Abstract
Syntrophic interspecies electron exchange is essential for the stable functioning of diverse anaerobic microbial communities. Hydrogen/formate interspecies electron transfer (HFIT), in which H2 and/or formate function as diffusible electron carriers, has been considered to be the primary mechanism for electron sharing because most common syntrophs were thought to lack biochemical components, such as electrically conductive pili (e-pili), necessary for direct interspecies electron transfer (DIET). Here we report that Syntrophus aciditrophicus, one of the most intensively studied microbial models for HFIT, produces e-pili and can grow via DIET. Pilin genes likely to yield e-pili were found in other genera of hydrogen/formate-producing syntrophs. The finding that DIET is a likely option for diverse syntrophs that are abundant in many anaerobic environments necessitates a reexamination of the paradigm that HFIT is the predominant mechanism for syntrophic electron exchange within anaerobic microbial communities of biogeochemical and practical significance.
Introduction
H2/formate interspecies electron transfer (HFIT) and direct interspecies electron transfer (DIET) are mechanisms for the electron-donating partner in syntrophic consortia to dispose of electrons released from the oxidation of key intermediates (organic acids, alcohols, aromatics) during the anaerobic degradation of complex organic matter 1-5. The relative proportion of electron flux through DIET or HFIT can influence the speed of interspecies electron transfer, the stability of anaerobic microbial communities, and their ability to adapt to environmental change 4,6,7. However, there are no accurate methods for measuring the rates that H2 and formate are transferred between microbes or for quantifying interspecies electrical currents in complex communities. Therefore, the importance of HFIT or DIET in microbial communities is typically inferred from the composition of the microbial community.
It has previously been considered that DIET is primarily restricted to environments in which Geobacter species are abundant 8-12, because Geobacter species are the only microbes in pure culture that have been definitively shown to function as electron-donating partners for DIET 9,13-17. It is assumed that HFIT predominates where microorganisms closely related to traditional syntrophs known to grow via HFIT are most abundant. However, most cultures of electron- donating syntrophs were characterized prior to the concept of DIET. Their capacity for DIET has not been fully explored.
For example, Syntrophus aciditrophicus is one of the most intensively studied pure culture models for HFIT 18-21. It was previously concluded that S. aciditrophicus would be unlikely to participate in DIET 21,22 because it lacks a gene homologous to the gene for the Geobacter pilin monomer that assembles into its electrically conductive pili (e-pili) 23, a conduit for extracellular electron transfer required for Geobacter species to function as electron-donating partners in DIET17. This gene would not be expected in S. aciditrophicus because Geobacter e- pili have evolved relatively recently and are primarily restricted to Geobacter species and close relatives 24. However, it has recently been found that e-pili, phylogenetically distinct from Geobacter e-pili have independently evolved multiple times 25.
Here we report that S. aciditrophicus expresses e-pili and is capable of growing via DIET. These results, and analysis of the pilin genes of other common syntrophs, indicate that the capacity for DIET should be considered as an option for microorganisms known to grow via HFIT and suggest that DIET may be more wide spread than previously considered.
Results and Discussion
Transmission electron microscopy of S. aciditrophicus revealed filaments with a morphology typical of type IV pili (Fig. 1a). A complement of genes consistent with type IV pili assembly is present in the genome (Fig. 1b). One gene, SYN 00814 encodes a N-terminal domain characteristic of PilA, the pilin monomer for Type IVa pili (Fig. 1c). This includes a short signal peptide (13 amino acids) which is cleaved by PilD at the G|FTLIE recognition site and a highly conserved, hydrophobic, transmembrane domain. Additional genes for pilus assembly (pilD, pilM, pilN, pilO, pilP, pilQ) and transcriptional control of pilus expression (pilS, pilR) are also present in the genome (Figure 1b). The amino acid sequence of the putative PilA protein fits the empirical criteria 25 for a pilin monomer likely to yield e-pili: 1) aromatic amino acids are located in the key positions required for conductivity in G. sulfurreducens e-pili; 2) the abundance of aromatic amino acids (10.9 % of amino acids) is above the minimum threshold of 9 % found to be necessary for high e-pili conductivity; and 3) no large gaps (> 35 amino acids) that lack aromatic amino acids (Fig. 1c).
Low culture densities of S. aciditrophicus prevented harvesting sufficient quantities of pili to measure pili conductance on the electrode arrays previously employed for the study of other e-pili 25. Therefore, the method initially employed to document the presence of e-pili in G. sulfurreducens 26 was adapted 27 as an alternative approach. Cultures of S. aciditrophicus were directly drop-cast on highly ordered pyrolytic graphite (HOPG), washed with deionized water, and examined with atomic force microscopy. Pili visualized with topographic imaging, (Fig. 2a), had a height/diameter of ca. 4 nm (Fig. 2b). Conductive imaging revealed that the pili were electrically conductive (Fig. 2b, c). Point-mode current-voltage (I-V) spectroscopy yielded an Ohmic-like response that was similar to G. sulfurreducens pili prepared in the same manner 27 (Fig. 2d) and in other similar studies of G. sulfurreducens pili 26. As previously reported 27, the pili from G. sulfurreducens strain Aro-5, which lack key aromatic amino acids required for high conductivity 28-31, had much lower conductance (Fig. 2d).
The lack of tools for genetic manipulation of S. aciditrophicus limited further functional analysis of the putative PilA gene predicted to yield its e-pili in the native organism. Therefore, the gene was heterologously expressed in G. sulfurreducens, replacing the native G. sulfurreducens pilA with an approach that has successfully yielded G. sulfurreducens strains that express a diversity of both highly conductive and poorly conductive heterologous pili 25,32-35. This new G. sulfurreducens strain, designated strain SP (for Syntrophus pili), expressed abundant pili (Fig. 3a) and produced electrical current at densities comparable to the control strain expressing the G. sulfurreducens wild-type pilA (Fig. 3b). Such high current densities are only possible when G. sulfurreducens expresses e-pili 25,28. As previously reported 25,28, G. sulfurreducens strain Aro-5, with its poorly conductive pili, produced much lower currents (Fig. 3b). Networks of pili sheared from the electrode-grown biofilm of strain SP, purified, and drop cast on electrode arrays as previously described 25, had a conductance comparable to G. sulfurreducens wild-type pili (Fig. 3c). These results further demonstrated that the S. aciditrophicus PilA gene yields a pilin monomer that can assemble into e-pili.
The presence of e-pili in S. aciditrophicus suggested it might be capable of establishing an electrical connection for DIET. To evaluate this, S. aciditrophicus was grown in co-culture with G. sulfurreducens, the microbe most intensively studied as an electron-accepting partner for DIET 4. G. sulfurreducens can also function as a H2- and formate-consuming partner for HFIT, providing a positive control for establishing the S. aciditrophicus/G. metallireducens co-culture if DIET was not possible 36. The electron donor was benzoate, a substrate that S. aciditrophicus can metabolize, but G. sulfurreducens cannot. The electron acceptor was fumarate, an electron acceptor only G. sulfurreducens can utilize. In the presence of a H2/formate-consuming partner S. aciditrophicus metabolizes benzoate to acetate with the production of either H2: or formate:
With electron transfer via DIET the relevant reaction is:
In addition to oxidizing H2 and formate, or consuming electrons released during DIET, G. sulfurreducens can also metabolize acetate, making the overall reaction expected for the oxidation of benzoate with the reduction of fumarate to succinate in the co-culture:
S. aciditrophicus/G. sulfurreducens co-cultures grew with repeated sub-culturing and, within the experimental error, exhibited the expected stoichiometry of benzoate consumption and succinate production (Fig. 4a). In these co-cultures HFIT, DIET, or a combination of the two, were feasible. Therefore, to eliminate the possibility of HFIT, S. aciditrophicus was co-cultured with the previously described strain of G. sulfurreducens 37, designated here as G. sulfurreducensHF, that can not utilize H2 or formate because the genes for the uptake hydrogenase and formate dehydrogenase were deleted. The S. aciditrophicus/ G. sulfurreducensHF co-culture had a longer initial lag period, but then metabolized benzoate with the reduction of fumarate almost as fast as the co-culture with wild-type G. sulfurreducens (Fig. 4b). These results demonstrate that S. aciditrophicus can grow via DIET.
Although some co-cultures form visible aggregates during growth via DIET 13, others produce small, relatively fragile aggregates 14. There were no visible aggregates in the S. aciditrophicus/G. sulfurreducens co-cultures but it appeared in scanning electron micrographs of cells collected on filters that co-cultures grown under conditions in which only DIET was possible have a greater tendency to form more small clumps than co-cultures grown under conditions in which HFIT was also an option (Fig. 4 d,e).
Granular activated carbon (GAC) can greatly reduce the initial lag time in establishing DIET-based co-cultures because both partners attach to GAC, which functions as an electrically conductive conduit 14,38. GAC significantly reduced the lag time of the S. aciditrophicus/ G. sulfurreducensHF co-cultures (Fig. 4c). As previously observed for other co-cultures in which GAC promoted DIET, cells from the S. aciditrophicus/ G. sulfurreducensHF co-culture heavily colonized the GAC (Fig. 4e), consistent with a GAC conduit for DIET.
In addition to e-pili, multi-heme outer-surface c-type cytochromes appear to play an important role in DIET in Geobacter species 4,5. However, the S. aciditrophicus genome encodes only a few putative c-type cytochromes 21 and cytochromes were not readily apparent in heme-stained preparations of cell protein (Supplementary Fig. 1). Not all microbes require cytochromes for effective electron transport to the outer cell surface 39. More detailed examination of the role of e-pili and other S. aciditrophicus components in DIET will require the development of methods for genetic manipulation of this microorganism.
S. aciditrophicus is the first isolate outside the genus Geobacter found to grow via DIET and is the first syntroph shown to have the option to grow via HFIT or DIET. Other Syntrophus species also have pilin genes likely to yield e-pili, as do other diverse genera of syntrophic microorganisms known to grow via HFIT in defined co-cultures (Fig. 5). Establishing conditions that favor DIET often enrich for microbes in these genera 6. For example, in enrichment cultures specifically designed to promote the metabolism of propionate or butyrate via DIET,
Smithella (propionate enrichment) or Syntrophomonas (butyrate enrichment) species were the most abundant bacteria 40-42. The greater energetic demands 43 required for synthesizing the abundant aromatic amino acids that are required for e-pili conductivity suggests that e-pili provide a strong selective advantage under some environmental conditions. Conferring the capacity for DIET is a likely explanation.
For decades, data on the functioning of complex anaerobic microbial communities has been interpreted through the lens of HFIT, but the available data does not rule out the possibility of DIET, which was not known to be an option at the time. For example, estimated H2 turnover rates in anaerobic digesters, rice paddy soils, and aquatic sediments were consistently less than 10 % of the independently determined rate of methane production derived from the reduction of carbon dioxide to methane 44-46, a result that is consistent with DIET providing most of the electrons for carbon dioxide reduction. The mismatch between measured H2 turnover rates and the concept of H2 as a primary interspecies electron carrier was rationalized with the suggestion that there was a separate pool of H2 within closely juxatapositioned assemblages of H2 producers and H2 consumers. However, the existence of two distinct pools of H2 that did not equilibrate over time via diffusion, a seemingly physical impossibility, was never verified. Attempts to accurately measure formate fluxes have also been problematic 47 and there is no method for directly measuring DIET-based electron fluxes in complex communities.
A fresh perspective and new analytical tools will be required to resolve the relative importance of HFIT and DIET in diverse anaerobic microbial communities. Just as electron- accepting partners have different gene expression patterns depending on whether they are participating in HFIT or DIET 37,48, it may be possible to determine whether electron-donating syntrophs are engaged in DIET or HFIT from metatranscriptomic analysis of anaerobic communities.
METHODS
Bacterial strains, plasmids and culture conditions
Syntrophus aciditrophicus, G. sulfurreducens wild-type, G. sulfurreducens strain Aro-5, and G. sulfurreducensHF were obtained from our laboratory culture collections. A strain of G. sulfurreducens expressing the PilA gene of S. aciditrophicus instead of the native G. sulfurreducens PilA gene was constructed as previously described 25. S. aciditrophicus and G. sulfurreducens strains were routinely grown under strict anaerobic conditions at 30 °C in the previously described 49 defined, bicarbonate-buffered medium with N2:CO2 (80:20) as the gas phase. For S. aciditrophicus the medium was amended with crotonate (20 mM) and for G. sulfurreducens strains acetate (10 mM) was the electron donor and fumarate (40 mM) was the electron acceptor. The presence of c-type cytochromes in whole cell lysates was evaluated with heme-staining of proteins separated on denaturing gels as previously described 17. G. sulfurreducens strains were grown with a graphite electrode as the electron acceptor as previously described 50.
Cocultures were established in 10 ml of culture medium 49 in anaerobic pressure tubes with benzoate (1 mM) as the electron donor and fumarate (40 mM) as the electron acceptor with cysteine (2 mM) and sulfide (1 mM) added as reducing agents. When noted cultures were amended with granular activated carbon (0.25 g; 8-20 mesh). Benzoate, acetate, and succinate were analyzed with high-performance liquid chromatography as previously described 16. Previously describe methods were employed for transmission electron microscopy 21 and scanning electron microscopy 26.
Pili conductivity
A 100 µl sample of cultures was drop cast onto highly oriented pyrolytic graphite (HOPG). After 10 min the HOPG was washed twice with 100 µl of deionized water, blotted dry to remove excess water, and dried for 12 hours at 24 °C in a desiccator. Samples were equilibrated with atmospheric humidity for at least 2 hours and then examined with an Oxford Instruments Cypher ES Environmental AFM in ORCA electrical mode equipped with a Pt/Ir- coated Arrow-ContPT tip with a 0.2 N/m force constant (NanoWorld AG, Neuchâtel, Switzerland). Pili were located in contact mode, with a set point of 0.002 V and a scan rate of 1.5 Hz. For conductive imaging, the grounded tip, attached to a transimpedance amplifier, served as a translatable top electrode to locally detect the current response of the individual pili to a 100 mV bias applied to the HOPG substrate. Individual pili conductivity was further characterized by lightly pressing the AFM tip (set point 0.002 V) to the top of the pili and applying a quadruplicate amplitude of ±0.6 V voltage sweep at a frequency of 0.99 Hz, receiving ca. 8,000 points of reference per measurement. Pili expressed in G. sulfurreducens were further analyzed with four probe conductivity measurements on films of pili purified from the cells as previously described 25.