Shewanella oneidensis to control extracellular

University of Minnesota – Twin Cities, St. Paul, MN, USA 55108. 19. * To whom correspondence should be addressed: [email protected]. 20. 21. Page 1 o...
1 downloads 0 Views 2MB Size
Letter pubs.acs.org/synthbio

Engineering a Native Inducible Expression System in Shewanella oneidensis to Control Extracellular Electron Transfer Elizabeth A. West,† Abhiney Jain,† and Jeffrey A. Gralnick*,†,‡ †

BioTechnology Institute and ‡Department of Plant and Microbial Biology, University of Minnesota − Twin Cities, St. Paul, Minnesota 55108, United States S Supporting Information *

ABSTRACT: Shewanella oneidensis MR-1 is a model organism for understanding extracellular electron transport, in which cells transfer intracellular electrons to an extracellular terminal electron acceptor such as insoluble minerals or poised electrodes. Biotechnological applications exploiting the respiratory capabilities of Shewanella species have led to their proposed use in wastewater treatment, bioremediation, and remote sensors. Transcriptional regulation tools can be used to rationally engineer S. oneidensis, optimizing performance in biotechnological applications, introducing new capabilities, or investigating physiology. Engineered gene expression in S. oneidensis has primarily involved the use of foreign regulatory systems from Escherichia coli. Here we characterize a native S. oneidensis pathway that can be used to induce gene expression with trimethylamine N-oxide, then engineer strains in which extracellular electron transfer is controlled by this compound. The ability to induce this pathway was assessed by measuring iron reduction over time and by analyzing anodic current produced by cells grown in bioreactors. KEYWORDS: trimethylamine N-oxide (TMAO), extracellular electron transport, regulation

M

described by Gao et al. are used to understand promoter characteristics to ultimately achieve a desired phenotype.6 A major S. oneidensis engineering aim has been directed at understanding and exploiting its respiratory capabilities. S. oneidensis is able to respire a variety of substrates, including oxygen, fumarate, nitrate, sulfite, dimethyl sulfoxide (DMSO), trimethylamine N-oxide (TMAO), and iron and manganese oxide minerals.1 Shewanella species typically grow at circumneutral pH where some electron acceptors, such as iron and manganese oxide, are insoluble. S. oneidensis has a metal reduction (Mtr) pathway that facilitates electron transfer from the cytoplasm to external substrates, a process known as extracellular electron transport (EET).7,8 At its core, EET in S. oneidensis requires five proteins spanning the inner membrane, periplasm and the outer membrane. Electrons from carbon oxidation enter the quinone pool and reduce the inner membrane-anchored c-type cytochrome, CymA.9 From CymA, electrons travel to the periplasmic c-type cytochrome MtrA via intermediary proteins. The fumarate reductase FccA or the small tetraheme cytochrome CctA are needed for Fe(III) respiration and likely facilitate the transfer of electrons from CymA to MtrA.10 Electrons from MtrA reduce MtrC, an outer membrane c-type cytochrome.11,12 The β barrel protein MtrB facilitates direct interaction between MtrA on the periplasmic side and MtrC outside the cell.13 Optimal iron oxide and

embers of the Shewanella genus thrive in aquatic environments throughout the world and are known for their respiratory versatility.1 In recent years, biotechnological interest in Shewanella species has grown due to their genetic tractability, robust growth in the laboratory, and respiratory capabilities. The ability of Shewanellae to respire extracellular compounds has inspired efforts to demonstrate and enhance their use in microbial fuel cells and bioremediation.1−3 Rational engineering of Shewanella species will be crucial for optimizing their performance in biotechnological applications. Transcriptional regulation is one engineering approach that has been achieved in Shewanella species using non-native promoters, however the use of native expression systems to rationally engineer Shewanella has not been reported.2,3 By increasing the number of genetic regulatory tools, both native and foreign, more sophisticated engineering can be accomplished by combining regulatory components in single species systems or in multispecies constructed communities. Arabinose and arsenic-inducible E. coli promoters have been used to develop S. oneidensis biosensors that produce current in a bioelectrochemical reactor in response to a specific inducer.2,3 The E. coli lac promoter and the hybrid tac promoter have also been used in S. oneidensis.4,5 These systems have been well characterized and utilized in a wide range of organisms. Implementation of native gene expression tools also requires characterization to quantify promoter strength and induction conditions. Reporter systems such as the LacZ system © XXXX American Chemical Society

Received: November 18, 2016 Published: May 31, 2017 A

DOI: 10.1021/acssynbio.6b00349 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Letter

ACS Synthetic Biology

Figure 1. Model of Tor pathway for TMAO respiration in S. oneidensis. A complete description of the genes and proteins can be found in the text.

been found in association with fish and fish spoilage and S. oneidensis has been shown to respire TMAO to trimethylamine (TMA) using the Tor pathway (Figure 1).23 The periplasmic protein, TorT likely binds TMAO and activates the histidine kinase, TorS.24 TorS activates the response regulator, TorR, that binds as a dimer to hexameric repeats in the promoter regions of torR, torF, and the torECAD operon.24,25 The torECAD operon is highly induced in the presence of TMAO and encodes the proteins needed for TMAO respiration, including: a membrane anchored c-type cytochrome (TorC), a terminal reductase (TorA), and a TorA specific chaperone (TorD).25 Electrons from lactate oxidation enter the ubiquinone pool and reduce TorC.26,27 TorA oxidizes TorC and transfers the electrons to TMAO. torE encodes a small (56 aa) protein that is needed for optimal TMAO respiration, however its exact function is unknown.28 torF encodes a protein that is predicted to localize to the outer membrane and while torF is highly upregulated in the presence of TMAO, its function is unknown.25 The torECAD and torF promoters were characterized using single-copy genomic integrated constructs aerobically and anaerobically in rich and minimal medium to understand promoter strength and induction conditions with varying TMAO concentrations. Expression levels in wild type cells, where inducer may be depleted via respiration, were compared to levels in mutants that are unable to respire TMAO. The results obtained from these characterization studies were then used to engineer S. oneidensis to control mtrCAB expression with TMAO. The ability to control EET with TMAO in the engineered strain was demonstrated by inducing Fe(III) reduction, and by inducing current production on a positively poised electrode.

electrode respiration requires flavins, which have been hypothesized to act as soluble electron shuttles and/or cofactors for outer membrane multiheme c-type cytochromes.8,14−17 Respiration of insoluble substrates and anodic current production requires the Mtr pathway.4,7,8 The ability of Shewanella and other species to perform EET and interface with an electrode has garnered interest from the biotechnology sector. Bioelectrochemical systems (BESs) show promise for wastewater treatment, desalination, powering of remote sensors, electrosynthesis, and studying the physiology of an organism.1,18−20 In a BES, an organism interacts with an electrode by either transferring electrons from carbon oxidation to the electrode, producing current in the process, or incorporating electrons from the electrode into biosynthesis. For certain applications, it may be necessary to control when the cells interface with the electrode and produce or draw current. Inducible S. oneidensis promoters derived from its TMAO respiration pathway present a method to induce gene expression using native components. As regulatory circuitry increases to accomplish complicated sensing regimes, native systems may present an important addition to the diversity of genetic circuits that can be applied to a given system. An additional advantage of a TMAO-based regulatory system is that the signal is consumed over time in a genetic background capable of TMAO respiration. Finally, we expect native systems to exhibit less off-target regulatory effects due to coevolution with its genome over time compared to foreign regulatory proteins that may interact unexpectedly with native regulatory pathways or bind promiscuously to undesired targets in the genome. High concentrations of TMAO are found in the tissues of some marine organisms where it protects their proteins from osmotic and hydrostatic stresses.21,22 Shewanella species have B

DOI: 10.1021/acssynbio.6b00349 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Letter

ACS Synthetic Biology



RESULTS AND DISCUSSION Eliminating Inducer Depletion. Regulating gene expression with TMAO-inducible promoters in an organism that respires TMAO poses the problem of inducer depletion. Decreased TMAO levels over time could lead to transient and differential transcriptional induction over time. We have observed that TMAO is utilized in parallel with a variety of electron acceptors (data not shown), further necessitating construction of a strain unable to respire our inducing molecule. Although the ability to induce and control shortterm expression changes could be useful in certain applications, a more stable system with continually induced transcription was also desired. In order to ensure constant inducer levels, torECAD was deleted to prevent TMAO respiration to TMA. However, when the ΔtorECAD mutant was grown anaerobically with TMAO as the electron acceptor, growth was observed, albeit more slowly than wild type (Figure 2).

remaining activity in a torA mutant was due to the DMSO respiratory pathway.28 Deletion of the DMSO respiration operon, dmsEFABGH, in the ΔtorECAD mutant abolished growth with TMAO as the electron acceptor. Deletion of only the genes needed for DMSO respiration did not impair growth on TMAO, confirming the Tor pathway is the primary route for TMAO reduction (Figure 2). The torECAD and torF Promoters Are Induced over a Range of TMAO Concentrations Aerobically and Anaerobically. The torECAD and torF promoters had previously been identified as part of the TorR regulon in S. oneidensis.25 To characterize the behavior of these promoters we drove expression of a chromosomal insertion of lacZ into three genetic backgrounds: wild type, ΔtorECAD (Δtor), and ΔtorECAD, ΔdmsEFABGH (ΔtorΔdms). The backgrounds differ in TMAO respiration ability where respiration is unimpaired in wild type, impaired in Δtor and eliminated in ΔtorΔdms (Figure 2). Miller assays were used to measure promoter activity by quantifying β-galactosidase, the enzyme encoded by lacZ. Expression levels were measured over a range of TMAO concentrations in minimal medium (SBM) and rich medium (LB), aerobically and anaerobically. PtorECAD and PtorF were induced by ∼300 μM TMAO in SBM. Expression increases with higher TMAO concentrations and plateaus at ∼1 mM TMAO (Figure 3). In LB, 30 μM TMAO is sufficient for inducing transcription by either promoter (Figure S1). Expression driven by the torECAD promoter is higher anaerobically than aerobically for each genetic background tested (Figure 3B). In the absence of TMAO, PtorECAD drives low levels of expression (Miller Unit ∼100). PtorECAD activity is not significantly different between the three genetic backgrounds, except anaerobically in SBM, where expression in the ΔtorΔdms mutant is slightly lower than the other backgrounds (Figure 3B). Unlike the torECAD promoter, the torF promoter has significantly lower expression in the WT background (Figure 3A). The lower activity in a TMAO-respiration proficient background by the PtorF promoter suggests that this promoter requires higher concentrations of TMAO to achieve robust induction. Because the WT background actively is removing TMAO, we observe less expression in this genetic background than in the background impaired in TMAO respiration. Replacement of the Native mtrCAB Promoter with PtorF Enables Induction of Extracellular Electron Transport with TMAO. PtorF was chosen for use in additional studies

Figure 2. Generating a strain of S. oneidensis unable to utilize TMAO requires elimination of both TMAO and DMSO respiratory pathways. Growth of wild type (black, ●), ΔdmsEFABGH (green, ■), ΔtorECAD (purple, ▲), and ΔtorECADΔdmsEFABGH (blue, ◆). OD600 was measured over time for anaerobic SBM cultures with 20 mM lactate and 40 mM TMAO. Error bars represent standard error of the mean (SEM) from three independent experiments performed in triplicate.

Although S. oneidensis has distinct pathways for DMSO and TMAO respiration, a previous study reported that while TorA was the primary TMAO reductase, a second mechanism also existed which facilitated growth in a torA mutant background.26,27 Given that some bacteria have a single pathway for TMAO and DMSO respiration, we hypothesized that the

Figure 3. Quantitation of TMAO-responsive promoters using LacZ activity. Miller assay data for strains with the PtorF::lacZ fusion (A) and PtorECAD::lacZ fusion (B) in the following genetic backgrounds: WT (red, ●), Δtor (blue, ■), and ΔtorΔdms (orange, ▲). Cells were grown in minimal medium (SBM) aerobically (open symbols) and anaerobically with fumarate as the electron acceptor (closed symbols). Error bars represent SEM from three independent experiments performed in triplicate. C

DOI: 10.1021/acssynbio.6b00349 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Letter

ACS Synthetic Biology

Figure 4. Promoter replacement scheme. The native mtrCAB promoter was replaced with the torF promoter by replacing the region 132 bp upstream of the mtrCAB transcriptional start site with the region 132 bp upstream of the torF transcriptional start site.

Figure 5. Iron reduction rate quantitation of engineered PtorF promoter replacements driving expression of mtrCAB. Reduction of Fe(III) citrate (left) and Fe(III) oxide (right) for WT (black, ●), PtorF::mtrCAB, ΔtorΔdms (orange, ◆); PtorF::mtrCAB, Δtor (blue, ▲); PtorF::mtrCAB, WT (green, ■); and ΔMtr (red, ×). Open symbols correspond to no TMAO and closed symbols correspond to addition of 1 mM TMAO. Error bars represent SEM from three independent experiments performed in triplicate (the wild type control was performed in duplicate).

wild type levels. Fe(II) levels are slightly lower in the presence of TMAO for PtorF::mtrCAB in the wild type background, possibly due to decreased expression driven by the torF promoter in the wild type background, consistent with the strain’s ability to remove the inducer via respiration. Fe(II) levels for wild type and ΔMtr were not significantly different in the presence of TMAO (data not shown). In the absence of TMAO, Fe(III) citrate reduction in the PtorF::mtrCAB strains was about 50% decreased compared to wild type and induced PtorF::mtrCAB strains, suggesting some low-level expression from the engineered promoter, or due to read-through from the upstream omcA promoter.29 We favor the latter explanation given the very low levels of LacZ activity we observed prior to induction (Figure 3). Additional features could be engineered

since it is highly induced in the presence of TMAO and drives minimal expression in the absence of TMAO. To demonstrate an application involving a tor promoter, the native mtrCAB promoter was replaced with PtorF in the WT, Δtor, and ΔtorΔdms backgrounds on the genome (Figure 4). To test the ability to induce the process of extracellular electron transport itself, ferrozine assays were used to quantify Fe(III) citrate and Fe(III) oxide reduction by monitoring Fe(II) accumulation over time. Fe(II) levels for induced and uninduced promoter replacements strains were compared to positive (wild type) and negative controls (ΔMtr, JG1453) (Figure 5). Fe(III) citrate reduction primarily occurs by the direct transfer of electrons from MtrC to Fe(III) citrate.4 In the presence of TMAO, Fe(III) citrate reduction in the PtorF::mtrCAB strains was near D

DOI: 10.1021/acssynbio.6b00349 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Letter

ACS Synthetic Biology

about 75% lower than the native promoter strain. Final current output was about 50% lower in the uninduced condition compared to the induced condition. While current production was successfully induced in the ΔtorΔdms mutant with PtorF::mtrCAB, current density was lower than the strain containing the native promoter for mtrCAB. Current plateaus at about 40 h and then slowly begins to drop over the next 60 h (Figure 6). The observed steady decrease is consistent with turnover of cells and turnover of the MtrCAB conduit within cells under conditions where the inducer is no longer present. In order to increase current production in the promoter replacement strains, it may be necessary to supply TMAO throughout the experiment, or supply a higher starting concentration. The reactor is continually flushed with argon gas, and although it is humidified to minimize evaporation, the volatile nature of TMAO may also facilitate it leaving the system. Alternatively, or in addition to, PtorF expression may be influenced differently under the bioreactor conditions compared to experiments using soluble or insoluble Fe(III) (Figure 5). The work presented in this study has expanded the number of transcriptional regulation tools available for use in S. oneidensis, characterized two native TMAO-dependent promoters and provided key background work on the physiology of TMAO respiration. We demonstrated an example of how a TMAO-inducible promoter can be used to control gene expression through promoter replacement with a new inducible, native promoter and characterized activity of MtrCAB in the engineered strain on soluble Fe(III), insoluble Fe(III) and on poised graphite electrodes. Future work will focus on the optimization of this system to control extracellular electron transport for applications in bioelectrochemical sensing.

into this gene cluster including modifications to the native omcA promoter or introduction of a robust terminator downstream of omcA. In the presence of TMAO, Fe(III) oxide reduction was significantly different in the three promoter replacement strains. Lower Fe(II) levels were observed for PtorF::mtrCAB in the wild type background than the mutant backgrounds. Reduction of TMAO to TMA in the wild type and Δtor strains may compete with Fe(III) oxide reduction and result in lower Fe(II) levels than ΔtorΔdms. Slower Fe(III) oxide reduction in the wild type background may also be a result of lower expression levels driven by PtorF as was previously observed with the PtorF::lacZ fusions (Figure 3A). Lower mtrCAB expression levels might impair Fe(III) oxide reduction more than Fe(III) citrate since Fe(III) oxide reduction is a slower process given that the substrate is insoluble and unable to diffuse. Fe(III) oxide reduction was slightly lower in the Δtor background, which may result from diverted electron flux through TMAO reduction via the DMSO pathway. Wild type and JG1453 Fe(III) oxide reduction were not significantly different in the presence of TMAO (data not shown). Inducing Current Production with TMAO in the ΔtorΔdms Mutant with PtorF::mtrCAB. The ability of S. oneidensis to perform EET enables respiration of poised electrodes, resulting in current production. To test the ability of the torF promoter to regulate current output, PtorF::mtrCAB in the ΔtorΔdms background was grown in a three electrode bioreactor. Current produced by the promoter replacement strain in the presence of TMAO was compared to the current produced in the absence of TMAO, and current produced by ΔtorΔdms with PmtrCAB in the absence of TMAO. Current produced by the native promoter strain and the induced promoter replacement strain increased steadily for about 30 h (Figure 6). After 40 h, current produced by the induced strain plateaued, and current produced by the strain with the native promoter continued to increase. The induced promoter replacement strain had a final current output that was



MATERIALS AND METHODS Bacterial Strains and Growth Conditions. Strains used in this study are listed in Table 1. Plates were struck with cells from −80 °C glycerol stocks. Escherichia coli strains were grown at 37 °C on Luria−Bertani (LB) plates supplemented with 100 μg/mL of kanamycin (Km) and 360 mM diaminopimelic acid (DAP) for E. coli WM3064. S. oneidensis strains were struck onto LB plates and grown at 30 °C. Overnight cultures were prepared by inoculating liquid medium with a single colony from a plate. S. oneidensis overnight cultures were either grown in LB or Shewanella basal medium (SBM). SBM was supplemented with 5 mL/L vitamin solution, 5 mL/L mineral solution, and 0.05% (wt/vol) Casamino acids (CAA).30 Sodium lactate was added to a final concentration of 20 mM. For anaerobic cultures, medium was flushed with nitrogen gas, and supplemented with fumarate or TMAO, to a final concentration of 40 mM. Plasmids and Strain Construction. Plasmids used in this study are listed in Table 1 and primers are listed in Supplementary Table 1. In frame gene deletions and insertions were generated as previously described.30 Briefly, 1 kb fragments upstream and downstream of the region to be deleted were amplified by colony PCR. The fragments were ligated and cloned into the suicide vector, pSMV3, which contains a kanamycin resistance cassette and sacB.31 The torF and torECAD promoters have been previously described.25 lacZ promoter fusions were generated by amplifying 161 bp upstream of the torECAD transcriptional start site (TSS) and 132 bp upstream of the torF TSS and ligating the fragments

Figure 6. Current production in the engineered PtorF::mtrCAB strain is induced with TMAO. Chronoamperometry of WT (blue, ●); PtorF::mtrCAB, ΔtorΔdms with 5 mM TMAO (green, ▲); and PtorF::mtrCAB, ΔtorΔdms without TMAO (red, ■). Strains were grown in three-electrode bioreactors with graphite working electrodes poised at 0.24 V vs SHE. Error bars represent SEM from three independent experiments performed in triplicate. E

DOI: 10.1021/acssynbio.6b00349 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Letter

ACS Synthetic Biology

with 75 μL of the resuspension, and filter sterilized TMAO was added to the desired concentration. The cultures were grown aerobically or anaerobically, while shaking, to an OD600 of about 0.1. One hundred microliters of the culture were transferred to Z-buffer lysis solution containing 3.5 μL/mL 2mercaptoethanol, 20 μL/mL chloroform, and 0.002% sodium dodecyl sulfate. Z-buffer has a pH of 7.0 and contains 16.1 g/L Na2HPO4·7H2O, 5.5 g/L NaH2PO4·1H2O, 75 g/L KCl, and 0.246 g/L MgSO4·7H2O. Two hundred microliters of 4 mg/ mL ortho-nitrophenyl-β-galactosidase (ONPG) were added to the lysed cells. Reactions were stopped with 2 M Na2CO3 and centrifuged at maximum speed for 4 min. One milliliter of the supernatant was transferred to a cuvette and the absorbance at 420 nm was measured. Miller units were calculated as follows: (1000·A420)/(OD600·T·V). T represents the number of minutes the ONPG reacted with the lysed cells. V represents the number of milliliters of culture added to the lysis solution. Values represent the average of at least three separate experiments in which each condition was performed in duplicate. Fold inductions were calculated relative to the Miller Units of uninduced cultures. Quantification of Fe(III) Respiration. Resting cell ferrozine assays were used to quantify Fe(II) over time.33 LB cultures were inoculated, grown throughout the day, and then used to inoculate fresh media, with or without 1 mM TMAO. Cultures were grown overnight. Cells from the overnight culture were pelleted, washed once and resuspended in SBM to an OD of 1.0. In 96-well plates, 270 μL of SBM with 20 mM lactate, 5 mL/L vitamins, 5 mL/L minerals, and 5 mM Fe(III) citrate or Fe(III) oxide, was inoculated with 30 μL of the resuspended cells. The plate was incubated in an airtight chamber made anaerobic by degassing for 15 min with nitrogen gas. To quantify Fe(II), 30 μL from the plate were transferred to 270 μL of 0.5 M HCl. Thirty microliters of the stopped reaction were added to 270 μL of 4 mM ferrozine, and the absorbance at 562 nm was determined. Fe(II) concentrations were determined using a standard curve of freshly made ferrous sulfate. Bioreactor Construction and Chronoamperometry. Reactors were assembled as previously described, with minor changes.14 Graphite carbon electrodes were cleaned by sonicating in deionized water twice. The salt bridge was filled with 0.1 M KCl in 1% agarose. 3 M KCl covered the interface between the reference electrode and agarose. Reactors were kept anaerobic by continual flushing with humidified argon gas. The electrodes were poised to a potential of +0.24 V vs SHE (standard hydrogen electrode) and current was measured every 120 s using a 16-channel VMP potentiostat (Bio-Logic, Claix, France). Culturing Conditions for Bioreactors. Fifty microliters from LB aerobic overnight cultures were transferred to anaerobic LB cultures supplemented with 20 mM lactate and 40 mM fumarate. Conditions with TMAO had a final concentration of 1 mM TMAO. Cells were grown to an OD600 of ∼0.5. Bioreactors containing 14 mL of SBM supplemented with 5 mL/L vitamins, 5 mL/L minerals, 0.5% (wt/vol) CAA, 60 mM sodium lactate, and 40 mM sodium fumarate were inoculated with 1 mL from the anaerobic LB culture. TMAO was added to a final concentration of 5 mM when indicated.

Table 1. Strains and Plasmids Used in This Work E. coli strains UQ950

WM3064

genotype/characteristic

reference

DH5α λ(pir) cloning host; F-Δ(argF-lac)169 Φ80dlacZ58ΔM15 gln V44(AS) rf bD1 gyrA96(NalR) recA1 endA1 spoT1 thi-1 hsdR17 deoR λpir+ Donor strain for conjugation; thrB1004 pro thi rpsL hsdS lacZΔM15 RP4−1360 Δ(araBAD)567 ΔdapA1341::[erm pir(wt)]

31

S. oneidensis strains

genotype/characteristic

JG274 JG3266

MR-1 ΔtorECAD

JG3516

ΔtorECADΔdmsEFABGH

JG3278

ΔtorECADF (Δtor)

JG2655

ΔdmsEFABGH

JG3544

ΔtorECADFΔdmsEFABGH (ΔtorΔdms)

JG3332

MR-1 with genomic PtorF::lacZ

JG3333

MR-1 with genomic PtorECAD::lacZ

JG3334

Δtor with genomic PtorF::lacZ

JG3335

Δtor with genomic PtorECAD::lacZ

JG3363

ΔtorΔdms with genomic PtorF::lacZ

JG3364

ΔtorΔdms with genomic PtorECAD::lacZ

JG3459

MR-1 with PtorF::mtrCAB

JG3460

Δtor with PtorF:mtrCAB

JG3631

ΔtorΔdms with PtorF::mtrCAB

JG1453

ΔmtrCAB, ΔmtrDEF, ΔdmsE, ΔcctA, ΔomcA, ΔSO4360 (ΔMtr) genotype/characteristic

plasmids pEB007 pSMV3

pSMV3 derivative used to insert promoter-lacZ fusions into the genome at the attTn7 site Suicide vector used to generate gene deletions

31

reference 34 This Study This Study This Study This Study This Study This Study This Study This Study This Study This Study This Study This Study This Study This Study 35 reference This Study 31

upstream of lacZ in the pSMV3 derivative, pEB007. The promoter fusions were inserted into the attTn7 site that has previously been targeted for gene insertions.32 Use of pEB007 enables insertion of the promoter fusions into a neutral site on the genome that is upstream of glmS. The mtrCAB promoter replacement was generated by ligating the torF promoter region between amplified 1 kb regions flanking the native mtrCAB promoter and inserted into pSMV3. Genomic insertions or deletions were sequenced verified using primers flanking the region of interest. Growth Curves. LB overnight cultures of S. oneidensis were pelleted and washed with SBM twice, and then normalized to an OD600 of 1.0. One hundred microliters of the resuspension were added to SBM supplemented with 20 mM lactate and 40 mM TMAO for a final volume of 10 mL. Cultures were grown anaerobically while shaking at 225 rpm. Growth over time was measured by recording OD600. β-Galactosidase Assays. Overnight LB cultures were pelleted and resuspended to an OD600 of 1.0 in either LB or SBM. Ten milliliter cultures of LB or SBM were inoculated F

DOI: 10.1021/acssynbio.6b00349 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Letter

ACS Synthetic Biology



(11) Pitts, K. E., Dobbin, P. S., Reyes-Ramirez, F., Thomson, A. J., Richardson, D. J., and Seward, H. E. (2003) Characterization of the Shewanella oneidensis MR-1 decaheme cytochrome MtrA: Expression in Escherichia coli confers the ability to reduce soluble Fe(III) chelates. J. Biol. Chem. 278, 27758−27765. (12) Beliaev, A. S., Saffarini, D. A., McLaughlin, J. L., and Hunnicutt, D. (2001) MtrC, an outer membrane decahaem c cytochrome required for metal reduction in Shewanella putrefaciens MR-1. Mol. Microbiol. 39, 722−730. (13) Myers, C. R., and Myers, J. M. (2002) MtrB is required for proper incorporation of the cytochromes OmcA and OmcB into the outer membrane of Shewanella putrefaciens MR-1. Appl. Environ. Microbiol. 68, 5585−5594. (14) Marsili, E., Baron, D. B., Shikhare, I. D., Coursolle, D., Gralnick, J. A., and Bond, D. R. (2008) Shewanella secretes flavins that mediate extracellular electron transfer. Proc. Natl. Acad. Sci. U. S. A. 105, 3968− 3973. (15) Von Canstein, H., Ogawa, J., Shimizu, S., and Lloyd, J. R. (2008) Secretion of flavins by Shewanella species and their role in extracellular electron transfer. Appl. Environ. Microbiol. 74, 615−623. (16) Okamoto, A., Hashimoto, K., Nealson, K. H., and Nakamura, R. (2013) Rate enhancement of bacterial extracellular electron transport involves bound flavin semiquinones. Proc. Natl. Acad. Sci. U. S. A. 110, 7856−61. (17) Edwards, M. J., White, G. F., Norman, M., Tome-Fernandez, A., Ainsworth, E., Shi, L., Fredrickson, J. K., Zachara, J. M., Butt, J. N., Richardson, D. J., and Clarke, T. A. (2015) Redox linked flavin sites in extracellular decaheme proteins involved in microbe-mineral electron transfer. Sci. Rep. 5 (11677), 1−11. (18) Rabaey, K., and Rozendal, R. A. (2010) Microbial electrosynthesis - revisiting the electrical route for microbial production. Nat. Rev. Microbiol. 8, 706−716. (19) Lu, L., and Ren, Z. J. (2016) Microbial electroylsis cells for waste biorefinery: A state of the art review. Bioresour. Technol. 215, 254−64. (20) Lovley, D. R. (2012) Electromicrobiology. Annu. Rev. Microbiol. 66, 391−409. (21) Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D., and Somero, G. N. (1982) Living with water stress: evolution of osmolyte systems. Science 217, 1214−1222. (22) Seibel, B. A., and Walsh, P. J. (2002) Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage. J. Exp. Biol. 205, 297−306. (23) Dos Santos, J.-P., Iobbi-Nivol, C., Couillault, C., and Méjean, V. (1998) Molecular analysis of the trimethylamine N-oxide (TMAO) reductase respiratory system from a Shewanella species. J. Mol. Biol. 284, 421−433. (24) Gon, S., Patte, J., Santos, J., Dos, and Méjean, V. (2002) Reconstitution of the trimethylamine oxide reductase regulatory elements of Shewanella oneidensis in Escherichia coli. J. Bacteriol. 184, 1262−1269. (25) Bordi, C., Ansaldi, M., Gon, S., Iobbi-nivol, C., Méjean, V., and Me, V. (2004) Genes regulated by TorR, the trimethylamine oxide response regulator of Shewanella oneidensis. J. Bacteriol. 186, 4502− 4509. (26) Lemaire, O. N., Honor, F. A., Vincent, M., Fons, M., and Iobbinivol, C. (2016) Efficient respiration on TMAO requires TorD and TorE auxiliary proteins in Shewanella oneidensis. Res. Microbiol. 167, 1− 8. (27) Bordi, C., Iobbi-Nivol, C., Méjean, V., and Patte, J. C. (2003) Effects of ISSo2 insertions in structural and regulatory genes of the trimethylamine oxide reductase of Shewanella oneidensis. J. Bacteriol. 185, 2042−2045. (28) McCrindle, S. L., Kappler, U., and McEwan, A. G. (2005) Microbial dimethylsulfoxide and trimethylamine-N-oxide respiration. Adv. Microb. Physiol. 50, 147−198. (29) Kasai, T., Kouzuma, A., Nojiri, H., and Watanabe, K. (2015) Transcriptional mechanisms for differential expression of outer

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.6b00349. Miller assay data for PtorF:: lacZ and PtorECAD::lacZ strains in LB, and table of primers used in this study (PDF)



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jeffrey A. Gralnick: 0000-0001-9250-7770 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the Office of Naval Research grants N000141210309 and N000141310552 to JAG. The authors would like to thank Dr. Evan Brutinel for contributing pEB007 and Dr. Brittany Bennett for contributing the ΔdmsEFABGH strain and the anonymous reviewers for their constructive feedback.



REFERENCES

(1) Hau, H. H., and Gralnick, J. A. (2007) Ecology and biotechnology of the genus Shewanella. Annu. Rev. Microbiol. 61, 237−258. (2) Golitsch, F., Bücking, C., and Gescher, J. (2013) Proof of principle for an engineered microbial biosensor based on Shewanella oneidensis outer membrane protein complexes. Biosens. Bioelectron. 47, 285−291. (3) Webster, D. P., TerAvest, M. A., Doud, D. F. R., Chakravorty, A., Holmes, E. C., Radens, C. M., Sureka, S., Gralnick, J. A., and Angenent, L. T. (2014) An arsenic-specific biosensor with genetically engineered Shewanella oneidensis in a bioelectrochemical system. Biosens. Bioelectron. 62, 320−324. (4) Coursolle, D., and Gralnick, J. A. (2010) Modularity of the Mtr respiratory pathway of Shewanella oneidensis strain MR-1. Mol. Microbiol. 77, 995−1008. (5) Thormann, K. M., Duttler, S., Saville, R. M., Hyodo, M., Shukla, S., Hayakawa, Y., and Spormann, A. M. (2006) Control of formation and cellular detachment from Shewanella oneidensis MR-1 biofilms by cyclic di-GMP. J. Bacteriol. 188, 2681−2691. (6) Gao, H., Wang, X., Yang, Z. K., Chen, J., Liang, Y., Chen, H., Palzkill, T., and Zhou, J. (2010) Physiological roles of ArcA, Crp, and EtrA and their interactive control on aerobic and anaerobic respiration in Shewanella oneidensis. PLoS One 5, e15295. (7) Bretschger, O., Obraztsova, A., Sturm, C. A., In, S. C., Gorby, Y. A., Reed, S. B., Culley, D. E., Reardon, C. L., Barua, S., Romine, M. F., Zhou, J., Beliaev, A. S., Bouhenni, R., Saffarini, D., Mansfeld, F., Kim, B. H., Fredrickson, J. K., and Nealson, K. H. (2007) Current production and metal oxide reduction by Shewanella oneidensis MR-1 wild type and mutants. Appl. Environ. Microbiol. 73, 7003−7012. (8) Coursolle, D., Baron, D. B., Bond, D. R., and Gralnick, J. A. (2010) The Mtr respiratory pathway is essential for reducing flavins and electrodes in Shewanella oneidensis. J. Bacteriol. 192, 467−474. (9) Myers, C. R., and Myers, J. M. (1997) Cloning and sequence of cymA, a gene encoding a tetraheme cytochrome c required for reduction of iron (III), fumarate, and nitrate by Shewanella putrefaciens MR-1. J. Bacteriol. 179, 1143−1152. (10) Sturm, G., Richter, K., Doetsch, A., Heide, H., Louro, R. O., and Gescher, J. (2015) A dynamic periplasmic electron transfer network enables respiratory flexibility beyond a thermodynamic regulatory regime. ISME J. 9, 1802−1811. G

DOI: 10.1021/acssynbio.6b00349 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Letter

ACS Synthetic Biology membrane cytochrome genes omcA and mtrC in Shewanella oneidensis MR-1. BMC Microbiol., DOI: 10.1186/s12866-015-0406-8. (30) Baron, D., LaBelle, E., Coursolle, D., Gralnick, J. A., and Bond, D. R. (2009) Electrochemical measurement of electron transfer kinetics by Shewanella oneidensis MR-1. J. Biol. Chem. 284, 28865− 28873. (31) Saltikov, C. W., and Newman, D. K. (2003) Genetic identification of a respiratory arsenate reductase. Proc. Natl. Acad. Sci. U. S. A. 100, 10983−10988. (32) Teal, T. K., Lies, D. P., Wold, B. J., and Newman, D. K. (2006) Spatiometabolic stratification of Shewanella oneidensis biofilms. Appl. Environ. Microbiol. 72, 7324−7330. (33) Stookey, L. L. (1970) Ferrozine—a new spectrophotometric reagent for iron. Anal. Chem. 42, 779−781. (34) Myers, C. M., and Nealson, K. H. (1988) Bacterial manganese reduction and growth with manganese oxide as the sole electron acceptor. Science 240, 1319−1321. (35) Coursolle, D., and Gralnick, J. A. (2012) Reconstruction of extracellular respiratory pathways for iron(III) reduction in Shewanella oneidensis strain MR-1. Front. Microbiol. 3, 1−11.

H

DOI: 10.1021/acssynbio.6b00349 ACS Synth. Biol. XXXX, XXX, XXX−XXX