Nucleotide sequence encoding the flavoprotein and iron-sulfur protein subunits of the Bacillus subtilis PY79 succinate dehydrogenase complex

Abstract

The nucleotide sequence of a 2.7-kilobase segment of DNA containing the sdhA and sdhB genes encoding the flavoprotein (Fp, sdhA) and iron-sulfur protein (Ip, sdhB) subunits of the succinate dehydrogenase of BaciUus subtilis was determined. This sequence extends the previously reported sequence encoding the cytochrome b558 subunit (sdhC) and completes the sequence of the sdh operon, sdhCAB. The The structural genes encoding the subunits of the SDH complex form an operon at 225°on the B. subtilis linkage map (42). For compatibility with Escherichia coli, the B. subtilis genes have been redefined as sdhC (cytb558, formerly sdhA), sdhA (Fp, formerly sdhB), and sdhB (Ip, formerly sdhC), so the operon is transcribed in the order sdhCAB (11,17,23,33,60 (40), and SDHD (32, 60). The nucleotide sequence of the sdh operon (sdhCDAB) of E. coli has been determined previously and compared with that of the frdABCD operon, which encodes the similar but genetically distinct and anaerobically derepressed enzyme fumarate reductase (FRD) (6,7,11,60). The Fp and Ip subunits of the two enzymes exhibit high degrees of mutual sequence homology both for the predicted amino acid sequences and for the corresponding genes. The nucleotide sequence revealed the presence of two hydrophobic subunits encoded by proximal (sdhCD) rather than distal (frdCD) genes, but despite similarities in size and hydropathy profile, there is little homology between the predicted amino acid sequences for the corresponding gene products. The present work is aimed at defining the differences between the 3-and 4-subunit SDH complexes and comparing the analogous complexes of gram-positive and -negative bacteria. Here the nucleotide sequence of the B. subtilis sdhCAB operon is extended by 2.5 kb to include the structural genes encoding the Fp (sdhA) and Ip (sdhB) subunits, and a comparison of the subunit amino acid sequences with those of the analogous subunits of the E. coli SDH and fumarate reductase is presented. MATERIALS AND METHODSBacteria and plasmids. Plasmid pSH1047 sdhC+ sdhA+ sdhB+ gerE+ has been described elsewhere (17). E. coli MV1OCh3/86 (C600 AtrpES carrying hybrid X prophage Ch3/86 with trfA and trfB of plasmid RK2) was used as a transformation host for preparing plasmid DNA (17). Strain

Processing of Bacillus subtilis succinate dehydrogenase and cytochrome &‐558 polypeptides

“…The three polypeptides of the B. subtilis SDH-cytochrome b-558 complex are expressed from plasmid pSH1047 in E. coli, but the functional complex is not assembled [9]. Fp and Ip are found in the cytoplasm, whereas cytochrome b-558 with normal physico-chemical properties is found in the inner membrane.…”

“…The cytochrome spans the lipid bilayer and anchors Correspondence address: L. Hederstedt, Dept of Microbiology, University of Lund, SGlvegatan 21, S-223 62 Lund, Sweden SDH to the inner surface of the cytoplasmic membrane [7]. The nucleotide sequence of the genes coding for Fp, Ip, and cytochrome b-558 was recently determined from the cloned B. subtilis sdhCAB operon [8,9]. To confirm each of the reading frames predicted from the DNA sequence and to determine possible N-terminal posttranslational processings of the subunits, we have studied the proteins by radiosequence analysis.…”

“…All three polypeptides of the B. subtilis SDH-cytochrome b-558 complex are expressed in Escherichia coli from the sdhCAB operon cloned in plasmid vectors [9,11,12]. However, the cultures (A~oo = 0.7) were collected by centrifugation (7000 x g, 20 min, WC) and were lysed in 1.5 ml cold 30 mM Tris-HCl, pH 8.0, containing 10 mM Na-EDTA, 2 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine-HCI, and 5 mg/l each of chymostatin, leupeptin, elastatinal and antipain (Sigma).…”

Structural studies on three flavin‐interacting regions of the flavoprotein subunit of complex II in Ascaris suum mitochondria

“…2B). Another segment interacting with the AMP moiety is the stretch from residues 357 to 386 in E. coli sdhA [8] and from residues 355 to 374 in B. subtilis sdhA [11]. To see if this segment is also detectable in the Ascaris Fp subunit, 20 out of the 40 tryptic peptides of the Fp subunit were partially sequenced, since the Lys residues in the amino terminal part of the segment in bacterial Fp (Lys-343 in E. coli sdhA and Lys-341 in B. subtilis sdhA) are well-conserved and trypsin specifically digests the C-terminal of Lys and Arg residues in the a From the results reported in [20] b From the results reported in [8] c From the results reported in [10] a Calculated according to [21] peptide.…”

“…It is a major component of Ascaris mitochondria (8o70 of mitochondrial protein) [7] and is composed of the following four subunits: a flavoprotein (Fp) subunit containing a flavin (Mr= 68 kDa), an Ip subunit associated with iron-sulfur centers (Mr = 26 kDa) and two hydrophobic, heme bcontaining polypeptides called CybL and Cybs (Mr = 15 and 13.5 kDa, respectively) [3,5]. In the case of bacteria, the genes of complex II have been cloned and sequenced from Escherichia coil (sdh and frd) [8][9][10], Bacillus subtilis (sdh ) [11,12] and Proteus vulgaris (frd) [13]. Structural information on mitochondrial complex…”