Abstract
The sdhCDAB operon, encoding succinate dehydrogenase, was cloned from the soybean symbiont Bradyrhizobium japonicum. Sdh from B. japonicum is phylogenetically related to Sdh from mitochondria. This is the first example of a mitochondrion-like Sdh functionally expressed in Escherichia coli.
In the Bradyrhizobium japonicum-soybean (Glycine max) symbiosis, the plant host provides the bacterial partner with the dicarboxylates succinate, fumarate, and malate as carbon and energy sources (5, 10, 11). Bradyrhizobial mutants that lack a C4-dicarboxylate uptake system develop nodules that are no longer able to fix nitrogen, clearly demonstrating a critical role for dicarboxylates in the symbiosis (6). Because dicarboxylates are assimilated via enzymes of the tricarboxylic acid (TCA) cycle, we have focused our attention on succinate dehydrogenase, a TCA cycle enzyme that is necessary for growth on succinate as a sole source of carbon and energy. Sdh and the related enzyme fumarate reductase (Frd) have been studied in a number of systems, most notably in Escherichia coli, Bacillus subtilis, and beef heart mitochondria (1). Sdh catalyzes the oxidation of succinate to fumarate, and this reaction is coupled to the reduction of ubiquinone to ubiquinol. Ubiquinol then transfers electrons to the electron transport chain for aerobic respiration. All Sdh complexes contain two catalytic subunits: SdhA, which forms the catalytic site for succinate oxidation and covalently binds flavin; and SdhB, which binds three iron sulfur centers (1). Most Sdh enzymes contain one (SdhC) or two (SdhC and SdhD) additional subunits that serve to attach the catalytic subunits to the inner side of the cytoplasmic membrane (1). The membrane-spanning subunits are also proposed to be involved in interaction of the enzyme with quinones (4, 13).
In this article, we report the cloning and sequencing of the sdhCDAB genes from B. japonicum. In addition, we investigate the close phylogenetic relationship between B. japonicum Sdh and eukaryotic Sdh and describe the functional expression of B. japonicum Sdh in E. coli.
Characterization of the succinate dehydrogenase (sdhCDAB) operon from B. japonicum.
A PCR product, amplified from B. japonicum genomic DNA with primers to regions of sdhA from Paracoccus denitrificans (PD1, 5′-TCGCACACGGTCGCGGCGCAAGGC-3′; and PD3, 5′-CCTTCGCCGCGCGCGCCTTC-3′) was used to identify a 5.2-kb EcoRV fragment that carried all four sdh genes in the gene order sdhCDAB (Fig. 1). There is an overlap between the last codon of the sdhC gene and the first codon of the sdhD gene, and there is a 103-base gap between the last codon of the sdhA gene and the first codon of the sdhB gene. The sdhB gene is followed by a rho-independent transcriptional terminator, consistent with sdhB as the final gene in the operon. The 5′ end of the B. japonicum sdhCDAB transcript was determined by primer extension analysis (7) to begin 56 bases before the predicted start codon of sdhC (Fig. 1). The −35 and −10 regions upstream of the transcriptional start bear some similarity to a proposed B. japonicum consensus promoter (3). Despite the similarity of the B. japonicum consensus sequences to those of E. coli, the B. japonicum sdh promoter was not functional in E. coli (data not shown). However, all four subunits could be synthesized when an E. coli promoter was placed upstream of the sdhC gene (see below), consistent with the four sdh genes forming an operon.
FIG. 1.
(A) Physical map of the 5.2-kb EcoRV fragment encoding the entire B. japonicum sdhCDAB operon. The top line indicates the size of the DNA fragment in kilobase pairs. The second line represents the relative position of the genes coding for the individual subunits of the Sdh enzyme plus an additional incomplete open reading frame which encodes a protein with similarity to the R. leguminosarum gstR gene product. The arrows indicate the direction of transcription. The last line shows the nucleotide sequence of the promoter region and 5′ untranslated leader sequence with the start of transcription indicated as +1 and the start of translation for the sdhC gene underlined. The consensus B. japonicum “housekeeping” promoter sequence is indicated beneath the sdhCDAB sequence with the six critical bases underlined. An asterisk marks bases within the sdhCDAB sequence that match the consensus. (B) Determination of the sdhCDAB transcriptional start site by primer extension analysis. Twenty micrograms of total RNA from cells grown under either iron-sufficient or iron-deficient conditions was hybridized to a primer complementary to the beginning of the sdhC coding region. The DNA sequencing ladder was generated by using the same primer. The nucleotide sequence of the coding strand is indicated to the left of the DNA sequencing ladder.
B. japonicum Sdh activity and sdh gene expression.
Membranes prepared from B. japonicum cells (4) grown under iron- or heme-limited conditions (8) lack measurable Sdh activity (Fig. 2). This is consistent with the known properties of other Sdhs, which contain multiple iron sulfur centers and b-type cytochromes. sdh mRNA is undetectable in iron-deficient cells (Fig. 1B), consistent with Sdh abundance being controlled at the level of gene expression. Surprisingly, B. japonicum cells grown under oxygen-limited conditions contain elevated levels of Sdh enzyme (Fig. 2). This expression pattern is in contrast to that seen for E. coli, in which sdhCDAB expression is decreased under oxygen limitation (9). This discrepancy may reflect a requirement for B. japonicum Sdh under oxygen-limited conditions for metabolism of succinate, a major source of carbon and energy in the nodules.
FIG. 2.
Sdh activity of membranes prepared from B. japonicum cells grown under various physiological conditions. Membranes were isolated from cells grown under the described growth conditions and assayed for succinate-dependent reduction of phenazinemethosulfate (PMS). Each data point represents the average of at least three independent assays. Standard error bars are shown.
The relationship of B. japonicum Sdh to mitochondrial Sdh.
The predicted amino acid sequences of all available SdhA and SdhB subunits were aligned, and phylogenetic relationships (displayed as cladograms) were estimated (Fig. 3). All eukaryotic sequences form a single clade (indicated in Fig. 3 by the boxes) that also includes sequences from prokaryotes belonging to the α-subgroup of the proteobacteria (boldface in Fig. 3), including B. japonicum. This similarity is consistent with the endosymbiont theory of mitochondrial origin, which proposes that mitochondria evolved from symbiotic bacteria belonging to the α-subgroup of the proteobacteria (14). Therefore, B. japonicum Sdh may serve as an excellent model enzyme for analysis of mitochondrial complex II.
FIG. 3.
Phylogenetic relationships of all publicly available, predicted SdhA or FrdA and SdhB or FrdB amino acid sequences. Sequences were aligned by using the GCG program PILEUP, distances were calculated by using PAUP, and trees were drawn by using neighbor joining. (A) The predicted SdhA or FrdA sequences used for the organism and subunit were as follows (with accession numbers in parentheses): Acidianus ambivalens SdhA (AJ005961), Aquifex aeolicus SdhA (AE000697), Arabidopsis thaliana SdhA (AJ001809), Archaeoglobus fulgidus SdhA (AE001057), Ascaris suum FrdA (D30650), Bacillus subtilis SdhA (P08065), Bos taurus SdhA (P31039), Bradyrhizobium japonicum SdhA (AF007569), Caenorhabditis elegans SdhA (Q09508), Caenorhabditis elegans FrdA (U23514), Candida albicans SdhA (Y10377), Chlamydia trachomatis SdhA (AE001330), Coxiella burnetii SdhA (P51054), Dirofilaria immitis SdhA (S78630), Drosophila melanogaster SdhA (Y09064), Escherichia coli FrdA (J01611), Escherichia coli SdhA (P10444), Haemophilus influenzae FrdA (P44894), Helicobacter pylori FrdA (O06913), Homo sapiens SdhA (P31040), Methanobacterium thermoautotrophicum FrdA (AE000910), Methanobacterium thermoautotrophicum Thiol:FrdA (AJ000941), Methanococcus jannaschii FrdA (Q60356), Mus musculus SdhA, Mycobacterium leprae SdhA (U00022), Mycobacterium tuberculosis FrdA (Q10760), Mycobacterium tuberculosis SdhA (AL021841), Natronobacterium pharaonis SdhA (Y07709), Paenibacillus macerans SdhA (Y08563), Paracoccus denitrificans SdhA (Q59661), Plasmodium falciparum SdhA (D86573), Proteus vulgaris FrdA (P20922), Rickettsia prowazekii SdhA (P31038), Rhodoferax fermentans FrdA (AB015757), Rhodospirillum rubrum SdhA (AB015756), Saccharomyces cerevisiae FrdA (JC5123), Saccharomyces cerevisiae SdhA (Q00711), Saccharomyces cerevisiae FrdA (JC5123), Schizosaccharomyces pombe SdhA (D89263), Schizosaccharomyces pombe FrdA (Z99292), Shewanella putrefaciens FrdA (Q02469), Shewanella putrefaciens SdhA (Y13760), Sulfolobus acidocaldarius SdhA (Y09041), Synechocystis sp. SdhA (D90906), Wolinella succinogenes FrdA (P17412), and Wolinella succinogenes FrdA (Y10581). (B) The predicted SdhB sequences used were as follows: Acidianus ambivalens SdhB (AJ005961), Agaricus bisporus SdhB (Y15942), Aquifex aeolicus FrdB (AE000695), Arabidopsis thaliana SdhB (P21915), Archaeoglobus fulgidus SdhB (AE001057), Ascaris suum SdhB (AB008568), Bacillus subtilus SdhB (P08066), Bradyrhizobium japonicum SdhB (AF007569), Caenorhabditis elegans SdhB (AB008569), Chlamydia trachomatis SdhB (AE001330), Chondrus crispus SdhB (P48932), Coxiella burnetii SdhB (P51053), Cyanidium caldarium SdhB (P48933), Drosophila melanogaster SdhB (P21914), Escherichia coli FrdB (P00364), Escherichia coli SdhB (P07014), Haemonchus contortus SdhB (X75822), Haemophilus influenzae FrdB (P44893), Helicobacter pylori FrdB (O06914), Homo sapiens SdhB (P21912), Methanococcus jannaschii FrdB (Q57557), Methanobacterium thermoautotrophicum FrdB (AE000937), Methanobacterium thermoautotrophicum Thiol:FrdA (AJ000942), Mus musculus SdhB, Mycobacterium leprae SdhB (S73040), Mycobacterium tuberculosis FrdB (Q10761), Mycobacterium tuberculosis SdhB (AL021841), Mycosphaerella graminicola SdhB (O42772), Natronobacterium pharaonis SdhB (Y07709), Neisseria gonorrhoeae SdhB (J03844), Paenibacillus macerans SdhB (Y08563), Paracoccus denitrificans SdhB (Q59662), Plasmodium falciparum SdhB (D86574), Pleurotus ostreatus SdhB (AB007361), Porphyra purpurea SdhB (P80477), Proteus vulgaris FrdB (P20921), Rattus norvegicus SdhB (P21913), Reclinomonas americana SdhB (P80480), Rickettsia prowazekii SdhB (3860614), Rhodoferax fermentans FrdB (AB015757), Saccharomyces cerevisiae SdhB (P21801), Schizosaccharomyces pombe SdhB (Z99091), Shewanella putrefaciens SdhB (Y13760), Sulfolobus acidocaldarius SdhB (Y09041), Synechococcus sp. strain PCC7002 SdhB (AF052290), Synechocystis sp. SdhB (D90909), Synechocystis sp. SdhB (D64003), Thermoplasma acidophilum SdhB (S34619), Ustilago maydis SdhB (P32420), Vibrio cholera SdhB (AJ231124), and Wolinella succinogenes FrdB (P17596). Note that the Mus musculus SdhA and SdhB sequences were derived by combining the sequences of several overlapping EST clones. The accession numbers for the various fragments are as follows: SdhA, W97337, W59232, AA048847, AA103522, AA028724, and W90791; and SdhB, AA050217 and AA109032.
Several organisms contain both Sdh and Frd enzymes, and it is proposed that the duplicate sequences arose from a gene duplication (1). The Sdh and Frd sequences are generally separated into distinct clades, but this trend is not consistent. However, sequences in the mostly Sdh clade have the gene order sdh- or frdCDAB and sequences in the primarily Frd clade have the gene order frd- or sdhABCD. In this respect, gene order correlates well with the cladogram structure. Outlying sequences have the gene order sdh- or frdCAB (these enzymes have one C subunit that is equivalent to the CD subunits of other enzymes), indicating that the four-subunit enzymes may have evolved from the three-subunit enzymes. The sdh genes from two organisms, Synechocystis sp. and Rickettsia prowazekii, are not found in an operon.
The archaeal sequences form a heterogeneous group appearing in at least three separate clades. The methanogenic archaea appear in one clade; Acidianus ambivalens, Sulfolobus acidocaldarius, and Aquifex aoelicus appear in a second clade; and Archaeoglobus fulgidis, Thermoplasma acidophilum, and Natronobacterium pharaonis appear in a third clade. In some of the archaea, the genes do not form an operon (Methanobacterium thermoautotrophicum, Methanococcus jannaschii, and A. aoelicus), two have the gene order sdhABCD (A. fulgidis and S. acidocaldarius), and another has the gene order sdhCDBA (N. pharaonis). M. thermoautotrophicum also has the unique distinction of having two fumarate reductase enzymes, one of which is a thiol/fumarate reductase. The predicted amino acid sequences of the M. thermoautotrophicum Frds are very closely related, indicating a recent gene duplication leading to two genes with different specificities.
The diversity of Sdh and Frd structure at the level of subunit composition, gene order, and activity suggests that these enzymes have evolved rapidly. The mechanism for this rapid evolution is not readily apparent, but could involve lateral gene transfer and warrants further investigation.
Complementation of an E. coli sdh frd double mutant.
The B. japonicum sdhCDAB operon under the control of a heterologous promoter (the IPTG [isopropyl-β-d-thiogalactopyranoside]-inducible trc promoter of plasmid pTrc99A) (2) complements E. coli DW35 (an sdh frd double mutant) (12) for growth on minimal succinate medium (Fig. 4). However, the doubling time is longer than the doubling time of DW35 transformed with the E. coli sdhCDAB operon (10 h versus 2 h). Even DW35 transformed with the E. coli frdABCD operon has a shorter doubling time in minimal succinate medium than the same strain transformed with B. japonicum sdhCDAB (Fig. 4). The doubling time was consistent over a wide range of IPTG concentrations (10 μM to 10 mM), indicating that insufficient expression levels are not the reason for slow growth (data not shown). This was confirmed by determination of abundance of membrane-associated flavin. (DW35 lacks membrane-associated covalent flavin—diagnostic for Sdh and Frd, the only membrane-associated enzymes with a covalent flavin.) Membrane-associated flavin indicates that the abundance of Sdh in the membrane of DW35 complemented with the B. japonicum sdhCDAB operon is equivalent to the abundance of Sdh in the membrane of DW35 complemented by the E. coli sdhCDAB operon (data not shown). The slow growth may simply reflect the respiration rate in the more slowly growing B. japonicum. The activity of respiratory enzymes may be optimized to the respiratory chain in which they operate and could act as a rate-limiting step when expressed in another bacterium.
FIG. 4.
Complementation of an E. coli sdh frd double mutant strain DW35. DW35 was transformed with plasmids carrying either the wild-type E. coli sdhCDAB operon (□), the wild-type E. coli frdABCD operon (▵), the B. japonicum sdhCDAB operon (▴), or a vector alone as a control (■). OD600, optical density at 600 nm.
Nucleotide sequence accession number.
The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned accession no. AF007569.
Acknowledgments
We thank Mark McPeek for help with analysis of sequence alignments and critical reading of the manuscript; Gary Cecchini for E. coli and P. denitrificans sdhCDAB-containing plasmids, the P. denitrificans sdhCDAB sequence, and many helpful suggestions; and Rob McClung for critically reading the manuscript.
This work was supported by a postdoctoral grant (NRICGP 94-37305-0620) from the U.S. Department of Agriculture to D.J.W.
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