In the late 1980's, Shewanella oneidensis MR-1 (Myers and Nealson, 1988a) and later several species of Geobacter (Lovley et al., 1993; Champine et al., 2000) were shown to be capable of electron transfer to insoluble electron acceptors (IEAs), such as insoluble metal oxides and/or charged electrodes: a process called extracellular electron transport (EET) (Myers and Nealson, 1988b; Venkateswaran et al., 1999; Bond and Lovley, 2003). This ability attracted considerable interest with regard to biogeochemical cycling, bioremediation, corrosion, nano-materials processing, and energy production (Bretschger et al., 2007; Kan et al., 2011; Hsu et al., 2012). While several groups of microbes are known to be capable of EET, major mechanistic studies have been done with only two model systems, Shewanella (Fredrickson et al., 2008; Shi et al., 2009), and Geobacter (Lovley et al., 2004).

Thus far, more than 100 other strains of Shewanella have been isolated from a wide variety of habitats including open water column, sandstone shale, marine and fresh water sediments, oil-pipelines, oil brine, and even algal communities atop Antarctic Ice (Hau and Gralnick, 2007). The genomes of more than 20 of these species have been fully sequenced (Fredrickson et al., 2008). Several of these species have been shown to be capable of EET to IEA, including S. oneidensis MR-1, Shewanella putrefaciens CN32, S. sp. ANA-3, S. sp. W3-18-1, Shewanella amazonensis SB2B, Shewanella frigidimarina NCIMB 400, and Shewanella loihica PV-4 (Fredrickson et al., 1998; Venkateswaran et al., 1998; Gao et al., 2006; Bretschger, 2008). Other members of the Shewanella genus such as Shewanella baltica OS217 and Shewanella denitrificans OS155 (Table 1) are not capable of EET (Brettar et al., 2002). Of all these strains, the congregation in response to IEA has only been studied for MR-1.

Within the genus Shewanella, the EET mechanism of MR-1 has been the most extensively characterized. MR-1 employs several approaches for insoluble IEAs reduction: (1) direct EET via extracellular multiheme cytochromes (Beliaev and Saffarini, 1998; Myers and Myers, 2001, 2002; Meyer et al., 2004; Mitchell et al., 2012; Kracke et al., 2015) (Figure 1A); (2) mediated EET using soluble electron shuttles bound to membrane cytochromes (Lovley et al., 1996; Marsili et al., 2008; Li et al., 2012; Kotloski and Gralnick, 2013; Okamoto et al., 2014); (3) mediated EET utilizing conductive outer membrane extensions that contain cytochromes (Gorby et al., 2006; El-Naggar et al., 2010); and (4) conductive extracellular matrices containing conductive and semiconductive minerals (Kato et al., 2010). Several genes have been identified in strain MR-1 and shown to be essential for EET (Figure 1A and Table 2), including the tetraheme cytochrome c cymA (SO_4591) and the combination of mtrBC/omcA (SO_1776, SO_1778, and SO_1779) that code for the decaheme cytochrome c component and tetraheme cytochrome c necessary for reduction of several anaerobic electron acceptors, including metal oxides (Myers and Myers, 2001, 2002; Schwalb et al., 2003). Because all these mechanisms rely on the cell proximity to IEA for EET, it is important to understand the cell sensing and net swimming migration toward the IEA. With regard to congregation, redox taxis or energy taxis, many studies have been conducted on MR-1 due to its versatile electron acceptor utilization (Bencharit and Ward, 2005; Baraquet et al., 2009; Harris et al., 2010). Energy taxis is a term that broadly encompasses aerotaxis, phototaxis, redox taxis, taxis to alternative electron acceptors, and chemotaxis to oxidizable substrates (Alexandre et al., 2004).

It is well-documented that the accumulation of MR-1 cells in response to soluble electron acceptors is a form of energy taxis, which depends on H⁺ flux and the establishment of a proton motive force (Baraquet et al., 2009) (Figure 1). A part of this response includes more rapid swimming, as also seen with electron shuttles such as riboflavin or anthraquinone 2.6-disulfonate (AQDS) (Bencharit and Ward, 2005; Harris et al., 2010; Li et al., 2012). In contrast, the accumulation of cells around IEA, which has been called congregation (Nealson et al., 1995), involves both increased swimming speed upon contact with the IEA (called electrokinesis), and increased swimming reversals upon a decrease in PMF. Swimming reversals allow multiple transient cell-IEA encounters (lasting for 1–100 ms), and the rate at which swimming cells transition to irreversible attachment to IEA during congregation has not yet been quantified.

The mechanism(s) that cells utilize to locate IEAs remain unclear (Nealson et al., 1995; Bencharit and Ward, 2005; Harris et al., 2010). Early reports proposed that the other studied model organism Geobacter, accumulates around IEAs by sensing a gradient of reduced metal ions (Childers et al., 2002), however, reduced metal ions are not involved with the sensing mechanism used by MR-1 (Bencharit and Ward, 2005). Bacterial congregation in response to poised electrodes was recently described, pointing to the redox sensing, rather than metal ion sensing, as the trigger for response (Harris et al., 2010). The positive applied potentials to electrode (200–600 mV vs. Ag/AgCl) caused MR-1 to congregate similar to that seen with metal oxides (Harris et al., 2012). Redox potentials of MnO₂ containing minerals range between 400 and 600 mV vs. Ag/AgCl and accept electrons more readily than Fe(OH)₃ minerals, which carry the equivalent poised potential of 100 to 300 mV vs. Ag/AgCl (Burdige, 1993). In a previous study, the characteristic swimming of MR-1 around IEA was hypothesized to be regulated by two self-sensing chemotaxis receptors, methyl-accepting proteins (MCPs) with, Ca⁺ channel chemotaxis receptor (Cache) domain (SO_2240) and Per/Arnt/Sim (PAS) domains (SO_1385), and by the chemotaxis signal transduction protein kinase CheA-3 (SO_3207) to allow the cell to sample the redox potential, or electron accepting ability of a surface (Table 2 and Figure 1B) (Harris et al., 2012). Before this study, the genes responsible for the motility driven attachment of cells on and around IEA were unknown.

In this report, we study the congregation mechanism of different Shewanella strains by monitoring swimming patterns and cell attachments to MnO₂ and Fe(OH)₃. In addition to screening the WT and several deletion mutants of MR-1, other Shewanella species (see Table 1) were screened. Many strains were shown to be capable of congregation around both MnO₂ and FeOH₃, while others responded selectively to MnO₂ (SB2B) or Fe(OH)₃ (W3-18-1). When cell attachment to the mineral surfaces was monitored we observed a strong correlation between the ability of the cell to congregate, and the attachment of the cells to the IEA surface. We then compared the genomes of these species to find candidate genes involved in the congregation swimming, accumulation, and cell attachment phenotypes in response to specific IEA surface.

Members of the genus Shewanella are comprised of heterotrophic, facultative aerobes capable of utilizing a wide range of organics and inorganics as energy sources. Many Shewanella strains have been isolated from water column and sediment habitats in locations all across the globe. In addition to soluble electron acceptors, many of these organisms can respire a wide assortment of naturally occurring insoluble metal oxides under anoxic conditions.

In our study of congregation behavior in eight Shewanella species, we used cell tracking with computer analysis and time series assay of cell attachment to MnO₂ or Fe(OH)₃ to demonstrate that five out of eight shewanellae were capable of this behavior CN32, ANA-3, W3-18-1, SB2B, and PV-4) and that some strains (W3-18-1, SB2B) show a preference for one metal oxide over the other under these conditions (Figure 4). Such findings are consistent with the notion that these bacteria have adapted to the prevalent insoluble electron acceptor found in the habitat from which they were isolated. Furthermore, three Shewanella strains that were isolated from the water column did not congregate in response to IEA. Whether these differences derive from the absence of genes involved in EET (as in the OS217) or other reasons will be a point for future studies.

In other studies, overnight growth of the Shewanella strains comparing reduction rates of various metal oxides in head to head comparison, have been shown to reduce metal oxide preferentially from their environmental niche (Bretschger et al., 2007). The results presented here are consistent with the hypothesis that Shewanella species have evolved a congregation and attachment behavior consistent with the environments from which they were isolated (Table 3). For example, W3-18-1 seems to reveal significant inclination for congregation in the presence of and attachment to Fe(OH)₃ minerals native to Pacific continental shelf (Harris et al., 2012) while SB2B prefers MnO₂ (Venkateswaran et al., 1998) similar to that of the Amazon river sediment.

The genomic comparison in Table 1 suggests that the presence of octaheme cytochrome (SO_4142) may be important for swimming in response to relatively “low” redox potentials (100–300 mV vs. Ag/AgCl) of Fe(OH)₃. While mtrF (SO_1780) may be needed for response to relatively “higher” redox potentials of MnO₂ (between 400 and 600 mV vs. Ag/AgCl). This hypothesis could then be tested with deletion mutants in MR-1. This work complements previous work of Harris et al. (2012), by revealing that genes involved with the net effect of the motility behavior toward IEA minerals or electrodes is cumulative through relevant time scales of 1–30 min (Figure 2) (Harris et al., 2012). Our findings with CAAP confocal microscopy technique, demonstrated that congregation leads to a slow migration of cells toward IEA minerals with eventual attachment. Thus, this behavior is appropriately termed congregation, as it describes the gathering or accumulation of motile bacteria around IEA. We showed here that genes mcp cache and mtrBC/omcA are essential for accumulation and attachment phenotype in MR-1, in addition to being responsible for the characteristic swimming patterns of increased speed and run-and-reversal type behavior that was identified previously (Harris et al., 2012).

The results of the experiments with ΔcymA, ΔmtrBC/omcA, and Δmcp_cache mutants in MR-1 show an inhibition of accumulation, attachment and congregation behavior in response to all IEAs. Therefore, it can be hypothesized that the presence of homologous EET genes (cymA, mtrB, mtrC, omcA), and methyl accepting chemotaxis gene (mcp_cache) determines the phenotypic responses we see in other Shewanella spp. (Table 3). Genetic comparison of strains, which could respond to “lower” redox potential IEA, indicates that peripheral outer membrane octaheme cytochromes (such as SO_4142) may play some role in responding to “lower” redox potential IEA. Hence these observations on sustained swimming around specific minerals corresponded with specific genotypes of the different species.

During many MFC (Kotloski and Gralnick, 2013) and metal reducing batch culture experiments, extracellular electron transfer-mediated energy taxis, or congregation ability, was not measured (Kotloski and Gralnick, 2013). In metal oxide reduction assays the 3D distribution of cells and cell motility could greatly influence metal reduction due to incubator shaker speed, culture flask dimensions, or mixing. Congregation behavior directly influences cell attachment to IEAs. The relationship between congregation and nanowire-like appendage formation is still unknown (Pirbadian et al., 2015), although the motility and congregation parameters are rarely monitored in these studies (Gorby et al., 2005; Reguera et al., 2005). Transforming these core congregation genes (cymA, mtrB, mtrA, omcA, mcp_pas, and mcp_cache) into other bacteria species, with single polar flagellum, may someday improve bioremediation capabilities by being able to induce bacterial attachment and colonization of surfaces that would otherwise be difficult or impossible.

HH: designed and performed the experiments; HH and IS-A: wrote and revised paper; ES, ME-N, and JM: helped design experiments; WT: helped to perform the data analysis; JM and KN: revised the manuscript.