SdhE Is a Conserved Protein Required for Flavinylation of Succinate Dehydrogenase in Bacteria^*

Bacterial Strains, Plasmids, Phage, and Culture Conditions

Bacterial strains and plasmids are listed in supplemental Table S1, primers are in supplemental Table S2, and details of strain and plasmid constructions are described in the supplemental Experimental Procedures. Serratia sp. strain ATCC 39006 and Escherichia coli strains were grown at 30 and 37 °C, respectively, in Luria broth (LB; 5 g/liter yeast extract, 10 g/liter Bacto Tryptone, and 5 g/liter NaCl), in minimal medium (0.1% (NH₄)₂SO₄, 0.41 mm MgSO₄,40 mm K₂HPO₄, 14.7 mm KH₂PO₄, and 0.2% w/v glucose or 0.4% w/v succinate buffered with 75 mm HEPES, pH 6.9–7.1) at 180 rpm, or on LB agar or minimal agar supplemented with 1.5% (w/v) agar. For growth in minimal medium, strains were first grown overnight in LB, subcultured overnight in minimal glucose medium (0.2%), and then used to start growth in minimal medium in flasks. Bacterial growth was measured at an optical density of 600 nm (A₆₀₀) in a Jenway 6300 spectrophotometer. When required, media were supplemented with antibiotics at the following concentrations unless otherwise stated: 50 μg/ml kanamycin, 50 μg/ml streptomycin, 50 μg/ml ampicillin, and 25 μg/ml chloramphenicol.

Biological Membrane Preparation

Membrane and soluble fractions for localization studies were prepared following protocols previously described with plasmids induced at an A₆₀₀ of 0.6 with either 1 mm IPTG or 0.1% arabinose (9). Membrane fractions for Blue Native (BN)-PAGE analysis were prepared using the same methods with the addition of 10 μl of protease inhibitor mixture (Sigma) and 5 μl of DNase (Promega) to the resuspended culture prior to cell lysis. Membrane pellets for BN-PAGE were resuspended in 50 mm imidazole with 10% glycerol, and 400 μg of protein aliquots were frozen at −80 °C for future use. Protein concentration was determined using the Thermo Scientific BCA assay. When required, 400-μg aliquots were thawed and solubilized on ice with 0.5% Triton X-100 (Sigma-Aldrich) using 4× Invitrogen BN-PAGE sample buffer and prepared for BN-PAGE as described previously (16). NativePAGE Novex Bis-Tris gels (4–16%) (Invitrogen) were run following the manufacturer's guidelines and visualized with Coomassie Blue staining as described previously (17).

Purification of His-tagged Proteins

Five-ml LB overnight cultures of strains were used to inoculate 100-ml LB cultures grown in 1-liter flasks at a ratio of 1:50. Cultures were grown to an A₆₀₀ of 0.6, induced with 1 mm IPTG, and grown overnight. Cultures were lysed under native conditions with 10 μl of protease inhibitor mixture (Sigma) and 5 μl of DNase (Promega) and purified using Ni-NTA resin and gravity feed columns using the batch purification protocol described in The QIAexpressionist (47). Proteins were eluted using an imidazole gradient of 45–250 mm imidazole.

Western Blotting

Western blotting of SDS-PAGE and BN-PAGE gels was carried out using a Tris/glycine buffer system following standard protocols. For BN-PAGE gels prior to immunodetection, PVDF membranes (0.45 μm) were washed in 20 ml of 8% acetic acid following Invitrogen protocols. Membranes were analyzed using mouse monoclonal anti-His (Sigma) or monoclonal anti-FLAG (Sigma) antibodies, and as a secondary antibody, goat anti-mouse IgG horseradish peroxidase (Santa Cruz Biotechnology). Bands were visualized by either the ChemiDoc imaging system (Bio-Rad) or x-ray film using the SuperSignal West Pico chemiluminescent substrate kit.

SDH and UV Fluorescence Flavin Assays

The enzymatic activity of SDH was measured by monitoring the decrease in absorbance at 600 nm over a period of 10 min in the presence of 0.1 m sodium succinate (pH 7.5) (Sigma), 0.05 mm 2,6-dichlorophenolindophenol (extinction co-efficient = 22 mm⁻¹ cm⁻¹) (Sigma), 0.1 m NaPO₄ buffer (pH 7), and 50–100 μg of resuspended biological membranes in a 1-ml reaction volume. Experiments were carried out in technical triplicates, and activity was expressed relative to the background reduction of 2,6-dichlorophenolindophenol (membranes without succinate).

For FAD-UV assays, His-tagged purified proteins were separated by SDS-PAGE (15% polyacrylamide gels for SdhA and 20% gels for SdhE), destained, and exposed to UV light (FAD-UV) as described previously (18). Aliquots of the same samples were separated on additional gels for visualization of proteins by Coomassie Blue staining or Western blotting.

FAD Binding Assays

Purified proteins were incubated with the stated amounts of exogenous FAD from a 1 mg/ml stock in a 100-μl reaction volume buffered with 0.1 m NaPO₄ (pH 7). Reactions were left overnight at room temperature and analyzed by FAD-UV and Coomassie Blue staining.

Cell lysates for FAD incorporation assays of wild type (WT) and ΔsdhE overexpressing SdhA N-His were prepared from 100-ml cultures grown in a 1-liter flask, induced with 1 mm IPTG at A₆₀₀ of 0.5 and grown overnight. Cultures were harvested, lysed by lysozymes (20 μg/ml) and sonication (6 × 10-s bursts) in 0.1 m NaPO₄ buffer (pH 7), and then centrifuged at 150,000 × g for 30 min with the resulting supernatant being kept at 4 °C for future use (19). FAD binding assays involved incubating 40 μl of WT + SdhA N-His (∼1 mg of total protein) or 83 μl of ΔsdhE + SdhA N-His (∼2 mg of total protein) with 10 μg of FAD with or without 35 μg of purified SdhE N-His in a 100-μl reaction buffered with 0.1 m NaPO₄ (pH 7). Reactions were incubated at room temperature for 30 min and analyzed by FAD-UV.

Sample Preparation for UV-visible Spectra

Two ml of purified SdhE N-His (pMAT10) at 0.85 mg/ml from WT Serratia (90 mm imidazole elution fraction) was incubated with or without 300 μg of FAD overnight at 4 °C. To remove imidazole and unbound FAD, SdhE N-His with or without FAD was dialyzed overnight at 4 °C into elution buffer without imidazole (50 mm NaH₂PO₄, 300 mm NaCl, pH 8.0) with at least three buffer changes. One-ml volumes of each sample were analyzed by UV-visible spectra. Samples were analyzed by FAD-UV to confirm the loss of free FAD. SdhE N-His with or without FAD was also analyzed by intact protein mass spectrometry as described below, with the exception that samples were dialyzed into 0.1 m NaPO₄ buffer (pH 7.0).

Trichloroacetic Acid Precipitation

Two-ml samples of purified SdhE N-His (pMAT10) at 0.6 mg/ml from WT Serratia (90 mm imidazole elution fraction) were incubated with or without 300 μg of FAD overnight at 4 °C. Free FAD was not removed from these samples. The UV-visible spectra of SdhE N-His with or without FAD in a total volume of 1 ml was measured. Sixty μl of 50% trichloroacetic acid was added to the 1-ml SdhE N-His samples, to give a final concentration of 3%, and incubated on ice for 30 min. The samples were centrifuged at 10,000 × g for 5 min. The resulting supernatants containing any free FAD were decanted, and the UV-visible spectra were measured as the acid precipitation spectra. The precipitated protein pellets were dissolved in 6 m guanidine hydrochloride in 0.1 m NaPO₄, pH 7.0, and the UV-visible spectra were measured. The mol of FAD that precipitated per mol of SdhE N-His was determined using the extinction coefficients of FAD (ϵ₄₅₀ = 11,300 m⁻¹ cm⁻¹) and SdhE N-His (ϵ₂₈₀ = 12,615 m⁻¹ cm⁻¹).

Co-immunoprecipitation

Cultures were grown as described for the purification of His-tagged proteins. Cultures were induced with 1 mm IPTG and 0.1% arabinose. Lysates were passed through a Ni-NTA column, washed 8× in 10 mm imidazole wash buffer, and eluted using an imidazole gradient as described previously for the purification of His-tagged proteins.

Intact Protein Mass Spectrometry

SdhE N-His ± FAD was rebuffered either by drying and resolubilization in 30% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid (TFA) in water or by ZipTip purification on C4 material (>50 kDa) or C18 material (<50 kDa). One μl of sample was premixed with 10 μl of matrix (10 mg/ml α-cyano-4-hydroxycinnamic acid dissolved in 65% (v/v) aqueous acetonitrile containing 0.1% (v/v) TFA). Each sample was then spiked with 1 μl of a polypeptide/protein standard for internal mass calibration. An aliquot of 0.8 μl was then spotted onto a MALDI sample plate (Opti-TOF 384-well plate, AB SCIEX) and air-dried. Samples were analyzed on a 4800 MALDI tandem time-of-flight analyzer (MALDI-TOF/TOF, AB SCIEX). All mass spectrometry (MS) spectra were acquired in linear, positive-ion mode with 4000 laser pulses per sample spot. The mass range between 1000 and 25,000 was calibrated on the average masses of a four-polypeptide/protein calibration mix containing porcine insulin 1⁺ ion (m/z 5777.6), ubiquitin 1⁺ ion (m/z 8564.9), protein G 2⁺ ion (m/z 10,800.0), α-lactalbumin 1⁺ ion (m/z 14,070.1), and protein G 1⁺ ion (m/z 21,599.0).

Protein Identification by Mass Spectrometry

Excised protein bands were subjected to in-gel digestion with trypsin following previously described protocols (20). Eluted peptides were dried using a centrifugal concentrator. Samples were resolubilized in 5% (v/v) acetonitrile, 0.2% (v/v) formic acid in water and injected onto an Ultimate 3000 nano-flow UHPLC system (Dionex, Thermo Scientific) that was in-line-coupled to the nanospray source of an LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Scientific). Peptides were separated on an in-house packed emitter-tip column (75-μm inner diameter PicoTip fused silica tubing (New Objectives, Woburn, MA) packed with C-18 material on a length of 8–9 cm) by a gradient developed from 5% (v/v) acetonitrile, 0.2% (v/v) formic acid to 80% (v/v) acetonitrile, 0.2% (v/v) formic acid in water over 35 min at a flow rate of 400 nl/min.

Full MS in a mass range between m/z 300–2000 was performed in the Orbitrap mass analyzer with a resolution of 60,000 at m/z 400. The strongest five signals were selected for collision-induced dissociation-MS/MS in the LTQ ion trap at a normalized collision energy of 35%.

For protein identification, MS/MS data were searched against both a user-defined and a Serratia amino acid sequence database (4417 sequence entries) using an in-house Mascot server. The search was set up for full tryptic peptides with a maximum of three missed cleavage sites. Carboxyamidomethyl cysteine, oxidized methionine, and pyroglutamate (Glu and Gln) were included as variable modifications where appropriate. The precursor mass tolerance threshold was 10 ppm, and the maximum fragment mass error was 0.8 Da. A conservative significance threshold using an individual ion score of greater than 36 was selected to account for the small database sizes.

Statistical Analysis

All experiments were performed in triplicate, and results are presented as the mean ± standard deviation. Statistical analysis was performed when appropriate using the Student's t test.

Construction of Phylogenetic Trees

Nucleotide sequences of sdhE (DUF339) homologues were imported into the MEGA4 software from MicrobesOnline (21, 22). Evolutionary history was inferred using the neighbor-joining method with bootstrap test (1000 replicates) used to determine the percentage of replicate trees in which the associated taxa clustered together. Branch lengths were represented in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated from the dataset. There were a total of 210 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 (21).

sdhEygfX Operon Is Conserved among Enterobacteriaceae

The Serratia ygfX gene encodes a predicted protein with 57% similarity/53% identity at the amino acid level to E. coli YgfX. The predicted translational start site of ygfX overlaps that of sdhE (previously termed ygfY) (encoding a protein with 84% similarity/80% identity at the amino acid level to the E. coli homologue), suggesting a putative operon (sdhEygfX) (supplemental Fig. S1A). Using primers specific to sdhE and ygfX and primers that span the predicted operon, RT-PCR analysis showed that sdhE and ygfX are transcribed as a bicistronic mRNA (supplemental Fig. S1B).

SdhE is an 88-amino acid protein, predicted to be cytoplasmic and a member of the DUF339 and COG2938 family of proteins, an uncharacterized domain present in eukaryotes and bacteria. YgfX is predicted to be a 143-amino acid inner membrane-bound protein and a member of the DUF1434 domain of unknown function. Use of Pfam and MicrobesOnline (22, 23) revealed that DUF1434 is present predominantly in the Enterobacteriaceae and several γ-proteobacterial families (Shewanellaceae and Vibrionaceae). Gene neighborhood analysis of DUF1434 (YgfX) using STRING (24) and the genome context viewer in MicrobesOnline (22) indicated that genes encoding DUF1434 proteins are found in putative operons with genes encoding DUF339 (SdhE) proteins in all Enterobacteriaceae species except Wigglesworthia glossinidia. This revealed that the sdhEygfX operon is highly conserved among this bacterial family.

SdhE Is Required for Succinate Dehydrogenase Activity

Putative functional linkages between SdhE and other proteins were identified using COG2938 and SdhE protein sequences in STRING (24). COG2938 proteins showed strong neighborhood and co-occurrence with SdhA (succinate dehydrogenase flavoprotein subunit) (supplemental Fig. S2A and supplemental Table S3). The Serratia SdhE sequence is absent from the STRING database, so E. coli K12 SdhE was chosen as a representative homologue. The strongest predicted functional partner was YgfX; however, multiple subunits of the SDH complex were present as putative functional partners (supplemental Fig. S2B and supplemental Table S3). Therefore, we hypothesized that SdhE had an involvement in succinate metabolism, by association or interaction with one or more of the subunits of SDH.

SDH is a key step in the TCA cycle, converting succinate to fumarate and donating electrons to the ETC (25, 26). If the loss of sdhE affected SDH activity, sdhE mutants would have impaired growth on minimal succinate, yet grow as well as wild type (WT; refers to parental strain LacA) on minimal glucose when glycolysis is the predominant means of energy generation and the sdhCDAB operon is repressed (26–28). To test this, clean single and double deletion mutations were constructed in sdhE and ygfX. Both ΔsdhE and ΔsdhEygfX deletion mutants had impaired growth on minimal succinate, yet grew as well as the WT on minimal glucose (Fig. 1, A and B). Impaired growth on minimal succinate could be complemented by sdhE in trans (supplemental Fig. S2C). The loss of YgfX alone did not impair growth on minimal succinate (Fig. 1B). SDH assays showed that the loss of SdhE led to a 90% reduction in SDH activity (Fig. 1C). This shows that the deletion of sdhE leads to a nonfunctional SDH complex.

SdhE Is Required for Incorporation of FAD into SdhA

FAD is a co-factor required for SDH activity (26, 29). During the course of our work, it was reported that a DUF339 protein (renamed Sdh5) in Saccharomyces cerevisiae was required for FAD incorporation into the SDH flavoprotein, Sdh1 (30). We hypothesized that SdhE homologues in bacteria would flavinylate the Sdh1 homologue SdhA. The presence of covalently bound FAD can be determined by running purified proteins on a denaturing gel as noncovalently linked FAD is released from the protein, whereas covalently linked FAD remains bound (31, 32). Visualization by UV light (FAD-UV) produces an illuminated band at the same molecular weight as the protein of interest (18, 31). To test FAD insertion, N-terminally His-tagged SdhA was overexpressed in WT or ΔsdhE Serratia, purified, and examined by FAD-UV. SdhA N-His purified from WT Serratia contained a detectable FAD co-factor, whereas SdhA N-His purified from ΔsdhE lacked FAD (Fig. 1D). This confirms that SdhE is required for the incorporation of FAD into SdhA.

Loss of SdhE Does Not Affect SDH Stability

In S. cerevisiae, mutation of sdh5 affected FAD incorporation and resulted in SDH destabilization (30). This prompted investigations into whether SdhE and FAD incorporation also influenced SDH stability in bacteria. The stability of SDH was first assessed by examining the cellular localization of the N-terminal His-tagged SDH subunits SdhA and SdhC. Total levels of SdhA N-His and SdhC N-His were at higher levels in ΔsdhE and ΔsdhEygfX relative to the WT and ΔygfX strains (Fig. 2A). In all strains, SdhC N-His was correctly localized to the membrane (Fig. 2A). All strains correctly localized SdhA N-His to the membrane, with SdhA N-His more abundant in the membrane fractions of ΔsdhE and ΔsdhEygfX relative to the WT and ΔygfX (Fig. 2A). SdhA N-His was present in the soluble fraction of the WT and ΔygfX strains, but not in ΔsdhE and ΔsdhEygfX (Fig. 2A).

The formation of the complete SDH complex was further assessed by separating membranes from WT and ΔsdhE strains expressing SdhA and SdhC N-His proteins by BN-PAGE. Both WT and ΔsdhE produced bands at ∼360 kDa for SdhA N-His in addition to a faint, but detectable, SdhC N-His (Fig. 2B). Crystal structure and BN-PAGE data have shown that functional SDH is trimeric with a molecular mass of 360 kDa (33, 34). Finally, native WT and ΔsdhE membranes were separated by BN-PAGE (Fig. 2C), and proteins present in bands at 360 kDa were identified by LC-coupled LTQ-Orbitrap MS. SdhA was the highest peptide match for both WT and ΔsdhE (protein score, 1537/1013; peptide matches, 40/24, respectively) with SdhB, SdhC, and SdhD also present at lower protein scores and peptide matches. Therefore, the loss of SdhE does not affect the stability or formation of SDH in Serratia.

SdhE Is a Soluble Monomer That Interacts with SdhA

To test whether SdhE was a soluble protein and YgfX was membrane-bound, C-terminal FLAG-tagged versions of both proteins were overexpressed in WT Serratia. SdhE C-FLAG was detected only in the soluble fraction, and YgfX C-FLAG was detected only in the membrane fraction (Fig. 3A). Previous structural analysis of E. coli SdhE has suggested that it is monomeric (35). Consistent with this, co-immunoprecipitation and bacterial two-hybrid studies revealed no self-interactions (data not shown).

S. cerevisiae Sdh5 interacted with Sdh1 (30). To test whether this occurred between the bacterial homologues, SdhE N-His was purified from WT Serratia under native conditions to determine whether native SdhA would co-purify. Analysis of all proteins co-eluting with SdhE N-His, by LC-coupled LTQ-Orbitrap MS, showed that although SdhA bound weakly to the Ni-NTA column in the vector control (peptide matches, 15; protein score, 584), a greater amount of SdhA was detected when SdhE N-His was purified on an Ni-NTA column (peptide matches, 47; protein score, 2017). FAD-UV analysis of SdhE N-His elution fractions provided further confirmation by showing a faint, but detectable, illuminating band at the size of SdhA (64 kDa), but absent from the vector control (Fig. 3B). However, when viewed by Coomassie Blue staining, a band at ∼64 kDa was present in both SdhE N-His and the vector control (Fig. 3B). MS of this band showed that the dominant product from both vector and SdhE N-His was glucosamine/fructose 6-phosphate aminotransferase (66 kDa). Interestingly, the SdhE N-His 64-kDa band contained SdhA (P score 916/peptide matches 12) but was absent from the vector control.

To further test this interaction, a reciprocal co-immunoprecipitation was used by purifying SdhA N-His (bait) on an Ni-NTA resin and screening the co-purification of co-expressed SdhE C-FLAG (prey). SdhE C-FLAG co-purified with SdhA N-His (Fig. 3C). However, the amount of SdhE C-FLAG that co-purified with SdhA N-His was low, suggesting that this might be a weak and/or transient interaction (Fig. 3C). Therefore, YgfX is membrane-bound, whereas SdhE is a soluble monomer that forms weak and/or transient interactions with the flavoprotein SdhA.

SdhE Binds FAD Co-factor

SdhE N-His purified from WT Serratia did not produce a detectable band when examined by FAD-UV (Fig. 4A). However, the addition of increasing amounts of exogenous FAD to SdhE N-His resulted in UV-illuminated bands at the same molecular mass, suggesting that SdhE is able to covalently bind the FAD co-factor (Fig. 4A). To try and detect very low levels of bound FAD, the FAD-UV method was altered by prolonging the incubation of the gel in acetic acid for 24 h. Using this method, low levels of bound FAD became visible in the SdhE sample without the addition of FAD (Fig. 4B). Binding of FAD to SdhE had not been previously observed for the S. cerevisiae homologue (30). Furthermore, SdhE lacks any conserved His, Tyr, or Cys residues, the usual amino acids for FAD binding (32, 36).

If FAD is bound to a protein, UV-visible spectral analysis should produce peaks at ∼375 and 450 nm. Purified SdhE N-His was incubated with or without FAD, and then all unbound FAD was removed by dialysis, as confirmed by FAD-UV (Fig. 4C). The UV-visible spectrum of purified SdhE N-His without FAD added contained very minor peaks at 375 and 450 nm (Fig. 4D). However, the UV-visible spectrum of SdhE N-His preincubated with FAD and dialyzed to remove unbound FAD had a greater absorbance at 375 and 450 nm (Fig. 4E). The difference between the UV-visible spectra of these two samples indicate binding of FAD to SdhE (Fig. 4E, inset). Interestingly, dialysis resulted in a reduction in intensity of the FAD-UV band for SdhE N-His as compared with the sample prior to dialysis (Fig. 4C), suggesting that dialysis removes some FAD bound to SdhE N-His.

To further investigate possible SdhE-FAD binding, these samples were analyzed by intact protein MALDI-TOF MS. Purified SdhE N-His showed peaks of the singly and doubly charged ions at m/z 11,884.7 and 5941.04, respectively, corresponding to measured masses of the uncharged molecule of 11,880 and 11,883.7 Da (Fig. 4F). These measured masses match the expected average mass of SdhE N-His (11,885 Da) within the instrument error range of 5–10 Da for intact protein measurement by linear TOF MS in the mass range used here. The intact mass of SdhE N-His incubated with FAD and dialyzed also had a major peak at the mass for SdhE N-His alone (Fig. 4G). However, a distinct peak shift was observed with an increased mass of 795.4 Da for the singly charged species and 787.8 for the doubly charged ions (Fig. 4G). A similar peak shift was reproduced in multiple experiments. Considering the instrument error, this measured mass increase was in the range of the estimated mass of FAD (785 Da), which is consistent with SdhE binding a single FAD.

Covalently and noncovalently bound FAD can be discriminated not only by SDS-PAGE and FAD-UV assays (Fig. 4) but also by trichloroacetic acid precipitation. Covalently bound FAD should precipitate with the protein, whereas noncovalently bound FAD will be present in the supernatant. To further investigate FAD binding, SdhE N-His was incubated with FAD. Following acid precipitation of SdhE N-His + FAD, as expected, the supernatant contained free unbound FAD (data not shown). The precipitated protein pellet was bright yellow in color as compared with the control (supplemental Fig. S3) and was subsequently dissolved in 6 m guanidine hydrochloride. The UV-visible spectrum of the dissolved SdhE N-His pellet demonstrated the presence of FAD with distinct peaks at 375 and 450 nm (Fig. 5A). FAD precipitated with SdhE N-His at a ratio of 0.1 mol of FAD/mol of protein. SdhE N-His not preincubated with FAD contained no detectable FAD present in the acid-precipitated protein pellet (Fig. 5A). These results show that the majority of SdhE N-His exists in a non-FAD-bound form but that SdhE N-His is able to bind FAD when it is present.

To determine whether FAD was interacting with SdhE or with the His tag, a tobacco etch virus-cleavable His tag was introduced. SdhE was still able to bind FAD despite the removal of the His tag, demonstrating that FAD binds to SdhE (supplemental Fig. S4). Furthermore, a conserved residue (Gly-16) that was required for Sdh5 interactions with Sdh1 in S. cerevisiae (30) was mutated (G16R) to investigate specific requirements for FAD binding. FAD-UV analysis of purified SdhE^WT and SdhE^G16R, with or without exogenous FAD, demonstrated that SdhE^G16R contained lower levels of detectable FAD, relative to SdhE^WT (Fig. 5B). Therefore, our data obtained from in-gel FAD-UV, UV-visible spectra, MS analysis, acid precipitation, and site-directed mutagenesis represent multiple lines of evidence that support the hypothesis that SdhE is able to bind FAD. However, it is hypothesized that SdhE exists predominately in a non-FAD-bound state, with the SdhE-FAD interaction possibly being weak or of low affinity.

SdhE Facilitates FAD Incorporation into SdhA

To determine whether SdhE directly incorporated FAD into SdhA, purified SdhE^WT N-His was added back to cell lysates of ΔsdhE expressing SdhA N-His with or without exogenous FAD. SdhA expressed in ΔsdhE was unable to bind FAD when incubated with FAD alone (Fig. 5C). When incubated with FAD and purified SdhE^WT, SdhA expressed in ΔsdhE was able to bind FAD (Fig. 5C). When SdhE^G16R and FAD were added, SdhA expressed in ΔsdhE was unable to bind FAD. In conclusion, SdhE facilitates the incorporation of FAD into SdhA.

SdhE Mutants Are Pleiotropic

The role of SdhE in the TCA cycle and the ETC prompted further investigations to determine the phenotypic consequences of mutations in sdhE and ygfX. Both ΔsdhE and ΔsdhEygfX mutants showed reduced A₆₀₀ throughout growth in LB liquid after ∼8 h growth, but continued to grow slowly until they reached an equivalent stationary phase A₆₀₀ as the WT (Fig. 6). However, there was no difference in viable count, as determined by colony-forming units (cfu)/ml, throughout growth (supplemental Fig. S5A). This difference in A₆₀₀ in stationary phase cultures (14 h) correlated with a small difference in cell length between WT and ΔsdhE (p < 0.05) as determined by transmission electron microscopy (supplemental Table S4). Similar differences in cell size were observed when grown in minimal glucose medium (supplemental Table S4).

Analysis of pig biosynthesis throughout growth showed that ΔsdhE, ΔygfX, and ΔsdhEygfX had a 50, 90, and 90% reduction in addition to a 70, 40, and 70% reduction in the transcription of the pig biosynthetic operon pigA::lacZ (supplemental Fig. S5, B and C). The reduction in pig biosynthesis could be complemented by adding back the deleted gene(s) in trans (supplemental Fig. S5D). Interestingly, ΔsdhE overexpressing SdhE produced a further 50% reduction in pig biosynthesis (supplemental Fig. S5D).

Analysis of other phenotypes showed that both ΔsdhE and ΔsdhEygfX had a 19 and 11% reduction in the production of the plant cell wall-degrading exoenzymes cellulase and pectate lyase, in addition to a 20% reduction in swimming motility and virulence in potato tuber rot assays (supplemental Table S4). Both ΔsdhE and ΔsdhEygfX showed reduced production of N-acyl homoserine lactones throughout growth (supplemental Table S4), whereas the production of the β-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid (a carbapenem) was completely abolished (supplemental Table S4 and supplemental Fig. S5, E and F). Caenorhabditis elegans survival curves showed no statistically significant decrease in virulence (supplemental Fig. S5G). Phenotypic alterations for ΔsdhE and ΔsdhEygfX could be complemented by adding sdhE in trans (supplemental Fig. S5, H–L). ΔygfX showed no difference in any phenotypes relative to the WT strain except for the reduction in pig. These results indicate that both genes affect pig biosynthesis at the level of transcription and that the lack of SdhE results in widespread defects.

SdhE Evolved Once Preceding the Evolution of Mitochondria

Analysis of DUF339 proteins using Pfam and the STRING occurrence viewer (COG2938 query) demonstrated that this domain is present in eukaryotes (124 species) and bacteria (593 species) (23, 24). Interestingly, bacteria that contain a DUF339 protein are restricted to the α-, β-, and γ-proteobacteria. Analysis of the mitochondrial proteome showed that a DUF339 protein (EM15/Sdh5) in S. cerevisiae is a nuclear-encoded mitochondrial protein (37). It is hypothesized that mitochondria are descendants of an ancestral α-proteobacterial Rickettsia-like species and that the proteobacteria have evolved in a linear manner following the progression of ϵ, δ, α, β, and γ (38, 39). We therefore hypothesized that sdhE evolved only once in the α-proteobacteria prior to the evolution of the mitochondria and remained in the majority of bacterial and eukaryotic descendants. Phylogenetic analysis of sdhE among bacterial species reveals three distinct clades representing the α-, β-, or γ-proteobacteria (Fig. 7A), with sdhE sequences from β and γ being the most closely related and α being distantly related (Fig. 7A). In agreement with our hypothesis, the Sdh5 sequence from S. cerevisiae groups with Rickettsia in the α-proteobacteria clade (Fig. 7A). These results suggest that DUF339 evolved once in an ancestral α-proteobacterial species and that it has remained in both bacterial and eukaryotic descendants.

Each of the proteobacterial classes displayed a dominant sdhE genomic organization as determined by using the STRING neighborhood viewer (COG2938 as a search query) and the MicrobesOnline genomic context viewer (Fig. 7B). The dominant organization in α-proteobacteria, recG-DUF339-mfd, places sdhE in an operon with the transcription repair factor mfd. The dominant organization in β-proteobacteria, sdhCDAB-DUF339-gltA, is consistent with an involvement in succinate dehydrogenase. Finally, the dominant genomic organization in γ-proteobacteria is ygfZ (COG354)-DUF339-conserved hypothetical (frequently DUF1434/YgfX).

sdhEygfX Is Functionally Conserved among Enterobacteriaceae

The sdhEygfX operon is highly conserved among the Enterobacteriaceae. To test the functional conservation of SdhE and YgfX, E. coli sdhE (Km^R marked) deletion mutants (40) were assessed for a functional SDH, and E. coli homologues of sdhE and ygfX were assessed for their ability to complement Serratia ΔsdhEygfX. E. coli sdhE mutants were impaired for growth in LB and minimal succinate (supplemental Fig. S6, A and B) as a result of an inactive SDH (supplemental Fig. S6C). Additionally, when added in trans, E. coli homologues complemented Serratia ΔsdhEygfX reduction in pig and A₆₀₀, i.e. SdhE complemented A₆₀₀, YgfX complemented pig, and SdhEYgfX complemented A₆₀₀ and pig (supplemental Fig. S6, D and E). This demonstrates that SdhE and YgfX are functionally conserved among the Enterobacteriaceae and are likely to function through the same mechanism. Furthermore, this supports the role of SdhE as a key protein in SDH function in bacteria, such as in the model bacterium, E. coli.