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Deletion of two homologous Geobacter sulfurreducens c-type cytochrome genes, omcG and omcH, decreased the rate of Fe(III) reduction and decreased the level of an outer membrane cytochrome critical for Fe(III) reduction, OmcB, without affecting its transcription. Expression of either gene restored Fe(III) reduction and OmcB expression, suggesting functional similarity.
The Geobacteraceae of the delta subdivision of the class Proteobacteria are a family of Fe(III)-reducing bacteria that have been intensively studied because of their important role in biological electricity production and in the bioremediation of organic contaminants, radionuclides, and toxic metals (1-3, 18, 24, 26). Geobacter sulfurreducens, the first Geobacter species for which a sequenced genome and a genetic system became available (6, 21), is one of the most intensively studied members of this environmentally important family.
One defining characteristic of Geobacter species is the expression of abundant c-type cytochromes. The genome of G. sulfurreducens contains over 100 putative c-type cytochrome-encoding genes (21). Although c-type cytochromes are generally involved in electron transport reactions, some heme-containing proteins have alternative physiological functions. Examples include catalase, peroxidases, bacterioferritin, and redox-sensing transcriptional regulators (5, 8, 10, 11, 29).
Genetic studies have implicated several G. sulfurreducens c-type cytochromes in Fe(III) reduction and electricity production (4, 16, 17, 20). Furthermore, G. sulfurreducens cytochromes can influence each other's expression. Deletion of an outer membrane-associated monoheme c-type cytochrome, OmcF, negatively affected Fe(III) reduction and decreased the levels of mRNAs for two previously characterized outer membrane cytochromes, OmcB and OmcC (14), one of which, OmcB, had been shown to play a critical role in Fe(III) reduction (16). Here we present another potential example of regulatory interactions between cytochromes. Two homologous multiheme cytochromes, OmcG and OmcH, were found to be involved in Fe(III) reduction and required for OmcB expression at the posttranscriptional level.
A cluster of three putative extracellular, multiheme c-type cytochrome-encoding genes, designated omcA, omcH, and omcG (Fig. (Fig.1A),1A), was targeted for deletion as part of a genetic screen intended to identify c-type cytochromes involved in Fe(III) reduction. Signal sequences were identified with SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) at the N termini of both OmcA and OmcH, and analysis of the sequence upstream of the published omcG start codon (21) revealed the presence of an in-frame start codon followed by a signal sequence, indicating that the actual omcG start codon was likely to be 66 bp upstream of the published start codon. Following reannotation of the omcG start codon, all three cytochromes were predicted to be extracellular by two subcellular localization software packages, Psortb v2.0 and Pence proteome analyst (9, 19). All three of these cytochromes contain multiple copies of two types of heme-binding motifs, CXXCH and CXXXXCH (31), i.e., 14 and 4 copies, 19 and 5 copies, and 24 and 3 copies for omcG, omcH, and omcA, respectively. The predicted molecular masses of OmcG, OmcH, and OmcA following signal peptide cleavage and heme incorporation are ca. 78.7, 103.0, and 118.04 kDa, respectively. The N-terminal halves of OmcG (32A to 391G) and OmcH (30A to 384G) are 84.4% identical. There is no homology between their C termini outside of the heme binding motifs. However, the C terminus of OmcH (P480 to R901) is 70% identical to that of OmcA (P622 to K1038).
An OmcA-, OmcH-, and OmcG-deficient triple mutant (strain DLBK03) (Fig. (Fig.1A)1A) was constructed by replacing the omcA-omcH-omcG cluster with a kanamycin resistance cassette via homologous recombination as previously described (14, 17, 22), using the primers indicated in Table Table1.1. The genotype of the triple mutant was confirmed by Southern blotting genomic DNA digested with KpnI (locations of KpnI sites are indicated in Fig. Fig.1A)1A) as previously described (6, 14). Growth of the triple mutant and that of the wild-type strain in medium in which acetate was the electron donor and fumarate was the electron acceptor (6) were indistinguishable (data not shown). However, when log-phase acetate-fumarate-grown cultures (A600 = ~0.5) were inoculated (3%) into acetate-Fe(III)-citrate medium (6), the triple mutant was impaired in Fe(III) reduction (Fig. (Fig.1B).1B). The wild-type strain completely reduced the Fe(III) in the medium within 2 days and had a doubling time of approximately 7 h, whereas the triple mutant had a doubling time of 18 h and required 6 days to complete Fe(III) reduction (Fig. (Fig.1B).1B). Fe(II) concentrations were determined by the ferrozine assay, and cell densities were determined by epifluorescence microscopy using acridine orange staining as previously described (12, 14, 25).
To determine which gene was responsible for this phenotype, three single mutants (strains DLBK09, DLBK10 and DLBK11) (Fig. (Fig.1A)1A) were constructed using the methods described above and the primers indicated in Table Table1.1. The rates of Fe(III) reduction by the three single mutants were comparable to that of the wild type (Fig. (Fig.1B),1B), indicating that deletion of more than one cytochrome was required for impairment of Fe(III) reduction. The high degree of identity between the N termini of OmcG and OmcH suggested that they might have similar physiological functions and thus be able to compensate for each other's absence. An omcH omcG::kan double mutant (strain DLBK15) (Fig. (Fig.1A;1A; Table Table1)1) was therefore constructed and screened as described above. The phenotype of this double mutant was comparable to that of the triple mutant (Fig. (Fig.1B1B).
Complementation studies were performed to investigate the functional redundancy of omcG and omcH. An omcG expression vector, pRG5-omcG, was constructed by amplifying the omcG coding sequence with primers Ex4776F and Ex4776R (Table (Table1)1) using previously described amplification conditions (14), inserting the coding sequence into pCR 2.1-TOPO (Invitrogen, Carlsbad, CA), excising it with EcoRI and HindIII, and ligating it into the EcoRI and HindIII sites of the expression vector pRG5 (14). The omcH expression vector, pRG5-omcH, was constructed via a similar strategy. The omcH coding sequence was amplified with primers Ex4779F and Ex4779R (Table (Table1),1), and flanking EcoRI and BamHI sites were utilized for insertion of the omcH coding sequence into pRG5. The omcG and omcH coding sequences were subsequently sequenced to screen for PCR artifacts. Following transformation of the triple mutant with the two expression vectors, spectinomycin-resistant colonies were screened for the simultaneous presence of both the plasmid and the omcA omcH omcG::kan mutation by using the primers indicated in Table Table1.1. A representative transformant of each type, DLBK03/pRG5-omcG and DLBK03/pRG5-omcH, was selected for phenotypic analysis. Expression of either omcG or omcH in trans in the triple mutant restored the wild-type phenotype (Fig. (Fig.1C),1C), confirming that omcG and omcH have overlapping physiological functions and play a critical role in Fe(III) reduction.
Although analysis of the G. sulfurreducens proteome by accurate mass and time tag validation methods indicated that both OmcG and OmcH are expressed during growth on both fumarate and Fe(III) citrate (Y. R. Ding, unpublished data), it was not possible to confirm their predicted localization by Tris-Tricine denaturing polyacrylamide gel electrophoresis and heme staining as previously described (14), possibly due to low expression levels.
The cytochrome contents of outer membrane-enriched fractions prepared from the wild-type and triple mutant strains by Sarkosyl extraction (23) were compared by electrophoresis and heme staining as previously described (14). A 78-kDa heme-containing protein which comigrated with OmcB, an outer membrane cytochrome previously demonstrated to play a critical role in Fe(III) reduction (16), was absent from the outer membrane fractions of the triple mutant during growth on both acetate-fumarate medium (Fig. (Fig.2A)2A) and acetate-Fe(III)-citrate medium (data not shown). This band reappeared at wild-type levels when either omcG or omcH was expressed in the triple mutant (Fig. (Fig.2A2A).
Western blot analysis was performed to determine whether the 78-kDa band was in fact OmcB. In order to generate an OmcB-specific antiserum, an 846-bp fragment of the omcB gene (A116 to N397) was amplified using primers Omcbpepnoti and Omcbpephindiii (Table (Table1),1), digested with NotI and HindIII, and inserted into the NotI and HindIII sites of the expression vector pET15b (Novagen, WI). Competent Escherichia coli strain Rosetta2 (DE3) (Novagen, WI) was transformed with the resulting plasmid, pET15b-omcBpep. The His-tagged OmcB peptide was successfully overexpressed and purified by Ni-nitrilotriacetic acid affinity chromatography as recommended by Novagen, and its identity was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Cross-reacting antibodies were removed by immunoabsorption using an acetone extract prepared from an OmcB-deficient mutant (16) as described by Sambrook et al. (27), except that cells were disrupted by sonication and cell debris and unbroken cells were removed by centrifugation at 10,000 × g for 10 min. The specificity of the antiserum was confirmed by probing immunoblots of wild-type, OmcB-deficient, and OmcC-deficient outer membrane-enriched fractions (Fig. (Fig.2B).2B). Outer membrane-enriched fractions were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). Immunoblots were probed with the OmcB-specific antiserum according to established protocols (details are at http://aroianlab.ucsd.edu/protocols/western.htm), and immunoreactive bands were visualized using an alkaline phosphatase-conjugated goat anti-rabbit secondary antibody and SigmaFast 5-bromo-4-chloro-3-indolylphosphatase/nitroblue tetrazolium tablets (Sigma, St. Louis, MO) according to the manufacturer's instructions.
Western blot analysis confirmed that the 78-kDa band missing from the outer membrane-enriched fraction of the triple mutant was OmcB (Fig. (Fig.2C).2C). The OmcB protein was also undetectable in the inner membrane-enriched and soluble fractions of the triple mutant (data not shown). In addition, OmcB was detected at wild-type levels in the two complemented strains, DLBK03/pRG5-omcG and DLBK03/pRG5-omcH (Fig. (Fig.2C).2C). Thus, expression of either omcG or omcH in trans fully restored both the ability to reduce Fe(III) (Fig. (Fig.1C)1C) and wild-type levels of OmcB expression (Fig. 2A and C) in the triple mutant.
In a previous study, deletion of OmcF, a low-molecular-weight outer membrane cytochrome, eliminated expression of OmcB by dramatically decreasing the levels of omcB transcripts (14). The levels of omcB mRNA in the triple mutant were therefore assessed by Northern analysis using an omcB-specific probe (amplified with primers 8916 and 8908-2) (Table (Table1)1) as previously described (14, 15). The omcB gene is transcribed from two independent promoters within a three-gene cluster (orf1-orf2-omcB), resulting in the production of two transcripts, a 5-kb transcript that includes all three genes and a 2.5-kb transcript consisting of omcB alone (15). Both omcB transcripts were detected at the wild-type level in the triple mutant (Fig. (Fig.2D),2D), indicating that deletion of OmcG and OmcH, unlike elimination of OmcF, affected OmcB expression posttranscriptionally.
This study provides additional evidence that Fe(III) reduction involves multiple interacting cytochromes. Both OmcG and OmcH play a role in Fe(III) reduction and influence the level of OmcB, a third cytochrome critical for Fe(III) reduction (16). It is likely that OmcG or OmcH may be involved in either the translation or stabilization of the OmcB protein in G. sulfurreducens. Unlike the case for the low-molecular-weight cytochrome OmcF, which was found to be required either for transcriptional activation of the omcB gene or for the stability its transcripts (14), deletion of the omcG and omcH genes did not affect levels of omcB mRNA. In the case of the c-type cytochrome nitrate reductases of Wolinella succinogenes and Thermus thermophilus, deletion of a second cytochrome which served as a membrane anchor led to accumulation of the nitrate reductase in the soluble rather than the membrane fraction (28, 33). However, failure to detect OmcB in the outer membrane of the triple mutant did not appear to be due to mistargeting or failure to incorporate heme, since OmcB could not be detected by Western blotting in either the inner membrane-enriched or soluble fractions of the triple mutant (data not shown).
One possible explanation for the effect of omcG and omcH deletion on the level of OmcB is that OmcB, OmcG, and OmcH are part of a complex required for Fe(III) reduction and the absence of OmcG and OmcH results in the accumulation and degradation of OmcB in the periplasmic space. The G. sulfurreducens genome contains two homologs (56 to 57% similar) of the periplasmic protease of E. coli, DegP, which degrades misfolded proteins, including outer membrane proteins, that accumulate in the periplasmic space (13, 30). Alternative explanations are also plausible, including direct or indirect interactions between OmcG or OmcH and the intracellular signaling network that affect expression of OmcB.
Further study will be required to determine how OmcG and OmcH influence the abundance of OmcB. However, the finding that three cytochromes OmcF, OmcG, and OmcH, which influence the rate at which cells develop the capacity for Fe(III) reduction, also influence OmcB expression highlights the central role of OmcB in Fe(III) reduction by wild-type G. sulfurreducens and points to interactions, either direct or indirect, between the four cytochromes. In addition, this study and the previous study on OmcF (14) clearly demonstrate that c-type cytochromes can be involved not only in electron transfer but also in transcriptional and posttranscriptional regulation or processing in G. sulfurreducens.
This research was supported by the Office of Science (BER), U.S. Department of Energy (cooperative agreement DE-FC02-02ER63446 and grant DE-FG02-02ER63423).
We are grateful for the excellent technical support provided by Betsy Blunt.