Environmental Microbiology
Research Article
26 May 2021

Proteolytic Maturation of the Outer Membrane c-Type Cytochrome OmcZ by a Subtilisin-Like Serine Protease Is Essential for Optimal Current Production by Geobacter sulfurreducens

ABSTRACT

An outer membrane c-type cytochrome (OmcZ) in Geobacter sulfurreducens is essential for optimal current production in microbial fuel cells. OmcZ exists in two forms, small and large, designated OmcZS and OmcZL, respectively. However, it is still not known how these two structures are formed. A mutant with a disruption of the GSU2075 gene encoding a subtilisin-like serine protease (designated ozpA for the OmcZ protease), which is located downstream of omcZ, produced low currents at a level similar to that of the omcZ-deficient mutant strain. Biochemical analyses revealed that the ozpA mutant accumulated OmcZL and did not produce OmcZS, which is thought to be a mature form that is essential for the extracellular electron transfer to the electrode. A heterologous expression system cell lysate from an Escherichia coli strain producing OzpA cleaved OmcZL and generated OmcZS as the proteolytic product. Among the culture supernatant, loosely bound outer surface, and intracellular protein fractions from wild-type G. sulfurreducens, only the culture supernatant protein fraction showed OmcZL cleavage activity, indicating that the mature form of OmcZ, OmcZS, can be produced outside the cells. These results indicate that OzpA is an essential protease for current production via the maturation of OmcZ, and OmcZS is the key to the extracellular electron transfer to electrodes. This proteolytic maturation of OmcZ is a unique regulation among known c-type cytochromes in G. sulfurreducens.
IMPORTANCE Microbial fuel cells are a promising technology for energy generation from various waste types. However, the molecular mechanisms of microbial extracellular electron transfer to the electrode need to be elucidated. G. sulfurreducens is a common key player in electricity generation in mixed-culture microbial fuel cell systems and a model microorganism for the study of extracellular electron transfer. Outer membrane c-type cytochrome OmcZ is essential for an optimal current production by G. sulfurreducens. OmcZ proteolytic cleavage occurs during maturation, but the underlying mechanism is unknown. This study identifies a subtilisin-like protease, OzpA, which plays a role in cleaving OmcZ and generating the mature form of OmcZ (OmcZS). OzpA is essential for current production and, thus, the proteolytic maturation of OmcZ. This is a novel regulation of the c-type cytochrome for G. sulfurreducens extracellular electron transfer. This study also provides new insights into the design strategy and development of microbial extracellular electron transfer for an efficient energy conversion from chemical energy to electricity.

INTRODUCTION

Iron-reducing bacteria can transfer electrons not only to metal ions but also to electrodes for respiration (1). Geobacter sulfurreducens produces a significantly high current as pure cultured microbial fuel cells (MFCs) (2, 3). An effective extracellular electron transfer (EET) is essential for current generation in MFCs. Understanding the critical components of the EET is necessary for improving the performance of MFCs and iron reduction. The MFC is a promising technology for electricity generation from organic compounds, including organic wastes. Various kinds of microorganisms have been identified as electroactive microorganisms that transfer electrons to electrodes. Among the electroactive microorganisms, G. sulfurreducens is generally the predominant bacteria on the anode in mixed-culture MFCs (1). G. sulfurreducens directly transfers electrons to the electrode via c-type cytochromes and electrically conductive nanowires (46). According to the genomic, transcriptomic, and proteomic analyses of G. sulfurreducens, it possesses 111 c-type cytochrome genes, and most of these genes are expressed (710). It has been proposed that electrons are transferred across the inner membrane via inner membrane cytochromes ImcH (GSU3259) or CbcL (GSU0274) to periplasmic cytochromes, such as PpcA (GSU0612) (11, 12). Porin-cytochrome complexes composed of a periplasmic cytochrome, a porin-like outer membrane protein, and an outer membrane cytochrome were demonstrated to be involved in electron transfer via the outer membrane (1315). Among the 5 gene clusters of the porin-cytochrome complex, OmbB, OmaB, and OmcB (GSU2737 to GSU2739) are essential for EET to iron oxide (16). Another porin-cytochrome complex composed of the extABCD gene cluster (GSU2645 to GSU2642) is required for an effective electron transfer to the electrodes (15). Some other outer membrane cytochromes and electrically conductive nanowires are involved in the electron transfer from the outer membrane to the extracellular electron acceptors. OmcS has been studied as an essential outer membrane cytochrome for iron reduction and is also important for current production (17). Immunolocalization analysis of OmcS (GSU2504), which is required for EET to iron oxide (18, 19), demonstrated its localization along with the electrically conductive nanowires (20). Recently, cryo-electron microscopic analysis of the conductive nanowires revealed that the nanowires were solely composed of OmcS (21, 22). Another c-type cytochrome OmcZ (product of GSU2076) also forms conductive nanowires with 1,000-fold higher conductivity than OmcS nanowires (23).
Among the characterized outer membrane c-type cytochromes, omcZ (GSU2076) is one of the essential genes for an optimal current production in microbial fuel cells (24). Transcriptome analysis revealed that omcZ was significantly upregulated under current-producing conditions, compared with fumarate-reducing conditions, and omcZ disruption significantly inhibited current production and biofilm formation on the anode. An unusual feature of OmcZ is that it exists in small and large forms (designated OmcZS [30 kDa] and OmcZL [50 kDa], respectively) (25). N- and C-terminal amino acid sequencing of purified OmcZS indicated that OmcZS was cleaved from OmcZL, which appeared as a full-length OmcZ, with the loss of the C-terminal part of OmcZL. According to molecular weight measurements using electrospray ionization mass spectrometry and heme quantification using pyridine hemochrome analysis, OmcZS retains all 8 hemes in its molecule with 7 typical heme-binding motifs (CXXCH) and an unusual motif, CX14CH (25). Subcellular fractioning, followed by Western blotting using an anti-OmcZ antibody, revealed that OmcZS was mainly localized in the outer membrane or the extracellular matrix. OmcZS is extremely insoluble in general biochemical buffers; thus, purified OmcZS was obtained from the insoluble fraction using a detergent extraction method, whereas the solubility of OmcZL was much higher than that of OmcZS (25). Subsequent immunoelectron microscopic localization of OmcZ using the anti-OmcZ antibody indicated that OmcZ (OmcZL or OmcZS) was dispersed in the extracellular matrix, highly accumulating at the anode surface when G. sulfurreducens grew on a graphite electrode as the sole electron acceptor (26). While these unique localization and structural features of OmcZ are important for current generation, the reasons behind them are not well understood.
The GSU2075 gene, which is located immediately downstream of omcZ (GSU2076) on the G. sulfurreducens chromosome, encodes a subtilisin-like serine protease with a 121-bp intergenic region between GSU2075 and omcZ. According to the transcriptome analysis, GSU2075 was also upregulated in the current-producing biofilms compared with fumarate-reducing biofilms (24, 27). Therefore, we hypothesized that the protease, the gene product of GSU2075, directly cleaves OmcZL to produce OmcZS. Here, we evaluated the proteolytic regulation of OmcZ and its effect on current production.

RESULTS AND DISCUSSION

Analysis of the OzpA sequence.

It is hypothesized that a putative protease encoded by GSU2075 (designated ozpA for the OmcZ protease) cleaves OmcZL to produce OmcZS. GSU2075 is located immediately downstream of the omcZ and encodes a subtilase, which is a member of the subtilisin-like serine proteases (also known as S8 family serine peptidases). OzpA consists of 485 amino acids and has a signal peptide with a cleavage site between amino acids 22 and 23, according to the results of signal peptide analysis obtained using SignalP server 3.0. The PSORTb server predicts that OzpA lacks a transmembrane region and localizes in the extracellular (score, 4.13) or outer membrane (certainty, 5.87) and not in the periplasm (0.00), inner membrane (0.00), or cytoplasm (0.00). Amino acid sequence alignments and phylogenetic analysis showed that OzpA belongs to the subtilisin family of the subtilisin-like superfamily, which is composed of six families, as follows: subtilisin, thermitase, proteinase K, lantibiotic peptidase, kexin, and pyrolysin (see Fig. S1 in the supplemental material) (28). Subtilisin-like superfamily proteins generally have a highly conserved catalytic triad (Asp, His, and Ser residues) in α/β protein scaffolds. A conserved domain search showed OzpA has putative catalytic triad (Asp133, His169, Ser322), which is commonly conserved among serine proteases (see Fig. S2 in the supplemental material). Amino acid residues comprising a putative active site, Ile212, Phe231, and Asn260, are also well conserved, similar to other members of the subtilase family (29). Many subtilases are synthesized as preproenzymes; subsequently, they are translocated across the cell membrane via the prepeptide (or signal peptide), and finally, they are activated by the cleavage of the propeptide (30). The subtilisin propeptides are known to function as intramolecular chaperones, assisting in the folding of the mature peptidase (31), but also act as “temporary inhibitors” (32). In the amino acid sequence of OzpA, amino acids 51 to 91 showed 51% similarity with corresponding region of the inhibitor I9 domain of subtilisin BPN' from Bacillus amylosacchariticus, which is an activation peptide from peptidases of the subtilisin family. These results suggest that OzpA consists of a prepeptide (signal peptide), propeptide, and a mature sequence, and it is activated by the cleavage of the propeptide region, similar to other subtilisins. In general, prokaryotic subtilases are secreted outside the cell (33). The closeness to other subtilases implies that OzpA is an endopeptidase that is localized in the extracellular space, as well as most of the other subtilisins.

Characterization of the ozpA-deficient mutant.

An ozpA-deficient mutant was generated to investigate the function of the ozpA gene. The ozpA-deficient mutant did not show any difference from the wild-type G. sulfurreducens strain in terms of growth, by using acetate and fumarate as the sole electron donor and acceptor, respectively (see Fig. S3 in the supplemental material). SDS-PAGE analysis and subsequent heme staining of the whole-cell lysate and the loosely bound outer membrane-enriched protein (LBOP) fractions revealed that OmcZS was detected in the fractions from the wild-type strain but not from the ozpA-deficient mutant (Fig. 1A). OmcZL was present in the ozpA-deficient mutant (Fig. 1A). There was no significant difference in the expression of the other abundant cytochromes between the wild-type and ozpA-deficient strains (Fig. 1A). Western blotting using an anti-OmcZ antibody verified that the ozpA-deficient mutant accumulated OmcZL (Fig. 1B). The results demonstrated that the ozpA-deficient mutant did not produce OmcZS, suggesting that OzpA is involved in the cleavage of OmcZL. In contrast, in the other outer membrane c-type cytochromes OmcB, OmcE, and OmcS, there were no significant differences between the wild-type and ozpA-deficient mutant strains (Fig. 1A). This finding indicates that OzpA is highly specific to OmcZL. OzpA homologs were commonly conserved in other Geobacteraceae, such as Geobacter metallireducens (34), Geobacter anodireducens (35), Geobacter bemidjiensis (36), Geobacter pickeringii (37), and Geobacter uraniireducens (38). Most of the ozpA homologs are identified immediately downstream of omcZ homologs. These results indicate that the ozpA gene is conserved with the omcZ gene and is required for a functional OmcZ.
FIG 1
FIG 1 Profiles of c-type cytochromes in the ozpA-deficient mutant. (A) Heme-stained gel image of whole-cell lysates of wild-type and ozpA-deficient mutant G. sulfurreducens strains and of LBOP fractions of wild-type and ozpA-deficient mutant strains. (B) Western blot using anti-OmcZ antibody for LBOP fractions from wild-type, omcZ-deficient mutant, and ozpA-deficient mutant G. sulfurreducens strains.
As OmcZ is important for current generation, current generation by the ozpA-deficient mutant was investigated using the single-chamber air-cathode system (Fig. 2). The maximal current density of the wild-type strain was 533 μA/cm2 during the operation, whereas that of the ozpA-deficient mutant was 3.55 μA/cm2. These results indicate that the ability of the ozpA-deficient mutant to produce current was significantly inhibited. The current density was comparable to that of the omcZ-deficient mutant (9.52 μA/cm2) (Fig. 2, inset), indicating that OmcZ maturation by the ozpA gene is required for current production. An ozpA-complemented strain that harbored ozpA in an expression vector produced a maximal current density of 152 μA/cm2. The ability of the ozpA-deficient mutant to produce current was partially complemented. The gene expression system of the plasmid vector was used for the ozpA expression in the complemented strain, which was not the intact expression system of G. sulfurreducens. Thus, the expression level of the ozpA-complemented strain might not be appropriate. Full complementation may require a better expression system for OzpA.
FIG 2
FIG 2 Current production of wild-type (black), ozpA-deficient mutant (red), ozpA-complemented (blue), and omcZ-deficient mutant (green) strains. Current production of only ozpA-deficient and omcZ-deficient mutant strains for the comparison are shown in the inset. Representation of triplicates is shown for ozpA-deficient mutant and ozpA-complemented strains.

Subcellular localization of the active form of OzpA.

Subcellular fractions, culture supernatant, LBOP, and intracellular fractions were collected from the wild-type strain, and their proteolytic activity to OmcZL was investigated. OmcZL-abundant LBOP from the ozpA-deficient mutant (Fig. 1A) was mixed with the subcellular fractions. SDS-PAGE and subsequent heme staining showed that OmcZL was significantly decreased, and instead, OmcZS was increased only for the culture supernatant fraction (Fig. 3), indicating that the active form of OzpA localizes extracellular but neither the outer membrane nor the intracellular membrane. This result supports the abovementioned localization analysis by PSORTb and demonstrates that OzpA is secreted outside the cells similarly to the other bacterial subtilisins (33).
FIG 3
FIG 3 Proteolytic cleavage of OmcZ by culture supernatant, loosely bound cell surface protein, and intracellular fraction protein from wild-type G. sulfurreducens. Heme-stained gel of LBOP of ozpA-deficient mutant treated with PBS buffer (negative control), culture supernatant, LBOP, and intracellular fraction protein from wild-type G. sulfurreducens.

In vitro proteolytic cleavage of OmcZL.

An in vitro reaction with the LBOP of the ozpA-deficient mutant, which contained OmcZL, but not OmcZS (Fig. 1A), and a crude extract of OzpA-expressing Escherichia coli was performed in order to examine whether OzpA catalyzes the cleavage of OmcZL. SDS-PAGE and heme staining revealed that OmcZS was produced after the treatment with the OzpA-expressing E. coli crude extract, with a decrease in OmcZL, whereas OmcZS was not detected after the treatment with a crude extract of E. coli lacking OzpA, which was used as a control (Fig. 4A). Western blotting using an anti-OmcZ antibody confirmed that there was OmcZS only after treatment with the crude extract of OzpA-expressing E. coli (Fig. 4B). The results indicate that OmcZL was directly cleaved by OzpA to produce OmcZS.
FIG 4
FIG 4 In vitro proteolytic cleavage of OmcZ by OzpA. Heme-564-stained gel (A) and Western blot using anti-OmcZ antibody (B) of LBOP fraction, LBOP fraction treated with a crude extract of E. coli lacking OzpA, and LBOP fraction treated with a crude extract of E. coli expressing OzpA.

Implications.

A previous study demonstrated that OmcZS was extremely insoluble, but the solubility of OmcZL was much higher than that of OmcZS (25). This study demonstrates that OzpA alters the solubility of OmcZ by forming OmcZS, which is insoluble, from soluble OmcZL. The highly insoluble property of OmcZS might increase the affinity for hydrophobic materials, such as graphite electrodes. If OmcZ is produced in its insoluble form inside the cells, it must be strongly attached to the cell membranes and may not be easily located outside the cells. However, OmcZL, the soluble form of OmcZ, can be secreted outside the cells and easily located far from the cells. Once OmcZL is cleaved by OzpA, it becomes highly insoluble and can be attached to the hydrophobic electrode surface, even when it is located far away from the cells. In this model, OzpA is likely to be extracellularly activated via self-cleavage, and the activated OzpA produces OmcZS, the mature form of OmcZ, via cleavage of OmcZL. This way, OmcZS can be highly concentrated at the biofilm-electrode interface in bioelectrochemical systems and directly transfer electrons to the electrode. In addition, the insolubility could facilitate the self-assembly for OmcZ nanowire formation. The unique maturation process, localization of OmcZ, and formation of highly conductive OmcZ nanowires may be the reason why OmcZ is essential for an optimal current production, unlike other outer membrane c-type cytochromes.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

G. sulfurreducens strain PCA (wild type) (39) and the omcZ-deficient mutant (24) were kindly gifted by D. R. Lovley (University of Massachusetts). G. sulfurreducens strains were cultured anaerobically at 30°C in NBAF medium (40) containing acetate (15 mM) and fumarate (40 mM) as the sole electron donor and acceptor, respectively. Kanamycin (200 μg/ml) and/or gentamicin (20 μg/ml) were added as needed. E. coli strain BL21 Star(DE3) (Invitrogen, Carlsbad, CA, USA) was used as a host for the pET101/D-TOPO vector (Thermo Fisher Scientific, Waltham, MA, USA) and its derivatives. Strain BL21 was cultured at 37°C shaking in Luria-Bertani medium (Nacalai Tesque, Kyoto, Japan) with ampicillin (50 μg/ml). Growth of PCA and the ozpA-deficient mutant in NBAF was analyzed by measuring the absorbance at 600 nm with an OD-Monitor C & T instrument (Taitec, Saitama, Japan).

Deletion mutant construction and complementation.

The ozpA gene (GSU2075) was disrupted using a single-step recombination method, as described previously (40, 41). A linear DNA fragment containing a kanamycin-resistance gene inserted between the upstream and downstream sequences of the ozpA gene was generated using recombinant PCR. A 618-bp upstream sequence of ozpA (−654 to −37; +1 at A of the start codon of ozpA) was amplified using primers 2075-1 and 2075-2 (Table 1). A 617-bp downstream sequence of ozpA (+1468 to +2084; ozpA ends at +1458) was amplified using primers 2075-3 and 2075-4 (Table 1). The kanamycin resistance gene (1,190 bp) was amplified from plasmid pBBR1MCS-2 (42) using PCR by using primers 2075-5 and 2075-6 (Table 1). The 3 fragments were combined using a recombinant PCR method (41) by using primers 2075-2 and 2075-3, which are located at both ends for the combined DNA fragment (Table 1). The amplified 2,384-bp DNA fragment was integrated into the pCR2.1 vector (Invitrogen) using the ligation high v2 reagent (Toyobo, Osaka, Japan), according to the manufacturer’s instructions. The resultant plasmid (designated pC2075) was introduced into One Shot Top10 chemically competent E. coli (Invitrogen) using heat shock at 42°C. The insert sequence of pC2075 was confirmed using sequencing analysis by using BigDye terminator v3.1 and the ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA). The DNA fragment containing the insert sequence of pC2075 was linearized using EcoRI (TaKaRa, Tokyo, Japan) digestion (EcoRI sites were derived from pCR2.1 vector and located at both inserts). The linearized pC2075 was transformed into competent cells of G. sulfurreducens strain PCA using electroporation according to the method described in reference 40. Electroporation was performed using a MicroPulser electroporator (Bio-Rad, Hercules, CA, USA) and Gene Pulser/MicroPulser electroporation cuvettes, with a 0.1-cm gap (Bio-Rad). To confirm that the target gene (ozpA) was properly disrupted by the kanamycin resistance marker insertion, Southern blotting was performed. Two different 618- and 171-bp DNA fragments, which are located upstream of ozpA (−654 to −37; +1 at A of the start codon of ozpA) and the kanamycin resistance gene (+170 to +340; +1 at A of the start codon of the kanamycin resistance gene), respectively, were amplified using primer sets 2075-1 and 2075-2 and Km1 and Km2 (Table 1). The DNA fragments were labeled using digoxigenin (DIG) by using the DIG DNA labeling detection kit (Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s instructions. The genomic DNA of strain PCA (wild type) and the ozpA-deficient mutant was purified using the genomic DNA extraction buffer set and Genomic-tip 100/G columns (Qiagen, Hilden, Germany). The genomic DNA of the strains was treated with restriction enzymes SacI, SphI, or EcoRI (TaKaRa). The digests of the genomic DNA were separated on 1% agarose gels. The transfer from the gel to a nitrocellulose membrane (Immobilon-Ny+ membrane; Merck Millipore, Burlington, MA, USA) was performed using a Model 785 vacuum blotter (Bio-Rad). Southern hybridization analysis using the abovementioned probes was performed according to previously described methods (43). To complement the ozpA mutation, an ozpA expression plasmid was constructed. DNA fragments containing full-length ozpA sequences were amplified using PCR by using primer sets 2075-compF1 and 2075-compR1 (Table 1), which contained XhoI and SpeI sites at the 5′ and 3′ ends, respectively. The amplified DNA fragment was digested using XhoI and SpeI and inserted into pBBR1MCS-5 (42) between XhoI and SpeI sites in its multiple-cloning site. The resultant plasmid (designated pBozpComp) was introduced into E. coli strain TOP10 using the method described above. The insert sequence of pBozpComp was confirmed via sequencing analysis using the method described above. pBozpComp was transformed into competent cells of the G. sulfurreducens ozpA mutant strain using electroporation, as described above. The transformants were confirmed using plasmid extraction by using standard alkaline lysis miniprep (44) and agarose gel electrophoresis, after EcoRI digestion.
TABLE 1
TABLE 1 Primers used in this study
Primer nameUsagePrimer sequence (5′–3′)a
2075-1ΔozpA constructionACATCGCGAATGCATGTAAA
2075-2ΔozpA constructionCAGAGCAGCTACACGGTCAC
2075-3ΔozpA constructionAATGCCCTCGATCTTTTTCC
2075-4ΔozpA constructionGCACGATGAAGACAGTACGC
2075-5ΔozpA constructionTCATTTACATGCATTCGCGATGTACCTGGGATGAATGTCAGCTAC
2075-6ΔozpA constructionGTTGCGTACTGTCTTCATCGTGCAGAAGGCGGCGGTGGAATCG
Km1Probe for Southern blottingTGAATGAACTGCAGGACGAG
Km2Probe for Southern blottingATACTTTCTCGGCAGGAGCA
2075-compF1ozpA complementationGGCCTCGAGCGTGAAGGGATACGTGCAC
2075-compR1ozpA complementationACTAGTTCACAGCTGCTCCGGAACG
Subt-TOPO-NOzp-overexpressing E. coli constructionCACCATGAGGTATCTGCTCGCCGTTAC
2075-COzp-overexpressing E. coli constructionTCACAGCTGCTCCGGAACGG
T7-forwardSequence check for ozpA-expression vectorTAATACGACTCACTATAGGG
T7-reverseSequence check for ozpA-expression vectorTAGTTATTGCTCAGCGGTGG
a
Italics represent complementary sequences to kanamycin resistance marker. Underlines represent recognition sequences to restriction enzyme.

Subcellular localization analysis of OzpA.

The loosely bound outer membrane-enriched protein (LBOP) fractions of the cells were prepared as previously described (17) with modifications described below. G. sulfurreducens was grown on NBAF medium, as described above, until the stationary phase. The cell culture was subjected to shearing using a laboratory blender (Waring J-SPEC 7010BUJ timer; Waring Laboratory Science, Winsted, CT, USA) at room temperature at 18,000 rpm for 2 min. G. sulfurreducens cells and insoluble materials were removed using centrifugation at 6,000 × g for 15 min at 4°C. The outer surface fraction proteins were precipitated using 50% ammonium sulfate for 12 h at 4°C and collected using ultracentrifugation at 100,000 × g for 2 h at 4°C. The pellet was suspended in 10 mM Tris-HCl (pH 7.6). LBOP from the ozpA-deficient mutant (OmcZL-enriched fraction) was used for investigating the OzpA activity of subcellular fractions from the wild-type strain. Subcellular fractions of the wild-type strain were collected from the cells grown on NBAF medium at 30°C to stationary phase. The cells were harvested by centrifugation at 4,670 × g for 15 min. The culture supernatant was collected and concentrated by ultrafiltration through a 10-kDa cutoff filter (Amicon Ultra-15; Merck Millipore). The cells were suspended in phosphate-buffered saline (PBS) and were subjected to shearing using a laboratory blender at room temperature at 18,000 rpm for 2 min. After centrifugation at 4,670 × g for 15 min, the supernatant was concentrated by ultrafiltration through a 10-kDa cutoff filter. The cells were suspended in PBS and centrifuged at 4,670 × g for 15 min. This procedure was repeated twice for washing. The washed cells were suspended in PBS and sonicated using a sonicator (Q125 sonicator; Qsonica, Newton, CT, USA) at 30 s three times. After centrifugation at 4,670 × g for 15 min, the supernatant was concentrated by ultrafiltration. Ten microliters of LBOP from the ozpA-deficient mutant (protein concentration, 1.38 mg/ml) was mixed with 10 μl of each fraction (0.5 mg/ml) and incubated at 37°C for 24 h.

In vitro reaction of OmcZL with OzpA.

The ozpA gene was amplified using PCR by using the primer set 2075-TOPO-N and 2075-C, which is listed in Table 1, and PCA genomic DNA as the template. The PCR product was ligated into the pET101/D-TOPO vector (Invitrogen) (designated pETC-Ozp). The sequence of pETC-Ozp was confirmed via sequencing analysis, as described above. pETC-Ozp was introduced into the E. coli strain BL21 Star(DE3) (Invitrogen). The OzpA-expressing E. coli cells were cultured with shaking in Luria-Bertani medium (Nacalai Tesque) with ampicillin (50 μg/ml) at 25°C, 30°C, or 37°C, and with 0.5 mM or 1.5 mM isopropylthio-β-galactoside (IPTG). Their expressions were compared using SDS-PAGE analysis but did not show significant differences among the examined conditions (data not shown). Thus, the OzpA-expressing E. coli was cultured at 30°C with 1.5 mM IPTG for further study. After 12 h of incubation, the cells were harvested using centrifugation at 15,000 × g for 3 min and were suspended in PBS. The cell suspension was sonicated using the sonicator at 30 s three times and centrifuged at 15,000 × g for 30 min. The supernatant was used as the crude cell extract for the reaction. The reaction of LBOP of the ozpA-deficient mutant (OmcZL enriched fraction) with the crude extract of OzpA-expressing E. coli was performed at 37°C for 24 h.

SDS-PAGE and Western blotting.

Protein concentrations were determined using the Bradford method and using bovine serum albumin as a standard (45), by using the Quick Start Bradford dye reagent (Bio-Rad). SDS-PAGE analyses were performed using 12.5% (wt/vol) polyacrylamide gels and Mini-Protean Tetra cell systems (Bio-Rad). Four micrograms of protein were applied per lane and stained with Bio-Safe Coomassie blue (Bio-Rad), and proteins containing hemes were stained as previously described (46) to identify the c-type cytochromes. A prestained protein ladder (Thermo Fisher Scientific) was used as a protein molecular mass standard. After SDS-PAGE, proteins were transferred to polyvinylidene difluoride membranes using the Mini Trans-Blot cell (Bio-Rad). Western blotting was performed using a One-Hour Western complete kit (GenScript), according to the manufacturer’s instructions. Anti-OmcZ antibodies were kindly gifted by D. R. Lovley (University of Massachusetts), which were produced against purified OmcZS as the antigen in rabbits (25). The antibody against OmcZS was purified from the obtained serum, as previously described (25).

Bioelectrochemical system operation.

Current production by G. sulfurreducens strains was investigated using a single-chamber air-cathode system (47, 48). A graphite electrode (102-cm2 solid graphite stick, 30 by 30 by 70 mm, Misumi Corporation, Tokyo, Japan) poised at +300 mV (versus Ag/AgCl electrode) with a potentiostat (Series GTM 300 potentiostat; Gamry Instruments, Philadelphia, PA, USA) was provided as the sole electron acceptor. The air cathode was prepared as previously described (48). It contained approximately 0.625 mg/cm2 of Pt catalyst and faced the reactor by 50 mm in diameter. A Ag/AgCl electrode (RE-11, saturated-KCl; EC Frontier, Kyoto, Japan) was prepared as the reference electrode. The inoculum strains were grown in NBAF medium using resazurin and 1 mM cysteine as a reductant. Forty-five milliliters of NBAF-grown culture were added to 450 ml of FWA medium (49) containing 10 mM acetate, 2 mM cysteine, and resazurin. The bioelectrochemical systems were operated at 30°C anaerobically. Once the current reached approximately 30 to 50 μA/cm2, the system was switched to a continuous flowthrough system at a flow rate of 0.5 ml/min.

Sequence alignment analysis.

The sequences were searched for using NCBI (https://www.ncbi.nlm.nih.gov/) and aligned using ClustalW (http://clustalw.ddbj.nig.ac.jp/). Molecular evolutionary genetics analysis (MEGA) software v7.0 was used to create a bootstrapped (1,000 trials) neighbor-joining phylogenetic tree of the subtilisin-like superfamily. The SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP-3.0/) was used to predict the signal peptide cleavage sites of the protein. The PSORTb server v3.0.2 (https://www.psort.org/psortb/) was used to predict protein localization. The amino acid sequence of OzpA was also analyzed using NCBI Conserved Domain Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).

Data availability.

The amino acid sequences used for the alignment analysis in Fig. S2 in the supplemental material were from the following proteins: OzpA from G. sulfurreducens (GenBank accession no. AAR35451.1), OzpA homolog from G. anodireducens (ANA40025.1), OzpA homolog from G. metallireducens (ABB31173.1), OzpA homolog from G. pickeringii (AJE02865.1), OzpA homolog from G. bemidjiensis (ACH40057.1), subtilisin E from Bacillus subtilis 168 (P04189.3), ubtilisin BPN' from Bacillus amyloliquefaciens (P00782.1), subtilisin Carlsberg from Bacillus licheniformis (CAB56500.1), Tk-subtilisin from Thermococcus kodakarensis KOD1 (P58502.1), thermitase from Thermoactinomyces vulgaris (1105242A), proteinase K from Parengyodontium album (P06873.2), stetterlysin from Thermococcus stetteri (AAC68832.1), pyrolysin from Pyrococcus furiosus DSM 3638 (AAB09761.1), lantibiotic peptidase from Bacillus cereus AH1273 (EEL90241.1), KEX2 from Saccharomyces cerevisiae S288c (NP_014161.1), furin from Homo sapiens (NP_001276752.1), and actin from Homo sapiens (P68032.1).

ACKNOWLEDGMENTS

This work was financially supported by the Program to Disseminate Tenure Tracking System from the Japanese Ministry of Education, Culture, Sports, Science, and Technology, a grant for Scientific Research on Priority Areas from the University of Miyazaki, a grant from the Grants-in-Aid for Scientific Research B (K.I.; 26850052), and the Institute for Fermentation, Osaka (IFO).

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REFERENCES

1.
Logan BE, Rossi R, Ragab A, Saikaly PE. 2019. Electroactive microorganisms in bioelectrochemical systems. Nat Rev Microbiol 17:307–319.
2.
Nevin KP, Richter H, Covalla SF, Johnson JP, Woodard TL, Orloff AL, Jia H, Zhang M, Lovley DR. 2008. Power output and columbic efficiencies from biofilms of Geobacter sulfurreducens comparable to mixed community microbial fuel cells. Environ Microbiol 10:2505–2514.
3.
Yi H, Nevin KP, Kim BC, Franks AE, Klimes A, Tender LM, Lovley DR. 2009. Selection of a variant of Geobacter sulfurreducens with enhanced capacity for current production in microbial fuel cells. Biosens Bioelectron 24:3498–3503.
4.
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. 2011. Geobacter: the microbe electric’s physiology, ecology, and practical applications. Adv Microb Physiol 59:1–100.
5.
Shi L, Fredrickson JK, Zachara JM. 2014. Genomic analyses of bacterial porin-cytochrome gene clusters. Front Microbiol 5:657.
6.
Lovley DR, Walker DJF. 2019. Geobacter protein nanowires. Front Microbiol 10:2078.
7.
Methé 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. 2003. Genome of Geobacter sulfurreducens: metal reduction in subsurface environments. Science 302:1967–1969.
8.
Qiu Y, Cho BK, Park YS, Lovley D, Palsson BO, Zengler K. 2010. Structural and operational complexity of the Geobacter sulfurreducens genome. Genome Res 20:1304–1311.
9.
Ding YH, Hixson KK, Giometti CS, Stanley A, Esteve-Núñez A, Khare T, Tollaksen SL, Zhu W, Adkins JN, Lipton MS, Smith RD, Mester T, Lovley DR. 2006. The proteome of dissimilatory metal-reducing microorganism Geobacter sulfurreducens under various growth conditions. Biochim Biophys Acta 1764:1198–1206.
10.
Ding YHR, Hixson KK, Aklujkar MA, Lipton MS, Smith RD, Lovley DR, Mester T. 2008. Proteome of Geobacter sulfurreducens grown with Fe(III) oxide or Fe(III) citrate as the electron acceptor. Biochim Biophys Acta 1784:1935–1941.
11.
Levar CE, Chan CH, Mehta-Kolte MG, Bond DR. 2014. An inner membrane cytochrome required only for reduction of high redox potential extracellular electron acceptors. mBio 5:e02034-14.
12.
Zacharoff L, Chan CH, Bond DR. 2016. Reduction of low potential electron acceptors requires the CbcL inner membrane cytochrome of Geobacter sulfurreducens. Bioelectrochemistry 107:7–13.
13.
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. 2014. A trans-outer membrane porin-cytochrome protein complex for extracellular electron transfer by Geobacter sulfurreducens PCA. Environ Microbiol Rep 6:776–785.
14.
Shi L, Dong H, Reguera G, Beyenal H, Lu A, Liu J, Yu HQ, Fredrickson JK. 2016. Extracellular electron transfer mechanisms between microorganisms and minerals. Nat Rev Microbiol 14:651–662.
15.
Otero FJ, Chan CH, Bond DR. 2018. Identification of different putative outer membrane electron conduits necessary for Fe(III) citrate, Fe(III) oxide, Mn(IV) oxide, or electrode reduction by Geobacter sulfurreducens. J Bacteriol 200:e00347-18.
16.
Liu Y, Fredrickson JK, Zachara JM, Shi L. 2015. Direct involvement of ombB, omaB, and omcB genes in extracellular reduction of Fe(III) by Geobacter sulfurreducens PCA. Front Microbiol 6:1075.
17.
Holmes DE, Chaudhuri SK, Nevin KP, Mehta T, Methé BA, Liu A, Ward JE, Woodard TL, Webster J, Lovley DR. 2006. Microarray and genetic analysis of electron transfer to electrodes in Geobacter sulfurreducens. Environ Microbiol 8:1805–1815.
18.
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.
19.
Aklujkar M, Coppi MV, Leang C, Kim BC, Chavan MA, Perpetua LA, Giloteaux L, Liu A, Holmes DE. 2013. Proteins involved in electron transfer to Fe(III) and Mn(IV) oxides by Geobacter sulfurreducens and Geobacter uraniireducens. Microbiology (Reading) 159:515–535.
20.
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.
21.
Wang F, Gu Y, O'Brien JP, Yi SM, Yalcin SE, Srikanth V, Shen C, Vu D, Ing NL, Hochbaum AI, Egelman EH, Malvankar NS. 2019. Structure of microbial nanowires reveals stacked hemes that transport electrons over micrometers. Cell 177:361–369.
22.
Filman DJ, Marino SF, Ward JE, Yang L, Mester Z, Bullitt E, Lovley DE, Strauss M. 2019. Cryo-EM reveals the structural basis of long-range electron transport in a cytochrome-based bacterial nanowire. Commun Biol 2:219.
23.
Yalcin SE, O’Brien JP, Gu Y, Reiss K, Yi SM, Jain R, Srikanth V, Dahl PJ, Huynh W, Vu D, Acharya A, Chaudhuri S, Varga T, Batista VS, Malvankar NS. 2020. Electric field stimulates production of highly conductive microbial OmcZ nanowires. Nat Chem Biol 16:1136–1142.
24.
Nevin KP, Kim BC, Glaven RH, Johnson JP, Woodard TL, Methé BA, DiDonato RJ, Jr., Covalla SF, Franks AE, Liu A, Lovley DR. 2009. Differences in physiology between current-producing and fumarate-reducing biofilms of Geobacter sulfurreducens: identification of a novel outer-surface cytochrome essential for electron transfer to anodes at high current densities. PLoS One 4:e5628.
25.
Inoue K, Qian X, Morgado L, Kim BC, Mester T, Izallalen M, Salgueiro CA, Lovley DR. 2010. Purification and characterization of OmcZ, an outer-surface, octaheme c-type cytochrome essential for optimal current production by Geobacter sulfurreducens. Appl Environ Microbiol 76:3999–4007.
26.
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.
27.
Krushkal J, Yan B, DiDonato LN, Puljic M, Nevin KP, Woodard TL, Adkins RM, Methé BA, Lovley DR. 2007. Genome-wide expression profiling in Geobacter sulfurreducens: identification of Fur and RpoS transcription regulatory sites in a relGsu mutant. Funct Integr Genomics 7:229–255.
28.
Siezen RJ, Leunissen JAM. 1997. Subtilases: the superfamily of subtilisin-like serine proteases. Protein Sci 6:501–523.
29.
Saeki K, Ozaki K, Kobayashi T, Ito S. 2007. Detergent alkaline proteases: enzymatic properties, genes, and crystal structures. J Biosci Bioeng 103:501–508.
30.
Power SD, Adams RM, Wells JA. 1986. Secretion and autoproteolytic maturation of subtilisin. Proc Natl Acad Sci U S A 83:3096–3100.
31.
Li Y, Hu Z, Jordan F, Inouye M. 1995. Functional analysis of the propeptide of subtilisin E as an intramolecular chaperone for protein folding. Refolding and inhibitory abilities of propeptide mutants. J Biol Chem 270:25127–25132.
32.
Kojima S, Minagawa T, Miura K. 1997. The propeptide of subtilisin BPN’ as a temporary inhibitor and effect of an amino acid replacement on its inhibitory activity. FEBS Lett 411:128–132.
33.
Siezen RJ, Renckens B, Boekhorst J. 2007. Evolution of prokaryotic subtilases: genome-wide analysis reveals novel subfamilies with different catalytic residues. Proteins 67:681–694.
34.
Kato S, Hashimoto K, Watanabe K. 2013. Iron-oxide minerals affect extracellular electron-transfer paths of Geobacter spp. Microbes Environ 28:141–148.
35.
Sun D, Call D, Wang A, Cheng S, Logan BE. 2014. Geobacter sp. SD-1 with enhanced electrochemical activity in high-salt concentration solutions. Environ Microbiol Rep 6:723–729.
36.
Aklujkar M, Young ND, Holmes D, Chavan M, Risso C, Kiss HE, Han CS, Land ML, Lovley DR. 2010. The genome of Geobacter bemidjiensis, exemplar for the subsurface clade of Geobacter species that predominate in Fe(III)-reducing subsurface environments. BMC Genomics 11:490.
37.
Badalamenti JP, Bond DR. 2015. Complete genome of Geobacter pickeringii G13T, a metal-reducing isolate from sedimentary kaolin deposits. Genome Announc 3:e00038-15.
38.
Butler JE, Young ND, Lovley DR. 2010. Evolution of electron transfer put of the cell: comparative genomics of six Geobacter genomes. BMC Genomics 11:40.
39.
Caccavo F, Jr., Lonergan DJ, Lovley DR, Davis M, Stolz JF, McInerney MJ. 1994. Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metal-reducing microorganism. Appl Environ Microbiol 60:3752–3759.
40.
Coppi MV, Leang C, Sandler SJ, Lovley DR. 2001. Development of a genetic system of Geobacter sulfurreducens. Appl Environ Microbiol 67:3180–3187.
41.
Lloyd JR, Leang C, Myerson ALH, Coppi MV, Cuifo S, Methe B, Sandler SJ, Lovley DR. 2003. Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens. Biochem J 369:153–161.
42.
Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, II, Peterson KM. 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176.
43.
Nojiri H, Sekiguchi H, Maeda K, Urata M, Nakai S, Yoshida T, Habe H, Omori T. 2001. Genetic characterization and evolutionary implications of a car gene cluster in the carbazole degrader Pseudomonas sp. strain CA10. J Bacteriol 183:3663–3679.
44.
Green MR, Sambrook J. 2012. Preparation of plasmid DNA by alkaline lysis with SDS: minipreps, p 11–14. In Molecular cloning: a laboratory manual, fourth edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
45.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.
46.
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.
47.
Bond DR, Lovley DR. 2003. Electricity production by Geobacter sulfurreducens attached to electrodes. Appl Environ Microbiol 69:1548–1555.
48.
Liu H, Logan BE. 2004. Electricity generation using an air-cathode single chamber microbial fuel cell in the presence and absence of a proton exchange membrane. Environ Sci Technol 38:4040–4046.
49.
Lovley DR, Philips 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.

Information & Contributors

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Published In

cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 87Number 1226 May 2021
eLocator: e02617-20
Editor: Robert M. Kelly, North Carolina State University
PubMed: 33837010

History

Received: 23 October 2020
Accepted: 19 March 2021
Accepted manuscript posted online: 9 April 2021
Published online: 26 May 2021

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Keywords

  1. Geobacter sulfurreducens
  2. c-type cytochrome
  3. subtilisin
  4. extracellular electron transfer
  5. electroactive microorganism

Contributors

Authors

Ayako Kai
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan
Takahiro Tokuishi
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan
Takashi Fujikawa
Interdisciplinary Graduate School of Agriculture and Engineering, University of Miyazaki, Miyazaki, Japan
Yoshihiro Kawano
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts, USA
Miyuki Nagamine
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan
Yoichi Sakakibara
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan
Masahito Suiko
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan

Editor

Robert M. Kelly
Editor
North Carolina State University

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