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
Updated information and services can be found at: http://aem.asm.org/content/80/3/1219 These include: SUPPLEMENTAL MATERIAL REFERENCES
CONTENT ALERTS
Supplemental material This article cites 32 articles, 10 of which can be accessed free at: http://aem.asm.org/content/80/3/1219#ref-list-1 Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more»
Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/
Downloaded from http://aem.asm.org/ on January 17, 2014 by ST ANDREWS UNIV
Xing Liu, Pier-Luc Tremblay, Nikhil S. Malvankar, Kelly P. Nevin, Derek R. Lovley and Madeline Vargas Appl. Environ. Microbiol. 2014, 80(3):1219. DOI: 10.1128/AEM.02938-13. Published Ahead of Print 2 December 2013.
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 Xing Liu,a,b Pier-Luc Tremblay,a* Nikhil S. Malvankar,a Kelly P. Nevin,a Derek R. Lovley,a Madeline Vargasa,c Department of Microbiology, University of Massachusetts, Amherst, Massachusetts, USAa; State Key Laboratory of Bioelectronics, Southeast University, Nanjing, People’s Republic of Chinab; Department of Biology, College of the Holy Cross, Worcester, Massachusetts, USAc
T
he capacity of Geobacter species for extracellular electron transfer allows them to play an important role in processes such as metal reduction in soils and sediments, bioremediation of organic and metal contaminants in the subsurface, the conversion of organic wastes to electricity, and interspecies electron transfer to promote methane production (1). Geobacter sulfurreducens, and presumably other Geobacter species, produce electrically conductive pili that have been proposed to serve as a conduit for extracellular electron transfer (2, 3), but the mechanisms for electron transfer along G. sulfurreducens pili is a matter of considerable debate (4–6). Laboratories that have experimentally investigated G. sulfurreducens pili have concluded that electron transport along the pili is an intrinsic function of the pili itself and does not involve electron hopping/tunneling between traditional electron transfer proteins, such as cytochromes (2, 3, 7, 8), even though the c-type cytochrome OmcS is associated with the pili (9, 10). This contrasts to the proposed cytochrome-to-cytochrome electron hopping/tunneling along conductive filaments of Shewanella oneidensis (11, 12). A potential explanation for cytochrome-independent electron transport along G. sulfurreducens pili is that the pili possess metallike conductivity, similar to that observed in carbon nanotubes and synthetic organic conducting polymers (2). Initial evidence for metal-like conductivity in pilus preparations included (i) temperature dependence of conductivity characteristic of metal-like conductivity and inconsistent with electron hopping/tunneling (2), (ii) pH dependence of conductivity characteristic of metallike conductivity (2), (iii) no impact of denaturing cytochromes on conductivity (2), and (iv) spacing of cytochromes on the pili too great for electron hopping/tunneling (9, 10). The metal-like conductivity was suggested to be due to overlapping pi-pi orbitals of aromatic amino acids in the carboxy terminus of PilA (2), which modeling studies suggest are exposed on
February 2014 Volume 80 Number 3
the outer surface of the pili (13). Replacing the gene for PilA, the structural pilin protein, with a gene encoding a PilA in which an alanine was substituted for each of five aromatic amino acids in the carboxyl terminus of PilA resulted in a strain, designated Aro5, which produced pili with properly localized OmcS but with greatly diminished conductivity (7). The Aro5 strain was unable to effectively reduce Fe(III) oxide or produce high current densities. Electrostatic force microscopy studies revealed that a charge injected into native individual wild-type pili, still attached to cells, propagated along the pili in a manner comparable to that observed in carbon nanotubes, even in regions in which no cytochromes were present (N. S. Malvankar, S. E. Yalcin, M. T. Tuominen, and D. R. Lovley, submitted for publication). But there was no such charge propagation in the Aro5 strain. We considered that an alternative approach to the study of pilus function might be to evaluate the phenotype when pili from other bacteria that are not effective in extracellular electron transfer, but possess other important pilus functions, such as attachment, biofilm formation, and motility (14, 15), were expressed in G. sulfurreducens. Type IV pili from Bacteroides nodosus (16), Moraxella bovis (17), and Neisseria gonorrhoeae (18) were cor-
Received 10 September 2013 Accepted 27 November 2013 Published ahead of print 2 December 2013 Address correspondence to Derek R. Lovley, [email protected]. * Present address: Pier-Luc Tremblay, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.02938-13. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.02938-13
Applied and Environmental Microbiology
p. 1219 –1224
aem.asm.org
1219
Downloaded from http://aem.asm.org/ on January 17, 2014 by ST ANDREWS UNIV
The conductive pili of Geobacter species play an important role in electron transfer to Fe(III) oxides, in long-range electron transport through current-producing biofilms, and in direct interspecies electron transfer. Although multiple lines of evidence have indicated that the pili of Geobacter sulfurreducens have a metal-like conductivity, independent of the presence of c-type cytochromes, this claim is still controversial. In order to further investigate this phenomenon, a strain of G. sulfurreducens, designated strain PA, was constructed in which the gene for the native PilA, the structural pilin protein, was replaced with the PilA gene of Pseudomonas aeruginosa PAO1. Strain PA expressed and properly assembled P. aeruginosa PilA subunits into pili and exhibited a profile of outer surface c-type cytochromes similar to that of a control strain expressing the G. sulfurreducens PilA. Surprisingly, the strain PA pili were decorated with the c-type cytochrome OmcS in a manner similar to the control strain. However, the strain PA pili were 14-fold less conductive than the pili of the control strain, and strain PA was severely impaired in Fe(III) oxide reduction and current production. These results demonstrate that the presence of OmcS on pili is not sufficient to confer conductivity to pili and suggest that there are unique structural features of the G. sulfurreducens PilA that are necessary for conductivity.
Liu et al.
sulfurreducens DL1. All the sequences start from the PilD cleavage site. Aromatic amino acids of G. sulfurreducens PilA essential for pilus conductivity are highlighted in red or yellow. The conserved sequences are underlined. The alignment was made with ClustalW2.
rectly assembled when the appropriate genes were expressed in Pseudomonas aeruginosa. However, in those instances, all the heterologous type IV pili had a structure similar to the P. aeruginosa type IV pili, whereas the PilA sequence of G. sulfurreducens differs significantly from the PilA of many bacteria (Fig. 1). Most intensively studied PilA have a similar structure with a highly conserved hydrophobic ␣-helical N-terminal region involved in pilin maturation and assembly and a globular domain in the carboxy terminus (14). However, after processing of the prepilin by PilD, the G. sulfurreducens PilA is much smaller (61 amino acids) than the processed PilAs of previously studied Gram-negative bacteria (140 to 161 amino acids), retaining the highly conserved ␣-helical N-terminal region but not the C-terminal globular domain, which is replaced by a small random-coiled segment (8). It has been suggested that these differences in sequence (Fig. 1) are a likely explanation for the previously described lack of conductivity of natively expressed P. aeruginosa pili (3). Here, we report that despite its much larger size and structural differences, the PilA of P. aeruginosa was not only expressed in G. sulfurreducens but also assembled into pili. Surprisingly, OmcS attached to these heterologous pili. However, the pili had low conductivity, and the strain expressing these pili was deficient in long-range extracellular electron transfer. MATERIALS AND METHODS Bacterial strains, plasmids, and culture conditions. All bacterial strains and plasmids used in this study are summarized in Table S1 in the supplemental material. The G. sulfurreducens strains were routinely cultured at 30°C under strict anaerobic conditions (80/20 N2-CO2) in mineralbased medium containing acetate (20 mM) as the electron donor and either Fe(III) citrate (56 mM), fumarate (40 mM), or Fe(III) oxide (100 mmol/liter) as the electron acceptor, as previously described (19). Fe(II) was determined with the ferrozine assay (20). Chemically competent E. coli TOP10 (Invitrogen, Grand Island, NY, USA) was used routinely for cloning and cultured at 37°C in Luria-Bertani medium with the appropriate antibiotic. Construction of strain PA. Strain PA was constructed from Geobacter sulfurreducens strain DL1. Primers used for construction of strain PA and
1220
aem.asm.org
relevant genotypes are listed in Table S2 in the supplemental material. To construct strain PA, three DNA fragments were generated independently by PCR. Primer pairs GspilAf/GspilAr and GspilACf/GspilACr amplified the 219 bp upstream and 500 bp downstream of the PilA gene (locus tag GSU1496), respectively, using G. sulfurreducens DL1 genomic DNA as the template. Primer pair PapilAf/PapilAr amplified the Pseudomonas aeruginosa PAO1 PilA gene (locus tag PA4525) from P. aeruginosa genomic DNA. The three PCR products were combined via recombinant PCR with primer pair GspilAf/GspilACr as previously described (21). After the sequence was verified, the recombinant PCR product was digested with XhoI and ligated with the vector pPLT173 using T4 DNA ligase (New England BioLabs, Ipswich, MA, USA). pPLT173 contains 500 bp upstream of the Geobacter pilA gene followed by a gentamicin resistance cassette and an XhoI restriction site. The final plasmid was linearized with NcoI (NEB) and electroporated into DL1 competent cells as previously described (19). Transformants were selected on acetate-fumarate agar plates (19) containing gentamicin (20 g/ml) and were verified by PCR using the primers listed in Table S1 in the supplemental material. Genomic DNA was extracted using the Epicentre MasterPure DNA purification kit (Epicentre Biotechnologies, Madison, WI, USA). Plasmid isolation and gel extraction were performed by using the QIAprep Spin miniprep kit and QIAquick gel extraction kit (Qiagen, Valencia, CA, USA), respectively, by following the manufacturer’s instructions. The high-fidelity JumpStart AccuTaq LA DNA polymerase (Sigma-Aldrich, St. Louis, MO, USA) was used routinely for PCRs. When needed, the PCR products were cloned into vector pCR2.1-TOPO with the TOPO TA cloning kit (Invitrogen). The correct sequence was selected after Sanger sequencing verification. Cell attachment test. G. sulfurreducens strains were cultured anaerobically in NBAF medium (19), which contains acetate as the electron donor and fumarate as the electron acceptor, and incubated at 25°C for 2 days after they reached the stationary phase. Planktonic cells were decanted, and the culture tubes were rinsed twice with Milli-Q water. After decanting the water, 0.1% crystal violet was added to stain the attached cells for 5 min. The stained cells were washed three times with Milli-Q water and then destained with 100% ethanol. The absorbance of the chromatic eluate was measured at 580 nm with a Genesys 2 spectrophotometer (Spectronic Instruments). Western blotting and heme staining analyses. Cell lysates were prepared from late-log-phase cells with B-PER protein extraction reagents
Applied and Environmental Microbiology
Downloaded from http://aem.asm.org/ on January 17, 2014 by ST ANDREWS UNIV
FIG 1 Alignment of mature PilA of Neisseria gonorrhoeae MS11, Moraxella bovis Epp63, Pseudomonas aeruginosa PAO1, Bacteroides nodosus H1, and Geobacter
Geobacter Expression of Pseudomonas Pili
(Thermo Scientific, Waltham, MA, USA), and outer membrane proteins were mechanically sheared from all the strains and isolated as previously described (22). Protein concentrations were measured with the bicinchoninic acid (BCA) assay (Micro BCA protein assay kit; Thermo Scientific). Equal amounts of protein were separated by SDS-PAGE using 12.5% Tris-tricine polyacrylamide gel (Amresco, Solon, OH, USA). For heme staining, reducing reagent was omitted from the SDS loading buffer (23). After electrophoresis, protein bands were semidry transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). The blotting membrane was treated with anti-PAO1 PilA rabbit polyclonal antibody (kindly provided by L. Burrows) and anti-rabbit secondary antibody tagged with horseradish peroxidase (GE Healthcare, Piscataway, NJ, USA) sequentially as described previously (24). The PilA band was visualized by chemiluminescence (SuperSignal West Pico kit; Thermo Scientific). As described previously (23), c-type cytochromes were heme stained using N,N,N=,N=-tetramethylbenzidine. Immunogold labeling and transmission electron microscopy (TEM). The presence of P. aeruginosa PilA pili was verified with immunogold labeling as previously described (9). Briefly, cultures were grown anaerobically in NBAF medium at 25°C. Late-log-phase cells were applied on 400-mesh carbon-coated copper grids for 5 min. The grids were blocked for 30 min in 1⫻ phosphate-buffered saline (PBS; pH 7.0) containing 3% bovine serum albumin (BSA), incubated with anti-PAO1 PilA rabbit antibody at a 1:10 dilution for 2 h, and rinsed three times in 1⫻ PBS. The samples were labeled with 10-nm-gold-conjugated anti-rabbit antibody at a 1:100 dilution (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature, washed three times in 1⫻ PBS, and negatively stained with 2% uranyl acetate. Samples were examined using a Tecnai 12 transmission electron microscope operated at 100 kV, and images were taken with a Teitz TCL camera. The c-type cytochrome OmcS was localized with the same procedure, but the primary antibody was substituted with anti-OmcS rabbit polyclonal antibody at a 1:50 dilution. Current production. The capacity for current production was evaluated in dual-chamber microbial fuel cells with 10 mM acetate as the electron donor and graphite electrodes poised at ⫹300 mV (Ag/AgCl) as the
February 2014 Volume 80 Number 3
electron acceptor as previously described (25). Once current production began, the anode chamber was switched into flowthrough mode at a dilution rate of 0.15/h. Current production was recorded with a Power Lab 4SP and plotted with Chart 5.0 software (ADI Instruments, Mountain View, CA). Confocal microscopy. Anode biofilms were imaged with confocal laser scanning microscopy using the LIVE/DEAD BacLight viability stain kit from Molecular Probes (Eugene, OR, USA) as previously described (26, 27). Images were processed and analyzed using the Leica LAS software (Leica). Measurements of the conductivity of pili. The conductivity of pili was determined as previously described (2). Briefly, cells were grown anaerobically in NBAF medium at 25°C to late log phase. Pili were sheared from cells by vortexing at high speed for 2 min in 150 mM ethanolamine buffer (pH 10.5). Cells and debris were separated by centrifugation (Sorvall RC 6 Plus; Thermo Scientific) at 17,000 ⫻ g for 30 min. The suspended pili were concentrated by ultracentrifugation (Beckman L8-70M) at 100,000 ⫻ g for 3 h. TEM imaging as previously described (2, 7) confirmed the presence of abundant pili in the preparations. The preparations of pili (5 g protein) were placed on split-gold electrodes (2) and air dried overnight in a desiccator, which previous studies (2, 3) demonstrated settles pili on the electrodes to form a network, while maintaining a thin water layer on the pili. A voltage ramp of 0 to 0.05 V was applied across split electrodes in steps of 0.025 V with a source meter (Keithley 2400). After the exponential decay of the transient ionic current, the steady-state electronic current for each voltage was measured every second over a minimum period of 100 s with a LabVIEW data acquisition program (National Instruments). The time-averaged current for each applied voltage was calculated to create the current-voltage (I-V) characteristics and to measure conductance (G), which is given by the relation G ⫽ I/V. As previously described (2, 7), the conductivity of pili was calculated from measured conductance with the formula ⫽ G(2a/gL), where L is the length of the electrodes (L ⫽ 2.54 cm), a is the half-spacing between the electrodes (2a ⫽ 50 m), and g is the film thickness (⬃5 m) measured using confocal microscopy.
aem.asm.org 1221
Downloaded from http://aem.asm.org/ on January 17, 2014 by ST ANDREWS UNIV
FIG 2 Expression and assembly of P. aeruginosa PilA pili in G. sulfurreducens. (A) Western blot analysis of cell-free lysates of wild-type P. aeruginosa PAO1, G. sulfurreducens control strain, and strain PA, in which the gene for the wild-type PilA was replaced with the gene for P. aeruginosa PilA. (B, C) Transmission electron micrographs of negatively stained strain PA, treated with anti-PAO1 PilA rabbit polyclonal antibodies followed by anti-rabbit 10-nm-gold-conjugated secondary antibody. Scale bars represent 100 nm (B) and 50 nm (C). The images in panels B and C are representative of multiple samples.
Liu et al.
RESULTS AND DISCUSSION
Expression and assembly of P. aeruginosa pili in G. sulfurreducens. G. sulfurreducens strain PA was generated (see Fig. S1 in the supplemental material) by deleting the native PilA (Gs-PilA) gene and replacing it on the chromosome with the gene for the PilA of P. aeruginosa PAO1 (Pa-PilA). Cell-free lysates of strain PA contained the Pa-PilA monomer (Fig. 2A). As expected, no Pa-PilA was detected in the control strain, which, as previously described (7), was constructed in the same manner as strain PA but retained the PilA sequence of G. sulfurreducens. Transmission electron microscopy coupled with immunogold labeling with antibodies raised against Pa-PilA demonstrated the assembly of mature Pa-
PilA pili (Fig. 2B and C). Thus, despite significant differences in the size and structure of the mature Pa-PilA and Gs-PilA (Fig. 1), Pa-PilA was expressed and properly assembled into pili in G. sulfurreducens. Furthermore, strain PA was highly effective in attachment. The A580 of crystal violet that bound to late-stationary-phase strain PA cells that attached to culture tubes was 6.2 ⫾ 0.33 (mean ⫾ standard deviation; n ⫽ 3 tubes), whereas the value for the control strain was 0.6 ⫾ 0.001. The 10-fold-higher-attached biomass of strain PA might be attributed to the larger globular domain of the Pa-PilA pili, because the globular domain of type IV pili are thought to be responsible for pilus-mediated cell attachment to surfaces (8, 28).
FIG 4 Rates of Fe(II) production from the reduction of Fe(III) citrate (A) or Fe(III) oxide (B) by strain PA and the control strain. Results are the means and standard deviations from triplicate cultures.
1222
aem.asm.org
Applied and Environmental Microbiology
Downloaded from http://aem.asm.org/ on January 17, 2014 by ST ANDREWS UNIV
FIG 3 Transmission electron micrographs of negatively stained cells successively incubated with anti-OmcS rabbit polyclonal antibodies and anti-rabbit IgG conjugated with 10-nm-gold-labeled secondary antibody. (A, B) Strain PA; (C) pilA-deficient strain of G. sulfurreducens. Scale bar represents 100 nM. Images are representative of multiple samples.
Geobacter Expression of Pseudomonas Pili
days and stained with LIVE/DEAD stain. Scale bar is 25 m. (B) Current production of strain PA and control strain. Results are representative of triplicate replicates.
Outer membrane cytochrome profile and alignment of OmcS along Pa-PilA pili. Outer surface preparations stained for heme revealed similar distributions of c-type cytochromes in strain PA and the control strain, with slightly higher intensity staining for some bands in the preparation from strain PA (see Fig. S2 in the supplemental material). These included OmcZ, a multiheme c-type cytochrome associated with the outer surface of the cell, which is required for optimal current production but not Fe(III) oxide reduction (25, 29). The multiheme c-type cytochrome, OmcS (30), which is required for Fe(III) oxide reduction (22), was also abundant in strain PA (see Fig. S2 in the supplemental material). Immunogold labeling revealed that OmcS was localized along the pili of strain PA (Fig. 3A and B), in a manner similar to that previously described for the specific localization of OmcS along the wild-type pili of strain DL1 (9). An association between OmcS and Pa-PilA pili is surprising, because P. aeruginosa does not produce OmcS. One possibility is that OmcS specifically interacts with PilA in the conserved N terminus region, which G. sulfurreducens and P. aeruginosa share. Another possibility is that the highly hydrophobic nature of OmcS (30) leads to a rather nonspecific association with hydrophobic regions of the pili. Long-range electron transfer. Strain PA reduced soluble Fe(III) citrate as well as the control strain (Fig. 4A), demonstrating that it had the ability to transfer electrons to the outer cell surface. However, strain PA could not effectively reduce Fe(III) oxide (Fig. 4B). The inability to reduce Fe(III) oxides persisted even after prolonged incubation of more than 100 days (data not shown). Strain PA grew as a biofilm on graphite anodes (Fig. 5A) but was deficient in current production compared with the current production of the previously described (7) control strain (Fig. 5B). There was a long lag period before strain PA produced a current, and current production plateaued at levels much lower than those of the control strain. Even after the extended incubation necessary to observe strain PA current production, the strain PA biofilms (Fig. 5A) were much thinner than the ca. 30-m-thick biofilms previously reported (7) for the control strain. The diminished current production and Fe(III) oxide reduction of strain PA could be attributed to the fact that the P. aeruginosa pili had low conductivity. The conductivity of pilus preparations sheared from strain PA were 14-fold lower (0.45 ⫾ 0.07
February 2014 Volume 80 Number 3
S/cm; mean ⫾ standard deviation, n ⫽ 3 preparations) than those of the control strain (6.49 ⫾ 3.56 S/cm), which is consistent with previous studies which suggested that the biofilms and pili of P. aeruginosa have poor conductivity (2, 3). Implications. The results demonstrate that the structure of PilA is crucial to pilus conductivity and the long-range electron transport capabilities of G. sulfurreducens. Even though strain PA produced abundant pili with OmcS attached, the pili had low conductivity, and the culture had diminished capacity for reduction of Fe(III) oxides and current production. These results are in accordance with the concept that OmcS does not confer pilus conductivity and that pili themselves must have an intrinsic conductivity for electron transfer to Fe(III) oxide and long-range electron transport through current-producing biofilms (31, 32). It is speculated that the OmcS localized on pili are required for Fe(III) oxide reduction because the multiple hemes of OmcS facilitate electron transfer from pili to Fe(III) oxides (31, 32). There is not yet sufficient understanding of the structural features of G. sulfurreducens pili that are responsible for conductivity to definitively state why the pili of P. aeruginosa PAO1 are not conductive. However, of the five aromatic amino acids implicated in contributing to metal-like conductivity of G. sulfurreducens pili (7), the PilA of P. aeruginosa PAO1 lacks aromatic amino acids 3, 4, and 5, and aromatic amino acid 1 is a tyrosine instead of a phenylalanine (Fig. 1). Furthermore, the large globular domain in the C terminus of P. aeruginosa PAO1 PilA may result in structural differences that prevent conductivity. The ability of G. sulfurreducens to assemble the structurally divergent type IV pili of P. aeruginosa PAO1 suggests that expression of PilAs of different structure in G. sulfurreducens may be a productive approach for screening the pili of other organisms for their capacity for long-range electron transport. Such studies are under way. ACKNOWLEDGMENTS We sincerely thank Lori Burrows for the anti-PAO1 PilA antibody. This research was supported by Office of Naval Research grant no. N00014-13-1-0550.
aem.asm.org 1223
Downloaded from http://aem.asm.org/ on January 17, 2014 by ST ANDREWS UNIV
FIG 5 Current production and anode biofilm formation. (A) Confocal scanning fluorescence microscope image of strain PA biofilm grown on an anode for 38
Liu et al.
REFERENCES
1224
aem.asm.org
17. 18. 19. 20. 21.
22.
23.
24. 25.
26.
27. 28. 29.
30.
31. 32.
JR. 1987. Morphogenetic expression of Bacteroides nodosus fimbriae in Pseudomonas aeruginosa. J. Bacteriol. 169:33– 41. Beard MK, Mattick JS, Moore LJ, Mott MR, Marrs CF, Egerton JR. 1990. Morphogenetic expression of Moraxella bovis fimbriae (pili) in Pseudomonas aeruginosa. J. Bacteriol. 172:2601–2607. Hoyne PA, Haas R, Meyer TF, Davies JK, Elleman TC. 1992. Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa. J. Bacteriol. 174:7321–7327. Coppi MV, Leang C, Sandler SJ, Lovley DR. 2001. Development of a genetic system for Geobacter sulfurreducens. Appl. Environ. Microbiol. 67:3180 –3187. http://dx.doi.org/10.1128/AEM.67.7.3180-3187.2001. Lovley DR, Phillips EJP. 1988. Novel mode of microbial energy metabolism: organic carbon oxidation coupled to dissimilatory reduction of iron or manganese. Appl. Environ. Microbiol. 54:1472–1480. Risso C, Methé BA, Elifantz H, Holmes DE, Lovley DR. 2008. Highly conserved genes in Geobacter species with expression patterns indicative of acetate limitation. Microbiology 154:2589 –2599. http://dx.doi.org/10 .1099/mic.0.2008/017244-0. Mehta T, Coppi MV, Childers SE, Lovley DR. 2005. Outer membrane c-type cytochromes required for Fe(III) and Mn(IV) oxide reduction in Geobacter sulfurreducens. Appl. Environ. Microbiol. 71:8634 – 8641. http: //dx.doi.org/10.1128/AEM.71.12.8634-8641.2005. Thomas PE, Ryan D, Levin W. 1976. An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Anal. Biochem. 75:168 –176. http: //dx.doi.org/10.1016/0003-2697(76)90067-1. Giltner CL, Habash M, Burrows LL. 2010. Pseudomonas aeruginosa minor pilins are incorporated into type IV pili. J. Mol. Biol. 398:444 – 461. http://dx.doi.org/10.1016/j.jmb.2010.03.028. Nevin KP, Kim B-C, Glaven RH, Johnson JP, Woodard TL, Methé BA, DiDonato RJ, Jr, Covalla SF, Franks AE, Liu A, Lovley DR. 2009. Anode biofilm transcriptomics reveals outer surface components essential for high density current production in Geobacter sulfurreducens fuel cells. PLoS One 4:e5628. http://dx.doi.org/10.1371/journal.pone.0005628. Franks AE, Nevin KP, Glaven RH, Lovley DR. 2010. Microtoming coupled to microarray analysis to evaluate the spatial metabolic status of Geobacter sulfurreducens biofilms. ISME J. 4:509 –519. http://dx.doi.org /10.1038/ismej.2009.137. Nevin KP, Zhang P, Franks AE, Woodard TL, Lovley DR. 2011. Anaerobes unleashed: aerobic fuel cells of Geobacter sulfurreducens. J. Power Sources 196:7514 –7518. http://dx.doi.org/10.1016/j.jpowsour.2011.05.021. Craig L, Li J. 2008. Type IV pili: paradoxes in form and function. Curr. Opin. Struct. Biol. 18:267–277. http://dx.doi.org/10.1016/j.sbi.2007.12.009. Inoue K, Leang C, Franks AE, Woodard TL, Nevin KP, Lovley DR. 2011. Specific localization of the c-type cytochrome OmcZ at the anode surface in current-producing biofilms of Geobacter sulfurreducens. Environ. Microbiol. Rep. 3:211–217. http://dx.doi.org/10.1111/j.1758-2229 .2010.00210.x. Qian X, Mester T, Morgado L, Arakawa T, Sharma ML, Inoue K, Joseph C, Salgueiro CA, Maroney MJ, Lovley DR. 2011. Biochemical characterization of purified OmcS, a c-type cytochrome required for insoluble Fe(III) reduction in Geobacter sulfurreducens. Biochim. Biophys. Acta 1807:404 – 412. http://dx.doi.org/10.1016/j.bbabio.2011.01.003. Lovley DR. 2012. Long-range electron transport to Fe(III) oxide via pili with metallic-like conductivity. Biochem. Soc. Trans. 40:1186 –1190. http: //dx.doi.org/10.1042/BST20120131. Lovley DR. 2012. Electromicrobiology. Annu. Rev. Microbiol. 66:391– 409. http://dx.doi.org/10.1146/annurev-micro-092611-150104.
Applied and Environmental Microbiology
Downloaded from http://aem.asm.org/ on January 17, 2014 by ST ANDREWS UNIV
1. Lovley DR, Ueki T, Zhang T, Malvankar NS, Shrestha PM, Flanagan KA, Aklujkar M, Butler JE, Giloteaux L, Rotaru A-E, Holmes DE, Franks AE, Orellana R, Risso C, Nevin KP. 2011. Geobacter: the microbe electric’s physiology, ecology, and practical applications. Adv. Microb. Physiol. 59:1–100. http://dx.doi.org/10.1016/B978-0-12-387661-4.00004-5. 2. Malvankar NS, Vargas M, Nevin KP, Franks AE, Leang C, Kim BC, Inoue K, Mester T, Covalla SF, Johnson JP, Rotello VM, Tuominen MT, Lovley DR. 2011. Tunable metallic-like conductivity in microbial nanowire networks. Nat. Nanotechnol. 6:573–579. http://dx.doi.org/10 .1038/nnano.2011.119. 3. Reguera G, McCarthy KD, Mehta T, Nicoll JS, Tuominen MT, Lovley DR. 2005. Extracellular electron transfer via microbial nanowires. Nature 435:1098 –1101. http://dx.doi.org/10.1038/nature03661. 4. Lovley DR. 2011. Live wires: direct extracellular electron exchange for bioenergy and the bioremediation of energy-related contamination. Energy Environ. Sci. 4:4896 – 4906. http://dx.doi.org/10.1039/c1ee02229f. 5. Bonanni PS, Massazza D, Busalmen JP. 2013. Stepping stones in the electron transport from cells to electrodes in Geobacter sulfurreducens biofilms. Phys. Chem. Chem. Phys. 15:10300 –10306. http://dx.doi.org/10 .1039/c3cp50411e. 6. Malvankar NS, Lovley DR. 2012. Microbial nanowires: a new paradigm for biological electron transfer and bioelectronics. ChemSusChem 5:1039 –1046. http://dx.doi.org/10.1002/cssc.201100733. 7. Vargas M, Malvankar NS, Tremblay P-L, Leang C, Smith JA, Patel P, Synoeyenbos-West O, Nevin KP, Lovley DR. 2013. Aromatic amino acids required for pili conductivity and long-range extracellular electron transport in Geobacter sulfurreducens. mBio 4(2):e00105-13. http://dx.doi .org/10.1128/mBio.00105-13. 8. Feliciano GT, da Silva AJR, Reguera G, Artacho E. 2012. Molecular and electronic structure of the peptide subunit of Geobacter sulfurreducens conductive pili from first principles. J. Phys. Chem. A 116:8023– 8030. http://dx.doi.org/10.1021/jp302232p. 9. Leang C, Qian X, Mester T, Lovley DR. 2010. Alignment of the c-type cytochrome OmcS along pili of Geobacter sulfurreducens. Appl. Environ. Microbiol. 76:4080 – 4084. http://dx.doi.org/10.1128/AEM.00023-10. 10. Malvankar NS, Tuominen MT, Lovley DR. 2012. Lack of cytochrome involvement in long-range electron transport through conductive biofilms and nanowires of Geobacter sulfurreducens. Energy Environ. Sci. 5:8651– 8659. http://dx.doi.org/10.1039/c2ee22330a. 11. Pirbadian S, El-Naggar MY. 2012. Multistep hopping and extracellular charge transfer in microbial redox chains. Phys. Chem. Chem. Phys. 14: 13802–13808. http://dx.doi.org/10.1039/c2cp41185g. 12. El-Naggar MY, Wanger G, Leung KM, Yuzvinsky TD, Southam G, Yang J, Lau WM, Nealson KH, Gorby YA. 2010. Electrical transport along bacterial nanowires from Shewanella oneidensis MR-1. Proc. Natl. Acad. Sci. U. S. A. 107:18127–18131. http://dx.doi.org/10.1073/pnas.1004880107. 13. Reardon PN, Mueller KT. 2013. Structure of the type IVa major pilin from the electrically conductive bacterial nanowires of Geobacter sulfurreducens. J. Biol. Chem. 288:29260 –29266. http://dx.doi.org/10.1074/jbc .M113.498527. 14. Craig L, Pique ME, Tainer JA. 2004. Type IV pilus structure and bacterial pathogenicity. Nat. Rev. Microbiol. 2:363–378. http://dx.doi.org/10.1038 /nrmicro885. 15. Burrows LL. 2012. Pseudomonas aeruginosa twitching motility: type IV pili in action. Annu. Rev. Microbiol. 66:493–520. http://dx.doi.org/10 .1146/annurev-micro-092611-150055. 16. Mattick JS, Bills MM, Anderson BJ, Dalrymple B, Mott MR, Egerton
Updated information and services can be found at: http://aem.asm.org/content/80/3/1219 These include: SUPPLEMENTAL MATERIAL REFERENCES
CONTENT ALERTS
Supplemental material This article cites 32 articles, 10 of which can be accessed free at: http://aem.asm.org/content/80/3/1219#ref-list-1 Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more»
Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/
Downloaded from http://aem.asm.org/ on January 17, 2014 by ST ANDREWS UNIV
Xing Liu, Pier-Luc Tremblay, Nikhil S. Malvankar, Kelly P. Nevin, Derek R. Lovley and Madeline Vargas Appl. Environ. Microbiol. 2014, 80(3):1219. DOI: 10.1128/AEM.02938-13. Published Ahead of Print 2 December 2013.
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 Xing Liu,a,b Pier-Luc Tremblay,a* Nikhil S. Malvankar,a Kelly P. Nevin,a Derek R. Lovley,a Madeline Vargasa,c Department of Microbiology, University of Massachusetts, Amherst, Massachusetts, USAa; State Key Laboratory of Bioelectronics, Southeast University, Nanjing, People’s Republic of Chinab; Department of Biology, College of the Holy Cross, Worcester, Massachusetts, USAc
T
he capacity of Geobacter species for extracellular electron transfer allows them to play an important role in processes such as metal reduction in soils and sediments, bioremediation of organic and metal contaminants in the subsurface, the conversion of organic wastes to electricity, and interspecies electron transfer to promote methane production (1). Geobacter sulfurreducens, and presumably other Geobacter species, produce electrically conductive pili that have been proposed to serve as a conduit for extracellular electron transfer (2, 3), but the mechanisms for electron transfer along G. sulfurreducens pili is a matter of considerable debate (4–6). Laboratories that have experimentally investigated G. sulfurreducens pili have concluded that electron transport along the pili is an intrinsic function of the pili itself and does not involve electron hopping/tunneling between traditional electron transfer proteins, such as cytochromes (2, 3, 7, 8), even though the c-type cytochrome OmcS is associated with the pili (9, 10). This contrasts to the proposed cytochrome-to-cytochrome electron hopping/tunneling along conductive filaments of Shewanella oneidensis (11, 12). A potential explanation for cytochrome-independent electron transport along G. sulfurreducens pili is that the pili possess metallike conductivity, similar to that observed in carbon nanotubes and synthetic organic conducting polymers (2). Initial evidence for metal-like conductivity in pilus preparations included (i) temperature dependence of conductivity characteristic of metal-like conductivity and inconsistent with electron hopping/tunneling (2), (ii) pH dependence of conductivity characteristic of metallike conductivity (2), (iii) no impact of denaturing cytochromes on conductivity (2), and (iv) spacing of cytochromes on the pili too great for electron hopping/tunneling (9, 10). The metal-like conductivity was suggested to be due to overlapping pi-pi orbitals of aromatic amino acids in the carboxy terminus of PilA (2), which modeling studies suggest are exposed on
February 2014 Volume 80 Number 3
the outer surface of the pili (13). Replacing the gene for PilA, the structural pilin protein, with a gene encoding a PilA in which an alanine was substituted for each of five aromatic amino acids in the carboxyl terminus of PilA resulted in a strain, designated Aro5, which produced pili with properly localized OmcS but with greatly diminished conductivity (7). The Aro5 strain was unable to effectively reduce Fe(III) oxide or produce high current densities. Electrostatic force microscopy studies revealed that a charge injected into native individual wild-type pili, still attached to cells, propagated along the pili in a manner comparable to that observed in carbon nanotubes, even in regions in which no cytochromes were present (N. S. Malvankar, S. E. Yalcin, M. T. Tuominen, and D. R. Lovley, submitted for publication). But there was no such charge propagation in the Aro5 strain. We considered that an alternative approach to the study of pilus function might be to evaluate the phenotype when pili from other bacteria that are not effective in extracellular electron transfer, but possess other important pilus functions, such as attachment, biofilm formation, and motility (14, 15), were expressed in G. sulfurreducens. Type IV pili from Bacteroides nodosus (16), Moraxella bovis (17), and Neisseria gonorrhoeae (18) were cor-
Received 10 September 2013 Accepted 27 November 2013 Published ahead of print 2 December 2013 Address correspondence to Derek R. Lovley, [email protected]. * Present address: Pier-Luc Tremblay, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.02938-13. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.02938-13
Applied and Environmental Microbiology
p. 1219 –1224
aem.asm.org
1219
Downloaded from http://aem.asm.org/ on January 17, 2014 by ST ANDREWS UNIV
The conductive pili of Geobacter species play an important role in electron transfer to Fe(III) oxides, in long-range electron transport through current-producing biofilms, and in direct interspecies electron transfer. Although multiple lines of evidence have indicated that the pili of Geobacter sulfurreducens have a metal-like conductivity, independent of the presence of c-type cytochromes, this claim is still controversial. In order to further investigate this phenomenon, a strain of G. sulfurreducens, designated strain PA, was constructed in which the gene for the native PilA, the structural pilin protein, was replaced with the PilA gene of Pseudomonas aeruginosa PAO1. Strain PA expressed and properly assembled P. aeruginosa PilA subunits into pili and exhibited a profile of outer surface c-type cytochromes similar to that of a control strain expressing the G. sulfurreducens PilA. Surprisingly, the strain PA pili were decorated with the c-type cytochrome OmcS in a manner similar to the control strain. However, the strain PA pili were 14-fold less conductive than the pili of the control strain, and strain PA was severely impaired in Fe(III) oxide reduction and current production. These results demonstrate that the presence of OmcS on pili is not sufficient to confer conductivity to pili and suggest that there are unique structural features of the G. sulfurreducens PilA that are necessary for conductivity.
Liu et al.
sulfurreducens DL1. All the sequences start from the PilD cleavage site. Aromatic amino acids of G. sulfurreducens PilA essential for pilus conductivity are highlighted in red or yellow. The conserved sequences are underlined. The alignment was made with ClustalW2.
rectly assembled when the appropriate genes were expressed in Pseudomonas aeruginosa. However, in those instances, all the heterologous type IV pili had a structure similar to the P. aeruginosa type IV pili, whereas the PilA sequence of G. sulfurreducens differs significantly from the PilA of many bacteria (Fig. 1). Most intensively studied PilA have a similar structure with a highly conserved hydrophobic ␣-helical N-terminal region involved in pilin maturation and assembly and a globular domain in the carboxy terminus (14). However, after processing of the prepilin by PilD, the G. sulfurreducens PilA is much smaller (61 amino acids) than the processed PilAs of previously studied Gram-negative bacteria (140 to 161 amino acids), retaining the highly conserved ␣-helical N-terminal region but not the C-terminal globular domain, which is replaced by a small random-coiled segment (8). It has been suggested that these differences in sequence (Fig. 1) are a likely explanation for the previously described lack of conductivity of natively expressed P. aeruginosa pili (3). Here, we report that despite its much larger size and structural differences, the PilA of P. aeruginosa was not only expressed in G. sulfurreducens but also assembled into pili. Surprisingly, OmcS attached to these heterologous pili. However, the pili had low conductivity, and the strain expressing these pili was deficient in long-range extracellular electron transfer. MATERIALS AND METHODS Bacterial strains, plasmids, and culture conditions. All bacterial strains and plasmids used in this study are summarized in Table S1 in the supplemental material. The G. sulfurreducens strains were routinely cultured at 30°C under strict anaerobic conditions (80/20 N2-CO2) in mineralbased medium containing acetate (20 mM) as the electron donor and either Fe(III) citrate (56 mM), fumarate (40 mM), or Fe(III) oxide (100 mmol/liter) as the electron acceptor, as previously described (19). Fe(II) was determined with the ferrozine assay (20). Chemically competent E. coli TOP10 (Invitrogen, Grand Island, NY, USA) was used routinely for cloning and cultured at 37°C in Luria-Bertani medium with the appropriate antibiotic. Construction of strain PA. Strain PA was constructed from Geobacter sulfurreducens strain DL1. Primers used for construction of strain PA and
1220
aem.asm.org
relevant genotypes are listed in Table S2 in the supplemental material. To construct strain PA, three DNA fragments were generated independently by PCR. Primer pairs GspilAf/GspilAr and GspilACf/GspilACr amplified the 219 bp upstream and 500 bp downstream of the PilA gene (locus tag GSU1496), respectively, using G. sulfurreducens DL1 genomic DNA as the template. Primer pair PapilAf/PapilAr amplified the Pseudomonas aeruginosa PAO1 PilA gene (locus tag PA4525) from P. aeruginosa genomic DNA. The three PCR products were combined via recombinant PCR with primer pair GspilAf/GspilACr as previously described (21). After the sequence was verified, the recombinant PCR product was digested with XhoI and ligated with the vector pPLT173 using T4 DNA ligase (New England BioLabs, Ipswich, MA, USA). pPLT173 contains 500 bp upstream of the Geobacter pilA gene followed by a gentamicin resistance cassette and an XhoI restriction site. The final plasmid was linearized with NcoI (NEB) and electroporated into DL1 competent cells as previously described (19). Transformants were selected on acetate-fumarate agar plates (19) containing gentamicin (20 g/ml) and were verified by PCR using the primers listed in Table S1 in the supplemental material. Genomic DNA was extracted using the Epicentre MasterPure DNA purification kit (Epicentre Biotechnologies, Madison, WI, USA). Plasmid isolation and gel extraction were performed by using the QIAprep Spin miniprep kit and QIAquick gel extraction kit (Qiagen, Valencia, CA, USA), respectively, by following the manufacturer’s instructions. The high-fidelity JumpStart AccuTaq LA DNA polymerase (Sigma-Aldrich, St. Louis, MO, USA) was used routinely for PCRs. When needed, the PCR products were cloned into vector pCR2.1-TOPO with the TOPO TA cloning kit (Invitrogen). The correct sequence was selected after Sanger sequencing verification. Cell attachment test. G. sulfurreducens strains were cultured anaerobically in NBAF medium (19), which contains acetate as the electron donor and fumarate as the electron acceptor, and incubated at 25°C for 2 days after they reached the stationary phase. Planktonic cells were decanted, and the culture tubes were rinsed twice with Milli-Q water. After decanting the water, 0.1% crystal violet was added to stain the attached cells for 5 min. The stained cells were washed three times with Milli-Q water and then destained with 100% ethanol. The absorbance of the chromatic eluate was measured at 580 nm with a Genesys 2 spectrophotometer (Spectronic Instruments). Western blotting and heme staining analyses. Cell lysates were prepared from late-log-phase cells with B-PER protein extraction reagents
Applied and Environmental Microbiology
Downloaded from http://aem.asm.org/ on January 17, 2014 by ST ANDREWS UNIV
FIG 1 Alignment of mature PilA of Neisseria gonorrhoeae MS11, Moraxella bovis Epp63, Pseudomonas aeruginosa PAO1, Bacteroides nodosus H1, and Geobacter
Geobacter Expression of Pseudomonas Pili
(Thermo Scientific, Waltham, MA, USA), and outer membrane proteins were mechanically sheared from all the strains and isolated as previously described (22). Protein concentrations were measured with the bicinchoninic acid (BCA) assay (Micro BCA protein assay kit; Thermo Scientific). Equal amounts of protein were separated by SDS-PAGE using 12.5% Tris-tricine polyacrylamide gel (Amresco, Solon, OH, USA). For heme staining, reducing reagent was omitted from the SDS loading buffer (23). After electrophoresis, protein bands were semidry transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). The blotting membrane was treated with anti-PAO1 PilA rabbit polyclonal antibody (kindly provided by L. Burrows) and anti-rabbit secondary antibody tagged with horseradish peroxidase (GE Healthcare, Piscataway, NJ, USA) sequentially as described previously (24). The PilA band was visualized by chemiluminescence (SuperSignal West Pico kit; Thermo Scientific). As described previously (23), c-type cytochromes were heme stained using N,N,N=,N=-tetramethylbenzidine. Immunogold labeling and transmission electron microscopy (TEM). The presence of P. aeruginosa PilA pili was verified with immunogold labeling as previously described (9). Briefly, cultures were grown anaerobically in NBAF medium at 25°C. Late-log-phase cells were applied on 400-mesh carbon-coated copper grids for 5 min. The grids were blocked for 30 min in 1⫻ phosphate-buffered saline (PBS; pH 7.0) containing 3% bovine serum albumin (BSA), incubated with anti-PAO1 PilA rabbit antibody at a 1:10 dilution for 2 h, and rinsed three times in 1⫻ PBS. The samples were labeled with 10-nm-gold-conjugated anti-rabbit antibody at a 1:100 dilution (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature, washed three times in 1⫻ PBS, and negatively stained with 2% uranyl acetate. Samples were examined using a Tecnai 12 transmission electron microscope operated at 100 kV, and images were taken with a Teitz TCL camera. The c-type cytochrome OmcS was localized with the same procedure, but the primary antibody was substituted with anti-OmcS rabbit polyclonal antibody at a 1:50 dilution. Current production. The capacity for current production was evaluated in dual-chamber microbial fuel cells with 10 mM acetate as the electron donor and graphite electrodes poised at ⫹300 mV (Ag/AgCl) as the
February 2014 Volume 80 Number 3
electron acceptor as previously described (25). Once current production began, the anode chamber was switched into flowthrough mode at a dilution rate of 0.15/h. Current production was recorded with a Power Lab 4SP and plotted with Chart 5.0 software (ADI Instruments, Mountain View, CA). Confocal microscopy. Anode biofilms were imaged with confocal laser scanning microscopy using the LIVE/DEAD BacLight viability stain kit from Molecular Probes (Eugene, OR, USA) as previously described (26, 27). Images were processed and analyzed using the Leica LAS software (Leica). Measurements of the conductivity of pili. The conductivity of pili was determined as previously described (2). Briefly, cells were grown anaerobically in NBAF medium at 25°C to late log phase. Pili were sheared from cells by vortexing at high speed for 2 min in 150 mM ethanolamine buffer (pH 10.5). Cells and debris were separated by centrifugation (Sorvall RC 6 Plus; Thermo Scientific) at 17,000 ⫻ g for 30 min. The suspended pili were concentrated by ultracentrifugation (Beckman L8-70M) at 100,000 ⫻ g for 3 h. TEM imaging as previously described (2, 7) confirmed the presence of abundant pili in the preparations. The preparations of pili (5 g protein) were placed on split-gold electrodes (2) and air dried overnight in a desiccator, which previous studies (2, 3) demonstrated settles pili on the electrodes to form a network, while maintaining a thin water layer on the pili. A voltage ramp of 0 to 0.05 V was applied across split electrodes in steps of 0.025 V with a source meter (Keithley 2400). After the exponential decay of the transient ionic current, the steady-state electronic current for each voltage was measured every second over a minimum period of 100 s with a LabVIEW data acquisition program (National Instruments). The time-averaged current for each applied voltage was calculated to create the current-voltage (I-V) characteristics and to measure conductance (G), which is given by the relation G ⫽ I/V. As previously described (2, 7), the conductivity of pili was calculated from measured conductance with the formula ⫽ G(2a/gL), where L is the length of the electrodes (L ⫽ 2.54 cm), a is the half-spacing between the electrodes (2a ⫽ 50 m), and g is the film thickness (⬃5 m) measured using confocal microscopy.
aem.asm.org 1221
Downloaded from http://aem.asm.org/ on January 17, 2014 by ST ANDREWS UNIV
FIG 2 Expression and assembly of P. aeruginosa PilA pili in G. sulfurreducens. (A) Western blot analysis of cell-free lysates of wild-type P. aeruginosa PAO1, G. sulfurreducens control strain, and strain PA, in which the gene for the wild-type PilA was replaced with the gene for P. aeruginosa PilA. (B, C) Transmission electron micrographs of negatively stained strain PA, treated with anti-PAO1 PilA rabbit polyclonal antibodies followed by anti-rabbit 10-nm-gold-conjugated secondary antibody. Scale bars represent 100 nm (B) and 50 nm (C). The images in panels B and C are representative of multiple samples.
Liu et al.
RESULTS AND DISCUSSION
Expression and assembly of P. aeruginosa pili in G. sulfurreducens. G. sulfurreducens strain PA was generated (see Fig. S1 in the supplemental material) by deleting the native PilA (Gs-PilA) gene and replacing it on the chromosome with the gene for the PilA of P. aeruginosa PAO1 (Pa-PilA). Cell-free lysates of strain PA contained the Pa-PilA monomer (Fig. 2A). As expected, no Pa-PilA was detected in the control strain, which, as previously described (7), was constructed in the same manner as strain PA but retained the PilA sequence of G. sulfurreducens. Transmission electron microscopy coupled with immunogold labeling with antibodies raised against Pa-PilA demonstrated the assembly of mature Pa-
PilA pili (Fig. 2B and C). Thus, despite significant differences in the size and structure of the mature Pa-PilA and Gs-PilA (Fig. 1), Pa-PilA was expressed and properly assembled into pili in G. sulfurreducens. Furthermore, strain PA was highly effective in attachment. The A580 of crystal violet that bound to late-stationary-phase strain PA cells that attached to culture tubes was 6.2 ⫾ 0.33 (mean ⫾ standard deviation; n ⫽ 3 tubes), whereas the value for the control strain was 0.6 ⫾ 0.001. The 10-fold-higher-attached biomass of strain PA might be attributed to the larger globular domain of the Pa-PilA pili, because the globular domain of type IV pili are thought to be responsible for pilus-mediated cell attachment to surfaces (8, 28).
FIG 4 Rates of Fe(II) production from the reduction of Fe(III) citrate (A) or Fe(III) oxide (B) by strain PA and the control strain. Results are the means and standard deviations from triplicate cultures.
1222
aem.asm.org
Applied and Environmental Microbiology
Downloaded from http://aem.asm.org/ on January 17, 2014 by ST ANDREWS UNIV
FIG 3 Transmission electron micrographs of negatively stained cells successively incubated with anti-OmcS rabbit polyclonal antibodies and anti-rabbit IgG conjugated with 10-nm-gold-labeled secondary antibody. (A, B) Strain PA; (C) pilA-deficient strain of G. sulfurreducens. Scale bar represents 100 nM. Images are representative of multiple samples.
Geobacter Expression of Pseudomonas Pili
days and stained with LIVE/DEAD stain. Scale bar is 25 m. (B) Current production of strain PA and control strain. Results are representative of triplicate replicates.
Outer membrane cytochrome profile and alignment of OmcS along Pa-PilA pili. Outer surface preparations stained for heme revealed similar distributions of c-type cytochromes in strain PA and the control strain, with slightly higher intensity staining for some bands in the preparation from strain PA (see Fig. S2 in the supplemental material). These included OmcZ, a multiheme c-type cytochrome associated with the outer surface of the cell, which is required for optimal current production but not Fe(III) oxide reduction (25, 29). The multiheme c-type cytochrome, OmcS (30), which is required for Fe(III) oxide reduction (22), was also abundant in strain PA (see Fig. S2 in the supplemental material). Immunogold labeling revealed that OmcS was localized along the pili of strain PA (Fig. 3A and B), in a manner similar to that previously described for the specific localization of OmcS along the wild-type pili of strain DL1 (9). An association between OmcS and Pa-PilA pili is surprising, because P. aeruginosa does not produce OmcS. One possibility is that OmcS specifically interacts with PilA in the conserved N terminus region, which G. sulfurreducens and P. aeruginosa share. Another possibility is that the highly hydrophobic nature of OmcS (30) leads to a rather nonspecific association with hydrophobic regions of the pili. Long-range electron transfer. Strain PA reduced soluble Fe(III) citrate as well as the control strain (Fig. 4A), demonstrating that it had the ability to transfer electrons to the outer cell surface. However, strain PA could not effectively reduce Fe(III) oxide (Fig. 4B). The inability to reduce Fe(III) oxides persisted even after prolonged incubation of more than 100 days (data not shown). Strain PA grew as a biofilm on graphite anodes (Fig. 5A) but was deficient in current production compared with the current production of the previously described (7) control strain (Fig. 5B). There was a long lag period before strain PA produced a current, and current production plateaued at levels much lower than those of the control strain. Even after the extended incubation necessary to observe strain PA current production, the strain PA biofilms (Fig. 5A) were much thinner than the ca. 30-m-thick biofilms previously reported (7) for the control strain. The diminished current production and Fe(III) oxide reduction of strain PA could be attributed to the fact that the P. aeruginosa pili had low conductivity. The conductivity of pilus preparations sheared from strain PA were 14-fold lower (0.45 ⫾ 0.07
February 2014 Volume 80 Number 3
S/cm; mean ⫾ standard deviation, n ⫽ 3 preparations) than those of the control strain (6.49 ⫾ 3.56 S/cm), which is consistent with previous studies which suggested that the biofilms and pili of P. aeruginosa have poor conductivity (2, 3). Implications. The results demonstrate that the structure of PilA is crucial to pilus conductivity and the long-range electron transport capabilities of G. sulfurreducens. Even though strain PA produced abundant pili with OmcS attached, the pili had low conductivity, and the culture had diminished capacity for reduction of Fe(III) oxides and current production. These results are in accordance with the concept that OmcS does not confer pilus conductivity and that pili themselves must have an intrinsic conductivity for electron transfer to Fe(III) oxide and long-range electron transport through current-producing biofilms (31, 32). It is speculated that the OmcS localized on pili are required for Fe(III) oxide reduction because the multiple hemes of OmcS facilitate electron transfer from pili to Fe(III) oxides (31, 32). There is not yet sufficient understanding of the structural features of G. sulfurreducens pili that are responsible for conductivity to definitively state why the pili of P. aeruginosa PAO1 are not conductive. However, of the five aromatic amino acids implicated in contributing to metal-like conductivity of G. sulfurreducens pili (7), the PilA of P. aeruginosa PAO1 lacks aromatic amino acids 3, 4, and 5, and aromatic amino acid 1 is a tyrosine instead of a phenylalanine (Fig. 1). Furthermore, the large globular domain in the C terminus of P. aeruginosa PAO1 PilA may result in structural differences that prevent conductivity. The ability of G. sulfurreducens to assemble the structurally divergent type IV pili of P. aeruginosa PAO1 suggests that expression of PilAs of different structure in G. sulfurreducens may be a productive approach for screening the pili of other organisms for their capacity for long-range electron transport. Such studies are under way. ACKNOWLEDGMENTS We sincerely thank Lori Burrows for the anti-PAO1 PilA antibody. This research was supported by Office of Naval Research grant no. N00014-13-1-0550.
aem.asm.org 1223
Downloaded from http://aem.asm.org/ on January 17, 2014 by ST ANDREWS UNIV
FIG 5 Current production and anode biofilm formation. (A) Confocal scanning fluorescence microscope image of strain PA biofilm grown on an anode for 38
Liu et al.
REFERENCES
1224
aem.asm.org
17. 18. 19. 20. 21.
22.
23.
24. 25.
26.
27. 28. 29.
30.
31. 32.
JR. 1987. Morphogenetic expression of Bacteroides nodosus fimbriae in Pseudomonas aeruginosa. J. Bacteriol. 169:33– 41. Beard MK, Mattick JS, Moore LJ, Mott MR, Marrs CF, Egerton JR. 1990. Morphogenetic expression of Moraxella bovis fimbriae (pili) in Pseudomonas aeruginosa. J. Bacteriol. 172:2601–2607. Hoyne PA, Haas R, Meyer TF, Davies JK, Elleman TC. 1992. Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa. J. Bacteriol. 174:7321–7327. Coppi MV, Leang C, Sandler SJ, Lovley DR. 2001. Development of a genetic system for Geobacter sulfurreducens. Appl. Environ. Microbiol. 67:3180 –3187. http://dx.doi.org/10.1128/AEM.67.7.3180-3187.2001. Lovley DR, Phillips EJP. 1988. Novel mode of microbial energy metabolism: organic carbon oxidation coupled to dissimilatory reduction of iron or manganese. Appl. Environ. Microbiol. 54:1472–1480. Risso C, Methé BA, Elifantz H, Holmes DE, Lovley DR. 2008. Highly conserved genes in Geobacter species with expression patterns indicative of acetate limitation. Microbiology 154:2589 –2599. http://dx.doi.org/10 .1099/mic.0.2008/017244-0. Mehta T, Coppi MV, Childers SE, Lovley DR. 2005. Outer membrane c-type cytochromes required for Fe(III) and Mn(IV) oxide reduction in Geobacter sulfurreducens. Appl. Environ. Microbiol. 71:8634 – 8641. http: //dx.doi.org/10.1128/AEM.71.12.8634-8641.2005. Thomas PE, Ryan D, Levin W. 1976. An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Anal. Biochem. 75:168 –176. http: //dx.doi.org/10.1016/0003-2697(76)90067-1. Giltner CL, Habash M, Burrows LL. 2010. Pseudomonas aeruginosa minor pilins are incorporated into type IV pili. J. Mol. Biol. 398:444 – 461. http://dx.doi.org/10.1016/j.jmb.2010.03.028. Nevin KP, Kim B-C, Glaven RH, Johnson JP, Woodard TL, Methé BA, DiDonato RJ, Jr, Covalla SF, Franks AE, Liu A, Lovley DR. 2009. Anode biofilm transcriptomics reveals outer surface components essential for high density current production in Geobacter sulfurreducens fuel cells. PLoS One 4:e5628. http://dx.doi.org/10.1371/journal.pone.0005628. Franks AE, Nevin KP, Glaven RH, Lovley DR. 2010. Microtoming coupled to microarray analysis to evaluate the spatial metabolic status of Geobacter sulfurreducens biofilms. ISME J. 4:509 –519. http://dx.doi.org /10.1038/ismej.2009.137. Nevin KP, Zhang P, Franks AE, Woodard TL, Lovley DR. 2011. Anaerobes unleashed: aerobic fuel cells of Geobacter sulfurreducens. J. Power Sources 196:7514 –7518. http://dx.doi.org/10.1016/j.jpowsour.2011.05.021. Craig L, Li J. 2008. Type IV pili: paradoxes in form and function. Curr. Opin. Struct. Biol. 18:267–277. http://dx.doi.org/10.1016/j.sbi.2007.12.009. Inoue K, Leang C, Franks AE, Woodard TL, Nevin KP, Lovley DR. 2011. Specific localization of the c-type cytochrome OmcZ at the anode surface in current-producing biofilms of Geobacter sulfurreducens. Environ. Microbiol. Rep. 3:211–217. http://dx.doi.org/10.1111/j.1758-2229 .2010.00210.x. Qian X, Mester T, Morgado L, Arakawa T, Sharma ML, Inoue K, Joseph C, Salgueiro CA, Maroney MJ, Lovley DR. 2011. Biochemical characterization of purified OmcS, a c-type cytochrome required for insoluble Fe(III) reduction in Geobacter sulfurreducens. Biochim. Biophys. Acta 1807:404 – 412. http://dx.doi.org/10.1016/j.bbabio.2011.01.003. Lovley DR. 2012. Long-range electron transport to Fe(III) oxide via pili with metallic-like conductivity. Biochem. Soc. Trans. 40:1186 –1190. http: //dx.doi.org/10.1042/BST20120131. Lovley DR. 2012. Electromicrobiology. Annu. Rev. Microbiol. 66:391– 409. http://dx.doi.org/10.1146/annurev-micro-092611-150104.
Applied and Environmental Microbiology
Downloaded from http://aem.asm.org/ on January 17, 2014 by ST ANDREWS UNIV
1. Lovley DR, Ueki T, Zhang T, Malvankar NS, Shrestha PM, Flanagan KA, Aklujkar M, Butler JE, Giloteaux L, Rotaru A-E, Holmes DE, Franks AE, Orellana R, Risso C, Nevin KP. 2011. Geobacter: the microbe electric’s physiology, ecology, and practical applications. Adv. Microb. Physiol. 59:1–100. http://dx.doi.org/10.1016/B978-0-12-387661-4.00004-5. 2. Malvankar NS, Vargas M, Nevin KP, Franks AE, Leang C, Kim BC, Inoue K, Mester T, Covalla SF, Johnson JP, Rotello VM, Tuominen MT, Lovley DR. 2011. Tunable metallic-like conductivity in microbial nanowire networks. Nat. Nanotechnol. 6:573–579. http://dx.doi.org/10 .1038/nnano.2011.119. 3. Reguera G, McCarthy KD, Mehta T, Nicoll JS, Tuominen MT, Lovley DR. 2005. Extracellular electron transfer via microbial nanowires. Nature 435:1098 –1101. http://dx.doi.org/10.1038/nature03661. 4. Lovley DR. 2011. Live wires: direct extracellular electron exchange for bioenergy and the bioremediation of energy-related contamination. Energy Environ. Sci. 4:4896 – 4906. http://dx.doi.org/10.1039/c1ee02229f. 5. Bonanni PS, Massazza D, Busalmen JP. 2013. Stepping stones in the electron transport from cells to electrodes in Geobacter sulfurreducens biofilms. Phys. Chem. Chem. Phys. 15:10300 –10306. http://dx.doi.org/10 .1039/c3cp50411e. 6. Malvankar NS, Lovley DR. 2012. Microbial nanowires: a new paradigm for biological electron transfer and bioelectronics. ChemSusChem 5:1039 –1046. http://dx.doi.org/10.1002/cssc.201100733. 7. Vargas M, Malvankar NS, Tremblay P-L, Leang C, Smith JA, Patel P, Synoeyenbos-West O, Nevin KP, Lovley DR. 2013. Aromatic amino acids required for pili conductivity and long-range extracellular electron transport in Geobacter sulfurreducens. mBio 4(2):e00105-13. http://dx.doi .org/10.1128/mBio.00105-13. 8. Feliciano GT, da Silva AJR, Reguera G, Artacho E. 2012. Molecular and electronic structure of the peptide subunit of Geobacter sulfurreducens conductive pili from first principles. J. Phys. Chem. A 116:8023– 8030. http://dx.doi.org/10.1021/jp302232p. 9. Leang C, Qian X, Mester T, Lovley DR. 2010. Alignment of the c-type cytochrome OmcS along pili of Geobacter sulfurreducens. Appl. Environ. Microbiol. 76:4080 – 4084. http://dx.doi.org/10.1128/AEM.00023-10. 10. Malvankar NS, Tuominen MT, Lovley DR. 2012. Lack of cytochrome involvement in long-range electron transport through conductive biofilms and nanowires of Geobacter sulfurreducens. Energy Environ. Sci. 5:8651– 8659. http://dx.doi.org/10.1039/c2ee22330a. 11. Pirbadian S, El-Naggar MY. 2012. Multistep hopping and extracellular charge transfer in microbial redox chains. Phys. Chem. Chem. Phys. 14: 13802–13808. http://dx.doi.org/10.1039/c2cp41185g. 12. El-Naggar MY, Wanger G, Leung KM, Yuzvinsky TD, Southam G, Yang J, Lau WM, Nealson KH, Gorby YA. 2010. Electrical transport along bacterial nanowires from Shewanella oneidensis MR-1. Proc. Natl. Acad. Sci. U. S. A. 107:18127–18131. http://dx.doi.org/10.1073/pnas.1004880107. 13. Reardon PN, Mueller KT. 2013. Structure of the type IVa major pilin from the electrically conductive bacterial nanowires of Geobacter sulfurreducens. J. Biol. Chem. 288:29260 –29266. http://dx.doi.org/10.1074/jbc .M113.498527. 14. Craig L, Pique ME, Tainer JA. 2004. Type IV pilus structure and bacterial pathogenicity. Nat. Rev. Microbiol. 2:363–378. http://dx.doi.org/10.1038 /nrmicro885. 15. Burrows LL. 2012. Pseudomonas aeruginosa twitching motility: type IV pili in action. Annu. Rev. Microbiol. 66:493–520. http://dx.doi.org/10 .1146/annurev-micro-092611-150055. 16. Mattick JS, Bills MM, Anderson BJ, Dalrymple B, Mott MR, Egerton
