Novel Extracellular Electron Transfer Channels in a Gram-Positive Thermophilic Bacterium

Abstract

Biogenic transformation of Fe minerals, associated with extracellular electron transfer (EET), allows microorganisms to exploit high-potential refractory electron acceptors for energy generation. EET-capable thermophiles are dominated by hyperthermophilic archaea and Gram-positive bacteria. Information on their EET pathways is sparse. Here, we describe EET channels in the thermophilic Gram-positive bacterium Carboxydothermus ferrireducens that drive exoelectrogenesis and rapid conversion of amorphous mineral ferrihydrite to large magnetite crystals. Microscopic studies indicated biocontrolled formation of unusual formicary-like ultrastructure of the magnetite crystals and revealed active colonization of anodes in bioelectrochemical systems (BESs) by C. ferrireducens. The internal structure of micron-scale biogenic magnetite crystals is reported for the first time. Genome analysis and expression profiling revealed three constitutive c-type multiheme cytochromes involved in electron exchange with ferrihydrite or an anode, sharing insignificant homology with previously described EET-related cytochromes thus representing novel determinants of EET. Our studies identify these cytochromes as extracellular and reveal potentially novel mechanisms of cell-to-mineral interactions in thermal environments.

Keywords: Gram-positive bacteria; biogenic magnetite crystals; electrogenesis; iron reduction; multiheme cytochromes; thermophilic prokaryotes.

FIGURE 1

SEM images of small clusters of C. ferrireducens cells grown with ferrihydrite as sole electron acceptor. Surrounding material—nanoparticulate magnetite. (A–C) consecutive magnification of the same microscopic field. (D) Another microscopic field with a cell connected with the bulk of the mineral by a single appendage.

FIGURE 2

SEM images of C. ferrireducens cell colonies associated with macroscopic magnetite crystals in the late logarithmic phase of growth with ferrihydrite. (A) Non-piliated and piliated cells on the surface of a large magnetite crystal. (B) Overview of a large magnetite crystal. The white arrow on (A) indicates a singular cell appendage connecting the cell entrapped into magnetite crystal with the mineral surface, almost all the other cells associated with the crystal are non-piliated. Black arrows on (A,B) indicate incremental growth steps and facets of macroscopic magnetite crystals. (C) Different cell aggregates—inside macroscopic magnetite crystals and on the surface of nanoparticulate mineral phase. The red box on (C) indicates the area magnified at (D). (D) Cells and cell-like cavities on the surface of a large (micron-scale) magnetite crystal. Black arrows on (D) point out different surfaces of the cells contacting the nanoparticulate mineral phase and the cells entrapped into large magnetite crystals (rough and smooth cell surfaces, respectively).

FIGURE 3

(A) Current vs. time of duplicate MFC reactors with C. ferrireducens biofilms pregrown at open circuit anodes with glycerol as the electron donor and bicarbonate as the electron acceptor. (B) Confocal image of pregrown C. ferrireducens biofilm used for reactor 1 (with more biomass pregrown), stained with SYBR Green fluorescent DNA stain. (C) Confocal image of pregrown C. ferrireducens biofilm used for reactor 2 (with less biomass pregrown). Stars indicate time periods where the circuit was opened to measure anodic and cathodic open-circuit potentials. Black arrows indicate full media exchanges. White arrows indicate additions of glycerol and yeast extract only. Red arrows indicate addition of glycerol, yeast extract, and ferrihydrite simultaneously. Scale bars on (B,C) are 200 μm.

FIGURE 4

Protein profiles of crude cell extracts of C. ferrireducens grown in the presence of ferrihydrite and alternative soluble electron acceptors. (A) Lanes1–3 show full-length Coomassie-stained gels for proteins. (B) Lanes 1–3 show the same gels, benzidine-stained to visualize c-type hemes. Lane numbers indicate different electron acceptors: 1—ferrihydrite; 2—ferric citrate; 3—fumarate. MW—molecular weight markers (top to bottom: 200, 150, 120, 100, 85, 70, 60, 50, 40, 30, 25, 20, 15, 10 kDa); white arrows indicate heme bands containing OmhA cytochrome, red arrows SmhA, yellow arrow SmhB, and green arrows SmhC (refer to the text for cytochromes’ description). The Coomassie and benzidine stained gels received the same protein loading (13–14 μg per lane).

FIGURE 5

Molar proportions (riBAQs) of selected protein groups determining housekeeping, respiratory, and EET processes in C. ferrireducens cells grown with different electron acceptors. For each protein group, summarized molar proportion (riBAQ) of all its proteins is presented. Whole set of proteins detected in a sample is taken as 100%. Error bars indicate standard deviations in summarized riBAQs of each group. Asterisks indicate the most significant, statistically supported differences in molar proportions of protein groups (refer to the text for detail). Electrogenic growth conditions imply utilization of a stainless steel anode as the electron acceptor. The cells at all the growth conditions were harvested at the late logarithmic growth phase. Target proteins are grouped as follows: Group I RPO—four proteins of α, β, β’, and ω subunits of DNA-directed RNA polymerase, Group II ATPase—six proteins of α, β, γ, δ, ε, and B subunits of F₀F₁-type ATP synthase, Group III ETC—totally 17 proteins of A to F and H to N subunits of proton-translocating type I NADH-dehydrogenase together with four subunits of membrane-bound succinate dehydrogenase/fumarate reductase, Group IV Pili—10 proteins of pili assembly encoded in the “pilin-cytochrome” cluster (“PilA-C,” PilBT, PilVMNO proteins, and three other proteins with type-IV pilin N-terminal methylation sites), Group V Multihemes—10 multiheme cytochromes which are predicted to be secreted or cell surface-associated proteins (refer to the legend of Figure 6 for detail). The full set of the proteins included in each group appears in Supplementary Table S6.

FIGURE 6

Molar proportions (riBAQs) of individual multiheme cytochrome proteins. The data are shown for all the putative EET-related cytochromes of C. ferrireducens, which were identified in protein profiles: OmhA, SmhA, SmhB, SmhC cytochromes, Ga0395992_01_217646_219709 and Ga0395992_01_222075_224618 encoded in the “pilin-cytochrome” cluster, as well as cytochromes Ga0395992_02_29558_30178, Ga0395992_02_30996_31874, and putative quinol-oxidizing cytochromes Ga0395992_01_187284_188528 and Ga0395992_01_684739_686151. Inset—molar proportions of minor cytochromes (with riBAQs below 0.1), predominating cytochromes are cross-hatched in the inset. Putative Q-oxidases (Ga0395992_02_135863_137242 and Ga0395992_01_187284_188528) are marked bold and underlined. Asterisks indicate significant, statistically supported differences, which are not visually obvious on the figure. Singular asterisks indicate the differences in molar proportions of SmhA cytochrome in the cells grown with ferric citrate, ferrihydrite, and on anode; double asterisks indicate the differences in SmhC representation in ferric citrate-reducing or ferrihydrite-reducing cells (refer to the text for detail). Hash symbols indicate the cytochromes Ga0395992_01_217646_219709 and Ga0395992_01_222075_224618, encoded in the “pilin-cytochrome” cluster and expressed only in electrogenic and ferric citrate-respiring cells. Clear differences in molar proportions (such as for OmhA in ferrihydrite-reducing and other cells) are not specially marked.

FIGURE 7

Consensus trees constructed after Bayesian inference of phylogeny from the MAFFT alignment of OmhA and SmhC (A), and SmhB (B) cytochromes of C. ferrireducens and their best blast hits (see section “Materials and Methods” for detail). Unrooted 50% majority rule consensus phylograms are displayed as rectangular trees, for which posterior probability values are shown; posterior values of 1 are omitted for clarity. Mean branch lengths are characterized by scale bars indicating the evolutionary distance between the proteins (changes per amino acid position). The branches are annotated with labels indicating the protein sequence accession number, the protein name as retrieved from the database and the source organism. Branches and labels of the proteins are colored pink for the proteins retrieved from monoderm cultured organisms, cyan for the proteins from diderm cultured organisms, and black for the proteins from uncultured organisms. The target sequences of OmhA, SmhC, SmhB, and homologous cytochromes of C. ferrireducens are labeled red and their homologs from previously described EET-related cytochromes of other organisms are labeled dark blue. Clusters of sequences are categorized and highlighted where appropriate as follows. Blue cluster combines sequences of DmsE family decaheme cytochromes. Light and deep yellow clusters combine related sequences by the number of predicted heme-binding sites in them. Green clusters contain related sequences of various heme numbers from distinct taxonomic or physiological groups of source organisms. Highlighted clusters are labeled whether by collapsed subtrees with annotations given in bold underlined font or by red Roman numerals denoting the following. Cluster I.A—OmhA and related 11-heme proteins from Carboxydothermus spp.; cluster II.A—various multihemes from Acidobacteria; cluster III.A—octahemes from Epsilonproteobacteria; cluster IV.A—OcwA from “T. potens” and related nonaheme proteins; cluster V.A—SmhC and related hexa- and heptaheme proteins; cluster VI.A—9- to 16-heme proteins from monoderm prokaryotes, methanogenic archaea; cluster VII.A—see the blue cluster above; cluster VIII.A—octahemes of unassigned function from various Proteobacteria and Bacteroidetes; cluster I.B—SmhB and its homologs from Carboxydothermus species; cluster II.B—hexaheme proteins with unassigned function from various Firmicutes.