TY - JOUR
AU - Lovley, Derek R
AB - Abstract Physiological studies and biotechnology applications of Geobacter species have been limited by a lack of genetic tools. Therefore, potential additional molecular strategies for controlling metabolism were explored. When the gene for citrate synthase, or acetyl-CoA transferase, was placed under the control of a LacI/IPTG regulator/inducer system, cells grew on acetate only in the presence of IPTG. The TetR/AT system could also be used to control citrate synthase gene expression and acetate metabolism. A strain that required IPTG for growth on d-lactate was constructed by placing the gene for d-lactate dehydrogenase under the control of the LacI/IPTG system. d-Lactate served as an inducer in a strain in which a d-lactate responsive promoter and transcription repressor were used to control citrate synthase expression. Iron- and potassium-responsive systems were successfully incorporated to regulate citrate synthase expression and growth on acetate. Linking the appropriate degradation tags on the citrate synthase protein made it possible to control acetate metabolism with either the endogenous ClpXP or exogenous Lon protease and tag system. The ability to control current output from Geobacter biofilms and the construction of an AND logic gate for acetate metabolism suggested that the tools developed may be applicable for biosensor and biocomputing applications. Electronic supplementary material The online version of this article (doi:10.1007/s10295-016-1836-5) contains supplementary material, which is available to authorized users. Introduction Geobacter species have biotechnological significance due to their ability to effectively exchange electrons with other species, metals, and electrodes as well as their role in the bioremediation of a diversity of organic and metal contaminants [37, 38]. Methods for deleting and inserting genes of interest in the chromosome of Geobacter sulfurreducens [11, 12] and Geobacter metallireducens [47, 57], as well as expressing genes on plasmids, have been described and have been useful in elucidating novel aspects of Geobacter physiology, such as the role of cytochromes and other redox proteins in extracellular electron transport [28, 32, 35, 36, 41, 50, 51, 64], electron transfer via electrically conductive pili [34, 48, 63], and the anaerobic degradation of benzene [70] as well as constructing strains for enhanced current production [25, 30, 39]. However, methods for specifically controlling expression of genes or the abundance of proteins, which are beneficial in strain design, are limited. The LacI/IPTG (isopropyl β-d-1-thiogalactopyranoside) system [67], which has been widely used in a diversity of bacteria, is effective for controlling gene expression in G. sulfurreducens [25, 29, 59, 60, 68]. A vanillate-inducible expression system is also available [11]. Regulation of gene expression can be divided into two modes, induction and repression [65]. Induction is increase in gene expression upon signal sensing and repression is decrease. Examples of the former include LacI/IPTG and AraC/arabinose systems and the latter Fur/Fe(II) and TrpR/tryptophan. Both modes of the regulation can be useful in design of genetic circuits. The Fur/Fe(II) system has been applied to the construction of a regulated gene expression system [17, 33]. This system has the potential for broad applicability in circuit design because it can function either as a repressible system with Fe(II) as the signal, or as an inducible system with Fe(II) chelators such as 2,2′-dipyridyl and EDTA. Two-component signal transduction systems are important molecular signaling devices for sensing and responding to environmental changes in bacteria. These systems generally consist of a sensor histidine kinase that senses an environmental signal and a response regulator that exerts necessary adaptation to the signal, such as transcription of genes for the adaptive response [21]. The histidine kinase becomes active upon sensing a signal via autophosphorylation and then phosphorylates the response regulator. In the case of the response regulator as the transcription factor, the phosphorylated form of the response regulator typically can activate transcription. Two-component systems have been applied to create genetic circuits [46]. The two-component system adds another level of regulation, namely phosphorylation, in controlling gene expression in comparison with the system consisting of a single transcription factor such as the LacI/IPTG system. Furthermore, the two-component system can be rewired to create an artificial signaling system because of the domain or module structures in the histidine kinase and response regulator. This increases versatility of the two-component system in engineering novel connectivity between an input signal and an output response. This was first demonstrated with the osmosensor histidine kinase EnvZ [61]. By replacing the sensory module of EnvZ with that of Tar, the chemoreceptor for aspartate, gene expression mediated through EnvZ was regulated via aspartate instead of osmolality. Since then, many two-component systems have been rewired [46]. Transcription of a gene of interest in a gene circuit can be turned on and off in a variety of ways. However, even after transcription is turned off, translation may continue if mRNA still remains. Furthermore, protein may persist. Therefore, degradation of protein of interest can be a more secure approach to turn off a system. In bacteria, ClpXP and/or Lon proteases are involved in protein degradation. Regulated protein degradation systems using these proteases have been developed. ClpX recognizes the ssrA-tag sequence in many bacteria [2] and proteins have been engineered for degradation with the addition of the ssrA-tag sequence [1, 40]. In Mesoplasma florum Lon, protease degrades protein with the ssrA-tag sequence, and a regulated protein degradation system with Lon and the ssrA-tag sequence from M. florum was engineered [18, 24]. The ssrA-tag sequence of M. florum is very different from those of bacteria other than Mycoplasma and, thus, the engineered Lon degradation system may be useful for more specific protein degradation in bacteria that do not have a M. florum-like tag sequence [18]. One application in which it may be desirable to finely control gene expression and protein levels in G. sulfurreducens is the construction of strains for biosensors and biological computing. Genetic circuits engineered by synthetic biology approaches have been applied to a variety of purposes in a wide range of organisms [8, 27]. Biosensors are considered to have the great potential as analytical tools to detect and quantify compounds of interest and can be created by engineering genetic circuits for sensing and responding to a target compound [55, 62]. The potential advantages of biosensors over conventional analytical techniques are miniaturization, portability, no or minimal sample preparation, simple and rapid measurement, on-site and real-time monitoring, and low cost. Engineered circuits can also be applied in biocomputing, in which a cell senses a molecule as an input signal, processes the input, and executes a molecular and/or cellular response as an output [4]. Biocomputing can be applied in basic physiological studies as well as biotechnological practice. Concepts of biocomputing can be implemented in engineering more sophisticated biosensors [26]. Most microbial strains that have been constructed as biosensors or for biological computing have an output that must further be processed into an electrical signal. Many biosensors use luminescence, fluorescence, or colorimetry as a detection method [55, 62]. There are biosensors that generate electrochemical outputs, but these electric signals are generated in most cases via enzymatic reactions involving a target signal (substrate) [52]. However, microorganisms that can directly donate electrons to electrodes have the potential to produce a direct electrical signal for biosensing and biocomputing [5, 56]. For example, G. sulfurreducens, which can transfer electrons to electrodes [37], functioned as an acetate biosensor with current values as the output [58]. Shewanella oneidensis was genetically engineered for biosensors and biocomputing with electrical outputs [16, 23, 66]. Here, we report on multiple molecular switches for controlling gene expression in G. sulfurreducens and demonstrate their potential for regulating cellular activities. We also demonstrate how the endogenous ClpXP protease system and an exogenous Lon protease system can be used to control degradation of a target protein via a tag sequence to control enzymatic activity. Materials and methods Strains and growth conditions Geobacter sulfurreducens strains were grown anaerobically in a defined medium with fumarate (40 mM) as the electron acceptor at 30 °C, as previously described [12]. Electron donors were either acetate (15 mM), lactate (10 mM), or hydrogen. For growth on hydrogen, the 80 % nitrogen/20 % carbon dioxide gas phase was replaced with an 80 % hydrogen/20 % carbon dioxide gas phase and acetate (4 mM) was provided as a carbon source. The medium was supplemented with appropriate antibiotics, when necessary. Cell growth was monitored by measuring the optical density at 600 nm (OD600). Plate manipulations were conducted at 30 °C in an anaerobic glove box containing an N2:CO2:H2 (73:20:7) atmosphere. Escherichia coli DH5α [19] was used for plasmid preparation and grown in LB medium [43] supplemented with appropriate antibiotics, when necessary. Construction of G. sulfurreducens mutants Introduction of a plasmid vector into G. sulfurreducens and deletion of a gene from the chromosome by the double-crossover homologous recombination in G. sulfurreducens were conducted as described previously [12]. Modification from this method, if necessary, is described in specific cases below. Antibiotics used for selection of mutants were spectinomycin (75 µg/ml), gentamicin (20 µg/ml), kanamycin (200 µM), and chloramphenicol (15 µg/ml). Expression vector pCDN2s pCDN2s was created by replacing the kanamycin resistance gene in pCDNdeII [60] with a spectinomycin resistance gene. The spectinomycin resistance gene was amplified by PCR with the primer pair Sp-F/R listed in Table S1 and pSJS985Q [53] as the template. The spectinomycin resistance gene was cloned at EagI and SphI sites in pCDNdeII. LacI/IPTG system The gltA gene was cloned in pCDN2s for controlling its expression by LacI and IPTG. The gltA gene was amplified by PCR with the primer pair gltA-F/R listed in Table S1 and cloned at NdeI and EcoRI sites. The resultant plasmid was designated gltA/pCDN2s. The gltA/pCDN2s plasmid was introduced into the G. sulfurreducens ∆gltA strain [59]. Transformants were isolated with hydrogen as the electron donor in the presence of spectinomycin. The presence of the plasmid vector was confirmed by plasmid preparation and PCR amplification. Acetyl-CoA transferase The ato1 gene encoding an acetyl-CoA transferase [54] was amplified by PCR with the primer pair ato1-F/R listed in Table S1 and cloned at NdeI and XbaI sites in pCDN2s. The resultant plasmid designated ato1/pCDN2s was introduced into the G. sulfurreducens ATO3 strain lacking ato1 and ato2 genes [54]. Transformants were isolated with hydrogen as the electron donor in the presence of spectinomycin. d-Lactate dehydrogenase The D-ldh genes were amplified by PCR with the primer pair D-ldh-P1/P2 listed in Table S1 and cloned at NdeI and EcoRI sites in pCDN2s. The resultant plasmid designated D-ldh/pCDN2s was introduced into the G. sulfurreducens ∆D-ldh strain (manuscript in preparation). Transformants were isolated in the presence of spectinomycin. Expression vector pJBGt pJBGt (Fig. 5a) was constructed by replacing the lac promoter region in pJBG [9] with a DNA fragment containing the tetR gene and the tet promoter/operator. The DNA fragment, tet-JBG, containing the tetR gene and the tet promoter/operator (Fig. S1) was synthesized with codon optimization for G. sulfurreducens by GenScript and cloned at the AseI and HindIII sites. TetR/AT system The gltA gene was cloned in pJBGt for controlling its expression by TetR and anhydrotetracycline (AT). The gltA gene was amplified by PCR with the primer pair gltA-RBS/R listed in Table S1 and cloned at XbaI and EcoRI sites. The resultant plasmid was designated gltA/pJBGt. The gltA/pJBGt plasmid was introduced into the G. sulfurreducens ∆gltA strain [59]. Transformants were isolated with hydrogen as the electron donor in the presence of spectinomycin. gltA/pJs A promoter-less gltA expression vector, gltA/pJs, was constructed as follows. The gltA gene was amplified by PCR with the primer pair gltA-RBS2/R2 listed in Table S1 and cloned at XbaI and SalI sites in pNEB193 (NEW ENGLAND BioLabs Inc.). XbaI–HindIII (located in pNEB193) DNA fragment containing the gltA gene was cloned in pJBG [9]. The resultant plasmid was designated gltA/pJBG. The gentamicin resistance gene in gltA/pJBG was replaced with the spectinomycin resistance gene, which was amplified by PCR with the primer pair Sp-F2/R2 listed in Table S1. The resultant plasmid was designated gltA/pJs. LrtR/d-lactate system The GSU1623/1624 genes encoding a putative d-lactate dehydrogenase (D-LDH) essential for d-lactate utilization (manuscript in preparation) were replaced in the G. sulfurreducens ∆gltA strain [59] with the gentamicin resistance gene. Flanking DNA fragments were amplified by PCR with primer pairs, D-ldh-P1/P2 and P3/P4 listed in Table S1 for the upstream and the downstream regions, respectively. The DNA fragment of the gentamicin resistance gene was amplified by PCR with the primer pair Gm-fwd/rev and pJBG [9] as the template. These PCR products were digested with restriction enzymes, ligated, and cloned in a plasmid. The plasmid thus constructed was linearized by XhoI. The GSU1623/1624 deletion strain was designated G. sulfurreducens ∆gltA∆D-ldh. Transformants were isolated with hydrogen as the electron donor in the presence of gentamicin. The promoter region of the GSU1622 gene responsive to d-lactate (manuscript in preparation) was amplified by PCR with the primer pair P1622-P1/P2 listed in Table S1 and cloned at the XhoI and XbaI sites in gltA/pJs. The resultant plasmid was designated P1622-gltA/pJs. The lrtR gene (GSU1626) encoding a putative lactate responsive transcription repressor (manuscript in preparation) was amplified by PCR with the primer pair lrtR-P1/P2 listed in Table S1 and cloned at the SphI site in P1622-gltA/pJs. The resultant plasmid was designated P1622-gltA/lrtR/pJs. The P1622-gltA/pJs or P1622-gltA/lrtR/pJs plasmids were introduced into the G. sulfurreducens ∆gltA∆D-ldh strain. Transformants were isolated with hydrogen as the electron donor in the presence of spectinomycin. Fur/Fe(II) system A putative promoter region of the GSU3274 operon (Fig. S2AB) responsive to Fe(II) limitation [14] was amplified by PCR with the primer pair P3274-P1/P2 listed in Table S1 and cloned at the XhoI and XbaI sites in pCMZKT [59]. The resultant plasmid was designated P3274/pCMZKT. P3274/pCMZKT was introduced into the G. sulfurreducens PCA strain [10]. Transformants were isolated in the presence of kanamycin. The resultant strain was designated G. sulfurreducens P3274/pCMZKT and used for a lacZ-reporter assay. The strain was grown in the acetate–fumarate medium supplemented with 200 µM kanamycin. For the Fe(II)-free medium, FeSO4 was omitted. β-Galactosidase activity was measured as described previously [59]. The region for the lacI gene, the tac/lac promoter, the lac operator, and the ribosome-binding site (RBS) in gltA/pCDN2s was replaced with the promoter region of the GSU3274 gene and the RBS of the gltA gene to control its expression by Fur and Fe(II). DNA fragment containing the GSU3274 promoter and the gltA RBS was amplified with the primer pair P3274-P3/RgltA-P4 listed in Table S1 and cloned at ApaI and NdeI sites. The resultant plasmid was designated P3274-gltA/pCDN2s. The fur gene was amplified with the primer pair fur-P1/P2 listed in Table S1 and cloned at the SphI site in P3274-gltA/pCDN2s. The resultant plasmid was designated P3274-gltA/fur/pCDN2s. The P3274-gltA/pCDN2s or P3274-gltA/fur/pCDN2s plasmids were introduced into the G. sulfurreducens ∆gltA strain [59]. Transformants were isolated with hydrogen as the electron donor in the presence of spectinomycin. KdpDE/K+ system A putative promoter region of the kdp operon (Fig. S3) responsive to K+ limitation was amplified by PCR with the primer pair Pkdp-P1/P2 listed in Table S1 and cloned at the XhoI and XbaI sites in pCMZKT [59] or gltA/pJs. The resultant plasmids were designated Pkdp/pCMZKT or Pkdp-gltA/pJs, respectively. Pkdp/pCMZKT was introduced into the G. sulfurreducens PCA strain [10]. Transformants were isolated in the presence of kanamycin. The resultant strain was designated G. sulfurreducens Pkdp/pCMZKT and used for a lacZ-reporter assay. The strain was grown in medium supplemented with 200 µM kanamycin. For the K+-free medium, KH2PO4 and K2HPO4 were replaced with NaH2PO4 and Na2HPO4 and KCl was omitted. β-Galactosidase activity was measured as described previously [59]. Pkdp-gltA/pJs was introduced into the G. sulfurreducens ∆gltA strain [59]. Transformants were isolated with hydrogen as the electron donor in the presence of spectinomycin. ClpXP/tag system The promoter region of the gltA gene was amplified by PCR with the primer pair PgltA-P1/P2 listed in Table S1 and cloned at HindIII and XbaI sites in pNEB193 (New England BioLabs Inc.). The resultant plasmid was designated PgltA/pN. The gltA gene was amplified by PCR with the primer pair gltA-RBS2/R3 listed in Table S1 and cloned at XbaI and SacI sites in PgltA/pN, resulting in PgltA-gltA/pN. A tag sequence was prepared by phosphorylating and annealing oligonucleotides tag-top/bot listed in Table S1. These oligonucleotides also include a transcription termination signal. The annealed tag oligonucleotides were cloned at the SacI and EcoRI sites in PgltA-gltA/pN, resulting in PgltA-gltA-tag/pN. The HindIII–EcoRI DNA fragment containing the gltA promoter and the tagged gltA gene was cloned in pCDN2s. The resultant plasmid was designated PgltA-gltA-tag/pCDN2s. The gltA gene without the tag sequence was amplified by PCR with the primer pair gltA-RBS2/R listed in Table S1. The PgltA-gltA/pCDN2s plasmid was constructed as described for PgltA-gltA-tag/pCDN2s. The PgltA-gltA/pCDN2s or PgltA-gltA-tag/pCDN2s plasmids were introduced into the G. sulfurreducens ∆gltA strain (Fig. 9a). Transformants were isolated with hydrogen as the electron donor in the presence of spectinomycin. The clpX gene was replaced in the G. sulfurreducens ∆gltA strain [59] with the chloramphenicol resistance gene. Flanking DNA fragments were amplified by PCR with primer pairs, clpX-P1/P2 and P3/P4 listed in Table S1 for the upstream and the downstream regions, respectively. The DNA fragment of the chloramphenicol resistance gene was amplified by PCR with the primer pair, Cm-fwd/rev, listed in Table S1 and pJIR750ai (Sigma) as the template. These PCR products were digested with restriction enzymes, ligated, and cloned in a plasmid. The plasmid thus constructed was linearized by XhoI. Transformants were isolated with hydrogen as the electron donor in the presence of chloramphenicol. The clpX deletion strain was designated G. sulfurreducens ∆gltA∆clpX. The clpX gene was amplified by PCR with the primer pair clpX-P5/P6 listed in Table S1 and cloned at NdeI and XhoI sites in PgltA-gltA-tag/pCDN2s. The resultant plasmid designated PgltA-gltA-tag/clpX/pCDN2s was introduced into the G. sulfurreducens ∆gltA∆clpX strain (Fig. 9d). Transformants were isolated with hydrogen as the electron donor in the presence of spectinomycin. Heterologous Lon/tag system The G. sulfurreducens gltA gene with the tag sequence for the Mesoplasma florum Lon (gltA-Mftag) (Fig. S4) was synthesized by GenScript. A lon gene encoding the Lon protease from M. florum (Mf-lon) was synthesized with codon optimization for G. sulfurreducens (Fig. S5) by GenScript. The original TGA codons, which code for tryptophan in M. florum, were replaced with TGG codons because the TGA codon is a stop codon in G. sulfurreducens. The NdeI–XbaI fragment of gltA-Mftag was cloned in pCDN2s, resulting in gltA-Mftag/pCDN2s. The DNA fragment, tet-CD, containing the tetR gene and the tet promoter/operator (Fig. S1) was synthesized with codon optimization for G. sulfurreducens and cloned in pUC57 by GenScript. The BamHI (located in pUC57)–XbaI fragment of tet-CD was cloned in pBluescript (Stratagene), resulting in pBt. The NdeI–XbaI fragment of Mf-lon was cloned in pBt. The HindIII–SalI fragment containing the tetR gene and Mf-lon under the control of the tet promoter/operator was cloned in gltA-Mftag/pCDN2s, resulting in tet-Mflon/gltA-Mftag/pCDN2s. The tet-Mflon/gltA-Mftag/pCDN2s plasmid was introduced into the G. sulfurreducens ∆gltA strain [59]. Transformants were isolated with hydrogen as the electron donor in the presence of spectinomycin. AND logic gate The sfrAB genes [13] were replaced in the G. sulfurreducens ∆gltA strain [59] with the chloramphenicol resistance gene. Flanking DNA fragments were amplified by PCR with primer pairs, sfrAB-P1/P2 and P3/P4 listed in Table S1 for the upstream and the downstream regions, respectively. The chloramphenicol resistance gene was prepared as described above. These PCR products were digested with restriction enzymes, ligated, and cloned in a plasmid. The plasmid thus constructed was linearized by XhoI. The sfrAB deletion strain was designated G. sulfurreducens ∆gltA∆sfrAB. Transformants were isolated with hydrogen as the electron donor in the presence of chloramphenicol. The sfrAB genes were amplified by PCR with primer pairs, sfrAB-P5/P6, -P7/P8, and -P9/P10 listed in Table S1 and cloned at NdeI and XbaI sites in pNEB193 (NEW ENGLAND BioLabs Inc.). The NdeI–XbaI fragment of sfrAB was cloned in pCDN2s, resulting in sfrAB/pCDN2s. The DNA fragment containing the gltA gene under the control of TetR/AT was amplified by PCR with the primer pair tet-R/gltA-R4 listed in Table S1 and gltA/pJBGt as the template and cloned at the XbaI site in sfrAB/pCDN2s, resulting in tet-gltA/sfrAB/pCDN2s. The tet-gltA/sfrAB/pCDN2s plasmid was introduced into the G. sulfurreducens ∆gltA∆sfrAB strain. Transformants were isolated with hydrogen as the electron donor in the presence of spectinomycin. Western blot analysis The ∆gltA strain maintaining gltA/pCDN2s was grown on hydrogen as the electron donor and fumarate as the electron acceptor and IPTG was added at the mid-log phase (OD600 ~0.3). The ∆gltA strains retaining PgltA-gltA/pCDN2s or PgltA-gltA-tag/pCDN2s were grown on hydrogen as the electron donor and fumarate as the electron acceptor. The ∆gltA∆clpX strain retaining PgltA-gltA-tag/clpX/pCDN2s was grown on hydrogen as the electron donor and fumarate as the electron acceptor in the absence or presence of 1 mM IPTG. Western blot analysis was conducted with the antibody against the citrate synthase [59]. Amount of the citrate synthase was quantified by ImageJ (http://imagej.nih.gov/ij/index.html). Current production Cells were grown in current-producing systems as described previously [44, 45] with the following modifications. Biofilms of the ∆gltA strain with gltA/pCDN2s were grown on either graphite (65 cm2) or plantium wire (11 mm2) anodes with hydrogen (gas phase hydrogen 7 %; nitrogen 73 %; carbon dioxide 20 %) as the electron donor and fumarate as the electron acceptor. The graphite anode in H type cell system was poised at +300 mV versus Ag/AgCl [44]. The platinum wire anode in ministack system had 560 O resistance and the cathode was submerged in 50 mM FeCN [45]. The medium was then exchanged with fumarate-free medium, resulting in initiation of current production with hydrogen as the electron donor with acetate (1–3 mM) as a carbon source. To determine the potential for controlling acetate-dependent current production with IPTG, the hydrogen in the gas phase was replaced with nitrogen and the acetate concentration was increased to 10 mM. Current production was monitored as previously described [44] in the absence or presence of 1 mM (graphite) or 2 mM (platinum wire) IPTG. Results and discussion Regulating citrate synthase expression and acetate metabolism activity with the LacI/IPTG system The citrate synthase encoded by the gene gltA controls entry of acetyl-CoA into the TCA cycle and is essential for growth of G. sulfurreducens with acetate as the electron donor, but not H2 [59]. Abundance of both gltA transcripts and citrate synthase is directly linked to rates of G. sulfurreducens growth and acetate metabolism [7, 22, 59, 69], suggesting that regulating citrate synthase expression may be an effective method for controlling rates of G. sulfurreducens respiration and growth. To evaluate this possibility, gltA was deleted from the chromosome and a copy of gltA under the control of the LacI/IPTG system was expressed on the plasmid pCDN2s (Fig. 1a). In the absence of IPTG, the ∆gltA-gltA/pCDN2s strain grew with H2 as the electron donor, but it could not grow on acetate (Fig. 1b). Cultures amended with IPTG grew on acetate (Fig. 1b). Fig. 1 Open in new tabDownload slide Regulation of acetate metabolism with the LacI/IPTG regulator/inducer system. a Construction of the ∆gltA strain with gltA/pCDN2s. b Growth on acetate in the presence (+) or absence (−) of 1 mM IPTG. The inoculum was cells grown with hydrogen as the electron donor. c Time course of induction of citrate synthase. The top panel presents Western blot analysis for citrate synthase and the bottom panel shows quantification of the intensity of the bands in the top panel. IPTG was added at 1 mM and cells were harvested at the indicated time points. d Induction of citrate synthase by different IPTG concentrations. The top panel presents western blot analysis for citrate synthase and the bottom panel shows quantification of the intensity of the bands in the top panel. Cells were harvested at 1 h after IPTG addition. e Growth rates at different IPTG concentrations. Cells were grown with hydrogen as the electron donor to mid-log phase (OD600 ~0.3), the headspace was exchanged with the hydrogen-free gas, and 15 mM acetate and IPTG (0, 0.001, 0.01, 0.1, 1, or 10 mM) were added. Data are a representative of duplicate cultures To evaluate response of the ∆gltA strain with gltA/pCDN2s to IPTG, expression of the citrate synthase by IPTG induction was measured with Western blot analysis. After addition of IPTG to the medium at the mid-log phase, expression of the citrate synthase was observed within 15 min after the addition and kept increasing (Fig. 1c). This suggests that the engineered strain rapidly responded to IPTG. The abundance of citrate synthase and the rate of acetate-dependent growth of the ∆gltA strain with gltA/pCDN2s depended upon the amount of IPTG added (Fig. 1d, e). As expected from the lack of detected citrate synthase within 1 h of induction (Fig. 1d), there was no acetate-dependent growth in the presence of 1 µM IPTG (Fig. 1e). However, even though citrate synthase was not detected with the addition of 10 µM IPTG within an hour (Fig. 1d), 10 µM IPTG stimulated growth as well as 100 µM IPTG, suggesting that some citrate synthase was expressed at the lower IPTG concentration. Growth was faster in the presence of 1 and 10 mM IPTG than with 10 or 100 µM IPTG (Fig. 1e), consistent with higher citrate synthase expression (Fig. 1d), but growth with 10 mM IPTG was not faster than with 1 mM IPTG, consistent with the comparable expression of citrate synthase at these higher IPTG concentrations. These results suggest that the citrate synthase in G. sulfurreducens can be a molecular switch for growth on acetate and that the LacI/IPTG system can tune the expression of the citrate synthase. Coupling regulation of acetate metabolism with current output The ability to control acetate metabolism with IPTG in the ∆gltA strain with gltA/pCDN2s suggested a strategy in which the current output of G. sulfurreducens anode biofilms could be controlled with environmental signals. The ∆gltA strain maintaining gltA/pCDN2s was grown on either a graphite stick anode poised with a potentiostat (Fig. 2a) or unpoised platinum wire anode (Fig. 2b) with hydrogen as the electron donor in a medium that also contained acetate as a carbon source. When the hydrogen in the headspace was replaced with nitrogen, the biofilms produced a low background current (Fig. 2). With the addition of IPTG, there was an immediate increase in current production from the biofilms grown on the graphite stick anode (Fig. 2a), whereas the response of the platinum wire anode was slower (Fig. 2b). Current output maximized within ca. 12 (graphite stick) to 24 (platinum wire) hours (Fig. 2). Fig. 2 Open in new tabDownload slide Current production from acetate of anode biofilms in response to the addition of IPTG. a Poised graphite anode (65 cm2) or b unpoised platinum wire anode (11 mm2) in fuel cell mode. IPTG was added at the time indicated by the arrows Regulating acetate metabolism by controlling expression of acetyl-CoA transferase To determine whether controlling the expression of genes for other steps in acetate metabolism might also be effective, the potential for regulating acetyl-CoA transferase was investigated. Acetyl-CoA transferase, which transfers CoA from succinyl-CoA to acetate to form acetyl-CoA and succinate in the TCA cycle, is necessary for growth on acetate as the electron donor but not hydrogen [54]. G. sulfurreducens has two genes, ato1 and ato2, for acetyl-CoA transferase [54]. The ato1 gene was placed under the control of the LacI/IPTG regulator/inducer system in the plasmid vector ato1/pCDN2s, which was introduced into the G. sulfurreducens ATO3 strain, which lacks both the ato1 and ato2 genes [54]. The ATO3 strain with ato1/pCDN2s could not grow on acetate as the electron donor in the absence of IPTG, but was able to grow on acetate in its presence (Fig. 3). It is likely that other enzymes in the TCA cycle can be used as a switch for growth on acetate. For example, malate dehydrogenase catalyzing the synthesis of oxaloacetate from malate was shown to be required for growth on acetate as the electron donor but not for growth on hydrogen [54]. Fig. 3 Open in new tabDownload slide Regulation of growth on acetate by controlling the expression of the gene for acetyl-CoA transferase with the LacI/IPTG system. The inoculum was cells grown with hydrogen as the electron donor. Cells were grown in the presence (+) or absence (−) of 1 mM IPTG. Data are a representative of duplicate cultures Regulating d-lactate metabolism by controlling expression of d-lactate dehydrogenase Lactate dehydrogenase (LDH), which converts lactate to pyruvate, is a key enzyme in growth on lactate as the electron donor in G. sulfurreducens (manuscript in preparation). G. sulfurreducens can utilize both d- and l-lactate and has a LDH specific for each form of lactate (manuscript in preparation). G. sulfurreducens strains lacking D- or L-LDH are incapable of growing on the respective lactate form as the electron donor (manuscript in preparation). To determine whether D-LDH could be used as a molecular switch for growth on d-lactate, the genes for D-LDH were put under the control of the LacI/IPTG system in the plasmid vector D-ldh/pCDN2s, which was introduced into the previously constructed (manuscript in preparation) G. sulfurreducens ∆D-ldh strain. The ∆D-ldh strain with D-ldh/pCDN2s only grew with d-lactate as the electron donor when IPTG was provided (Fig. 4). This result demonstrates that the D-LDH can be a switch for metabolism of d-lactate. L-LDH could be adopted in a similar fashion. Fig. 4 Open in new tabDownload slide IPTG-inducible growth on d-lactate of a strain in which the d-lactate dehydrogenase was controlled with the LacI/IPTG regulator/inducer system. The inoculum was cells grown with acetate as the electron donor. Cells were grown in the presence (+) or absence (−) of 1 mM IPTG. Data are a representative of duplicate cultures TetR/AT system for controlling gene expression The transcription factor TetR is widely used with tetracycline or its analogs as the inducer for regulation of gene expression [6]. To evaluate whether or not the TetR-based system could be used in G. sulfurreducens, an expression plasmid vector, pJBGt, containing a tetR gene and tet promoter/operator sequence was constructed (Fig. 5a). The gltA gene was placed under the control of the tet promoter/operator in the plasmid vector pJBGt and introduced into the G. sulfurreducens ∆gltA strain. Anhydrotetracycline (AT), an analog of tetracycline, was used as the inducer because it has no antibiotic activity. The ∆gltA strain harboring gltA/pJBGt was able to grow on acetate as the electron donor in the presence of AT, but not in the absence of AT (Fig. 5b), demonstrating that the TetR/AT system can function as an induction system for gene expression in G. sulfurreducens. Fig. 5 Open in new tabDownload slide Application of the TetR/AT system to control the citrate synthase switch for controlling acetate metabolism. a Map of pJBGt. tetR, repressor; POtet, tet promoter/operator; MCS, multiple cloning site; gen, gentamicin resistance gene. b Growth on acetate in presence (+) or absence (−) of 60 nM AT. The inoculum was cells grown with hydrogen as the electron donor. Data are a representative of duplicate cultures d-Lactate responsive system for controlling gene expression Geobacter sulfurreducens more highly expresses genes involved in lactate utilization in the presence of lactate (manuscript in preparation). To determine whether d-lactate could serve as an inducer to control gene expression, D-ldh, the gene for lactate dehydrogenase was deleted from the ∆gltA strain, generating a strain that could not metabolize acetate and d-lactate. The promoter region of gene GSU1622, which is responsive to d-lactate (manuscript in preparation), was used to generate plasmid vector P1622-gltA/pJs in which gltA was under the control of the GSU1622 promoter and this plasmid was introduced into the G. sulfurreducens ∆gltA∆D-ldh strain. The G. sulfurreducens ∆gltA∆D-ldh strain with P1622-gltA/pJs grew on acetate in the absence of d-lactate (Fig. 6a). Therefore, the gene for the transcription repressor LrtR, which was predicted to be responsive to lactate (manuscript in preparation), was included in the plasmid vector P1622-gltA/pJs, yielding P1622-gltA/lrtR/pJs. The G. sulfurreducens ∆gltA∆D-ldh strain with P1622-gltA/lrtR/pJs grew with acetate as the electron donor in the presence of d-lactate but not in its absence (Fig. 6b). These results indicate that LrtR is a d-lactate-responsive transcription repressor and that LrtR and d-lactate can be used to control gene expression and cellular activity in G. sulfurreducens. It should be possible to construct a system in which l-lactate serves as the inducer in a similar manner with the appropriate l-lactate responsive promoter. Fig. 6 Open in new tabDownload slide Application of the LrtR/d-lactate system to control the citrate synthase switch for controlling acetate metabolism. a Growth on acetate without an additional copy of lrtR. b d-Lactate-dependent growth on acetate of the strain containing an additional copy of lrtR with 1 mM d-lactate as the inducer. The presence and absence of the inducer are indicated by + and −, respectively. The inoculum was cells grown with hydrogen as the electron donor. Data are a representative of duplicate cultures Fur/Fe(II) system To further expand the repertoire of genetic tools for regulation of gene expression in G. sulfurreducens, application of the transcription factor Fur (ferric uptake regulator) was evaluated. Fur often functions as an Fe(II)-responsive repressor [15, 31]. Gene GSU3274 was one of the most highly induced genes under Fe(II) limitation [14]. Therefore, the GSU3274 promoter was selected for the evaluation (Fig. 7). The lacZ-reporter assay showed that the DNA fragment containing a putative promoter region of the GSU3274 gene was transcribed in the absence of Fe(II) but not in the presence of Fe(II) (Fig. 7a). A circuit in which Fur/Fe(II) regulated the expression of gltA was designed (Fig. 7b). The gltA gene was put under the control of the GSU3274 promoter by replacing the IPTG-inducible promoter with the GSU3274 promoter in the plasmid vector gltA/pCDN2s, creating P3274-gltA/pCDN2s, which was introduced into the G. sulfurreducens ∆gltA strain (Fig. 7b, c). Fig. 7 Open in new tabDownload slide Application of Fur/Fe(II) system to control the citrate synthase switch. a lacZ-reporter assay. The strain containing P3274/pCMZKT was grown on acetate as the electron donor and fumarate as the electron acceptor in the absence or presence of Fe(II). Cells were harvested at the mid-log phase (OD600 ~0.3) for the assay. b Expected impact of the presence of Fe(II) on transcription of the citrate synthase gene. c Construct of the G. sulfurreducens ∆gltA strain containing P3274-gltA/fur/pCDN2s. The G. sulfurreducens ∆gltA strain containing P3274-gltA/pCDN2s does not contain fur on the plasmid. d Growth of the ∆gltA strain containing P3274-gltA/pCDN2s without the fur gene on the plasmid. e Effect of an additional copy of the fur gene on acetate growth. The inoculum was cells grown with hydrogen in the presence of Fe(II) at the normal concentration (11 µM). Cells were grown with different Fe(II) concentrations as indicated in the figures. Data are a representative of duplicate cultures Fe(II) did not inhibit the growth on acetate of the ∆gltA strain containing P3274-gltA/pCDN2s (Fig. 7d), indicating that the GSU3274 promoter for the gltA gene was not repressed in the presence of Fe(II). It was considered that this might be the result of insufficient Fur, which is required to bind to the promoters of many other genes [14]. To increase the abundance of Fur, fur was included in the plasmid vector P3274-gltA/pCDN2s, generating P3274-gltA/fur/pCDN2s. With increased fur expression, Fe(II) inhibited the growth of the G. sulfurreducens ∆gltA strain with P3274-gltA/fur/pCDN2s, with increased growth inhibition at higher Fe(II) concentrations (Fig. 7e). These results demonstrate that gene expression can be negatively controlled with the Fur system. It is likely that other metal responsive systems can be adopted for controlling gene expression in G. sulfurreducens. For instance, the Ni(II)-dependent transcription factor NikR was shown to regulate nik(MN)1 and nik(MN)2 genes for nickel transporters in G. uraniireducens [3]. The genome of G. sulfurreducens was predicted to encode a homolog of NikR as well as other metal responsive transcription factors [49]. K+ as the inducer with the two-component KdpDE system To determine if signals other than Fe(II) could be used to repress gene expression and to explore whether two-component systems might be useful tools for the design of switches, the possibility of repressing gene expression with K+ via a KdpDE two-component system [20] was investigated. Analysis of the G. sulfurreducens genome identified a homolog of the Kdp system (Fig. S3), which consists of the histidine kinase KdpD and the response regulator KdpE [20]. When the putative promoter region of the kdp operon (Fig. S3B) was incorporated into a lacZ-reporter, the promoter was inactive in the presence of K+, but active in its absence (Fig. 8a). The plasmid vector Pkdp-gltA/pJs in which gltA was placed downstream of the kdp promoter was constructed to control gltA expression with the kdp promoter and this plasmid was introduced into the G. sulfurreducens ∆gltA strain (Fig. 8b). The ∆gltA strain possessing Pkdp-gltA/pJs was capable of growing on acetate as the electron donor in the absence of K+, but not in its presence (Fig. 8c), demonstrating that the KdpDE two-component system and K+ can be applied to manipulate gene expression and metabolism of G. sulfurreducens. Fig. 8 Open in new tabDownload slide Application of KdpDE/K+ system to control the citrate synthase switch. a lacZ-reporter assay. The strain possessing Pkdp/pCMZKT was grown on acetate in the absence or presence of K+. Cells were harvested at the mid-log phase (OD600 ~0.3) for the assay. b Design of regulation of the gltA gene by KdpDE. c Growth on acetate. The inoculum was cells grown with hydrogen and growth on acetate in the presence (+) or absence (−) of K+ (9.4 mM) was monitored Controlling metabolism at the protein level with the ClpXP/tag protein degradation system As detailed in “Introduction”, for some strain constructions, it is desirable to reduce protein stability or to induce removal of the protein. Therefore, the potential of controlling protein degradation with the ClpXP protease complex and the ssrA-tag sequence was investigated. Analysis of the G. sulfurreducens genome identified a homolog of ClpXP (GSU1791, 1792) and the ssrA-tag sequence (GSUR031). The base pairs required for the tag sequence ADNYDYAVAA were added to gltA to yield the tag fused to the citrate synthase at the C-terminal end (Fig. 9a). The gene for the tagged citrate synthase under the control of the native promoter of the gltA gene on the plasmid (PgltA-gltA-tag/pCDN2s) was introduced into the G. sulfurreducens ∆gltA strain (Fig. 9a). A control strain was constructed in the similar manner, but without the fused tag. When cells were grown with hydrogen as the electron donor, citrate synthase could be detected in the control strain, but not the strain expressing the tagged citrate synthase (Fig. 9b). When transferred to medium with acetate as the electron donor the control strain grew, but the strain with the tagged citrate synthase did not (Fig. 9c). These results are consistent with degradation of the tagged citrate synthase. Fig. 9 Open in new tabDownload slide Application of ClpXP/tag protease system to control acetate metabolism. a Construct of the ∆gltA strain containing PgltA-gltA-tag/pCDN2s. b Degradation of the tagged citrate synthase (CS). The ∆gltA strains with either PgltA-gltA/pCDN2s (-tag) or PgltA-gltA-tag/pCDN2s (+tag) were harvested at the mid-log phase (OD600 ~0.3) and citrate synthase was assayed with western blot analysis. c Lack of growth on acetate of the strain with the tagged citrate synthase (+tag). Growth of the strain with the wild-type citrate synthase (−tag) is also shown. The inoculum was cells grown with hydrogen as the electron donor. d Construct of strain with a tagged citrate synthase and inducible ClpX protease. e Lower abundance of citrate synthase when ClpX protease gene was induced. Cells were grown in the absence (−) or presence (+) of IPTG (1 mM) and harvested at the mid-log phase (OD600 ~0.3) for western blot analysis. f Lack of growth when the ClpX protease gene was induced with IPTG. The inoculum was cells grown with hydrogen as the electron donor. Cells were grown in the presence (+) or absence (−) of 1 mM IPTG. Data are a representative of duplicate cultures Then to control when the tagged citrate synthase was degraded the gene for the ClpX subunit of the ClpXP complex was deleted from the ∆gltA strain (Fig. 9d). The clpX gene was placed under the control of the LacI/IPTG system on the plasmid containing the gene for the tagged citrate synthase under the control of the native gltA promoter in the plasmid vector PgltA-gltA-tag/clpX/pCDN2s, which was introduced into the ∆gltA∆clpX strain (Fig. 9d). In the absence of IPTG, the ∆gltA∆clpX strain with PgltA-gltA-tag/clpX/pCDN2s expressed the tagged citrate synthase during growth on hydrogen, but citrate synthase was not detected in the presence of IPTG (Fig. 9e). These results demonstrated that presence of citrate synthase could be controlled via the expression of ClpX. When transferred to the medium with acetate as the electron donor and fumarate as the electron acceptor, the ∆gltA∆clpX strain with PgltA-gltA-tag/clpX/pCDN2s grew in the absence of IPTG, but not in the presence of IPTG (Fig. 9f). These further indicated that the tagged citrate synthase was degraded via ClpX. These results demonstrated that the ClpXP/tag system can be applied to degradation of a desired protein and regulation of cellular activity. Exogenous Lon/tag system The ClpXP protease has natural substrates and thus application of endogenous ClpXP to an artificial substrate may cause an undesired cellular phenotype. Adoption of an exogenous system should provide specific degradation of a target protein. Therefore, the Lon/tag system from M. florum was evaluated in G. sulfurreducens. The tag sequence of M. florum, AANKNEENTNEVPTFMLNAGQANYAFA, was fused to the C-terminal end of the citrate synthase of G. sulfurreducens (Fig. S4). The gene for the tagged citrate synthase (gltA-Mftag) and the gene for the M. florum Lon (Mflon) (Fig. S5) were under the control of the LacI/IPTG and TetR/AT systems, respectively, in the plasmid vector tet-Mflon/gltA-Mftag/pCDN2s (Fig. 10a, b). When transferred to medium with acetate as the electron donor, this strain could grow only in the presence of IPTG and in the absence of AT (Fig. 10c, d). This suggests that the exogenous Lon/tag degradation system can be used to regulate protein degradation and cellular activity in G. sulfurreducens. Fig. 10 Open in new tabDownload slide Application of the exogenous Lon/tag protease system to control acetate metabolism. a Construct of the ∆gltA strain with an IPTG-inducible gene for citrate synthase with a protease tag and an AT-inducible protease gene. Expected response of gene expression (b) and growth (c) to inducers. d Growth on acetate with different combinations of IPTG and AT. The inoculum was cells grown with hydrogen as the electron donor. The presence (+) and absence (−) of IPTG (1 mM) and AT (60 nM) are indicated in the figure. Data are a representative of duplicate cultures Application of genetic tools to biocomputing The ability to control gene expression with multiple inducers offers the potential for biocomputing applications [8, 26, 27]. For example, an AND logic gate with growth as the output was constructed by controlling the expression of two sets of genes required for acetate metabolism, gltA, and the sfrAB genes encoding alpha and beta subunits of NADPH oxidoreductase, which are also essential for growth on acetate, but not hydrogen [13]. A ∆gltA∆sfrAB strain, which was unable to grow on acetate as the electron donor but able to grow on hydrogen, was first constructed and then the plasmid tet-gltA/sfrAB/pCDN2s was introduced, yielding a strain in which both AT and IPTG should be required for growth on acetate. This strain only grew when both the AT and IPTG inputs were present (Fig. 11), demonstrating the possibility of biocomputing with G. sulfurreducens. Fig. 11 Open in new tabDownload slide Creation of AND logic gate with IPTG-dependent expression of gltA and AT-dependent expression of sfrAB. Growth on acetate was only possible in the presence of both inducers. The inoculum was cells grown with hydrogen as the electron donor. The presence (+) and absence (−) of IPTG (1 mM) and AT (60 nM) are indicated in the figure. Data are a representative of duplicate cultures With the other regulatory systems developed here, it should be possible to create more complex genetic computational circuits and incorporate these into a bioelectrochemical system in which the output is a current signal. For instance, construction of a 3-input AND gate seems possible. AT, IPTG, and d-lactate can be used as the inputs to control gltA, sfrAB, and ato genes for growth on acetate as the output. In addition to AND gates, it should be possible to generate other logic gates in G. sulfurreducens. For example, an NOR gate could be generated. Fe(II) and K+ can serve as the inputs for controlling gltA and sfrAB for growth on acetate as the output. In this gate, growth on acetate can be achieved only in the absence of both Fe(II) and K+. It should also be possible to use these logic gates to control the extent of current output for biological computing. Implications The expansion of genetic tools for G. sulfurreducens described here is expected to be helpful for basic physiological studies as well as biotechnological applications. Further development of genetic tools for G. sulfurreducens should be feasible. For instance, the genome of G. sulfurreducens encodes an unusually large number of the two-component signal transduction systems [42], which could be adapted as novel gene regulation systems. Incorporation of exogenous regulatory systems from other organisms is also a possibility. The ability of G. sulfurreducens to directly exchange electrons with electrodes to either produce or consume electrical current [37] offers the possibility of constructing biosensors or biocomputing devices that can accept electrical inputs and/or produce electrical outputs. The approaches described here for inducing or repressing gene expression and controlling protein stability are expected to be useful tools in the further development of such devices. Acknowledgments We thank J. Ward for technical support. This work was supported by Semiconductor Research Corporation (SRC) SemiSynBio (SSB) Program. References 1. Abo T , Inada T, Ogawa K, Aiba H SsrA-mediated tagging and proteolysis of LacI and its role in the regulation of lac operon EMBO J 2000 19 3762 3769 10.1093/emboj/19.14.3762 313975 Google Scholar Crossref Search ADS PubMed WorldCat 2. Baker TA , Sauer RT ClpXP, an ATP-powered unfolding and protein-degradation machine Biochim Biophys Acta 2012 1823 15 28 10.1016/j.bbamcr.2011.06.007 Google Scholar Crossref Search ADS PubMed WorldCat 3. Benanti EL , Chivers PT Geobacter uraniireducens NikR displays a DNA binding mode distinct from other members of the NikR family J Bacteriol 2010 192 4327 4336 10.1128/JB.00152-10 2937385 Google Scholar Crossref Search ADS PubMed WorldCat 4. Benenson Y Biomolecular computing systems: principles, progress and potential Nat Rev Genet 2012 13 455 468 10.1038/nrg3197 Google Scholar Crossref Search ADS PubMed WorldCat 5. Bereza-Malcolm LT , Mann G, Franks AE Environmental sensing of heavy metals through whole cell microbial biosensors: a synthetic biology approach ACS Synth Biol 2015 4 535 546 10.1021/sb500286r Google Scholar Crossref Search ADS PubMed WorldCat 6. Bertram R , Hillen W The application of Tet repressor in prokaryotic gene regulation and expression Microb Biotechnol 2008 1 2 16 Google Scholar Crossref Search ADS PubMed WorldCat 7. Bond DR , Mester T, Nesbo CL, Izquierdo-Lopez AV, Collart FL, Lovley DR Characterization of citrate synthase from Geobacter sulfurreducens and evidence for a family of citrate synthases similar to those of eukaryotes throughout the Geobacteraceae Appl Environ Microbiol 2005 71 3858 3865 10.1128/AEM.71.7.3858-3865.2005 1169064 Google Scholar Crossref Search ADS PubMed WorldCat 8. Brophy JA , Voigt CA Principles of genetic circuit design Nat Methods 2014 11 508 520 10.1038/nmeth.2926 4230274 Google Scholar Crossref Search ADS PubMed WorldCat 9. Butler JE , Kaufmann F, Coppi MV, Nunez C, Lovley DR MacA, a diheme c-type cytochrome involved in Fe(III) reduction by Geobacter sulfurreducens J Bacteriol 2004 186 4042 4045 10.1128/JB.186.12.4042-4045.2004 419948 Google Scholar Crossref Search ADS PubMed WorldCat 10. Caccavo F Jr, Lonergan DJ, Lovley DR, Davis M, Stolz JF, McInerney MJ Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metal-reducing microorganism Appl Environ Microbiol 1994 60 3752 3759 201883 Google Scholar Crossref Search ADS PubMed WorldCat 11. Chan CH , Levar CE, Zacharoff L, Badalamenti JP, Bond DR Scarless genome editing and stable inducible expression vectors for Geobacter sulfurreducens Appl Environ Microbiol 2015 81 7178 7186 10.1128/AEM.01967-15 4579418 Google Scholar Crossref Search ADS PubMed WorldCat 12. Coppi MV , Leang C, Sandler SJ, Lovley DR Development of a genetic system for Geobacter sulfurreducens Appl Environ Microbiol 2001 67 3180 3187 10.1128/AEM.67.7.3180-3187.2001 92998 Google Scholar Crossref Search ADS PubMed WorldCat 13. Coppi MV , O’Neil RA, Leang C, Kaufmann F, Methe BA, Nevin KP, Woodard TL, Liu A, Lovley DR Involvement of Geobacter sulfurreducens SfrAB in acetate metabolism rather than intracellular, respiration-linked Fe(III) citrate reduction Microbiology 2007 153 3572 3585 10.1099/mic.0.2007/006478-0 Google Scholar Crossref Search ADS PubMed WorldCat 14. Embree M , Qiu Y, Shieu W, Nagarajan H, O’Neil R, Lovley D, Zengler K The iron stimulon and fur regulon of Geobacter sulfurreducens and their role in energy metabolism Appl Environ Microbiol 2014 80 2918 2927 10.1128/AEM.03916-13 3993298 Google Scholar Crossref Search ADS PubMed WorldCat 15. Fillat MF The FUR (ferric uptake regulator) superfamily: diversity and versatility of key transcriptional regulators Arch Biochem Biophys 2014 546 41 52 10.1016/j.abb.2014.01.029 Google Scholar Crossref Search ADS PubMed WorldCat 16. Golitsch F , Bucking C, Gescher J Proof of principle for an engineered microbial biosensor based on Shewanella oneidensis outer membrane protein complexes Biosens Bioelectron 2013 47 285 291 10.1016/j.bios.2013.03.010 Google Scholar Crossref Search ADS PubMed WorldCat 17. Guan L , Liu Q, Li C, Zhang Y Development of a Fur-dependent and tightly regulated expression system in Escherichia coli for toxic protein synthesis BMC Biotechnol 2013 13 25 10.1186/1472-6750-13-25 3621691 Google Scholar Crossref Search ADS PubMed WorldCat 18. Gur E , Sauer RT Evolution of the ssrA degradation tag in Mycoplasma: specificity switch to a different protease Proc Natl Acad Sci USA 2008 105 16113 16118 10.1073/pnas.0808802105 2570983 Google Scholar Crossref Search ADS PubMed WorldCat 19. Hanahan D Studies on transformation of Escherichia coli with plasmids J Mol Biol 1983 166 557 580 10.1016/S0022-2836(83)80284-8 Google Scholar Crossref Search ADS PubMed WorldCat 20. Heermann R , Jung K The complexity of the ‘simple’ two-component system KdpD/KdpE in Escherichia coli FEMS Microbiol Lett 2010 304 97 106 10.1111/j.1574-6968.2010.01906.x Google Scholar Crossref Search ADS PubMed WorldCat 21. Hoch JA , Silhavy TJ Two-component signal transduction 1995 Washington, DC ASM Press Google Scholar Crossref Search ADS Google Preview WorldCat COPAC 22. Holmes DE , Nevin KP, O’Neil RA, Ward JE, Adams LA, Woodard TL, Vrionis HA, Lovley DR Potential for quantifying expression of the Geobacteraceae citrate synthase gene to assess the activity of Geobacteraceae in the subsurface and on current-harvesting electrodes Appl Environ Microbiol 2005 71 6870 6877 10.1128/AEM.71.11.6870-6877.2005 1287699 Google Scholar Crossref Search ADS PubMed WorldCat 23. Hu Y , Yang Y, Katz E, Song H Programming the quorum sensing-based AND gate in Shewanella oneidensis for logic gated-microbial fuel cells Chem Commun (Camb) 2015 51 4184 4187 10.1039/C5CC00026B Google Scholar Crossref Search ADS PubMed WorldCat 24. Huang D , Holtz WJ, Maharbiz MM A genetic bistable switch utilizing nonlinear protein degradation J Biol Eng 2012 6 9 10.1186/1754-1611-6-9 3439342 Google Scholar Crossref Search ADS PubMed WorldCat 25. Izallalen M , Mahadevan R, Burgard A, Postier B, Didonato R Jr, Sun J, Schilling CH, Lovley DR Geobacter sulfurreducens strain engineered for increased rates of respiration Metab Eng 2008 10 267 275 10.1016/j.ymben.2008.06.005 Google Scholar Crossref Search ADS PubMed WorldCat 26. Katz E Biocomputing—tools, aims, perspectives Curr Opin Biotechnol 2015 34 202 208 10.1016/j.copbio.2015.02.011 Google Scholar Crossref Search ADS PubMed WorldCat 27. Khalil AS , Collins JJ Synthetic biology: applications come of age Nat Rev Genet 2010 11 367 379 10.1038/nrg2775 2896386 Google Scholar Crossref Search ADS PubMed WorldCat 28. Leang C , Coppi MV, Lovley DR OmcB, a c-type polyheme cytochrome, involved in Fe(III) reduction in Geobacter sulfurreducens J Bacteriol 2003 185 2096 2103 10.1128/JB.185.7.2096-2103.2003 151516 Google Scholar Crossref Search ADS PubMed WorldCat 29. Leang C , Krushkal J, Ueki T, Puljic M, Sun J, Juarez K, Nunez C, Reguera G, DiDonato R, Postier B, Adkins RM, Lovley DR Genome-wide analysis of the RpoN regulon in Geobacter sulfurreducens BMC Genom 2009 10 331 10.1186/1471-2164-10-331 Google Scholar Crossref Search ADS WorldCat 30. Leang C , Malvankar NS, Franks AE, Nevin KP, Lovley DR Engineering Geobacter sulfurreducens to produce a highly cohesive conductive matrix with enhanced capacity for current production Energy Environ Sci 2013 6 1901 1908 10.1039/c3ee40441b Google Scholar Crossref Search ADS WorldCat 31. Lee JW , Helmann JD Functional specialization within the Fur family of metalloregulators Biometals 2007 20 485 499 10.1007/s10534-006-9070-7 Google Scholar Crossref Search ADS PubMed WorldCat 32. Levar CE , Chan CH, Mehta-Kolte MG, Bond DR An inner membrane cytochrome required only for reduction of high redox potential extracellular electron acceptors MBio 2014 5 e02034 10.1128/mBio.02034-14 4251993 Google Scholar Crossref Search ADS PubMed WorldCat 33. Lim JM , Hong MJ, Kim S, Oh DB, Kang HA, Kwon O Iron chelator-inducible expression system for Escherichia coli J Microbiol Biotechnol 2008 18 1357 1363 Google Scholar PubMed OpenURL Placeholder Text WorldCat 34. Liu X , Tremblay PL, Malvankar NS, Nevin KP, Lovley DR, 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 10.1128/AEM.02938-13 3911229 Google Scholar Crossref Search ADS PubMed WorldCat 35. Liu Y , Wang Z, Liu J, Levar C, Edwards MJ, Babauta JT, Kennedy DW, Shi Z, Beyenal H, Bond DR, Clarke TA, Butt JN, Richardson DJ, Rosso KM, Zachara JM, Fredrickson JK, Shi L A trans-outer membrane porin-cytochrome protein complex for extracellular electron transfer by Geobacter sulfurreducens PCA Environ Microbiol Rep 2014 6 776 785 10.1111/1758-2229.12204 4282303 Google Scholar Crossref Search ADS PubMed WorldCat 36. Lloyd JR , Leang C, Hodges Myerson AL, Coppi MV, Cuifo S, Methe B, Sandler SJ, Lovley DR Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens Biochem J 2003 369 153 161 10.1042/bj20020597 1223068 Google Scholar Crossref Search ADS PubMed WorldCat 37. Lovley DR , Ueki T, Zhang T, Malvankar NS, Shrestha PM, Flanagan KA, Aklujkar M, Butler JE, Giloteaux L, Rotaru AE, Holmes DE, Franks AE, Orellana R, Risso C, Nevin KP Geobacter: the microbe electric’s physiology, ecology, and practical applications Adv Microb Physiol 2011 59 1 100 10.1016/B978-0-12-387661-4.00004-5 Google Scholar Crossref Search ADS PubMed WorldCat 38. Malvankar NS , Lovley DR Microbial nanowires for bioenergy applications Curr Opin Biotechnol 2014 27 88 95 10.1016/j.copbio.2013.12.003 Google Scholar Crossref Search ADS PubMed WorldCat 39. Malvankar NS , Mester T, Tuominen MT, Lovley DR Supercapacitors based on c-type cytochromes using conductive nanostructured networks of living bacteria Chem Phys Chem 2012 13 463 468 Google Scholar Crossref Search ADS PubMed WorldCat 40. McGinness KE , Baker TA, Sauer RT Engineering controllable protein degradation Mol Cell 2006 22 701 707 10.1016/j.molcel.2006.04.027 Google Scholar Crossref Search ADS PubMed WorldCat 41. Mehta T , Coppi MV, Childers SE, Lovley DR Outer membrane c-type cytochromes required for Fe(III) and Mn(IV) oxide reduction in Geobacter sulfurreducens Appl Environ Microbiol 2005 71 8634 8641 10.1128/AEM.71.12.8634-8641.2005 1317342 Google Scholar Crossref Search ADS PubMed WorldCat 42. Methe BA , Nelson KE, Eisen JA, Paulsen IT, Nelson W, Heidelberg JF, Wu D, Wu M, Ward N, Beanan MJ, Dodson RJ, Madupu R, Brinkac LM, Daugherty SC, DeBoy RT, Durkin AS, Gwinn M, Kolonay JF, Sullivan SA, Haft DH, Selengut J, Davidsen TM, Zafar N, White O, Tran B, Romero C, Forberger HA, Weidman J, Khouri H, Feldblyum TV, Utterback TR, Van Aken SE, Lovley DR, Fraser CM Genome of Geobacter sulfurreducens: metal reduction in subsurface environments Science 2003 302 1967 1969 10.1126/science.1088727 Google Scholar Crossref Search ADS PubMed WorldCat 43. Miller JH Experiments in molecular genetics 1972 Cold Spring Harbor Cold Spring Harbor Laboratory Press Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 44. Nevin KP , Kim BC, Glaven RH, Johnson JP, Woodard TL, Methe BA, Didonato RJ, Covalla SF, Franks AE, Liu A, Lovley DR Anode biofilm transcriptomics reveals outer surface components essential for high density current production in Geobacter sulfurreducens fuel cells PLoS One 2009 4 e5628 10.1371/journal.pone.0005628 2680965 Google Scholar Crossref Search ADS PubMed WorldCat 45. Nevin KP , Richter H, Covalla SF, Johnson JP, Woodard TL, Orloff AL, Jia H, Zhang M, Lovley DR Power output and columbic efficiencies from biofilms of Geobacter sulfurreducens comparable to mixed community microbial fuel cells Environ Microbiol 2008 10 2505 2514 10.1111/j.1462-2920.2008.01675.x Google Scholar Crossref Search ADS PubMed WorldCat 46. Ninfa AJ Use of two-component signal transduction systems in the construction of synthetic genetic networks Curr Opin Microbiol 2010 13 240 245 10.1016/j.mib.2010.01.003 3547608 Google Scholar Crossref Search ADS PubMed WorldCat 47. Oberender J , Kung JW, Seifert J, von Bergen M, Boll M Identification and characterization of a succinyl-coenzyme A (CoA):benzoate CoA transferase in Geobacter metallireducens J Bacteriol 2012 194 2501 2508 10.1128/JB.00306-12 3347176 Google Scholar Crossref Search ADS PubMed WorldCat 48. Reguera G , McCarthy KD, Mehta T, Nicoll JS, Tuominen MT, Lovley DR Extracellular electron transfer via microbial nanowires Nature 2005 435 1098 1101 10.1038/nature03661 Google Scholar Crossref Search ADS PubMed WorldCat 49. Rodionov DA , Dubchak I, Arkin A, Alm E, Gelfand MS Reconstruction of regulatory and metabolic pathways in metal-reducing delta-proteobacteria Genome Biol 2004 5 R90 10.1186/gb-2004-5-11-r90 545781 Google Scholar Crossref Search ADS PubMed WorldCat 50. Rollefson JB , Levar CE, Bond DR Identification of genes involved in biofilm formation and respiration via mini-Himar transposon mutagenesis of Geobacter sulfurreducens J Bacteriol 2009 191 4207 4217 10.1128/JB.00057-09 2698481 Google Scholar Crossref Search ADS PubMed WorldCat 51. Rollefson JB , Stephen CS, Tien M, Bond DR Identification of an extracellular polysaccharide network essential for cytochrome anchoring and biofilm formation in Geobacter sulfurreducens J Bacteriol 2011 193 1023 1033 10.1128/JB.01092-10 Google Scholar Crossref Search ADS PubMed WorldCat 52. Ronkainen NJ , Halsall HB, Heineman WR Electrochemical biosensors Chem Soc Rev 2010 39 1747 1763 10.1039/b714449k Google Scholar Crossref Search ADS PubMed WorldCat 53. Sandler SJ , Clark AJ RecOR suppression of recF mutant phenotypes in Escherichia coli K-12 J Bacteriol 1994 176 3661 3672 205555 Google Scholar Crossref Search ADS PubMed WorldCat 54. Segura D , Mahadevan R, Juarez K, Lovley DR Computational and experimental analysis of redundancy in the central metabolism of Geobacter sulfurreducens PLoS Comput Biol 2008 4 e36 10.1371/journal.pcbi.0040036 2233667 Google Scholar Crossref Search ADS PubMed WorldCat 55. Su L , Jia W, Hou C, Lei Y Microbial biosensors: a review Biosens Bioelectron 2011 26 1788 1799 10.1016/j.bios.2010.09.005 Google Scholar Crossref Search ADS PubMed WorldCat 56. TerAvest MA , Li ZJ, Angenent LT Bacteria-based biocomputing with cellular computing circuits to sense, decide, signal, and act Energy Environ Sci 2011 4 4907 4916 10.1039/c1ee02455h Google Scholar Crossref Search ADS WorldCat 57. Tremblay PL , Aklujkar M, Leang C, Nevin KP, Lovley D A genetic system for Geobacter metallireducens: role of the flagellin and pilin in the reduction of Fe(III) oxide Environ Microbiol Rep 2012 4 82 88 10.1111/j.1758-2229.2011.00305.x Google Scholar Crossref Search ADS PubMed WorldCat 58. Tront JM , Fortner JD, Plotze M, Hughes JB, Puzrin AM Microbial fuel cell biosensor for in situ assessment of microbial activity Biosens Bioelectron 2008 24 586 590 10.1016/j.bios.2008.06.006 Google Scholar Crossref Search ADS PubMed WorldCat 59. Ueki T , Lovley DR Genome-wide gene regulation of biosynthesis and energy generation by a novel transcriptional repressor in Geobacter species Nucleic Acids Res 2010 38 810 821 10.1093/nar/gkp1085 Google Scholar Crossref Search ADS PubMed WorldCat 60. Ueki T , Lovley DR Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens Nucleic Acids Res 2010 38 7485 7499 10.1093/nar/gkq652 2995071 Google Scholar Crossref Search ADS PubMed WorldCat 61. Utsumi R , Brissette RE, Rampersaud A, Forst SA, Oosawa K, Inouye M Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate Science 1989 245 1246 1249 10.1126/science.2476847 Google Scholar Crossref Search ADS PubMed WorldCat 62. van der Meer JR , Belkin S Where microbiology meets microengineering: design and applications of reporter bacteria Nat Rev Microbiol 2010 8 511 522 10.1038/nrmicro2392 Google Scholar Crossref Search ADS PubMed WorldCat 63. Vargas M , Malvankar NS, Tremblay PL, Leang C, Smith JA, Patel P, Snoeyenbos-West O, Nevin KP, Lovley DR Aromatic amino acids required for pili conductivity and long-range extracellular electron transport in Geobacter sulfurreducens MBio 2013 4 e00105 e00113 10.1128/mBio.00210-13 3604773 Google Scholar Crossref Search ADS PubMed WorldCat 64. Voordeckers JW , Kim BC, Izallalen M, Lovley DR Role of Geobacter sulfurreducens outer surface c-type cytochromes in reduction of soil humic acid and anthraquinone-2,6-disulfonate Appl Environ Microbiol 2010 76 2371 2375 10.1128/AEM.02250-09 2849258 Google Scholar Crossref Search ADS PubMed WorldCat 65. Wall ME , Hlavacek WS, Savageau MA Design of gene circuits: lessons from bacteria Nat Rev Genet 2004 5 34 42 10.1038/nrg1244 Google Scholar Crossref Search ADS PubMed WorldCat 66. Webster DP , TerAvest MA, Doud DF, Chakravorty A, Holmes EC, Radens CM, Sureka S, Gralnick JA, Angenent LT An arsenic-specific biosensor with genetically engineered Shewanella oneidensis in a bioelectrochemical system Biosens Bioelectron 2014 62 320 324 10.1016/j.bios.2014.07.003 Google Scholar Crossref Search ADS PubMed WorldCat 67. Wilson CJ , Zhan H, Swint-Kruse L, Matthews KS The lactose repressor system: paradigms for regulation, allosteric behavior and protein folding Cell Mol Life Sci 2007 64 3 16 10.1007/s00018-006-6296-z Google Scholar Crossref Search ADS PubMed WorldCat 68. Yun J , Malvankar NS, Ueki T, Lovley DR Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation ISME J 2015 10 310 320 10.1038/ismej.2015.113 Google Scholar Crossref Search ADS PubMed WorldCat 69. Yun J , Ueki T, Miletto M, Lovley DR Monitoring the metabolic status of geobacter species in contaminated groundwater by quantifying key metabolic proteins with Geobacter-specific antibodies Appl Environ Microbiol 2011 77 4597 4602 10.1128/AEM.00114-11 3127707 Google Scholar Crossref Search ADS PubMed WorldCat 70. Zhang T , Tremblay PL, Chaurasia AK, Smith JA, Bain TS, Lovley DR Identification of genes specifically required for the anaerobic metabolism of benzene in Geobacter metallireducens Front Microbiol 2014 5 245 4033198 Google Scholar PubMed OpenURL Placeholder Text WorldCat © Society for Industrial Microbiology 2016 This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) © Society for Industrial Microbiology 2016
TI - Genetic switches and related tools for controlling gene expression and electrical outputs of Geobacter sulfurreducens
JF - Journal of Industrial Microbiology and Biotechnology
DO - 10.1007/s10295-016-1836-5
DA - 2016-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/genetic-switches-and-related-tools-for-controlling-gene-expression-and-TOlkmgGofA
SP - 1561
EP - 1575
VL - 43
IS - 11
DP - DeepDyve
ER -