| 1. |
- Fridén, H, et al.
(författare)
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Deletion of the Bacillus subtilis sdh operon
- 1987
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 1574-6968 .- 0378-1097. ; 41:2, s. 206-206
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Tidskriftsartikel (refereegranskat)abstract
- Plasmid pKIM2 carries the Bacillus subtilis sdh operon and adjacent regions of the bacterial chromosome. The plasmid replicates in Escherichia coli but not in B. subtilis. Different portions of the sdh operon were removed from pKIM2 and replaced by a cat gene derived from pC194. A series of plasmids carrying sdh deletions was thus derived. Plasmid DNA was linearized at restriction sites within the vector part and used to transform B. subtilis to chloramphenicol resistance. The majority of the transformants had a succinate dehydrogenase-negative phenotype and were deleted in the sdh operon as verified by Southern blotting. The B. subtilis deletion mutants were used to determine the functional integrity of cloned sdh genes.
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| 2. |
- Fridén, H, et al.
(författare)
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Genetic and biochemical characterization of Bacillus subtilis mutants defective in expression and function of cytochrome b-558
- 1987
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 168, s. 695-701
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Tidskriftsartikel (refereegranskat)abstract
- Bacillus subtilis succinate dehydrogenase is bound to the cytoplasmic membrane by cytochrome b-558, a 23-kDa transmembrane protein which also functions as electron acceptor to the dehydrogenase. The structural gene for the apocytochrome, sdhC, has previously been cloned and sequenced. In this work the structure and translation of cytochrome b-558 was studied in different sdhC mutants. Mutant cytochrome was analyzed both in B. subtilis and after amplification in Escherichia coli. It is concluded that amino acid substitutions in the C-terminal half of the cytochrome can prevent the binding of succinate dehydrogenase without affecting membrane binding of the cytochrome protein or heme ligation. Mutagenesis of His-113 excludes this residue as an axial heme ligand. A base-pair exchange of G to A in the ribosome-binding sequence of sdhC was found to reduce cytochrome b-558 translation about tenfold in B. subtilis, whereas the mutation had no effect on translation in E. coli. Translation of the two succinate dehydrogenase genes from the sdhCAB polycistronic transcript does not seem to be coupled to translation of sdhC. Less than 10% of the wild-type amount of membrane-bound succinate dehydrogenase in B. subtilis still allows growth on non-fermentable substrate, but makes the dehydrogenase a limiting enzyme in the tricarboxylic acid cycle and leads to succinate accumulation.
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| 3. |
- Hederstedt, Lars, et al.
(författare)
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New properties of Bacillus subtilis succinate dehydrogenase altered at the active site
- 1989
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 260:2, s. 491-497
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Tidskriftsartikel (refereegranskat)abstract
- Mammalian and Escherichia coli succinate dehydrogenase (SDH) and E. coli fumarate reductase apparentlycontain an essential cysteine residue at the active site, as shown by substrate-protectable inactivation withthiol-specific reagents. Bacillus subtilis SDH was found to be resistant to this type of reagent and containsan alanine residue at the amino acid position equivalent to the only invariant cysteine in the flavoproteinsubunit of E. coli succinate oxidoreductases. Substitution of this alanine, at position 252 in the flavoprotein subunit of B. subtilis SDH, by cysteine resulted in an enzyme sensitive to thiol-specific reagents and protectable by substrate. Other biochemical properties of the redesigned SDH were similar to those of the wild-type enzyme. It is concluded that the invariant cysteine in the flavoprotein of E. coli succinate oxidoreductases corresponds to the active site thiol. However, this cysteine is most likely not essential for succinate oxidation and seemingly lacks an assignable specific function. An invariant arginine in juxtaposition to Ala-252 in the flavoprotein of B. subtilis SDH, and to the invariant cysteine in the E. coli homologous enzymes, is probably essential for substrate binding.
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| 4. |
- Hederstedt, Lars, et al.
(författare)
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Progress in succinate:quinone oxidoreductase research
- 1992
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Ingår i: Molecular Mechanisms in Bioenergetics. - 9780444895530 ; 23, s. 163-198
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Bokkapitel (refereegranskat)abstract
- This chapter discusses the progress in succinate:quinone oxidoreductase research. It reviews the progress made mainly within the last decade in understanding of the genetics, biogenesis, structure and functions of succinate:quinone oxidoreductases. The work on this class of enzymes has involved a vast amount of experimental efforts in many laboratories. As in many other fields of biological research, rapid advances have resulted from the increased use of a molecular biologist's approach, that is, a combination of molecular genetics, biochemistry and biophysical techniques. Succinate:quinone oxidoreductases are membrane bound enzymes that can catalyze the oxidation of succinate to fumarate coupled to the reduction of a quinone and the reduction of fumarate to succinate coupled to the oxidation of quinol. Succinate:quinone reductase (SQR), is present in strictly aerobic cells, and in vivo predominantly catalyzes the oxidation of succinate. Continued investigations of the structure of SQR and QFR enzymes will provide detailed, three-dimensional structural information, which is required for the better understanding of mechanisms of catalysis at the dicarboxylate and the quinone active sites and in the intra-molecular electron transfer.
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| 5. |
- Petricek, M, et al.
(författare)
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Cloning and characterization of the hemA region of the Bacillus subtilis chromosome
- 1990
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Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; , s. 2250-2258
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Tidskriftsartikel (refereegranskat)abstract
- A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. The first is hemA, encoding a protein of 50.8 kilodaltons. The primary defect of a B. subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteine-to-tyrosine substitution in the HemA protein. The predicted amino acid sequence of the B. subtilis HemA protein showed 34% identity with the Escherichia coli HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis. The B. subtilis HemA protein also complements the defect of an E. coli hemA mutant. The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function. It is not the proposed hemB gene product porphobilinogen synthase. The third open reading frame is hemC, coding for porphobilinogen deaminase. The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen III cosynthase. Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon.
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| 6. |
- Petricek, M, et al.
(författare)
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The structural gene for aspartokinase II in Bacillus subtilis is closely linked to the sdh operon
- 1989
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Ingår i: FEMS Microbiology Letters. - 1574-6968. ; 61:1-2, s. 85-87
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Tidskriftsartikel (refereegranskat)abstract
- The aecA and aecB loci map at 250 and 290 degrees, respectively, on the Bacillus subtilis chromosomal genetic map. The aecB locus has been proposed as the structural gene for aspartokinase II. From DNA sequence analyses and comparisons to the sequence of the aspartokinase II gene, it can be concluded that the structural gene for aspartokinase II is located close to sdh at 250 degrees and cannot be aecB. A detailed map over 7 kbp in the 250 degree region is presented.
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| 7. |
- Resnekov, Orna, et al.
(författare)
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Organization and regulation of the Bacillus subtilis odhAB operon, which encodes two of the subenzymes of the 2-oxoglutarate dehydrogenase complex
- 1992
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Ingår i: Molecular and General Genetics. - 1432-1874. ; , s. 285-296
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Tidskriftsartikel (refereegranskat)abstract
- The primary structure of Bacillus subtilis 105 kDa 2-oxoglutarate dehydrogenase (E1o) was deduced from the nucleotide sequence of the odhA gene and confirmed by N-terminal sequence analysis. The protein is highly homologous to E1o of Azotobacter vinelandii and Escherichia coli and of bakers' yeast cells. The 5′ end of the odhAB mRNA was determined and the promoter region for the odhAB operon was localized to a 375 by DNA fragment. The cellular concentration of the 4.5 kb odhAB transcript was found to be growth stage dependent; its concentration during growth in nutrient sporulation medium decreased abruptly at the end of the exponential growth phase and it was not detectable in early stationary phase. This decrease in the cellular concentration of the transcript is not the result of an increased rate of decay of the full-length odhAB mRNA, suggesting that transcription is down-regulated at the end of the exponential growth phase. The cellular concentration of the odhA and odhB gene products, E1o and dihydrolipoamide transsuccinylase (E2o), remains essentially constant throughout the growth curve in nutrient sporulation medium, indicating that both are rather stable proteins. In exponentially growing cells, glucose in nutrient sporulation medium repressed the cellular concentration of the odhAB mRNA, as well as that of E1o and E2o, about four-fold. This effect is most likely the result of a decreased rate of transcription from the odhAB promoter, since neither the stability nor the 5′-end of the transcript were affected by glucose in the medium. It is concluded that the cellular concentration of the 2-oxoglutarate dehydrogenase multienzyme complex (E1o and E2o) is regulated mainly at the transcriptional level.
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| 8. |
- Schröder, Ingrid, et al.
(författare)
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The hemX gene of the Bacillus subtilis hemAXCDBL operon encodes a membrane protein, negatively affecting the steady-state cellular concentration of HemA (glutamyl-tRNA reductase).
- 1994
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Ingår i: Microbiology. - : Microbiology Society. - 1465-2080 .- 1350-0872. ; 140
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Tidskriftsartikel (refereegranskat)abstract
- The Bacillus subtilis hemAXCDBL operon encodes enzymes for the biosynthesis of uroporphyrinogen III from glutamyl-tRNA. The function of the hemX gene product was studied in this work. The deduced amino acid sequence suggests HemX to be an integral 32 kDa membrane protein. This was confirmed by experiments using Escherichia coli minicells and hemX-phoA gene fusions. Deletion of the hemX gene from the Bacillus subtilis chromosome demonstrated that this gene is net required for haem synthesis. However, the deletion strain was found to overexpress the hemA gene product, glutamyl-tRNA reductase. A combination of results obtained with B. subtilis hemA and hemX in Escherichia coli and Bacillus subtilis shows that HemX negatively affects the steady-state cellular concentration of HemA protein. The mechanism by which HemX affects the HemA concentration is unclear.
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| 9. |
- Aevarsson, A, et al.
(författare)
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Ligands to the 2Fe iron-sulfur center in succinate dehydrogenase
- 1988
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Ingår i: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 232:2, s. 298-302
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Tidskriftsartikel (refereegranskat)abstract
- Membrane-bound succinate oxidoreductases are flavoenzymes containing one each of a 2Fe, a 3Fe and a 4Fe iron-sulfur center. Amino acid sequence homologies indicate that all three centers are located in the Ip (B) subunit. From polypeptide and gene analysis of Bacillus subtillis succinate dehydrogenase-defective mutants combined with earlier EPR spectroscopic data, we show that four conserved cysteine residues in the first half of Ip are the ligands to the [2Fe-2S] center. These four residues have previously been predicted to be the ligands. Our results also suggest that the N-terminal part of B. subtilis Ip constitutes a domain which can incorporate separately the 2Fe center and interact with Fp, the flavin-containing subunit of the dehydrogenase.
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| 10. |
- Ahuja, Umesh, et al.
(författare)
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Haem-delivery proteins in cytochrome c maturation System II
- 2009
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 73:6, s. 1058-1071
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Tidskriftsartikel (refereegranskat)abstract
- P>Cytochromes of the c-type function on the outer side of the cytoplasmic membrane in bacteria where they also are assembled from apo-cytochrome polypeptide and haem. Two distinctly different systems for cytochrome c maturation are found in bacteria. System I present in Escherichia coli has eight to nine different Ccm proteins. System II is found in Bacillus subtilis and comprises four proteins: CcdA, ResA, ResB and ResC. ResB and ResC are poorly understood polytopic membrane proteins required for cytochrome c synthesis. We have analysed these two B. subtilis proteins produced in E. coli and in the native organism. ResB is shown to bind protohaem IX and haem is found covalently bound to residue Cys-138. Results in B. subtilis suggest that also ResC can bind haem. Our results complement recent findings made with Helicobacter CcsBA supporting the hypothesis that ResBC as a complex translocates haem by attaching it to ResB on the cytoplasmic side of the membrane and then transferring it to an extra-cytoplasmic location in ResC, from where it is made available to the apo-cytochromes.
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