Electron Communication of Bacillus subtilis in Harsh Environments.

Elucidating the effect of harsh environments on the activities of microorganisms is important in revealing how microbes withstand unfavorable conditions or evolve mechanisms to counteract those effects, many of which involve electron transfer phenomena. Here we show that the non-acidophilic and non-thermophilic Bacillus subtilis is able to maintain activity after being subjected to extreme temperatures (100°C for up to 8 h) and acidic environments (pH = 1.50 for over 2 years). In the process, our results suggest that B. subtilis utilizes an extracellular electron transfer as an electron communication pathway between B. subtilis and the environment that involves the cofactor nicotinamide adenine dinucleotide as an essential participant to maintain viability. Elucidation of the capability of the non-acidophilic and non-thermophilic strain to maintain viability under these extreme conditions could aid in understanding the cell responses to different environments from the perspective of energy conservation pathways.

Introduction

Whether a microorganism is autotrophic or heterotrophic, free living or obligate parasite, energy generation is essential for the cell to survive (Hernandez and Newman, 2001). Energy metabolism is dominated by oxidation-reduction reactions, in which electron transfer plays a fundamental role, supplying the reducing power and maintaining the intracellular redox balance through the regeneration of the redox cofactor nicotinamide adenine dinucleotide (NAD) (Li et al., 2018, Nealson and Rowe, 2016). Extracellular electron transfer (EET) is the process by which electrons generated by microbial metabolism are transported to extracellular substrates that act as electron acceptors. Different EET mechanisms have been identified, including direct electron transfer via redox proteins, such as membrane-bound c-type cytochromes (Pirbadian et al., 2014, Reguera et al., 2005), or indirect electron transfer via secreted redox molecules, such as flavins (Marsili et al., 2008), phenazines (Wang et al., 2010), and quinones (Sasaki et al., 2014).

Harsh environments will, in general, denature proteins and suppress microbial activity, and even subtle changes in the structure of sensitive proteins may result in loss of the cell's ability to communicate with the environment. Bacteria are often subjected to various environmental stresses, among which the most important variables are temperature and pH. Both strongly affect bacterial metabolism and electron transfer (Cournet et al., 2010), but the latter has not been studied in detail for bacteria at high temperatures and low pH conditions. Elucidation of the mechanisms involved in EET has been the subject of widespread attention, as it is essential in the understanding of natural processes such as biogeochemical cycling as well as in the development and optimization of many applications, ranging from biofuel production to bioelectrochemical systems (Chen et al., 2012, Collier and Mrksich, 2006, Lan et al., 2018, Marsili et al., 2008, Nielsen et al., 2010, Pfeffer et al., 2012, Wang et al., 2018, Wu et al., 2018). Hence, it is imperative to understand how microorganisms manage to retain electron transfer capabilities between intracellular and extracellular environments under extreme conditions.

B. subtilis is an aerobic, gram-positive bacterium, with an ample metabolic repertoire, is widely present in soil and aquatic environments, and is a key player in environmental processes and in applications in processes of medical and biotechnological interest (Beauregard et al., 2013). B. subtilis provides an accessible model for investigating the response of electron transfer of gram-positive bacteria to environmental stress. Sporulation initiates in response to harsh environment through a six-stage process that lasts approximately 8 h. Even though spores have very low metabolic activity (Church and Halvorson, 1957, Ghosh et al., 2015, Segev et al., 2012), they still have to possess the ability to communicate with the environment to initiate germination and become a vegetative cell. So we conjecture that EET is a pathway used by B. subtilis to communicate with the environment as this knowledge will also contribute to elucidate the EET pathway in gram-positives in general. Despite the large corpus of metabolic and physiological studies, there is limited information on energy metabolism and EET when non-acidophilic and non-thermophilic B. subtilis is subjected to harsh conditions.

In this work, we analyzed the electrochemical activity of B. subtilis under two conditions: low pH and high temperatures. In a series of controlled experiments, we assessed the electrochemical activity of B. subtilis kept at pH = 1.50 over 2 years, whereas in another series of experiments we evaluated the electrochemical activity of B. subtilis in suspensions incubated at 100°C for various periods. Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and chronoamperometry (CA) were used to determine the electrochemical activity and the EET ability of B. subtilis, whereas ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF MS) was employed to identify low-molecular-weight redox molecules. Our results show that the electrogenic activity of B. subtilis remains after long-term exposure to harsh environmental conditions, and they also demonstrate the role of NAD in the EET mechanism.

Results

Redox Activity after High-Temperature Treatment

To investigate the effect of high temperature on electron transfer between the microbial cells and electrodes, a series of tests were performed. B. subtilis was incubated at 26°C and 100°C, and its electrochemical activity was assessed by CV (Figure 1). Bare glassy carbon electrode showed no redox peaks (Figure 1, curve a), whereas oxidation peaks at +0.39 V and +0.09 V and reduction peaks at +0.26 V and −0.06 V were observed for B. subtilis incubated at 26°C (Figure 1, curve b). Interestingly, significant redox currents were produced by B. subtilis after treatment at 100°C for 3 h (Figure 1, curve c), but the redox peaks at −0.06 V and +0.09 V disappeared after 8 h at 100°C (Figure 2A). These observations suggest that the mechanism responsible for the changes in current with temperature involves redox compounds or proteins that retain electrochemical activity after being subjected to high temperatures (Figure 2B).

Figure 1

Cyclic Voltammogram of B. subtilis Subjected to Different Temperature Treatments for 3 h

Curve a: bare glassy carbon electrode. Curve b: B. subtilis incubated at 26°C. Curve c: B. subtilis incubated at 100°C.

Figure 2

Effect of Exposure Time on the CV Peak Currents for B. subtilis Treated at 100°C

(A) (1) Oxidation current at +0.09 V, (2) reduction current at −0.06 V.

(B) (3) Oxidation current at +0.39 V, (4) reduction current at +0.26 V. Redox activity in conditions at differing pH values.

Although temperature has a significant effect on bacterial physiology and metabolism, other environmental factors, such as pH, severely affect bacterial functions. To further elucidate the effect of harsh environments on electron transfer, we studied the redox activity of B. subtilis at various pH values also. Experiments were carried out over a range of pH values in the range 1.50–12.00. When B. subtilis is grown in the medium used in the experiments, the pH of the culture reaches a stable value of 4.68, so we first use this condition for the electrochemical tests. At pH = 4.68, redox peaks were observed at +0.39 V and +0.26 V and at +0.09 V and −0.06 V, whereas at pH = 1.50 these redox peaks showed a positive shift (Figure 3). The shift in the negative direction upon an increase in pH value is consistent with the involvement of protons in the reaction studied, as indicated by the Nernst equation (Table S2). The electrochemical activity of the redox peaks at +0.39 V and +0.26 V remained stable within the pH range 1.50–10.60, but no redox peaks were observed at pH = 12.00 (Figure S5). These results suggest that extreme alkaline conditions may cause inhibition of the redox substance. However, this inhibition seems to be reversible; when the electrode was placed again in pH = 4.68 buffer after electrochemical testing at pH 12.00, all the redox peaks reappeared at the same positions as in earlier experiments. This feature may play a role in the mechanisms that ensure the survival of B. subtilis subjected to extreme conditions.

Figure 3

Cyclic Voltammograms of B. subtilis at Extreme Acidic Conditions for 736 Days

Electron communication of B. subtilis.

To investigate the long-term stability in acidic conditions, B. subtilis was suspended in pH = 1.50 buffer for 736 days. After this prolonged treatment, the low-pH redox peak currents at +0.57 V and +0.41 V and at +0.12 V and +0.06 V were still present (Figures 3 and S6). These data showed that B. subtilis redox ability remains stable even after very long-term exposure to low-pH conditions.

Our data show that B. subtilis is able to maintain the redox ability after treatment at high temperature and for long term in low pH, but it cannot directly demonstrate the presence of EET, i.e., the redox reaction of DPV or CV does not always mean the active state of live bacteria. To this end, CA was used to evaluate the effect of acetate as a substrate on B. subtilis, and because only viable bacteria or spores can respond to acetate, the data will confirm live B. subtilis contributing to current change.

Based on the CV data, potentials at +0.15 V and +0.50 V were chosen to perform CA analysis. When the potential was held at +0.15 V, the current due to the oxidation reaction at +0.09 V changed when acetate was added to the system, i.e., we held +0.15 V to determine if the electrons from acetate degradation are able to transport at +0.09 V (not at +0.39 V). In control experiments with a bare glassy carbon electrode, the stable current is 0.50 nA at 1800 s. Upon addition of acetate to the system, an increase in current of 0.10 nA was observed, caused by the perturbation of the electrolyte. In the experiments with the B. subtilis electrode, the stable current was 1.50 nA, which increased to 3.50 nA after addition of acetate (Figure 4A). This increase could be explained by changes in the bacterial capacitance, suggesting that the oxidation peak at + 0.09 V has a limited contribution to the EET pathway.

Figure 4

Current Responses of B. subtilis to Addition of 30 mM Acetate in pH 4.68 Phosphate Buffer

(A) The potential for CA measurements (not stirred) is +0.15 V versus Ag/AgCl.

(B) The potential for CA measurements (not stirred) is +0.50 V versus Ag/AgCl. The arrow indicates addition of 200 μL sodium acetate solution (inset: current response of a bare electrode).

When the potential is held at +0.50 V, a current change may be caused by the two oxidation reactions, i.e., at +0.09 V and +0.39 V, or only one of them. As a control, a stable current of 2.90 nA was observed at 2,120 s in the glassy carbon held at +0.50 V, which increased by ca. 0.50 nA after addition of acetate. However, the current measured in the B. subtilis electrode was 24 nA, increasing to 39 nA upon addition of acetate (Figure 4B). In the B. subtilis electrode system, the current measured at +0.50 V is therefore 10 times higher than that at +0.15 V. Compared with the data with B. subtilis at +0.15 V, the redox reaction at +0.39 V plays a major role in the observed current increase, strongly suggesting that the EET pathway in B. subtilis involves the oxidation peak at +0.39 V. Hence the redox substance involved at +0.39 V needs to be identified.

Lysozyme test was used for verifying whether the redox peaks come from vegetative cells, spores, or both. Spores are resistant to lysozyme, whereas vegetative cells are disrupted by the enzyme. Lysozyme does not show any redox activity in this condition as shown in the Figure 5, curve a. After lysozyme treatment the remnant of cell debris or contents of cellular materials enter into the supernatant, and their electrochemistry activity cannot be detected, so the electrochemistry signal originates from B. subtilis. In Figure 5, curve b, only the redox peaks at −0.06 and +0.09 V are observed in the lysozyme-treated supernatant, suggesting that the peaks at −0.06 V and +0.09 V may be due to the redox activity of the spores, whereas the disappearance of the peaks at +0.39 V and +0.26 V indicates that these originate by the redox activity of intact vegetative cells.

Figure 5

Cyclic Voltammograms of Lysozyme Treated B. subtilis

Cyclic voltammograms of lysozyme at bare glassy carbon electrode (curve a) and lysozyme-treated B. subtilis supernatant (curve b).

The results in Figure 6 show that both supernatant and pellet present the same redox peaks after treatment. All the redox peaks were observed in the supernatant after a high-temperature or acid treatment (Figure S7), suggesting that a molecule with redox activity is secreted to the extracellular medium. To avoid interferences, the identification of redox substances was performed only in supernatants. After exposure to low pH for 736 days, the electrochemical response shown by B. subtilis implies the existence of an electron transport that permits the adaptation of the microorganism to harsh environments.

Figure 6

Cyclic Voltammograms of B. subtilis Pellets and Supernatant

Cyclic voltammograms of pellets and supernatant. B. subtilis were centrifuged at 5,000 g for 10 min to obtain the pellets and then washed three times, and the resuspended pellets were then incubated for 5 h at 26°C: pellets (black), supernatant (red). Identifying redox substances of B. subtilis.

To identify redox substances that might be involved in EET, we analyzed the composition of the supernatant by UPLC-Q-TOF MS. The fragment ion at m/z 663.4566 corresponds to NAD+, whereas the fragment ion at m/z 685.4349 corresponds to NAD+-H + Na, In Figure 7B NAD+ ion intensity is slightly lower than that in Figure 7C, compatible with NAD+ being a heat-stable molecule (Chini et al., 2017) with higher stability in acidic conditions than at pH = 7.00 (Gorton and Domínguez, 2007). The fragment ions at m/z 664.4574 and 665.4590 correspond to NAD+ isotopic peak NAD++H and NAD++2H, respectively; The fragment ions 686.4415 and 687.4434 correspond to NAD+-H + Na isotopic peaks NAD+ +Na and NAD+ +Na + H, respectively. These results are consistent with the mass spectrum observed using NAD+ as standard (Figure S8).

Figure 7

UPLC-Q-TOF-MS Analysis of B. subtilis Supernatants

(A–C) UPLC-Q-TOF-MS analysis of supernatants of (A) B. subtilis resuspended in ultrapure water for 5 h at 26°C, (B) B. subtilis resuspended in ultrapure water for 5 h at 100°C, and (C) B. subtilis resuspended in pH 1.50 buffer for 5 h at 26°C.

To confirm the participation of NAD+ in the electrochemical activity of B. subtilis, we performed DPV tests in supernatants spiked with NAD+ standard (Figures 8 and S9). The supernatant showed an obvious oxidation peak at +0.30 V. After addition of 20 μM NAD+ at pH = 4.68, the current at the peak increased. The combined data from MS and DPV strongly suggest that NAD+ is indeed involved in EET.

Figure 8

DPV Analysis of Redox Substances

(A and B) (A) The oxidation peak of supernatants from B. subtilis resuspended for 5 h at 26°C, pH 4.68 and (B) the oxidation peak of the supernatants from B. subtilis resuspended in pH 1.50 PBS for 5 h. All results are from a minimum of three biological replicates. Experiments were performed under N2(G).

Our results show that the gram-positive B. subtilis is able to maintain electron transfer activity after being subjected to extreme temperatures and acidic environments for very long times. The results observed after exposure at low pH suggest that the bacteria are able to keep their electron communication pathways active, adapting to the acid environment to maintain their viability. Our analysis suggests that the redox peaks at +0.39 V and +0.26 V can be assigned to NAD+/NADH, −0.06 V and +0.09 V being assigned to cytochrome c, and that the EET in B. subtilis involves NAD as an essential participant in the process.

Discussion

High-Temperature Effect

Most Bacillus species present a certain degree of thermostability and are able to respond to environmental stresses by triggering metabolic and physiological changes. One of these involves the release of extracellular polymeric substances (EPS). EPS can be proteins, nucleic acids, lipids, or other biopolymers, with some of the proteins presenting enzymatic activities and redox functions (Bengtsson et al., 1999, Los and Murata, 2004, Morokutti et al., 2005, Rothschild and Mancinelli, 2001, Xiao et al., 2017). The redox peaks observed at −0.06 V and +0.09 V in Figure 1 (curve b) may be attributed to membrane-bound redox proteins, as we have previously reported (Xiao et al., 2017). Raman spectroscopy revealed cytochrome c at the surface of bacteria (Table S1). The addition of EDTA, a known Fe3+ chelator, caused the inactivation of cytochrome c (Figure S1). Finally, SDS-PAGE analysis shows a band of molecular weight approximately 38 kDa (Figure S2), which may correspond to subunit II of cytochrome c (Bengtsson et al., 1999). These results are consistent with the structural features of cytochrome c.

Heating can enhance molecular movements, which may accelerate EPS disaggregation or dissolution (Figure S4). This could facilitate direct contact between membrane-bound cytochrome c and the electrode, thus enabling electron transfer. However, increasing exposure time at 100°C leads to decreased peak currents, which then disappeared at long-term exposure. This may be explained by the fluidization of membranes caused by high temperatures and the consequent disintegration of the lipid bilayer (Los and Murata, 2004, Rothschild and Mancinelli, 2001), resulting in the decline of the redox activity of cytochrome c.

Low-pH Treatment

Under stress conditions, B. subtilis is able to initiate many survival mechanisms such as motility, uptake of exogenous DNA, and sporulation (Tan and Ramamurthi, 2014). When Bacillus is under extreme nutrient deprivation, it differentiates into spores, extremely resistant to potentially damaging environmental conditions (Barney and Austin, 2017). In the case of low-pH stress, to maintain metabolic activity, bacteria need to keep pH homeostasis. This mechanism requires more energy than in neutral pH environment, provided by the activity of the electron transport chain (Lund et al., 2014). Low pH can trigger spore germination and initiation of vegetative growth in B. subtilis (Wilks et al., 2009) and is linked to an increase in NADH oxidase activity in germinated spores and in the upregulation of NAD(P)-dependent dehydrogenases. These phenomena accelerate electron transfer and the pumping of protons out of the cell to maintain internal pH homeostasis (Wilks et al., 2009).

Electron Communication Pathway

In an attempt to elucidate the electron transport mechanism, we performed experiments to identify the redox molecule(s) involved in EET. B. subtilis can secrete flavins (Morokutti et al., 2005), which have been reported as electron transfer mediators for other bacteria, e.g., Shewanella. However, in this study, flavins may not appear to make a significant contribution to electron transfer because the culture medium contains Cu2+, which is known to form a stable complex with flavin through d-π back donation and may make inactive the redox reaction of flavin. When CuSO4 and guaiacol are added to lysogeny broth medium, flavin was not detected in our experiments.

Most energy-producing processes require coenzymes such as NAD, a highly abundant cellular component of bacteria that participates in electron transfer during oxidation-reduction reactions, which convert the oxidized form NAD into NADH, a strong reducing agent (Chini et al., 2017, Kido et al., 2015). NAD is oxidized or reduced by the loss or gain of two electrons, in reactions involving the removal of two hydrogen atoms (a “hydride ion” and a proton). NAD also regulates numerous NAD+/NADH-dependent enzymes, including dehydrogenases. NADH and NAD+ can be transported across cell plasma membranes, and it has been suggested that extracellular NAD+ may act as a signaling molecule (Ying, 2006). NAD+ is a heat-stable molecule (Chini et al., 2017), presenting higher stability in acidic conditions than at pH = 7.00 (Gorton and Domínguez, 2007), The nicotinamide moiety in NAD presents a planar structure, whereas it is puckered in NADH (Fjeld et al., 2003). The difference in structure leads to inhibition of the redox function at extreme alkaline conditions, which is consistent with the results observed in the CV at different pH values (Figure S5).

The MS results shown above suggest that NAD+ was secreted into the solution. Temperature not only influences bacterial growth rate, enzyme activity, cell composition, and nutritional requirements but also has effects on the solubility of solute molecules, ion transport and diffusion, osmotic effects on membranes, surface tension, and electron transfer (Beales, 2004). NAD is present in Bacillus vegetative cell and spores (Setlow and Setlow, 1977). B. subtilis spores show resistance to high temperature, and NAD+ is a small heat-stable molecule (Chini et al., 2017). NAD, a key class of cofactors, serves as essential electron donor or acceptor in all biological organisms (Liu et al., 2018) and drives major catabolic and anabolic reactions to maintain cellular redox homeostasis and energy metabolism (Xiao et al., 2018). NAD(H/+) is a considerable source of the intracellular electron pool from which intracellular electrons are transferred to extracellular electron acceptors via EET pathways (Li et al., 2018).

Intact bacteria can release NADH into the extracellular medium, and up to 70% of NADH release occurs during the exponential growth phase (Wos and Pollard, 2009). Secretion of NAD to the extracellular milieu has been reported in diverse species, such as E. coli, Rhodobacter capsulatus (Ying, 2006), and microorganisms in activated sludge (Wos and Pollard, 2009), as well as by wood-degrading fungi and animal cells (Kido et al., 2015, Xiao et al., 2018). The redox activity of this molecule suggests its participation in efficient electron transport and extracellular turnover of NADH to NAD+ (Wos and Pollard, 2009).

The study of EET is essential owing to its importance in biological fuel cells, biogeochemical cycling, and bioremediation processes. Elucidation of the capability of B. subtilis to maintain viability under extreme conditions could help in understanding bacterial responses to different environments from the perspective of energy conservation and electron transfer.

Limitations of the Study

In harsh environments, we studied the electron communication between B. subtilis and environment and finally identified NAD as an essential participant in this process. Further work is needed to confirm if cytochrome c plays a key role in direct electron transfer. Moreover, flavins may appear to make a contribution to electron transfer when the culture medium of B. subtilis does not contain Cu2+.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.