Microbiol. 60 (1994) 3752–3759. [66] C. Godfrey, A. Coddington, C. Greenwood, A.J. Thomson, P.M.A. Gadsby, Purification and properties of formate dehydrogenase from Pseudomonas aeruginosa - characterization of haem and iron-sulfur centers by magnetic-circular-dichroism and electron-paramagnetic-resonance spectroscopy, Biochem. J. 243 (1987) 225–233. [67] H. Mikkelsen, R. McMullan, A. Filloux, The Pseudomonas aeruginosa reference strain PA14 displays increased virulence due to a mutation in ladS, PLoS One 6 (2011). [68] Y. Wang, S.E. Kern, D.K. Newman, Endogenous phenazine antibiotics promote anaerobic survival of Pseudomonas aeruginosa via extracellular electron transfer, J. Bacteriol. 192 (2010) 365–369. [69] E.M. Bosire, L.M. Blank, M.A. Rosenbaum, Strain and substrate dependent redox mediator and electricity production by Pseudomonas aeruginosa, Appl. Environ. Microbiol. 82 (2016) 5026–5038. [70] K. Rabaey, N. Boon, M. Hofte, W. Verstraete, Microbial phenazine production enhances electron transfer in biofuel cells, Environ. Sci. Technol. 39 (2005) 3401–3408. [71] N.J. Hare, C.Z. Soe, B. Rose, C. Harbour, R. Codd, J. Manos, S.J. Cordwell, Proteomics of Pseudomonas aeruginosa Australian epidemic strain 1 (AES-1) cultured under conditions mimicking the cystic fibrosis lung reveals increased iron acquisition via the siderophore pyochelin, J. Proteome Res. 11 (2012) 776–795. [72] K. Bagramyan, N. Mnatsakanyan, A. Poladian, A. Vassilian, A. Trchounian, The roles of hydrogenases 3 and 4, and the F0F1-ATPase, in H2 production by Escherichia coli at alkaline and acidic pH, FEBS Lett. 516 (2002) 172–178. [73] R. Bohm, M. Sauter, A. Bock, Nucleotide sequence and expression of an operon in Escherichia coli coding for formate hydrogenylase components, Mol. Microbiol. 4 (1990) 231–243. [74] M. Sauter, R. Bohm, A. Bock, Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli, Mol. Microbiol. 6 (1992) 1523–1532. [75] M. Takacs, A. Toth, B. Bogos, A. Varga, G. Rakhely, K.L. Kovacs, Formate hydrogenlyase in the hyperthermophilic archaeon, Thermococcus litoralis, BMC Microbiol. 8 (2008). [76] K. Rabaey, N. Boon, S.D. Siciliano, M. Verhaege, W. Verstraete, Biofuel cells select for microbial consortia that self-mediate electron transfer, Appl. Environ. Microbiol. 70 (2004) 5373–5382. [77] L. Semenec, A.E. Franks, Delving through electrogenic biofilms: from anodes to cathodes to microbes, AIMS Bioeng. 2 (2015) 222–248. [78] P.M. Shrestha, A.E. Rotaru, M. Aklujkar, F.H. Liu, M. Shrestha, Z.M. Summers, N. Malvankar, D.C. Flores, D.R. Lovley, Syntrophic growth with direct interspecies electron transfer as the primary mechanism for energy exchange, Environ. Microbiol. Rep. 5 (2013) 904–910.

159

[79] M. Aklujkar, M.V. Coppi, C. Leang, B.C. Kim, M.A. Chavan, L.A. Perpetua, L. Giloteaux, A. Liu, D.E. Holmes, Proteins involved in electron transfer to Fe(III) and Mn(IV) oxides by Geobacter sulfurreducens and Geobacter uraniireducens, Microbiology 159 (2013) 515–535. [80] P.L. Tremblay, Z.M. Summers, R.H. Glaven, K.P. Nevin, K. Zengler, C.L. Barrett, Y. Qiu, B.O. Palsson, D.R. Lovley, A c-type cytochrome and a transcriptional regulator responsible for enhanced extracellular electron transfer in Geobacter sulfurreducens revealed by adaptive evolution, Environ. Microbiol. 13 (2011) 13–23. [81] J.A. Smith, P.L. Tremblay, P.M. Shrestha, O.L. Snoeyenbos-West, A.E. Franks, K.P. Nevin, D.R. Lovley, Going wireless: Fe(III) oxide reduction without pili by Geobacter sulfurreducens strain JS-1, Appl. Environ. Microbiol. 80 (2014) 4331–4340. [82] K. Inoue, C. Leang, A.E. Franks, T.L. Woodard, K.P. Nevin, D.R. Lovley, Specific localization of the c-type cytochrome OmcZ at the anode surface in current-producing biofilms of Geobacter sulfurreducens, Environ. Microbiol. Rep. 3 (2011) 211–217. [83] M.V. Coppi, R.A. O'Neil, D.R. Lovley, Identification of an uptake hydrogenase required for hydrogen-dependent reduction of Fe(III) and other electron acceptors by Geobacter sulfurreducens, J. Bacteriol. 186 (2004) 3022–3028. [84] H.B. Hu, Q.X. Yu, C. Feng, H.Z. Xue, B.K. Hur, Isolation and characterization of a new fluorescent Pseudomonas strain that produces both phenazine 1-carboxylic acid and pyoluteorin, J. Microbiol. Biotechnol. 15 (2005) 86–90. [85] R.S. Kumar, N. Ayyadurai, P. Pandiaraja, A.V. Reddy, Y. Venkateswarlu, O. Prakash, N. Sakthivel, Characterization of antifungal metabolite produced by a new strain Pseudomonas aeruginosa PUPa3 that exhibits broad-spectrum antifungal activity and biofertilizing traits, J. Appl. Microbiol. 98 (2005) 145–154. [86] P.R. Naik, N. Sakthivel, Functional characterization of a novel hydrocarbonoclastic Pseudomonas sp. strain PUP6 with plant-growth-promoting traits and antifungal potential, Res. Microbiol. 157 (2006) 538–546.

Lucie Semenec is a PhD candidate at Dr. Ashley Franks' Applied and Environmental Laboratory in the in the Department of Physiology, Anatomy and Microbiology, School of Life Sciences at La Trobe University. Her research focuses on the community dynamics of electrogenic biofilms and the underlying mechanisms of syntrophic and competitive microbial interactions.

Andrew E Laloo is a PhD student at the Advanced Water Management Centre at the University of Queensland. His research focuses on using ‘omic’ technologies in understanding the response of different microorganisms to biocides in wastewater treatment processes.

Benjamin L Schulz is a Senior Lecturer in the School of Chemistry and Molecular Biosciences at The University of Queensland. His doctoral studies at the ETH Zurich in Switzerland investigated microbial protein glycosylation. His research focuses on developing analytical methods for the analysis of complex proteomes and their modifications, and using these to understand the mechanisms controlling protein biosynthesis and post-translational modification.

Ismael A Vergara is a postdoctoral researcher in the Bioinformatics and Cancer Genomics group at the Peter MacCallum Cancer Centre. He graduated from his PhD in Genomics in 2011 and worked for 3 years in the Biotechnology industry in Canada focused on prostate cancer research. In 2014, he moved to Australia and joined the Peter Mac as a postdoctoral researcher in the field of melanoma genomics. His research as a computational biologist at the Peter Mac has been mainly focused on the molecular evolution of metastatic melanoma.

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L. Semenec et al. / Bioelectrochemistry 119 (2018) 150–160 Philip L Bond is Senior Research Fellow at the Advanced Water Management Centre, The University of Queensland. He has a BSc Hons from the University of Melbourne and PhD from The University of Queensland in 1997. After spending 9 years abroad (USA and the UK) he returned to the University of Queensland in 2006. He collaborates to perform multidisciplinary research to determine microbial ecosystem function. Especially through application of metagenomic and metaproteomic approaches. Specialising in the microbiology of water supply, wastewater treatment, sewer corrosion, anaerobic digestion, biofilm control, acid leaching environments and bioelectrochemistry.

Ashley E Franks is head of the Applied and Environmental Microbiology Laboratory at La Trobe University. He completed his PhD at the University of New South Wales investigating fungal inhibition by marine bacteria, including time at the University of Exeter in the UK. He then moved to the Biomerit Research Centre in Cork, Ireland to work on bacterial plant interactions as a Government of Ireland Fellow in Science Technology and Engineering. Later, as part of the Geobacter Project at the University of Massachusetts Amherst in the USA he worked on electricity producing microbes. His current research interests include microbial interactions in the soil, mammalian guts and electrodes.