Decorating the Outer Surface of Microbially Produced Protein

Subscriber access provided by UNIV OF SOUTHERN INDIANA

Article

Decorating the Outer Surface of Microbially Produced Protein Nanowires with Peptides Toshiyuki Ueki, David Jeffrey Fraser Walker, Pier-Luc Tremblay, Kelly Patricia Nevin, Joy E Ward, Trevor L. Woodard, Stephen S. Nonnenmann, and Derek R. Lovley ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00131 • Publication Date (Web): 12 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

Decorating the Outer Surface of Microbially Produced Protein Nanowires with Peptides

Toshiyuki Ueki1,2, David J.F. Walker1,2, Pier-Luc Tremblay1,3, Kelly P. Nevin1, Joy E. Ward1, Trevor L. Woodard1, Stephen S. Nonnenmann2,4, and Derek R. Lovley1,2*

1Department 2Institute 3Current

of Microbiology, University of Massachusetts-Amherst, Amherst, MA, USA

for Applied Life Sciences, University of Massachusetts-Amherst address: School of Chemistry, Chemical Engineering and Life Science, Wuhan

University of Technology, Wuhan 430070, PR China 4Department

of Mechanical and Industrial Engineering, University of Massachusetts-Amherst

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 29

Abstract The potential applications of electrically conductive protein nanowires (e-PNs) harvested from Geobacter sulfurreducens might be greatly expanded if the outer surface of the wires could be modified to confer novel sensing capabilities or to enhance binding to other materials. We developed a simple strategy for functionalizing e-PNs with surface-exposed peptides. The G. sulfurreducens gene for the monomer that assembles into e-PNs was modified to add peptide tags at the carboxyl terminus of the monomer. Strains of G. sulfurreducens were constructed that fabricated synthetic e-PNs with a six-histidine ‘His-tag’ or both the His-tag and a nine-peptide ‘HA-tag’ exposed on the outer surface. Addition of the peptide tags did not diminish e-PN conductivity. The abundance of HA-tag in e-PNs was controlled by placing expression of the gene for the synthetic monomer with the HA-tag under transcriptional regulation. These studies suggest broad possibilities for tailoring e-PN properties for diverse applications.

Keywords: bioelectronic materials, e-biologics, pili, Geobacter, sustainable electronics


2

Page 3 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Microbially produced, electrically conductive protein nanowires (e-PNs) possess properties, and possibilities for functionalization, not found in other electronic nanowire materials 1-5. For example, microorganisms can produce e-PNs from renewable feedstocks with low energy inputs. No toxic chemicals are required for microbial e-PN synthesis and the final product is robust, yet also biodegradable. Unlike silicon nanowires 6, e-PNs do not dissolve in water or bodily fluids, a distinct advantage for wearable and environmental electronic sensor applications, as well as implantable electronics. A dramatic change in conductivity in response to pH observed in some e-PNs 7, 8 suggests that they may be readily adapted for diverse sensor functions 2. Some e-PNs have evolved for making cell-to-cell electrical connections 1, 9, which may facilitate biocompatability. Microbial e-PNs offer the possibility of facile tuning of nanowire properties with the design of synthetic genes that modify microbial e-PN structure 2. Diverse microorganisms in both the Bacteria 10-14 and Archaea 15 naturally produce e-PNs. The most intensively studied microbially produced e-PNs are the electrically conductive pili (e-pili) of Geobacter sulfurreducens 7, 8, 10, 11, 16-23. Functional analysis of G. sulfurreducens e-pili has demonstrated that, when attached to cells, they serve as conduits for long-range electron exchange with other cells or minerals 1, 9. Filaments comprised of the multi-heme c-type cytochrome OmcS can also be recovered from preparations of outer-surface proteins of some strains of G. sulfurreducens 24, 25. However, the in vivo relevance of OmcS filaments is not clear and little is known about their electronic properties 26. G. sulfurreducens strain KN400, which produces the e-pilin monomer PilA in abundance, and very little OmcS, provides the opportunity to grow cells that express copious e-pili, but apparently not OmcS filaments 20, 26-28. The available data indicates that G. sulfurreducens assembles its thin (3 nm), long (ca. 10 µm) e-pili from multiple copies of a short (61 amino acids) pilin monomer peptide 10, 16, 29. The


3


Page 4 of 29

simplicity of e-pili composition offers the potential for broad possibilities in the design of novel wires through genetic manipulation of the monomer peptide gene. For example, the conductivity of e-PNs produced with G. sulfurreducens has been tuned over six orders of magnitude (ca. 4 x 10-5 - 3 x 102 S/cm at pH 7) 8, 11 by genetically manipulating the abundance of aromatic amino acids in the monomer. Modifying the surface properties of e-PNs with the addition of short, surface-exposed peptides could expand possibilities for attaching wires to substrates, enhance binding of desired analytes for sensor functions, or enable covalent bonding of e-PNs to polymeric materials to produce conductive composites 2. Inspiration for this possibility comes from previous studies that successfully expressed E. coli curli fibers modified with peptides to confer novel functions such as improved attachment to surfaces as well as metal, mineral, and protein binding 30, 31. However, curli fibers have a significantly different structure than e-pili and self-assemble outside the cell 32, 33, whereas pili are assembled from pilin monomers through a complex intracellular process 34. Modeling of the G. sulfurreducens e-pili structure predicted that amino acids at the carboxyl terminal of the pilin monomer are likely to be exposed on the outer surface of the e-pili 21.

This suggested that peptides added at the carboxyl terminus might have minimal interference

on e-pili structure and thus not destroy e-pili conductive properties, which are hypothesized to rely on a core of closely packed aromatic amino acids 21. Therefore, we examined whether G. sulfurreducens would express e-pili containing monomers in which peptides were added to the carboxyl terminus; whether the peptides introduced would be accessible on the outer surface of the e-pili; and the potential impact of the added peptides on e-pili conductivity. The results


4

Page 5 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


suggest that e-PNs decorated with outer surface peptides to introduce new binding properties can readily be produced with G. sulfurreducens. Results and Discussion Wires decorated with a six-histidine peptide (His-tag) In order to evaluate the possibility of displaying peptides on the outer surface of e-PNs, the wild-type G. sulfurreducens gene for the pilin monomer (PilA) was modified (Supplementary Figure 1) to encode six histidines (i.e. a ‘His-tag’) at the carboxyl end (Figure 1a). The His-tag was chosen for these proof-of-concept studies because highly selective reagents to detect its localization are commercially available. This synthetic gene was inserted into the chromosome of G. sulfurreducens strain KN400 (Figure 1b), along with the gene for the protein Spc that is required for pilin monomer stability35. The resultant strain, which contained genes for the wildtype PilA as well as the histidine-modified PilA pilin monomer (PilA-6His), was designated strain PilA-WT/PilA-6His. Western analysis with anti-6His antibody of cell lysates of strain PilA-WT/PilA-6His separated with SDS-PAGE revealed a single protein band at the molecular weight expected for the PilA-6His monomer (Figure 1c). There was no corresponding band in lysates of wild-type cells. Western analysis with antibody that detected wild-type PilA detected a single band in wild-type cell lysates and two bands in lysates of strain PilA-WT/PilA-6His (Figure 1c). The additional band in the strain PilA-WT/PilA-6His lysate was positioned at the higher molecular weight position detected with the anti-6His antibody. Transmission electron microscopy of cells labeled with the anti-6His antibody and a secondary antibody conjugated with gold revealed abundant His-tag loci along the wires emanating from the cells, demonstrating that the added His-tag was accessible to the antibody (Figure 2 a, b). There was no immunogold labeling of wild-type cells (Supplemental Figure 2).


5


Page 6 of 29

No unlabeled filaments were observed emanating from cells of strain PilA-WT/PilA-6His, indicating that no other wires, such as those comprised of the cytochrome OmcS, were expressed. This finding is consistent with previous studies that have demonstrated that the KN 400 strain that was used for constructing strain PilA-WT/PilA-6His produces little OmcS and that the filaments emanating from the cells are e-pili 20, 26, 27, 36. When strain PilA-WT/PilA-6His cells were treated with a Ni2+-NTA-gold reagent designed to label His-tags, the gold nanoparticles were specifically localized along the wires (Figure 2 c,d,e). Wild-type cells were not labeled (Supplemental Figure 3). These results demonstrate that the His-tag was accessible on the outer surface of the wires. G. sulfurreducens can only produce high current densities on graphite electrodes if its pili are electrically conductive 18, 29, 37. As previously demonstrated

38,

a strain in which the gene for

PilA was deleted produced low currents (Figure 3). In contrast, G. sulfurreducens strain PilAWT/PilA-6His produced maximum currents comparable to the wild-type strain (Figure 3), indicating the pili retained the capacity to promote long-range electron transport. There was a slightly longer lag period in the initiation of current production in strain PilA-WT/PilA-6His (Figure 3), but this is a period of initial growth in which attachment to the anode and short-range cell-to-anode electron transport are important 1 and thus not indicative of a change in the capacity of the cells for long-range electron transport via e-pili. Conductivity of individual wires was more directly evaluated with conductive atomic force microscopy (c-AFM), as previously described 15. The wires were readily identified in topographic imaging in contact mode (Figure 4a) and height measurements (Figure 4c,e) indicated a diameter of 3.1 + 0.3 nm (mean + standard deviation; n= 18, 6 points on 3 wires). This corresponds with the 3 nm diameter previously reported for individual e-pili from wild-type


6

Page 7 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


G. sulfurreducens 8, 10 and the diameter of individual e-pili when the G. sulfurreducens pilin monomer gene is heterologously expressed in Pseudomonas aeruginosa 39. The conductive tip was translated to the top of the wire, and the point-mode current-voltage response (I-V) spectroscopy revealed a conductance of 7.2 + 1.5 nS (mean + standard deviation; n =9) under a load force setpoint (1 nN) (Figure 4g, Supplemental Figure 4). This is slightly higher than the previously observed 15 conductance of 4.5 + 0.3 nS for e-PNs comprised solely of the wild-type monomer and much higher than the previously reported 15 conductance of the e-PNs from strain G. sulfurreducens strain Aro-5, which lacks key aromatic amino acids required for high conductivity. Wires decorated with two different peptides In order to determine whether two peptides with different functions could be displayed on one e-PN, a gene (Supplemental Figure 1) encoding the previously described 40 nine-peptide ‘HA-tag’ (YPYDVPDYA) at the carboxyl end of the wild-type PilA pilin monomer (Figure 5a) was incorporated into the chromosome along with the PilA-6His and wild-type (WT) genes (Figure 5b). Like the His-tag, the HA-tag was convenient for proof-of-concept studies because a HA-tag is commercially available. The gene for the PilA with the HA-tag (PilA-HA) was located downstream of the IPTG-inducible lac promoter/operator in order to provide the option of controlling the stoichiometry of incorporation of the PilA-HA monomer in the e-PNs (Figure 5b). This strain was designated G. sulfurreducens strain PilA-WT/PilA-6His/PilA-HA. Western blot analysis demonstrated that, in the presence of 1 mM IPTG, monomers of WT-PilA, PilA6His, and PilA-HA were expressed in this strain (Figure 5c). Immunogold labeling for just the His-tag (Figure 6a,b) or the HA-tag (Figure 6c,d) demonstrated that both tags were abundant in the e-PNs from strain PilA-WT/PilA-6His/PilA-


7


Page 8 of 29

HA grown in the presence of 1 mM IPTG. Dual labeling with secondary antibodies with different size gold particles demonstrated that both tags were present in the same e-PNs (Figure 6 e,f). e-PNs of strain PilA-WT/PilA-6His cells were not immunogold labeled with the anti-HA antibody (Supplementary Figure 5). The current production of strain PilA-WT/PilA-6His/PilAHA was similar to that of strain PilA-WT/PilA-6His, indicating the addition of the peptide tag did not significantly diminish pili conductivity (Figure 3). AFM analysis of the e-PNs (Figure 4b) revealed that incorporation of the HA-tag increased the diameter (height) to 4 nm (Figure 4d, f), suggesting an influence on the pilus assembly. Previous studies have demonstrated that making small changes in amino acid content can dramatically influence pilus diameter, but attempts to determine e-pili structure with cryoelectron microscope approaches have as yet been unsuccessful, making it difficult to interpret this phenomenon 22. Individual e-PNs of strain PilA-WT/PilA-6His/PilA-HA (Figure 4g, Supplemental Figure 6) yielded higher currents at equivalent applied voltages than observed with the e-PNs with just the His-tag, with an estimated conductance of 27.2 + 1.0 nS (n=9). The mechanisms for electron transport within e-pili are still a matter of conjecture, but aromatic amino acids play a key role 1. Thus, a potential explanation is that the multiple aromatic amino acids in the HA-tag, improve electron transport between the conductive AFM tip and e-pili. Some PilA-HA was expressed in strain PilA-WT/PilA-6His/PilA-HA even in the absence of the IPTG inducer (Figure 7a). However, the concentration of PilA-HA monomer in the cells was greater with added IPTG (Figure 7a). Increased pools of PilA-HA were associated with ePNs that labeled more heavily with immunogold labeling for the HA-tag (Figure 7b,c,d). These results demonstrate that it is possible to control the abundance of a specific peptide displayed on e-PNs with transcriptional control of the expression of the monomer modified with that peptide.


8

Page 9 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


It is likely that stoichiometric control of peptide display could be improved by fine-tuning the transcriptional regulation, possibly by increasing expression of LacI repressor and adding more LacI binding sites. Furthermore, multiple additional systems for inducing or repressing gene expression in G. sulfurreducens are available which provide other options for controlling the stoichiometry of expression of synthetic pilins decorated with peptide tags 41. Concluding Comments These results demonstrate that e-PNs produced with G. sulfurreducens can be decorated with one or more peptides while maintaining, or possibly increasing, their conductivity. The stoichiometry of peptide display can be controlled with transcriptional regulation. These capabilities greatly expand the potential applications of e-PNs in electronic devices and for the fabrication of electrically conductive composite materials. For example, sensors developed from other nanowire materials show promise for providing highly sensitive and specific, real-time electrical response for detection of diverse chemicals and biologics 42-44. Analytes of interest are detected as a change in nanowire conductivity that results from changes in pH associated with the activity of enzymes incorporated into the sensors, or binding of analytes to nanowires functionalized with antibodies, peptides, or other ligands. The conductivity of G. sulfurreducens e-PNs has already been shown to be highly responsive to pH 7, 8. Short peptides for binding enzymes and antibodies 45-50 displayed on the outer-surface of e-PNs could be an effective method for functionalizing e-PNbased sensors. Both peptides investigated in this study were effective in promoting antibody binding to e-pili. Furthermore, peptides can be designed to function as ligands for a wide diversity of chemical and biological analytes or to enhance attachment to cells 51-53. The His-tag demonstrated the potential for a peptide tag to serve as metal ligand. Thus, the simplicity of


9


Page 10 of 29

modifying the peptides displayed on e-PNs, and controlling the abundance of peptide display, could provide unprecedented flexibility in nanowire sensor design not readily achieved with other materials such as silicon nanowires or carbon nanotubes. In a similar manner, modifying the surface chemistry of e-PNs with short peptides may enable chemical linkages with polymers or enhance binding to materials to aid in e-PN alignment in electronic devices 1. The studies described here demonstrate that peptides of up to 9 amino acids can be added to the 61 amino acid monomer backbone of G. sulfurreducens e-PNs. However, it may be possible to decorate e-PNs with much larger peptides because the monomers of other e-pili have an N-terminal region homologous to the G. sulfurreducens monomer, but are comprised of over 100 amino acids 14, 54. These broad possibilities for modifying e-PNs with peptides coupled with the advantages of e-PNs as a ‘green’ sustainable material suggest that further investigation into the development of e-PN-based electronic devices and materials is warranted.

Materials and Methods Strains and growth conditions G. sulfurreducens strains were grown under anaerobic conditions at 30°C in a defined medium with acetate as the electron donor and fumarate as the electron acceptor as previously described 55, unless otherwise described. Escherichia coli was cultivated with Luria-Bertani medium with or without antibiotics 56. Construction of G. sulfurreducens PilA-WT/PilA-6His strain G. sulfurreducens PilA-WT/PilA-6His strain was constructed with G. sulfurreducens KN400 27. A gene for PilA-6His, the gene KN400_3442, which is located downstream of the pilA gene (KN400_1523) on the chromosome, and the putative transcription terminator were


10

Page 11 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


integrated at a non-coding region between KN400_0788 and KN400_0787 in the chromosome (Figure 1b). The primer pair upKpnI (CTAGGTACCGTGGTGGACCCCCTTACCGGT)/upSpeI (CGAACTAGTTGTGACCGCTGCCGGCTCCG) was used to amplify by PCR ca. 550 bp of KN400_0788 upstream of the integration site with the genomic DNA as template. This PCR product was digested with KpnI/SpeI and ligated with the vector pCR2.1GmrloxP 57, resulting in pCR2.1UP-GmrloxP. The 6His tag was fused to the C-terminal end of PilA by PCR with the primer pair pilAdnNotI (CACGCGGCCGCAAGAGGAGCCAGTGACGAAAATC)/pilACHis (GAGTTAGTGGTGGTGGTGGTGGTGACTTTCGGGCGGATAGGTTTGATC). For the construction of pilA-6His-KN400_3442, two PCR products were generated before being combined by recombinant PCR. pilA-6His was amplified with the primer pair pilAdnNotI/pilAHisrecup (CTCCAGTATGTATTTAATCAATTAGTGGTGGTGGTGGTGGTG) while KN400_3442 was amplified with the primer pair pilAHisrecdn (CACCACCACCACCACCACTAATTGATTAAATACATACTGGAG)/GSU1497XhoIAvrII (CTGCTCGAGGATACCTAGGCTATTCCGACAACTACGAGAC). The primer pair pilAdnNotI/GSU1497XhoIAvrII was then used to amplify pilA-6His-KN400_3442 by recombinant PCR. pilA-6His-KN400_3442 was cloned at NotI/XhoI sites in pCR2.1UP-GmrloxP downstream of GmrloxP, resulting in pCR2.1UP-GmrloxP-pilAHis-3442. The primer pair dnAvrII (CATCCTAGGAGGGCAGACATTGCGGAACGT)/dnXhoI (CATCTCGAGCGGGTTCCGCTGCCGTCGTAC) was used to amplify by PCR ca. 530 bp of KN400_0787 downstream of the integration site. This PCR product was cloned at AvrII/XhoI sites in pCR2.1UP-GmrloxP-pilAHis-3442, resulting in pCR2.1UP-GmrloxP-pilAHis-3442-DN. The final plasmid was linearized with XhoI for transformation as previously described 55.


11


Page 12 of 29

Transformants were selected with the medium containing gentamicin (20 μg/ml) and were verified by PCR. Construction of G. sulfurreducens PilA-WT/PilA-6His/PilA-HA strain G. sulfurreducens PilA-WT/PilA-6His/PilA-HA strain was constructed by introducing a gene encoding PilA monomer with the HA tag (pilA-HA) together with the gene KN400_3442 in the chromosome of the G. sulfurreducens PilA-WT/PilA-6His strain (Figure 5b). The pilA-HA gene was amplified by PCR with a primer pair TCTGGATCCAGGAGGAGACACTTATGCTTCAGAAAC/GTATTTAATCAATTACGCGT AGTCCGGCACGTCGTACGGGTAACTTTCGGGCGGATAG. The gene KN400_3442 was amplified by PCR with a primer pair CTATCCGCCCGAAAGTTACCCGTACGACGTGCCGGACTACGCGTAATTGATTAAATA C/TCTGAATTCCGATATGACTACTGCGAC. The pilA-HA gene and the KN400_3442 gene were connected by PCR with a primer pair TCTGGATCCAGGAGGAGACACTTATGCTTCAGAAAC/TCTGAATTCCGATATGACTA CTGCGAC. The PCR product of pilA-HA/KN400_3442 was digested with BamHI/EcoRI and cloned in the plasmid pKIkan, which is a derivative of pKIapr 58 and has a kanamycin-resistance gene instead of the apramycin-resistance gene. The sequences of KN400_1082 and 1083 used for homologous recombination for introduction of pilA-HA/KN400_3442 are same as those of GSU1106 and 1107 for homologous recombination sequences in pKIkan, respectively. The plasmid thus constructed was linearized with XhoI for electroporation. Western blot analysis The wild-type, PilA-WT/PilA-6His, and PilA-WT/PilA-6His/PilA-HA strains were grown with acetate and fumarate at 25 ºC. IPTG was added at 1 mM for the PilA-WT/PilA-


12

Page 13 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6His/PilA-HA strain with the exception of the study of the impact on IPTG concentrations on incorporation of PilA-HA in filaments. Cell extracts were prepared with B-PER Complete Bacterial Protein Extraction Reagent (Thermo Fisher Scientific) and the amount of protein was measured with the Bradford Protein Assay (Bio-Rad) as instructed by the manufacturer. Cell extracts were separated on 16.5% Tris-Tricine gel (Bio-Rad). An anti-PilA antibody was obtained against peptide, ESAFADDQTYPPES, corresponding to the C-terminal end of PilA (New England Peptide). An anti-6His antibody (6x-His Tag Polyclonal Antibody) and an antiHA antibody (HA Tag Polyclonal Antibody) were purchased from Invitrogen. Western blot analysis was conducted as described previously 59. The molecular weight standard markers were Precision Plus Protein Standards, all blue prestained (Bio-Rad). The amount of PilA-HA was quantified by ImageJ (http://imagej.nih.gov/ij/index.html). Immunogold labeling The strains were grown with acetate and fumarate at 25ºC. The PilA-WT/PilA-6His/PilAHA strain was grown with 1 mM IPTG unless otherwise specified. Immunogold labeling was conducted as previously described 59 with the modification that after the reaction with second gold antibody the grid was washed with PBS three times and water once before staining with 2% uranyl acetate. For immunogold labeling of just one type of peptide tag, the 6x-His Tag Polyclonal Antibody or HA Tag Polyclonal Antibody was the primary antibody and the antirabbit IgG-gold (10 nm) antibody (Sigma-Aldrich) was the secondary. Dual immunogold labeling was conducted with 6x-His Tag Monoclonal Antibody (Invitrogen) and the HA Tag Polyclonal Antibody as primary antibodies and an anti-mouse IgG-gold (40 nm) antibody (40 nm Goat Anti-Mouse IgG gold conjugate, Expedeon) and the anti-rabbit IgG-gold (10 nm) antibody


13


Page 14 of 29

as secondary antibodies. Samples were examined with transmission electron microscopy as described previously 11. Ni2+-binding assay The wild-type and PilA-WT/PilA-6His strains were grown with acetate and fumarate at 25ºC. Ni2+-binding assay was conducted with Ni-NTA-Nanogold (5 nm) (Nanoprobes). Seven µl of the culture was placed on a copper grid and incubated for 5 min. The grid was floated upside down in phosphate-buffered saline (PBS) for 5 min, in PBS containing 3% bovine serum albumin (BSA) and 40 mM imidazole for 15 min, and in PBS containing 0.3% BSA, 40 mM imidazole, and the Ni-NTA-Nanogold for 30 min at room temperature. The grid was washed with PBS containing 40 mM imidazole three times and with water once. Samples were stained with 2% uranyl acetate and examined by transmission electron microscopy as described previously 11. Current production The capacity to produce current was determined in the two-chambered H-cell system with a continuous flow of medium with acetate (10 mM) as the electron donor and graphite stick anode (65 cm2) poised at 300 mV versus Ag/AgCl as the electron acceptor as described previously 60. Briefly, cells were initially grown in the anode chamber in a medium containing 20 mM acetate and 40 mM fumarate. When the current reached 0.5 mA fresh medium containing 20 mM acetate and no fumarate was continuously pumped into the anode chamber at 5% of the anode chamber per hour. Conductive atomic force microscopy (c-AFM) of individual e-PNs As previously described 15, an aliquot (100 µl) of cell culture was drop-cast onto highly oriented pyrolytic graphite (HOPG). Conductive atomic force microscopy was preformed using


14

Page 15 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


an Oxford Instruments/Asylum Research Cypher ES atomic force microscope with a Pt/Ir-coated Arrow-ContPT tip (NanoWorld AG, Neuchâtel, Switzerland). Topographic imaging was performed in contact mode with a force of 0.1 nN. Point-mode current-voltage response (I-V) spectroscopy was achieved by applying a 1 nN force to the top of the wire and conducting quadruplicate voltage sweeps of -0.6 to 0.6 V at 0.99 Hz. The voltage sweep was averaged for each of the I-V curves and conductance was calculated from the linear portion of the I-V curve (0.2 to 0.2 V) (Supplemental Figure 4). Average conductance and standard deviation were calculated using 3 independent points on 3 independent e-PNs of each strain. Average height and standard deviation were calculated from 6 independent points on 3 independent e-PNs.

Funding Sources This research was supported by exploratory research development funds from the University of Massachusetts.

References 1. Lovley, D. R., Electrically conductive pili: biological function and potential applications in electronics. Curr. Opin. Electrochem. 2017, 4, 190-198. 2. Lovley, D. R., e-Biologics: Fabrication of sustainable electronics with ‘green’ biological materials. mBio 2017, 8, e00695-17. 3. Gutermann, T.; Gazit, E., Toward peptide-based bioelectronics: reductionist design of conductive pili mimetics. Bioelectron. Med. 2018, 1, 131-137. 4. Creasey, R. C. G.; Mostert, A. B.; Nguyen, T. A. H.; Virdis, B.; Freguia, S.; Laycock, B., Microbial nanowires −electron transport and the role of synthetic analogues Acta Biomater. 2018, 69, 1-30. 5. Ing, N. L.; El-Naggar, M. Y.; Hochbaum, A. I., Going the distance: long-range conductivity in protein and peptide bioelectronic materials. J. Phys. Chem. B 2018, 122, 104310423. 6. Zhou, W.; Dai, X.; Fu, T.-M.; Xie, C.; Liu, J.; Lieber, C. M., Long term stability of nanowire nanoelectronics in physiological environments. Nano Lett. 2014, 14, 1614-1619. 7. Malvankar, N. S.; Vargas, M.; Nevin, K. P.; Franks, A. E.; Leang, C.; Kim, B.-C.; Inoue, K.; Mester, T.; Covalla, S. F.; Johnson, J. P.; Rotello, V. M.; Tuominen, M. T.; Lovley,


15


Page 16 of 29

D. R., Tunable metallic-like conductivity in nanostructured biofilms comprised of microbial nanowires. Nat. Nanotechnol. 2011, 6, 573-579. 8. Adhikari, R. Y.; Malvankar, N. S.; Tuominen, M. T.; Lovley, D. R., Conductivity of individual Geobacter pili. RSC Adv. 2016, 6, 8354-8357. 9. Lovley, D. R., Syntrophy goes electric: direct interspecies electron transfer. Ann. Rev. Microbiol. 2017, 71, 643-664. 10. Reguera, G.; McCarthy, K. D.; Mehta, T.; Nicoll, J. S.; Tuominen, M. T.; Lovley, D. R., Extracellular electron transfer via microbial nanowires. Nature 2005, 435, 1098-1101. 11. Tan, Y.; Adhikari, R. Y.; Malvankar, N. S.; Ward, J. E.; Woodard, T. L.; Nevin, K. P.; Lovley, D. R., Expressing the Geobacter metallireducens PilA in Geobacter sulfurreducens yields pili with exceptional conductivity. mBio 2017, 8, e02203-16. 12. Holmes, D. E.; Dang, Y.; Walker, D. J. F.; Lovley, D. R., The electrically conductive pili of Geobacter species are a recently evolved feature for extracellular electron transfer. Microb. Genomics 2016, 2, doi: 10.1099/mgen.0.000072. 13. Sure, S.; Ackland, M. L.; Torriero, A. J.; Adholeya, A.; Kochar, M., Microbial nanowires: an electrifying tale. Microbiol. 2016, 162, 2017-2028. 14. Walker, D. J. F.; Adhikari, R. Y.; Holmes, D. E.; Ward, J. E.; Woodard, T. L.; Nevin, K. P.; Lovley, D. R., Electrically conductive pili from genes of phylogenetically diverse microorganisms. ISME J. 2018, 12, 48-58. 15. Walker, D. J. F.; Martz, E.; Holmes, D. E.; Zhou, Z.; Nonnenmann, S. S.; Lovley, D. R., The archaellum of Methanospirillum hungatei is electrically conductive. mBio 2019, 10, e00579-19. 16. Cologgi, D. L.; Lampa-Pastirk, S.; Speers, A. M.; Kelly, S. D.; Reguera, G., Extracellular reduction of uranium via Geobacter conductive pili as a protective cellular mechanism. Proc. Natl. Acad. Sci. U S A 2011, 108, 15248–15252. 17. Veazey, J. P.; Reguera, G.; Tessmer, S. H., Electronic properties of conductive pili of the metal-reducing bacterium Geobacter sulfurreducens probed by scanning tunneling microscopy. Phys. Rev. 2011, 84, 060901. 18. Vargas, M.; Malvankar, N. S.; Tremblay, P.-L.; Leang, C.; Smith, J. A.; Patel, P.; Snoeyenbos-West, O.; Nevin, K. P.; Lovley, D. R., Aromatic amino acids required for pili conductivity and long-range extracellular electron transport in Geobacter sulfurreducens mBio 2013, 4, e00105-13. . 19. Lampa-Pastirk, S.; Veazey, J. P.; Walsh, K. A.; Feliciano, G. T.; Steidl, R. J.; Tessmer, S.; Reguera, G., Thermally activated charge transport in microbial protein nanowires. Sci. Rep. 2016, 6, 23517. 20. Malvankar, N. S.; Yalcin, S. E.; Tuominen, M. T.; Lovley, D. R., Visualization of charge propagation along individual pili proteins using ambient electrostatic force microscopy. Nat. Nanotechnol. 2014, 9, 1012-1017. 21. Malvankar, N. S.; Vargas, M.; Nevin, K. P.; Tremblay, P.-L.; Evans-Lutterodt, K.; Nykypanchuk, D.; Martz, E.; Tuominen, M. T.; Lovley, D. R., Structural basis for metallic-like conductivity in microbial nanowires. mBio 2015, 6, e00084-15. 22. Tan, Y.; Adhikari, R. Y.; Malvankar, N. S.; Pi, S.; Ward, J. E.; Woodard, T. L.; Nevin, K. P.; Xia, Q.; Tuominen, M. T.; Lovley, D. R., Synthetic biological protein nanowires with high conductivity. Small 2016, 12, 4481–4485. 23. Ing, N. L.; Nusca, T. D.; Hochbaum, A. I., Geobacter sulfurreducens pili support ohmic electronic conduction in aqueous solution. Phys. Chem. Chem. Phys. 2017, 19, 21791-21799.


16

Page 17 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


24. Wang, F.; Gu, Y.; O'Brien, J. P.; Yi, S. M.; Yalcin, S. E.; Srikanth, V.; Shen, C.; Vu, D.; Ing, N. L.; Hochbaum, A. I.; Egelman, E. H.; Malvankar, N. S., Structure of microbial nanowires reveals stacked hemes that transport electrons over micrometers. Cell 2019, 177, 361– 369. 25. Filman, D. J.; Marino, S. F.; Ward, J. E.; Yang, L.; Mester, Z.; Bullitt, E.; Lovley, D. R.; Strauss, M., Cryo-EM reveals the structural basis of long-range electron transport in a cytochrome-based bacterial nanowire. Commun. Biol. 2019, 2, 219. 26. Lovley, D. R.; Walker, D. J. F., Geobacter protein nanowires. PeerJ Prepr. 2019, 7, e27773v1. 27. Yi, H.; Nevin, K. P.; Kim, B.-C.; Franks, A. E.; Klimes, A.; Tender, L. M.; Lovley, D. R., Selection of a variant of Geobacter sulfurreducens with enhanced capacity for current production in microbial fuel cells. Biosens. Bioelectron. 2009, 24, 3498-3503. 28. Malvankar, N. S.; Tuominen, M. T.; Lovley, D. R., Lack of involvement of c-type cytochromes in long-range electron transport in microbial biofilms and nanowires. Energ. Environ. Sci. 2012, 5, 8651 - 8659. 29. Tan, Y.; Adhikari, R. Y.; Malvankar, N. S.; Ward, J. E.; Nevin, K. P.; Woodard, T. L.; Smith, J. A.; Snoeyenbos-West, O. L.; Franks, A. E.; Tuominen, M. T.; Lovley, D. R., The low conductivity of Geobacter uraniireducens pili suggests a diversity of extracellular electron transfer mechanisms in the genus Geobacter. Front. Microbiol. 2016, 7, 980. 30. Nguyen, P. Q.; Botyanszki, Z.; Tay, P. K. R.; Joshi, N. S., Programmable biofilm-based materials from engineered curli nanofibres. Nat. Commun. 2014, 5, 4945 31. Chen, A. Y.; Deng, Z.; Billings, A. N.; Seker, U. O. S.; Lu, M. Y.; Citorik, R. J.; Zakeri, B.; Lu, T. K., Synthesis and patterning of tunable multiscale materials with engineered cells. Nat. Mater. 2014, 13, 515-523. 32. Wang, Y.; Pu, J.; An, B.; Lu, T. K.; Zhong, C., Emerging paradigms for synthetic design of functional amyloids. J. Mol. Biol. 2018, 430, 3720-3734. 33. Nguyen, P. Q.; Dorval Courchesne, N.-M.; Duraj-Thatte, A.; Praveschotinunt, P.; Joshi, N. S., Engineered living materials: prospects and challenges for using biological systems to direct the assembly of smart Materials. Adv. Mater. 2018, 30, 1704847. 34. Hospenthal, M. K.; Costa, T. R. D.; Waksman, G., A comprehensive guide to pilus biogenesis in Gram-negative bacteria. Nat. Rev. Microbiol. 2017, 15, 365-379. 35. Liu, X.; Zhou, S.; Lovley, D. R., A pilin chaperone required for the expression of electrically conductive Geobacter sulfurreducens pili Environ. Microbiol. 2019, 21, 2511-2522. 36. Malvankar, N. S.; Mester, T.; Tuominen, M. T.; Lovley, D. R., Supercapcitors based on c-type cytochromes using conductive nanostructured networks of living bacteria. ChemPhysChem 2012, 13, 463-468. 37. Liu, X.; Tremblay, P.-L.; Malvankar, N. S.; Nevin, K. P.; Lovley, D. R.; Vargas, M., A Geobacter sulfurreducens strain expressing Pseudomonas aeruginosa type IV pili localizes OmcS on pili but is deficient in Fe(III) oxide reduction and current production. Appl. Environ. Microbiol. 2014, 80, 1219-1224. 38. Smith, J. A.; Tremblay, P.-L.; Shrestha, P. M.; Snoeyenbos-West, O. L.; Franks, A. E.; Nevin, K. P.; Lovley, D. R., Going wireless: Fe(III) oxide reduction without pili by Geobacter sulfurreducens strain JS-1. Appl. Environ. Microbiol. 2014, 80, 4331-4340. 39. Liu, X.; Wang, S.; Xu, A.; Zhang, L.; Liu, H.; Ma, L. Z., Biological synthesis of highconductive pili in aerobic bacterium Pseudomonas aeruginosa. Appl. Microbiol. Biotechnol. 2019, 103, 1535-1544.


17


Page 18 of 29

40. Field, J.; Nikawa, J.; Broek, D.; MacDonald, B.; Rodgers, L.; Wilson, I. A.; Lerner, R. A.; Wigler, M., Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol. Cell. Biol. 1988, 8, 215965. 41. Ueki, T.; Nevin, K. P.; Woodard, T. L.; Lovley, D. R., Genetic switches and related tools for controlling gene expression and electrical outputs of Geobacter sulfurreducens. J. Ind. Microbiol. Biotechnol. 2016, 43, 1561-1575. 42. Ahmad, R.; Mahmoudi, T.; Ahn, M. S.; Hahn, Y. B., Recent advances in nanowiresbased field-effect transistors for biological sensor applications. Biosens. Bioelectron. 2018, 100, 312-325. 43. Zhang, A.; Lieber, C. M., Nano-Bioelectronics. Chem. Rev. 2016, 116, 215-57. 44. Zhu, C.; Yang, G.; Li, H.; Du, D.; Lin, Y., Electrochemical sensors and biosensors based on nanomaterials and nanostructures. Anal. Chem. 2015, 87, 230-49. 45. Lu, Y.; Huang, F.; Wang, J.; Xia, J., Affinity-guided covalent conjugation reactions based on PDZ-peptide and SH3-peptide interactions. Bioconjugate Chem. 2014, 25, 989-99. 46. Yan, X.; Zhou, H.; Zhang, J.; Shi, C.; Xie, X.; Wu, Y.; Tian, C.; Shen, Y.; Long, J., Molecular mechanism of inward rectifier potassium channel 2.3 regulation by tax-interacting protein-1. J. Mol. Biol. 2009, 392, 967-76. 47. Huang, J.; Nagy, S. S.; Koide, A.; Rock, R. S.; Koide, S., A peptide tag system for facile purification and single-molecule immobilization. Biochemistry 2009, 48, 11834-6. 48. Gaidamakova, E. K.; Backer, M. V.; Backer, J. M., Molecular vehicle for targetmediated delivery of therapeutics and diagnostics. J Controlled Release 2001, 74, 341-7. 49. Zakeri, B.; Fierer, J. O.; Celik, E.; Chittock, E. C.; Schwarz-Linek, U.; Moy, V. T.; Howarth, M., Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc. Natl. Acad. Sci. U S A 2012, 109 (12), E690-7. 50. Skerra, A.; Schmidt, T. G., Use of the Strep-Tag and streptavidin for detection and purification of recombinant proteins. Methods Enzymol. 2000, 326, 271-304. 51. Pavan, S.; Berti, F., Short peptides as biosensor transducers. Anal. Bioanal. Chem. 2012, 402. 52. Liu, Q.; Wang, J.; Boyd, B. J., Peptide-based biosensors. Talanta 2015, 136, 114-127. 53. Chen, H.; Huang, J.; Palaniappan, A.; Wang, Y.; Liedberg, B.; Platt, M.; Tok, A. L. Y., A review on electronic bio-sensing approaches based on non-antibody recognition elements. Analyst 2016, 141, 2335. 54. Walker, D. J. F.; Nevin, K. P.; Nonnenmann, S. S.; Holmes, D. E.; Woodard, T. L.; Ward, J. E.; Rotaru, A.-E.; McInerney, M. J.; Lovley, D. R., Syntrophus conductive pili demonstrate that common hydrogen-donating syntrophs can have a direct electron transfer option bioRxiv 2018, https://www.biorxiv.org/content/early/2018/11/28/479683. 55. Coppi, M. V.; Leang, C.; Sandler, S. J.; Lovley, D. R., Development of a genetic system for Geobacter sulfurreducens. Appl. Environ. Microbiol. 2001, 67, 3180-3187. 56. Sambrook, J. a. R., D.W., Molecular cloning: a laboratory manual 3rd ed. Cold Spring Harbor: Cold Spring Harbor, NY, 2001. 57. Aklujkar, M.; Lovley, D. R., Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of Pelobacter carbinolicus. BMC Evol. Biol. 2010, 10, 230.


18

Page 19 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


58. Ueki, T.; DiDonato, L. N.; Lovley, D. R., Toward establishing minimum requirements for extracellular electron transfer in Geobacter sulfurreducens FEMS Microbiol. Lett. 2017, 364, fnx093. 59. Leang, C.; Qian, X.; Mester, T.; Lovley, D. R., Alignment of the c-type cytochrome OmcS along pili of Geobacter sulfurreducens. Appl. Environ. Microbiol. 2010, 76, 4080-4084. 60. Nevin, K. P.; Kim, B.-C.; Glaven, R. H.; Johnson, J. P.; Woodard, T. L.; Methé, B. A.; DiDonato Jr, R. J.; Covalla, S. F.; Franks, A. E.; Liu, A.; Lovley, D. R., Anode biofilm transcriptomics reveals outer surface components essential for high current power production in Geobacter sulfurreducens fuel cells. PLoS One 2009, 4, e5628.


19


Page 20 of 29

Figure 1. Construction of G. sulfurreducens strain PilA-WT/PilA-6His and expression of pilin monomers. (a) Amino acid sequence of the wild-type PilA and PilA-6His with the added histidines highlighted in red. (b) Location of wild-type and pilA-6His genes on the chromosome. Numbers are gene numbers from the genome sequence (NCBI, NC_017454.1). genR is the gentamicin resistance gene. (c) Western blot analysis for the wild-type (lane 1) and PilA-WT/PilA-6His (lane 2) strains. Lane M is molecular weight standard markers. Headings designate the antibody employed. Figure 2. Transmission electron micrographs of immunogold (a, b) and Ni2+NTA-gold (c, d, e) labeling of the 6His-tag incorporated into e-PNs of G. sulfurreducens strain PilA-WT/PilA-6His. Figure 3. Current production of wild-type G. sulfurreducens, G. sulfurreducens strains PilA-WT/PilA-6His, G. sulfurreducens PilA-WT/PilA-6His/PilA-HA and a strain in which the gene for PilA was deleted. As detailed in the Material and Methods, cells were grown in the anode chamber of a two-chambered bioelectrochemical system. Figure 4. Current-voltage response of synthetic e-PNs decorated with peptide tags. Representative contact-mode imaging reported from the deflection channel of e-PNs from (a) G. sulfurreducens strain PilA-WT/PilA-6His and (b) strain PilA-WT/PilA-6His/PilA-HA. Height channel topographical imaging from (c) G. sulfurreducens strain PilA-WT/PilA-6His and (d) strain PilA-WT/PilA6His/PilA-HA. Height profile cross-section taken from the height images above at the red line for (e) G. sulfurreducens strain PilA-WT/PilA-6His and (f) strain


20

Page 21 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PilA-WT/PilA-6His/PilA-HA. (g) Point-mode current response (I-V) spectroscopy of e-PNs from PilA-WT/PilA-6His and PilA-WT/PilA-6His/PilAHA, compared to previously published data 15 on e-PNs from the wild-type strain of G. sulfurreducens, and strain Aro-5, which expresses poorly conductive pili. Figure 5. Construction of G. sulfurreducens strain PilA-WT/PilA-6His/PilA-HA and expression of pilin monomers. (a) Amino acid sequence of the PilA-HA with the HA tag highlighted in orange. (b) Location of wild-type, pilA-6His, and pilAHA genes on the chromosome. lacI is the Lac repressor gene. kanR is the kanamycin resistance gene. (e) Western blot analysis of cells lysates of the wildtype (lane 1), PilA-WT/PilA-6His (lane 2), and PilA-WT/PilA-6His/PilA-HA (lane 3) strains. Lane M is molecular weight standard markers. Headings designate the antibody employed. Figure 6. Transmission electron micrographs of immunogold labeling G. sulfurreducens strain PilA-WT/PilA-6His/PilA-HA for 6His-tag (a, b), HA-tag (c, d), and both tags (e, f). Gold nanoparticles were 10 nm diameter throughout with the exception of the dual labeling in panels e and f in which the 6His-tag was labeled with 40 nm diameter gold. Figure 7. Transcriptional control of PilA-HA expression and incorporation into ePNs in strain PilA-WT/PilA-6His/PilA-HA evaluated with anti-HA antibody. (a) Western blot analysis of cell lysates. Lane M is standard markers. Inset-relative abundance of PilA-HA as quantified by ImageJ (http://imagej.nih.gov/ij/index.html). (b, c, d) Immunogold labeling of HA-tag in e-PNs during growth in the presence of the designated concentration of IPTG.


21

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Page 22 of 29

a 1

11

21

31

41

51

61

Wild-Type PilA FTLIELLIVVAIIGILAAIAIPQFSAYRVKAYNSAASSDLRNLKTALESAFADDQTYPPES

PilA-6His FTLIELLIVVAIIGILAAIAIPQFSAYRVKAYNSAASSDLRNLKTALESAFADDQTYPPESHHHHHH

b

c pilA promoter

pilA promoter 0787

genR

pilA-6His

3442

0788

pilA

Anti-6His

Anti-PilA

M

M

1

2

1

2

3442

50 kDa 37 kDa 25 kDa 20 kDa 15 kDa 10 kDa


PilA-6His PilA-WT

Page 23 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

a

b


100 nm

200 nm

c

d

100 nm

e

100 nm


100 nm

20

Page 24 of 29

Strain PilA-WT/PilA-6His/PilA-HA

18

Wild-type Strain

16 14

Current (mA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41


12 10 8

Strain PilA-WT/PilA-6His

6 4

PilA Deletion Strain

2 0 0

0.25

0.5

0.75

1

1.25

1.5


Days

1.75

2

2.25

e-PNs from strain PilA-WT/PilA-6His

e-PNs from strainPilA-WT/PilA-6His/ b Biology ACSnm Synthetic

nm

PilA-HA

3

4

2

3

1

0

0

5 µm

c

e-PNs from strain PilA-WT/PilA-6His

5 µm 3 2 01 0

g 10

2 µm

-1

d

-4

e-PNs from strainPilA-WT/PilA-6His/ PilA-HA

nm

nm

8

nm 8 6

6

5 6

4

4 4 3 00

2

22 1

0

f

2 µm

0

4 3 02 1 0

Strain PilA-WT/PilA-6His/PilA-HA

5

0

-2

Height (nm)

e Height (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

a

Current (nA)

Page 25 of 29

Strain PilA-WT/ PilA-6His

Wild-type strain Strain Aro-5

-5 -10 - 0.6

ACS Paragon Plus Environment - 0.4

-0.2

0 Voltage (V)

0.2

0.4

0.6

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

a

Page 26 of 29

PilA-HA 1 11 21 31 41 51 61 FTLIELLIVVAIIGILAAIAIPQFSAYRVKAYNSAASSDLRNLKTALESAFADDQTYPPESYPYDVPDYA

b

c

pilA promoter 0787

pilA-6His

3442

genR

Anti-HA

0788

M 1 lac promoter/operator 1082

lacI

pilA-HA

3442

pilA promoter pilA

kanR

1083

50 kDa 37 kDa 25 kDa 20 kDa 15 kDa 10 kDa

3442


2

Anti-6His 3

M 1

2

3

Anti-PilA M 1

2

3

PilA-HA PilA-6His PilA-WT

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 100 nm 36 37 38 39 40 41


a

e

b

100 nm

c

d

100 nm

100 nm

f

100 nm

40 nm His-tag Label ACS Paragon Plus Environment

100 nm

10 nm HA-tag Label

a

M 0


0.01 1 IPTG (mM) 5000

50 kDa 37 kDa 25 kDa 20 kDa 15 kDa

4000 3000 2000 1000 0

b

No IPTG

PilA-HA (Arbitrary units)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Anti-HA

10 kDa 100 nm

c

0.01 mM IPTG

d

1 mM IPTG

100 nm ACS Paragon Plus Environment

100 nm

Page 28 of 29

Page 29 of 29


7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

> 5 µm length wires

Ni

Y

Introduction of Synthetic Pilin Genes 1 with Peptide Tags Yields 2 3 Conductive Protein Nanowires 4 5 with New Functionalities 6

Applications of Modified Wires Metal binding Antibody conjugation

Pilus assembly

WT pilA

Monomers With HA-Tag pilA-HA

Chromosome DNA pilA-6His

ACS Monomer Paragon Plus Environment Pilin Synthesis

Monomers With His-Tag

Decorating the Outer Surface of Microbially Produced Protein

Recommend Documents