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. 2020 Jul 3:11:1506.
doi: 10.3389/fmicb.2020.01506. eCollection 2020.

Quantification of Cable Bacteria in Marine Sediments via qPCR

Jeanine S Geelhoed  1 , Sebastiaan J van de Velde  2 , Filip J R Meysman  1   3
Affiliations

Quantification of Cable Bacteria in Marine Sediments via qPCR

Jeanine S Geelhoed et al. Front Microbiol. .

Abstract

Cable bacteria (Deltaproteobacteria, Desulfobulbaceae) are long filamentous sulfur-oxidizing bacteria that generate long-distance electric currents running through the bacterial filaments. This way, they couple the oxidation of sulfide in deeper sediment layers to the reduction of oxygen or nitrate near the sediment-water interface. Cable bacteria are found in a wide range of aquatic sediments, but an accurate procedure to assess their abundance is lacking. We developed a qPCR approach that quantifies cable bacteria in relation to other bacteria within the family Desulfobulbaceae. Primer sets targeting cable bacteria, Desulfobulbaceae and the total bacterial community were applied in qPCR with DNA extracted from marine sediment incubations. Amplicon sequencing of the 16S rRNA gene V4 region confirmed that cable bacteria were accurately enumerated by qPCR, and suggested novel diversity of cable bacteria. The conjoint quantification of current densities and cell densities revealed that individual filaments carry a mean current of ∼110 pA and have a cell specific oxygen consumption rate of 69 fmol O2 cell-1 day-1. Overall, the qPCR method enables a better quantitative assessment of cable bacteria abundance, providing new metabolic insights at filament and cell level, and improving our understanding of the microbial ecology of electrogenic sediments.

Keywords: Desulfobulbaceae; amplicon sequencing; cable bacteria; current density; marine sediment; oxygen consumption rate; quantitative PCR.

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Figures

FIGURE 1
FIGURE 1
Pore water geochemistry of sediment in unamended incubations (upper row, open symbols) and stimulated incubations (bottom row, filled symbols). (A,E) Microsensor profiles of O2, ΣH2S and pH (t = 22 days). In the stimulated incubations a suboxic zone of ∼10 mm wide was present where both oxygen and sulfide were below detection limit, indicative of the activity of cable bacteria. (B–D,F–H) Pore water concentrations (t = 24 days) of Ca, Fe, and Mn were much higher in the stimulated compared to the unamended incubations, indicating dissolution of minerals stimulated by acidification of the suboxic zone through anodic sulfide oxidation by cable bacteria.
FIGURE 2
FIGURE 2
qPCR quantification of 16S rRNA gene copies of (A) total bacteria (TB), (B) Desulfobulbaceae (DSB) and (C) cable bacteria (CB). (D) CB 16S copies relative to DSB 16S copies and (E) CB 16S copies relative to TB 16S copies. Data is for two replicate cores each from unamended incubations (open symbols) and stimulated incubations (filled symbols). Data in panels (A–C) shows the mean with error bars denoting the standard deviation (n = 2). 16S rRNA gene copies are expressed per gram of wet sediment.
FIGURE 3
FIGURE 3
Ratio of the relative abundance of (A) cable bacteria and (B) Desulfobulbaceae quantified by qPCR and by classification of amplicon sequences. For panel (A), relative abundance of cable bacteria by qPCR (y-axis) was calculated as CB 16S copies/TB 16 copies (%), and relative abundance by amplicon sequencing (x-axis) as Σreads classified as Ca. Electrothrix/Σreads classified as Bacteria (%). For panel (B), calculations were similar, using DSB 16S copies, and reads classified as Desulfobulbaceae. Each data point shows the ratio for one depth layer in one of the replicate cores of the unamended incubation (open symbols) and stimulated incubation (filled symbols). The inset in panel (A) shows a close-up for unamended incubation N1. Ratios for depth layers with cable bacteria present are shown in green, and for depth layers without cable bacteria in red.
FIGURE 4
FIGURE 4
Phylogenetic tree of 16S rRNA gene sequences of cable bacteria, cultured members of the Desulfobulbaceae and selected clones from sediment (the origin of the sediments is indicated in square brackets) and the amplicon sequence variants (ASVs) classified as Ca. Electrothrix detected in the incubations of marine sediment (in bold). The ASV sequences (indicated with * followed by the length of the sequence) were inserted using the evolutionary placement algorithm. Bootstrap values (%) were calculated for 1000 re-samplings; only values ≥60 are shown.
FIGURE 5
FIGURE 5
FISH images of filamentous bacteria hybridized with DSB706 from sediment of core N1 of the unamended incubation. The images show the overlay of false colored images for the DSB706 (red) and DAPI (blue) signal. The scale bars denote 10 μm.
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