Activeness of c-type cytochrome genes at transcriptional levels

Recently, proteomic measurements reveal 23 of all S. oneidensis c-type cytochromes in cells grown with ferric citrate (soluble Fe(III)) or MnO₂ (insoluble Mn(IV)) as electron acceptors under anaerobic conditions. This observation implicates that production of a considerable number of c-type cytochromes in physiologically relevant amounts is condition-specific [17]. Such information not only provides insights into their physiological functions but also may help to understand technological difficulties encountered during construction of a whole set of c-type cytochrome single-gene mutants. In our previous study, we failed to generate in-frame deletions for scyA (SO0264), torC (SO1233), SO1748, ccpA (SO2178) or SO3056 after multiple attempts [10].

To understand expression characteristics of c-type cytochromes in cells grown under aerobic and anaerobic conditions, we examined the mRNA abundance of their coding genes in exponentially growing cells using quantitative reverse transcription PCR (qRT-PCR) (Figure 1). The cymA gene, as reported repeatedly [18,19], showed a constant high level of transcription that is oxygen-independent. This is not surprising because the protein plays a key role in mediating electron transport in multiple respiratory pathways [20,21]. Interestingly, impacts of oxygen on transcription of the major components of the metal reduction pathway mtrA, mtrC, and omcA were also negligible. Combining growth defects of the ∆mtrA and ∆mtrC strains under aerobic conditions [10], these data suggest that these proteins and/or the pathway may be implicated in other physiological processes of general importance. Consistent with findings that the cbb₃ oxidase plays a predominant role in oxygen respiration [22], both ccoP and ccoO (encoding two essential subunits of the cbb₃ oxidase) were transcribed at significantly higher levels in cultures grown with oxygen than in those without oxygen. Other genes displaying the same pattern include scyA, cctA, and cytcB, implicating that these small electron shuttling c-type cytochromes may be more likely to function under aerobic conditions. In contrast, fccA (fumarate reductase) and dmsE (subunit of the DMSO reductase) were transcribed at higher levels in anaerobic than aerobic cultures. It is worth mentioning that transcription of a number of genes, most of which encode proteins of unknown function, was extremely low regardless of growth conditions. While one of possible explanations is that the culturing conditions do not favor transcription of these genes, at least one alternative has been reported. In S. oneidensis, the caa₃ oxidase has a negligible role in oxygen respiration because its coding operon (including coxB) is not expressed to a physiologically relevant level [22].

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Figure 1. Expression of genes encoding c-type cytochromes in S. oneidensis.

qRT-PCR analysis of RNA extracted from mid-logarithmic growing cells (OD₆₀₀, ~0.4 and ~0.15 for aerobic and anaerobic cultures respectively). All data were normalized to expression of the arcA and recA genes, which were constant during the exponential growth phase. Numbers reported are standardized to expression of the arcA gene, but the same trends are observed when standardized to expression of the recA gene. Error bars represent standard deviation for triplicate cultures. SO1778 is displayed as mtrC/omcB because both names have been used in previous publications, but only mtrC is used in the rest of this article since it is accepted more widely.

https://doi.org/10.1371/journal.pone.0075610.g001

Development of an att-based mutagenesis system and generation of a complete set of S. oneidensis c-type cytochrome mutants

In S. oneidensis, in-frame single-gene deletion strains were successfully constructed for 36 out of 41 c-type cytochrome genes using either the fusion-PCR- or cre-lox-based mutagenesis approach [10]. Among five c-type cytochrome genes that we failed to remove, only scyA was highly expressed aerobically as shown above, a condition under which the previous attempts are made [10], suggesting that most of these genes may not play an important role in aerobiosis. In addition, these genes do not carry common characteristics in sequences and possible secondary structures that may interfere with PCR amplification or recombination. As a consequence, the failure is likely ascribable to the mutagenesis approaches. Indeed, both the fusion-PCR-based and cre-lox-based methods proved inefficient and sometimes even ineffective because of multiple rounds of PCR and conventional cloning. Additionally, the mutagenesis delivery vector pDS3.0 is rather large (^~10 kb), resulting in few unique restriction enzyme sites available for cloning, especially for large open reading frames (ORFs). We therefore sought to develop a new mutagenesis system free of conventional cloning for Shewanella and other organisms in which plasmids with a pR6K origin (ori_R6K) could not replicate. A PCR fragment containing bacteriophage lambda attP sequences bracketing the Cm^R cassette and toxic gene ccdB, generated using pMK2010 as the template, was introduced into pDS3.0, resulting in pHGM01 [23] (Figure 2A). To produce an in-frame deletion construct for ORF of interest, two PCR fragments flanking the ORF were generated using primers containing the attB sequences (outside primer) and linking sequences (inside primers) with the genomic DNA as the template by PCR and then joined together by the second round of PCR with primers containing the attB sequences (fusion PCR step) (Figure 2B). The resulting fusion PCR product was transferred into pHGM01 by lambda BP recombination. It is worth noting that the recombination also occurred effectively in the presence of unwanted smaller DNA fragments, which are common, often inevitable, byproducts at the fusion PCR step and regarded to be the major obstacle for conventional cloning. The correct mutagenesis vector, verified by DNA sequencing, was then transferred into S. oneidensis by conjugation for the subsequent steps of the fusion-PCR-based mutagenesis procedure [24].

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Figure 2. The att-based mutagenesis developed in this study.

A. Mutational construct delivery vector pHGM01. The vector was created by introducing into the SacI site of pDS3.0 a fragment containing attP1, ccdB, Cm^R, and attP2 amplified from pMK2010. B. Generation of a fusion PCR product for BP recombination. Outside primers 5-O and 3-O contain attB1 and attB2 sequences, respectively. Inside primers 5-I and 3-I contain linking sequences respectively, which are complementary to each other.

https://doi.org/10.1371/journal.pone.0075610.g002

With this new system, we obtained in-frame deletion strains for the scyA, torC, SO1748, ccpA, and SO3056 genes. Physiological characterization of these five mutants was then carried out to assess impacts of each mutation as previously described [10]. To support growth, oxygen or one of following chemical agents was used as the sole electron acceptor: DMSO, fumarate, trimethylamine N-oxide (TMAO), NaNO₃, Fe-Citrate, and MnO₂ under anaerobic conditions. Results showed that none of mutations had a statistically significant effect on growth under any test condition except for the ∆torC strain grown on TMAO (Table 1). Consistent with the essentiality of the torECAD operon for TMAO reductase activity [25], the loss of the torC gene prevented S. oneidensis from growing on TMAO.

In silico analysis of Ccm system of S. oneidensis

S. oneidensis is distinct from other γ-proteobacteria in organization of the ccm genes (Figure 3). According to the genome annotation, S. oneidensis has two loci for ccm genes, SO0259-0269 and SO0476-0478. The first locus includes operons ccmABCDE, scyA, SO0265, and ccmF₁-dsbE-ccmH-SO0269. The ccmABCDE operon exists as a single copy in all 27 sequenced Shewanella and the essentiality of their products for cytochrome c maturation have been firmly established [1–3]. Within the ccmF₁-dsbE-ccmH-SO0269 operon, the dsbE gene (SO0267) has been proposed to encode a real CcmG protein, whose homologues are implicated in the reduction of disulphide bonds of the apocytochrome c prior to haem ligation in a variety of bacteria [15]. This proposal is supported by sequence analysis. SO0267 shares a highest sequence similarity with well-characterized E. coli CcmG proteins: 50%/72% (identity/positive), with an expect value of 2e^-38. We therefore renamed dsbE as ccmG, rendering a syntenic consistency to the ccmF ₁GH-SO2069 operon as the order of the ccmFGH(I) genes are perfectly preserved in γ-proteobacteria. However, the last gene of the ccmF ₁GH-SO0269 operon is a mystery. Although the E. coli CcmH_EC is 350 amino acids (aa) in length, a size equivalent to a combination of S. oneidensis CcmH (159 aa) and SO0269 (194 aa), SO0269 shows a modest sequence similarity to CcmG_EC (32%/52%, 3e^-8), but not to CcmH_EC. In addition, SO0269 has no sequence similarity to P. aeruginosa CcmI_PA (407 aa). Moreover, an analogue of SO0269 is completely missing from two sequenced Shewanella strains, S. denitrificans OS217 and S. violacea DSS12, implying that SO0269 may not be required for cytochrome c maturation. Two remaining genes in this locus are scyA and SO0265. It is interesting to note that SO0265 (415 aa) is a homologue of CcmI_PA with sequence similarity of 33%/54% and expect value of 2e^-37, implicating a possible role for this protein in cytochrome c maturation.

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Figure 3. Organization of the ccm genes in γ-proteobacteria.

The most common gene arrangement is represented by Pseudomonas aeruginosa, in which CcmH and CcmI are separate from each other. The less common one is represented by E. coli, whose CcmH is a fusion protein between CcmH and the C-terminal portion of CcmI found in other bacteria. In S. oneidensis, two loci for predicted ccm genes were shown. Genes renamed in this study were labeled with an asterisk mark. Genes are drawn to scale. The data were from http://img.jgi.doe.gov/cgi-bin/w/main.cgi.

https://doi.org/10.1371/journal.pone.0075610.g003

The second locus consists of a single operon, ccmF₂-nrfF-SO0476, which is perfectly preserved in all sequenced Shewanella. Like CcmF₁, CcmF₂ is highly homologous to CcmF_EC with sequence similarity of 40%/60% and expect value of 2e^-137. Such a high level of sequence conservation strongly suggests a close evolutionary relationship between the two proteins. Moreover, NrfF (SO0477) and SO0476 are homologues of CcmH (37%/69%, 2e^-16) and SO0269 (38%/63%, 3e^-37), respectively.

Maturation of c-type cytochromes is independent of ccmF₂-nrfF-SO0476

The in silico analysis raised questions about the role played by two CcmF components as well as uncertain SO0265 and SO0269 in S. oneidensis. To evaluate the impact of these proteins on cytochrome c maturation, we constructed mutants defective in one of their coding genes. Deletion of ccmF₂ did not elicit any difference from the parental wild type strain but the loss of ccmF₁ resulted in light-colored colonies, an indicator for the reduced amount of c-type cytochromes. Quantification of intracellular heme c content confirmed that the ∆ccmF₁ strain was severely impaired in heme c production whereas the ∆ccmF₂ strain had a heme level identical to that of the wild type (Figure 4). Importantly, the deficient in heme c resulted from the ccmF1 deletion was corrected by its expression in trans, indicating that the phenotype observed was due to the intended mutation.

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Figure 4. Heme c levels in S. oneidensis strains.

wild type, ∆ccmF1, and ∆ccmF2 strains. Mid-logarithmic growing cells (OD₆₀₀, ^~0.4 and ^~0.15 under aerobic and anaerobic conditions respectively) were collected for the heme c assay. ∆ccmF₁^c and ∆SO0265^c represents the mutants carrying pHG102-ccmF₁ and pHG102-SO0265, respectively. All other strains carry empty vector pHG102. Three independently collected samples were assayed and the averaged levels were presented in mM per g of proteins. Error bars represent the standard deviations of the data.

https://doi.org/10.1371/journal.pone.0075610.g004

To assess impact of loss of the ccmF₁ and ccmF₂ genes on respiration of various electron acceptors, the wild type and mutant strains were inoculated into M1 medium supplemented with one of tested electron acceptors and growth was monitored. With all tested electron acceptors, the wild type and ∆ccmF₂ strains were indistinguishable but the ∆ccmF₁ strain was distinct (Table 1). In the absence of CcmF₁, the bacterium lost ability to grow on DMSO, fumarate, TMAO, or NaNO₃ completely but was still capable of growing on oxygen, Fe-Citrate, and MnO₂, albeit significantly impaired. To examine impact of the entire ccmF₂-nrfF-SO0476 operon on cytochrome c maturation, we removed all of these three genes from the wild type and characterized the resulting mutants as described above. Similar results were obtained compared to the ∆ccmF₂ strain. These data, collectively, indicate that the ccmF₂-nrfF-SO0476 operon is not involved in cytochrome c maturation in S. oneidensis. We, therefore, renamed the ccmF₁ gene as ccmF.

Maturation of c-type cytochromes requires SO0265 and CcmH but not SO0269

The scyA gene locates in the middle of the ccm cluster and has been suggested to be involved in cytochrome c maturation [19]. However, our data from the physiological characterization as shown above imply that the protein may not have a role in the process, at least not significantly. This notion is further supported by that the ∆scyA strain had similar levels of heme c production compared to the wild type (Figure 4).

We then made an attempt to determine whether three remaining genes in the cluster, SO0265, ccmH, and SO0269, are relevant to cytochrome c maturation in S. oneidensis. As shown above, the ccmH gene of S. oneidensis is much shorter than its E. coli counterpart. Therefore, whether this protein is essential for cytochrome c maturation merits an investigation. Interestingly, phenotypes resulting from deletions of these genes were different (Figure 4 and Table 1). Apparently, the ccmH gene is totally essential for the process evidenced by that its deletion resulted in a ccmF phenotype. Loss of the SO0265 gene not only caused significant reduction in the bacterial growth on DMSO, fumarate, NaNO₃, Fe-Citrate or MnO₂, but also impaired growth with oxygen. However, relevance of the gene to TMAO respiration remained to be determined. In line with these growth phenotypes, the SO0265 mutant produced heme c approximately 35% relative to the wild type strain. In contrast, deletion of the SO0269 gene had little impact on aerobic and anaerobic growth as well as heme c production. As a result, whether this protein is part of the Ccm system in S. oneidensis remains to be determined.

Bacterial strains, plasmids, and growth conditions

A list of all bacterial strains and plasmids used in this study is given in Table 2. Information of primers used for PCR amplification in this study is available upon request. For genetic manipulations, E. coli and S. oneidensis strains were grown in Luria-Bertani (LB, Difco, Detroit, MI) medium at 37 and 30^oC, respectively. Where required, the growth medium was supplemented with chemical agents at the following concentrations: 2, 6-diaminopimelic acid (DAP), 30 µM; ampicillin, 50 µg/mL; kanamycin, 50 µg/mL; and gentamycin, 15 µg/mL.

qRT-PCR

qRT-PCR analysis of RNA extracted from mid-logarithmic growing cells (OD₆₀₀, ^~0.4 and ^~0.15 for aerobic and anaerobic cultures respectively) were carried out with an ABI7300 96-well qRT-PCR system (Applied Biosystems) essentially as described previously [37]. The expression of each gene was determined from three replicas in a single real-time qRT-PCR experiment. The Cycle threshold (C_T) values for each gene of interest were averaged and normalized against the C_T value of the arcA and recA genes, whose abundance was constant during the exponential phase [38,39]. Relative abundance (RA) of each gene was standardized to the C_T values of both the arcA and recA genes using the equation RA = 2^-∆CT, yielding similar fold differences.

Construction of in-frame mutants with a new mutagenesis-construct delivery plasmid employing BP recombination

The fusion PCR method used for construction of S. oneidensis in-frame deletion mutants involves with multiple rounds of PCR and conventional cloning, both of which greatly reduce its efficiency and effectiveness [24]. To make an improvement, in this study we developed a new mutagenesis-construct delivery vector employing attB-attP (BP) recombination based on the system reported before [24]. This plasmid, namely pHGM01, was created by introduction of a DNA fragment containing attP1, ccdB, Cm^R, and attP2 from pMK2010 into the SacI site of pDS3.0 [23,24] (Figure 2A). pHGM01 was propagated in E. coli strain DB3.1 λpir, which is able to maintain plasmids possessing a ccdB gene and an R6K γ origin of replication [23]. To construct an in-frame deletion mutant using pHGM01, attB1 and attB2 sequences were arranged next to gene specific sequences within the 5-O and 3-O primers, which located at each end of the fusion PCR product (in-frame deletion construct) for subsequent site specific recombination, which was performed using the BP Clonase (Invitrogen) according to the manufacturer’s instruction (Figure 2B). The resulting recombination mixture was transformed into E. coli strain WM3064, which permits the replication from the R6K γ origin but is sensitive to the ccdB gene. After recombination between attB and attP sequences, the fragment flanked by attP1 and attP2 within pHGM01 was replaced by the in-frame deletion construct, removing the ccdB gene. As a consequence, resultant destination vectors in which the recombination occurred enabled WM3064 cells to grow on plates containing ampicillin (and/or gentamycin) whereas the ccdB gene carried on the pHGM01 was toxic to WM3064. The correct destination vector for mutagenesis, verified by DNA sequencing, was then transferred into S. oneidensis by conjugation and the rest of the mutagenesis procedure was carried out the same as described before [24]. The final in-frame deletion mutant was confirmed by sequencing the mutated region.

Complementation of in-frame deletion mutants

Plasmids pHG101 and pHG102 were used in genetic complementation of mutants [40]. For complementation of genes next to their promoter, a fragment containing the gene of interest and its native promoter was generated by PCR and cloned into pHG101. For the rest genes, the gene of interest was amplified and inserted into MCS of pHG102 under the control of the arcA promoter, which is constitutively active [38]. Introduction of each verified complementation vector into the corresponding mutant was done by conjugation, and verified by PCR and restriction enzyme mapping.

Physiological characterization of mutant strains

For growth measurements under aerobic and anaerobic conditions, M1 defined medium containing 0.02% (w/v) of vitamin-free Casamino Acids was used as described previously [41,42]. Anaerobic media and cultures were prepared as reported earlier [18,41]. Growth of mutant strains was measured using a Bioscreen C microbiology reader (Labsystems Oy, Helsinki, Finland) by recording optical densities of cultures at 600 nm every 15 minutes. Generation times were calculated during the exponential growth phase.

Biochemical methods

Cells of the mid-exponential phase were harvested and then were lysed with lysis buffer (0.25 M Tris/HCl, (pH 7.5), 0.5% Trion-X100). Protein concentration was determined with a bicinchoninic acid assay kit with bovine serum albumin (BSA) as a standard according to the manufacturer’s instructions (Pierce Chemical). The amount of heme c was measured following the procedure described elsewhere [43].

Statistical analyses

Statistical significance of the difference between experimental groups was assessed by two-way analysis of variance (ANOVA) followed by Bonferroni posttests.