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. 2020 Apr 7;11(2):e02875-19.
doi: 10.1128/mBio.02875-19.

Adaptive Evolution of Geobacter sulfurreducens in Coculture with Pseudomonas aeruginosa

Affiliations

Adaptive Evolution of Geobacter sulfurreducens in Coculture with Pseudomonas aeruginosa

Lucie Semenec et al. mBio. .

Abstract

Interactions between microorganisms in mixed communities are highly complex, being either syntrophic, neutral, predatory, or competitive. Evolutionary changes can occur in the interaction dynamics between community members as they adapt to coexistence. Here, we report that the syntrophic interaction between Geobacter sulfurreducens and Pseudomonas aeruginosa coculture change in their dynamics over evolutionary time. Specifically, Geobacter sp. dominance increases with adaptation within the cocultures, as determined through quantitative PCR and fluorescence in situ hybridization. This suggests a transition from syntrophy to competition and demonstrates the rapid adaptive capacity of Geobacter spp. to dominate in cocultures with P. aeruginosa Early in coculture establishment, two single-nucleotide variants in the G. sulfurreducensfabI and tetR genes emerged that were strongly selected for throughout coculture evolution with P. aeruginosa phenazine wild-type and phenazine-deficient mutants. Sequential window acquisition of all theoretical spectra-mass spectrometry (SWATH-MS) proteomics revealed that the tetR variant cooccurred with the upregulation of an adenylate cyclase transporter, CyaE, and a resistance-nodulation-division (RND) efflux pump notably known for antibiotic efflux. To determine whether antibiotic production was driving the increased expression of the multidrug efflux pump, we tested Pseudomonas-derived phenazine-1-carboxylic acid (PHZ-1-CA) for its potential to inhibit Geobacter growth and drive selection of the tetR and fabI genetic variants. Despite its inhibitory properties, PHZ-1-CA did not drive variant selection, indicating that other antibiotics may drive overexpression of the efflux pump and CyaE or that a novel role exists for these proteins in the context of this interaction.IMPORTANCEGeobacter and Pseudomonas spp. cohabit many of the same environments, where Geobacter spp. often dominate. Both bacteria are capable of extracellular electron transfer (EET) and play important roles in biogeochemical cycling. Although they recently in 2017 were demonstrated to undergo direct interspecies electron transfer (DIET) with one another, the genetic evolution of this syntrophic interaction has not been examined. Here, we use whole-genome sequencing of the cocultures before and after adaptive evolution to determine whether genetic selection is occurring. We also probe their interaction on a temporal level and determine whether their interaction dynamics change over the course of adaptive evolution. This study brings to light the multifaceted nature of interactions between just two microorganisms within a controlled environment and will aid in improving metabolic models of microbial communities comprising these two bacteria.

Keywords: competition; evolution; mutualism; syntrophs.

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Figures

FIG 1
FIG 1
Proportion of G. sulfurreducens to P. aeruginosa in cocultures. Black and gray bars represent log copy numbers of DNA from early-stationary-phase cocultures using G. sulfurreducens-specific primers (Gsulf_F and Gsulf_R) and Pseudomonas-specific primers (Pse435F and Pse686R), respectively, via qPCR. Solid bars represent initial (s0) cocultures, and dotted bars represent adapted (s13) cocultures. *, P ≤ 0.05.
FIG 2
FIG 2
Proportion of G. sulfurreducens (DL-1) to P. aeruginosa (PAO1) in adapted (s13) cocultures via FISH. (A to C) All bacteria were probed with EUB338-ATTO633 probe (yellow) (A), the P. aeruginosa PAO1 strain was probed with PseaerA-ATTO488 probe (green) (B), and the G. sulfurreducens DL-1 strain was probed with GEO2-ATTO565 probe (red) (C). The white circle shows the localization of P. aeruginosa in flocs of G. sulfurreducens. Images are representative of triplicate samples taken during the early-stationary-growth phase of cocultures.
FIG 3
FIG 3
Genetic variants found in G. sulfurreducens from cocultures with P. aeruginosa initial (s0) and adapted (s13) via whole-genome sequencing. Heat map indicates the proportion of reads supporting the mutation in the specified gene, and numbers within each box represent the genetic frequency value. Three biological replicates per coculture were sequenced. The left column represents DL-1 pure culture in NBAF, and reads were aligned against the assembly of the Geobacter sulfurreducens PCA genome (NCBI assembly no. GCF_000007985.2).
FIG 4
FIG 4
Correlation between G. sulfurreducens tetR SNP mutation frequency and downstream operon protein abundance. Blue cells indicate the average frequency of genetic reads across replicates obtained from whole-genome sequencing (WGS) that contain the single-nucleotide insertion in GSU0951 (tetR). Pink cells indicate the log2 fold change (log2FC) of protein abundance in the cocultures versus DL-1 pure cultures. Gray cells indicate proteins absent from the proteome. The yellow dashed box indicates a previously predicted operon (40). Genes depicted by pink arrows represent those likely under the control of TetR (in blue), as confirmed by this study. Genes depicted by green arrows represent those absent from the proteomes.

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