| 1. |
- Fridén, H, et al.
(författare)
-
Deletion of the Bacillus subtilis sdh operon
- 1987
-
Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 1574-6968 .- 0378-1097. ; 41:2, s. 206-206
-
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.
|
|
| 2. |
- Fridén, H, et al.
(författare)
-
Genetic and biochemical characterization of Bacillus subtilis mutants defective in expression and function of cytochrome b-558
- 1987
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 168, s. 695-701
-
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.
|
|
| 3. |
- Hederstedt, Lars, et al.
(författare)
-
New properties of Bacillus subtilis succinate dehydrogenase altered at the active site
- 1989
-
Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 260:2, s. 491-497
-
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.
|
|
| 4. |
- Hederstedt, Lars, et al.
(författare)
-
Progress in succinate:quinone oxidoreductase research
- 1992
-
Ingår i: Molecular Mechanisms in Bioenergetics. - 9780444895530 ; 23, s. 163-198
-
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.
|
|
| 5. |
- Petricek, M, et al.
(författare)
-
Cloning and characterization of the hemA region of the Bacillus subtilis chromosome
- 1990
-
Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; , s. 2250-2258
-
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.
|
|
| 6. |
- Petricek, M, et al.
(författare)
-
The structural gene for aspartokinase II in Bacillus subtilis is closely linked to the sdh operon
- 1989
-
Ingår i: FEMS Microbiology Letters. - 1574-6968. ; 61:1-2, s. 85-87
-
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.
|
|
| 7. |
- Resnekov, Orna, et al.
(författare)
-
Organization and regulation of the Bacillus subtilis odhAB operon, which encodes two of the subenzymes of the 2-oxoglutarate dehydrogenase complex
- 1992
-
Ingår i: Molecular and General Genetics. - 1432-1874. ; , s. 285-296
-
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.
|
|
| 8. |
- Schröder, Ingrid, et al.
(författare)
-
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
-
Ingår i: Microbiology. - : Microbiology Society. - 1465-2080 .- 1350-0872. ; 140
-
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.
|
|
| 9. |
- Aevarsson, A, et al.
(författare)
-
Ligands to the 2Fe iron-sulfur center in succinate dehydrogenase
- 1988
-
Ingår i: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 232:2, s. 298-302
-
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.
|
|
| 10. |
- Ahuja, Umesh, et al.
(författare)
-
Haem-delivery proteins in cytochrome c maturation System II
- 2009
-
Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 73:6, s. 1058-1071
-
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.
|
|
| 11. |
- Al-Karadaghi, Salam, et al.
(författare)
-
Crystal structure of ferrochelatase: the terminal enzyme in heme biosynthesis
- 1997
-
Ingår i: Structure. - 0969-2126. ; 5:11, s. 1501-1510
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The metallation of closed ring tetrapyrroles resulting in the formation of hemes, chlorophylls and vitamin B12 is catalyzed by specific enzymes called chelatases. Ferrochelatase catalyzes the terminal step in heme biosynthesis by inserting ferrous ion into protoporphyrin IX by a mechanism that is poorly understood. Mutations in the human gene for ferrochelatase can result in the disease erythropoietic protoporphyria, and a further understanding of the mechanism of this enzyme is therefore of clinical interest. No three-dimensional structure of a tetrapyrrole metallation enzyme has been available until now. RESULTS: The three-dimensional structure of Bacillus subtilis ferrochelatase has been determined at 1.9 A resolution by the method of multiple isomorphous replacement. The structural model contains 308 of the 310 amino acid residues of the protein and 198 solvent molecules. The polypeptide is folded into two similar domains each with a four-stranded parallel beta sheet flanked by alpha helices. Structural elements from both domains build up a cleft, which contains several amino acid residues that are invariant in ferrochelatases from different organisms. In crystals soaked with gold and cadmium salt solutions, the metal ion was found to be coordinated to the conserved residue His 183, which is located in the cleft. This histidine residue has previously been suggested to be involved in ferrous ion binding. CONCLUSIONS: Ferrochelatase seems to have a structurally conserved core region that is common to the enzyme from bacteria, plants and mammals. We propose that porphyrin binds in the identified cleft; this cleft also includes the metal-binding site of the enzyme. It is likely that the structure of the cleft region will have different conformations upon substrate binding and release.
|
|
| 12. |
- Andersson, Jessica, 1967-
(författare)
-
Activation, reaction mechanism and allosteric regulation of the anaerobic ribonucleotide reductase from bacteriophage T4
- 2000
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ribonucleotide reductase (RNR) catalyse the conversion of ribonucleotides to their corresponding deoxyribonucleotides in all organisms. The deoxyribonucleotides are the building blocks for DNA. Three different classes of RNR are found, class I, II and III. The class I RNRs operate under aerobic conditions, the class III RNRs operate under anaerobic conditions and the class II RNRs are indifferent to oxygen. All classes of RNR catalyse the reaction using a free radical mechanism. The free radical is generated to initiate the reaction mechanism but the generation differs between the classes.I have worked with the anaerobic class III RNR from bacteriophage T4 and the work presented in this thesis involves several different aspects of the enzyme. The class III RNR from phage T4 can be used as a model for other class III RNRs.From isotope labelling experiments, we show that a stable glycyl radical forms in the phage T4 class III RNR. I used site-directed mutagenesis to locate the glycyl radical to Gly580 in the NrdD protein of the T4 class III RNR. The glycyl radical is absolutely required for enzymatic activity.Also using protein engineering, I show for the first time, the importance of cysteines in radical generation and the reaction mechanism of the class III RNRs. Four cysteines in the C-terminal of T4 NrdD are responsible for the last step in the generation of the glycyl radical at Gly580. Two cysteines in the active site of T4 NrdD, Cys79 and Cys290 are required for the reaction mechanism of the enzyme. A third residue within the active site, Asn311 is most likely also important for catalytic activity. A reaction mechanism that is different from the class I and II RNRs has been proposed.The first crystal structure of a class III RNR, the class III RNR from phage T4 is presented. Structural relationships with the known class I RNR structure is discussed as well as similarities with another glycyl-radical enzyme.Finally, the allosteric regulation of the class III RNR from phage T4 was characterized. Almost all RNRs are allosterically regulated to keep the deoxynucleotide pools balanced in the cell. Similarities to other RNRs as well as a unique feature of the class III RNR from phage T4 is discussed.
|
|
| 13. |
- Azarkina, Natalia, et al.
(författare)
-
A bb´-type quinol oxidase in Bacillus subtilis strain 168
- 1999
-
Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 1083-351X .- 0021-9258. ; 274, s. 32810-32817
-
Tidskriftsartikel (refereegranskat)abstract
- The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa3, which is a cytochrome c oxidase, and cytochrome aa3 and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa3 and caa3 terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a 'cytochrome o'-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence or B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase. It is concluded that the novel bb'- type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b558b595b'.
|
|
| 14. |
- Baureder, Michael, et al.
(författare)
-
Contribution of catalase to hydrogen peroxide resistance in Enterococcus faecalis.
- 2012
-
Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 1574-6968 .- 0378-1097. ; 331:2, s. 160-164
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Enterococcus faecalis exhibits high resistance to oxidative stress. Several enzymes are responsible for this trait. The role of alkyl hydroperoxide reductase (Ahp), thiol peroxidase (Tpx), and NADH peroxidase (Npr) in oxidative stress defense was recently characterized. Enterococcus faecalis, in contrast to many other streptococci, contains a catalase (KatA), but this enzyme can only be formed when the bacterium is supplied with heme. We have used this heme dependency of catalase activity and mutants deficient in KatA and Npr to investigate the role of the catalase in resistance against exogenous and endogenous hydrogen peroxide stress. The results demonstrate that in the presence of environmental heme catalase contributes to the protection against toxic effects of hydrogen peroxide.
|
|
| 15. |
- Baureder, Michael, et al.
(författare)
-
Genes Important for Catalase Activity in Enterococcus faecalis.
- 2012
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:5
-
Tidskriftsartikel (refereegranskat)abstract
- Little in general is known about how heme proteins are assembled from their constituents in cells. The Gram-positive bacterium Enterococcus faecalis cannot synthesize heme and does not depend on it for growth. However, when supplied with heme in the growth medium the cells can synthesize two heme proteins; catalase (KatA) and cytochrome bd (CydAB). To identify novel factors important for catalase biogenesis libraries of E. faecalis gene insertion mutants were generated using two different types of transposons. The libraries of mutants were screened for clones deficient in catalase activity using a colony zymogram staining procedure. Analysis of obtained clones identified, in addition to katA (encoding the catalase enzyme protein), nine genes distributed over five different chromosomal loci. No factors with a dedicated essential role in catalase biogenesis or heme trafficking were revealed, but the results indicate the RNA degradosome (srmB, rnjA), an ABC-type oligopeptide transporter (oppBC), a two-component signal transducer (etaR), and NADH peroxidase (npr) as being important for expression of catalase activity in E. faecalis. It is demonstrated that catalase biogenesis in E. faecalis is independent of the CydABCD proteins and that a conserved proline residue in the N-terminal region of KatA is important for catalase assembly.
|
|
| 16. |
- Baureder, Michael, et al.
(författare)
-
Heme proteins in lactic Acid bacteria.
- 2013
-
Ingår i: Advances in Microbial Physiology. - 2162-5468. ; 62:1, s. 1-43
-
Forskningsöversikt (refereegranskat)abstract
- Lactic acid bacteria (LAB) are of profound importance in food production and infection medicine. LAB do not rely on heme (protoheme IX) for growth and are unable to synthesize this cofactor but are generally able to assemble a small repertoire of heme-containing proteins if heme is provided from an exogenous source. These features are in contrast to other bacteria, which synthesize their heme or depend on heme for growth. We here present the cellular function of heme proteins so far identified in LAB and discuss their biogenesis as well as applications of the extraordinary heme physiology of LAB.
|
|
| 17. |
- Baureder, Michael, et al.
(författare)
-
In Vitro Assembly of Catalase.
- 2014
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 289:41, s. 28411-28420
-
Tidskriftsartikel (refereegranskat)abstract
- Most aerobic organisms contain catalase which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process.
|
|
| 18. |
- Baureder, Michael, et al.
(författare)
-
Production, purification and detergent exchange of isotopically labeled Bacillus subtilis cytochrome b(558) (SdhC)
- 2011
-
Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928. ; 80:1, s. 97-101
-
Tidskriftsartikel (refereegranskat)abstract
- Cytochrome 6558 of the gram-positive bacterium Bacillus subtilis is the membrane anchor subunit of the succinate:quinone oxidoreductase of the citric acid cycle. The cytochrome consists of the SdhC polypeptide (202 residues) and two protoheme IX groups that function in transmembrane electron transfer to menaquinone. The general structure of the cytochrome is known from extensive experimental studies and by comparison to Wolinella succinogenes fumarate reductase for which the X-ray crystal structure has been determined. Solution state NMR can potentially be used to identify the quinone binding site(s) and study, e.g. redox-linked, dynamics of cytochrome b(558). In this work we present an efficient procedure for the isolation of preparative amounts of isotopically labeled B. subtilis cytochrome 6558 produced in Escherichia coli. We have also evaluated several detergents suitable for NMR for their effectiveness in maintaining the cytochrome solubilized and intact for days at room temperature. (C) 2011 Elsevier Inc. All rights reserved.
|
|
| 19. |
- Bendz, Maria, et al.
(författare)
-
Quantification of Membrane Proteins Using Nonspecific Protease Digestions
- 2009
-
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 8:12, s. 5666-5673
-
Tidskriftsartikel (refereegranskat)abstract
- We present a mass spectrometry-based method for the identification and quantification of membrane proteins using the low-specificity protease Proteinase K, at very high pH, to digest proteins isolated by a modified SDS-PAGE protocol. The resulting peptides are modified with a fragmentation-directing isotope labeled tag. We apply the method to quantify differences in membrane protein expression of Bacillus subtilis grown in the presence or absence of glucose.
|
|
| 20. |
- Bengtsson, Jenny, et al.
(författare)
-
Bacillus subtilis contains two small c-type cytochromes with homologous heme-domains but different types of membrane-anchors
- 1999
-
Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 1083-351X .- 0021-9258. ; 274, s. 26179-26184
-
Tidskriftsartikel (refereegranskat)abstract
- We demonstrate that the cccB gene, identified in the Bacillus subtilis genome sequence project, is the structural gene for a 10-kDa membrane-bound cytochrome c551 lipoprotein described for the first time in B. subtilis. Apparently, CccB corresponds to cytochrome c551 of the thermophilic bacterium Bacillus PS3. The heme domain of B. subtilis cytochrome c551 is very similar to that of cytochrome c550, a protein encoded by the cccA gene and anchored to the membrane by a single transmembrane polypeptide segment. Thus, B. subtilis contains two small, very similar, c-type cytochromes with different types of membrane anchors. The cccB gene is cotranscribed with the yvjA gene, and transcription is repressed by glucose. Mutants deleted for cccB or yvjA-cccB show no apparent growth, sporulation, or germination defect. YvjA is not required for the synthesis of cytochrome c551, and its function remains unknown.
|
|
| 21. |
- Bengtsson, Jenny, et al.
(författare)
-
CtaG is required for formation of active cytochrome C oxidase in Bacillus subtilis
- 2004
-
Ingår i: Microbiology. - : Microbiology Society. - 1465-2080 .- 1350-0872. ; 150, s. 415-425
-
Tidskriftsartikel (refereegranskat)abstract
- The Gram-positive bacterium Bacillus subtilis contains two respiratory oxidases of the haem-copper superfamily: cytochrome aa(3), which is a quinol oxidase, and cytochrome caa(3), which is a cytochrome c oxidase. Cytochrome c oxidase uniquely contains a di-copper centre, Cu-A. B. subtilis CtaG is a membrane protein encoded by the same gene cluster as that which encodes the subunits of cytochrome c oxidase. The role of B. subtilis CrtaG and orthologous proteins present in many other Gram-positive bacteria has remained unexplored. The sequence of CtaG is unrelated to that of CtaG/Cox11p, of proteobacteria and eukaryotic cells. This study shows that B. subtilis CtaG is essential for the formation of active cytochrome caa(3) but is not required for assembly of the core subunits I and II with haem in the membrane and it has no role in the synthesis of active cytochrome aa(3). B. subtilis YpmQ, a homologue to Sco1p of eukaryotic cells, is also a membrane-bound cytochrome c oxidase-specific assembly factor. Properties of CtaG- and YpmQ-deficient mutants were compared. Cells lacking YpmQ showed a low cytochrome c oxidase activity and this defect was suppressed by the supplementation of the growth medium with copper ions. It has previously been proposed that YpmQ/Sco1p is involved in synthesis of the Cu-A centre. The results of this study are consistent with this proposal but the exact role of YpmQ in assembly of cytochrome c oxidase remains to be elucidated.
|
|
| 22. |
- Bengtsson, Jenny, et al.
(författare)
-
Subunit II of Bacillus subtilis cytochrome c oxidase is a lipoprotein
- 1999
-
Ingår i: Journal of Bacteriology. - 0021-9193. ; 181, s. 685-688
-
Tidskriftsartikel (refereegranskat)abstract
- The sequence of the N-terminal end of the deduced ctaC gene product of Bacillus species has the features of a bacterial lipoprotein. CtaC is the subunit II of cytochrome caa3, which is a cytochrome c oxidase. Using Bacillus subtilis mutants blocked in lipoprotein synthesis, we show that CtaC is a lipoprotein and that synthesis of the membrane-bound protein and covalent binding of heme to the cytochrome c domain is not dependent on processing at the N-terminal part of the protein. Mutants blocked in prolipoprotein diacylglyceryl transferase (Lgt) or signal peptidase type II (Lsp) are, however, deficient in cytochrome caa3 enzyme activity. Removal of the signal peptide from the CtaC polypeptide, but not lipid modification, is seemingly required for formation of functional enzyme.
|
|
| 23. |
- Brandsch, Roderich, et al.
(författare)
-
Expression and flavinylation of Arthrobacter oxydans 6-hydroxy-D-nicotine oxidase in Bacillus subtilis
- 1989
-
Ingår i: Journal of General Microbiology. - : Microbiology Society. - 0022-1287. ; 135:1093-1099
-
Tidskriftsartikel (refereegranskat)abstract
- 6-Hydroxy-d-nicotine oxidase (6-HDNO) of Arthrobacter oxydans, an enzyme inducible by dl-nicotine, contains FAD covalently bound via an 8α-N(3)His linkage. Expression of the gene encoding 6-HDNO and flavinylation of the protein were studied in Bacillus subtilis. In this heterologous system the following findings were made. 1. An enzymically active covalently flavinylated 6-HDNO of normal size can be expressed in B. subtilis. 2. The natural promoter of the 6-HDNO gene appeared inefficient in B. subtilis. The B. subtilis sdh promoter, when inserted upstream of the A. oxydans promoter, increased 6-HDNO expression >50-fold. 3. Expression of the 6-HDNO gene from plasmids in B. subtilis was, independently of the promoter construct used, stimulated more than fivefold by dl-nicotine in the growth medium. It is concluded that flavinylation of 6-HDNO is possibly autocatalytic and mediated by factors generally found in bacterial cells.
|
|
| 24. |
- Brugna, Myriam, et al.
(författare)
-
In vivo production of catalase containing haem analogues.
- 2010
-
Ingår i: The FEBS Journal. - : Wiley. - 1742-464X. ; 277:12, s. 2663-2672
-
Tidskriftsartikel (refereegranskat)abstract
- Haem (protohaem IX) analogues are toxic compounds and have been considered for use as antibacterial agents, but the primary mechanism behind their toxicity has not been demonstrated. Using the haem protein catalase in the Gram-positive bacterium Enterococcus faecalis as an experimental system, we show that a variety of haem analogues can be taken up by bacterial cells and incorporated into haem-dependent enzymes. The resulting cofactor-substituted proteins are dysfunctional, generally resulting in arrested cell growth or death. This largely explains the cell toxicity of haem analogues. In contrast to many other organisms, E. faecalis does not depend on haem for growth, and therefore resists the toxicity of many haem analogues. We have exploited this feature to establish a bacterial in vivo system for the production of cofactor-substituted haem protein variants. As a pilot study, we produced, isolated and analysed novel catalase variants in which the iron atom of the haem prosthetic group is replaced by other metals, i.e. cobalt, gallium, tin, and zinc, and also variants containing meso-protoheme IX, ruthenium meso-protoporphyrin IX and (metal-free) protoporphyrin IX. Engineered haem proteins of this type are of potential use within basic research and the biotechnical industry. Structured digital abstract * MINT-7722358, MINT-7722368: katA (uniprotkb:Q834P5) and katA (uniprotkb:Q834P5) physically interact (MI:0915) by copurification (MI:0025).
|
|
| 25. |
- Bukowska-Faniband, Ewa, et al.
(författare)
-
Cortex synthesis during Bacillus subtilis sporulation depends on the transpeptidase activity of SpoVD.
- 2013
-
Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 1574-6968 .- 0378-1097. ; 346:1, s. 65-72
-
Tidskriftsartikel (refereegranskat)abstract
- The nonessential process of peptidoglycan synthesis during Bacillus subtilis sporulation is one model to study bacterial cell wall biogenesis. SpoVD is a class B high-molecular weight penicillin-binding protein that is specific for sporulation. Strains lacking this protein produce spores without the peptidoglycan cortex layer and are heat-sensitive. The detailed functions of the four different protein domains of the SpoVD protein are unknown and the observed phenotype of strains lacking the entire protein could be an indirect defect. We therefore inactivated the transpeptidase domain by substitution of the active site serine residue. Our results demonstrate that endospore cortex synthesis depends on the transpeptidase activity of SpoVD specifically. This article is protected by copyright. All rights reserved.
|
|
| 26. |
- Bukowska-Faniband, Ewa, et al.
(författare)
-
The PASTA domain of penicillin-binding protein SpoVD is dispensable for endospore cortex peptidoglycan assembly in Bacillus subtilis.
- 2015
-
Ingår i: Microbiology. - : Microbiology Society. - 1465-2080 .- 1350-0872. ; 161:Online 6 January, 2014, s. 330-340
-
Tidskriftsartikel (refereegranskat)abstract
- Peptidoglycan is the major structural component of the bacterial cell wall. Penicillin-binding proteins (PBPs), located at the exterior of the cytoplasmic membrane, play a major role in peptidoglycan synthesis and remodelling. A PASTA domain (penicillin-binding protein and serine/threonine kinase associated domain) of about 65 residues is found at the C-terminal end of some PBPs and eukaryotic-like protein serine/threonine kinases in a variety of bacteria. The function of PASTA domains is not understood but some of them are thought to bind uncrosslinked peptidoglycan. Bacillus subtilis has sixteen different PBPs but only two of them, Pbp2b and SpoVD, contain a PASTA domain. SpoVD is specific for sporulation and essential for endospore cortex peptidoglycan synthesis. We have studied the role of the PASTA domain in SpoVD by deleting this domain and analyzed effects on endospore formation and subcellular localization of SpoVD. Our results demonstrate that the PASTA domain in SpoVD is not essential for cortex synthesis and not important for targeting SpoVD to the forespore outer membrane during sporulation.
|
|
| 27. |
- Carlsson Möller, Mirja, et al.
(författare)
-
Extracytoplasmatic processes impaired by inactivation of the trxA (thioredoxin gene) in Bacillus subtilis
- 2008
-
Ingår i: Journal of Bacteriology. - 0021-9193. ; 190:13, s. 4660-4665
-
Tidskriftsartikel (refereegranskat)abstract
- The trxA gene is regarded as essential in Bacillus subtilis, but the roles of the TrxA protein in this gram-positive bacterium are largely unknown. Inactivation of trxA results in deoxyribonucleoside and cysteine or methionine auxotrophy. This phenotype is expected if the TrxA protein is important for the activity of the class Ib ribonucleotide reductase and adenosine-5'-phosphosulfate/3'-phosphoadenosine-5'-phosphosulfate reductase. We demonstrate here that a TrxA deficiency in addition causes defects in endospore and cytochrome c synthesis. These effects were suppressed by BdbD deficiency, indicating that TrxA in the cytoplasm is the primary electron donor to several different thiol-disulfide oxidoreductases active on the outer side of the B. subtilis cytoplasmic membrane.
|
|
| 28. |
- Carlsson Möller, Mirja, et al.
(författare)
-
Role of membrane-bound thiol-disulfide oxidoreductases in endospore-forming bacteria
- 2006
-
Ingår i: Antioxidants & Redox Signaling. - : Mary Ann Liebert Inc. - 1557-7716 .- 1523-0864. ; 8:5-6, s. 823-833
-
Forskningsöversikt (refereegranskat)abstract
- Thiol-disulfide oxidoreductases catalyze formation, disruption, or isomerization of disulfide bonds between cysteine residues in proteins. Much is known about the functional roles and properties of this class of redox enzymes in vegetative bacterial cells but their involvement in sporulation has remained unknown until recently. Two membrane-embedded thiol-disulfide oxidoreductases, CcdA and StoA/SpoIVH, conditionally required for efficient production of Bacillus subtilis heat-resistant endospores, have now been identified. Properties of mutant cells lacking the two enzymes indicate new aspects in the molecular details of endospore envelope development. This mini-review presents an overview of membrane-bound thiol-disulfide oxidoreductases in the Gram-positive bacterium B. subtilis and endospore synthesis. Accumulated experimental findings on CcdA and StoA/SpoIVH are reviewed. A model for the role of these proteins in endospore cortex biogenesis in presented.
|
|
| 29. |
- Carlsson, Peter, et al.
(författare)
-
Bacillus subtilis citM, the structural gene for dihydrolipoamide transsuccinylase: cloning and expression in Escherichia coli
- 1987
-
Ingår i: Gene. - : Elsevier BV. - 1879-0038 .- 0378-1119. ; 61:2, s. 217-224
-
Tidskriftsartikel (refereegranskat)abstract
- The 2-oxoglutarate dehydrogenase multienzyme complex is composed of three different subenzymes: 2-oxoglutarate dehydrogenase (E1o), dihydrolipoamide transsuccinylase (E2o), and dihydrolipoamide dehydrogenase (E3). Bacillus subtilis E1o and E2o are encoded by the citK and citM genes, respectively. A 3.4-kb BamHI DNA fragment containing citK and citM markers was isolated from a library of B. subtilis DNA in Escherichia coli. Functional E2o was expressed from the cloned DNA both in B. subtilis and E. coli. E2o had an apparent Mr of 60000 when expressed in E. coli. The B. subtilis E2o component complemented an E. coli E2o-defective mutant in vivo and in vitro. It is concluded that functional B. subtilis E2o can be produced in E. coli and can interact with E. coli and E1o and E3 to form an active chimeric enzyme complex.
|
|
| 30. |
- Carlsson, P., et al.
(författare)
-
Genetic Characterization of Bacillus subtilis odhA and odhB, encoding 2-oxoglutarate dehydrogenase and dihydrolipoamide transsuccinylase, respectively
- 1989
-
Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 171:7, s. 3667-3672
-
Tidskriftsartikel (refereegranskat)abstract
- The 2-oxoglutarate dehydrogenase complex consists of three different subenzymes, the E1o (2-oxoglutarate dehydrogenase) component, the E2o (dihydrolipoyl transsuccinylase) component, and the E3 (dihydrolipoamide dehydrogenase) component. In Bacillus subtilis, the E1o and E2o subenzymes are encoded by odhA and odhB, respectively. A plasmid with a 6.8-kilobase-pair DNA fragment containing odhA and odhB was isolated. Functional E1o and E2o are expressed from this plasmid in Escherichia coli. Antisera generated against B. subtilis E1o and E2o expressed in E. coli reacted with antigens of the same size from B. subtilis. The nucleotide sequence of odhB and the terminal part of odhA was determined. The deduced primary sequence of B. subtilis E2o shows striking similarity to the corresponding E. coli protein, which made it possible to identify the lipoyl-binding lysine residue as well as catalytic histidine and aspartic acid residues. An mRNA of 4.5 kilobases hybridizing to both odhA and odhB probes was detected, indicating that odhA and odhB form an operon.
|
|
| 31. |
- Carlsson, Peter, et al.
(författare)
-
In vitro complementation of Bacillus subtilis and Escherichia coli 2-oxoglutarate dehydrogenase complex mutants and genetic mapping of B. subtilis citK and citM mutations
- 1986
-
Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 1574-6968 .- 0378-1097. ; 37:3, s. 373-378
-
Tidskriftsartikel (refereegranskat)abstract
- AbstractThe 2-oxoglutarate dehydrogenase complex of the tricarboxylic acid cycle (TCA) consists of multiple copies of 3 different subenzymes; E1, E2 and E3. The E3 subenzyme is also a component of the pyruvate dehydrogenase complex. Bacillus subtilis 2-oxoglutarate dehydrogenase mutants were studied. The mutants defective in E1, E2 and E3 subenzyme activity, respectively, could be separated into 3 groups by biochemical complementation analyses. The groups correspond to the citK, citM and citL genes. A B. subtilis subenzyme defect, probably E1, could be complemented with the corresponding Escherichia coli wild-type subenzyme and vice versa. Mutations in citK and citM are closely linked. The gene order kauA--- ---citK-citM was determined from 3-factor transformation crosses. It is concluded that the gene organization and the subenzyme structure of the 2-oxoglutarate dehydrogenase complex are similar in B. subtilis and E. coli.
|
|
| 32. |
- Crow, Allister, et al.
(författare)
-
Crystal structure and biophysical properties of Bacillus subtilis BdbD: An oxidizing thiol:disulfide oxidoreductase containing a novel metal site
- 2009
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 284:35, s. 23719-23733
-
Tidskriftsartikel (refereegranskat)abstract
- BdbD is a thiol: disulfide oxidoreductase (TDOR) from Bacillus subtilis that functions to introduce disulfide bonds in substrate proteins/peptides on the outside of the cytoplasmic membrane and, as such, plays a key role in disulfide bond management. Here we demonstrate that the protein is membrane-associated in B. subtilis and present the crystal structure of the soluble part of the protein lacking its membrane anchor. This reveals that BdbD is similar in structure to Escherichia coli DsbA, with a thioredoxin-like domain with an inserted helical domain. A major difference, however, is the presence in BdbD of a metal site, fully occupied by Ca2+, at an inter-domain position some 14 angstrom away from the CXXC active site. The midpoint reduction potential of soluble BdbD was determined as -75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pK(a), consistent with BdbD being an oxidizing TDOR. Equilibrium unfolding studies revealed that the oxidizing power of the protein is based on the instability introduced by the disulfide bond in the oxidized form. The crystal structure of Ca2+-depleted BdbD showed that the protein remained folded, with only minor conformational changes. However, the reduced form of Ca2+-depleted BdbD was significantly less stable than reduced Ca2+-containing protein, and the midpoint reduction potential was shifted by approximately -20 mV, suggesting that Ca2+ functions to boost the oxidizing power of the protein. Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR.
|
|
| 33. |
- Crow, Allister, et al.
(författare)
-
Structure and Functional Properties of Bacillus subtilis Endospore Biogenesis Factor StoA
- 2009
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 284:15, s. 10056-10066
-
Tidskriftsartikel (refereegranskat)abstract
- Bacillus subtilis StoA is an extracytoplasmic thiol-disulfide oxidoreductase (TDOR) important for the synthesis of the endospore peptidoglycan cortex protective layer. Here we demonstrate that StoA is membrane-associated in B. subtilis and report the crystal structure of the soluble protein lacking its membrane anchor. This showed that StoA adopts a thioredoxin-like fold with N-terminal and internal additions that are characteristic of extracytoplasmic TDORs. The CXXC active site of the crystallized protein was found to be in a mixture of oxidized and reduced states, illustrating that there is little conformational variation between redox states. The midpoint reduction potential was determined as -248 mV versus normal hydrogen electrode at pH 7 consistent with StoA fulfilling a reductive role in endospore biogenesis. pKa values of the active site cysteines, Cys-65 and Cys-68, were determined to be 5.5 and 7.8. Although Cys-68 is buried within the structure, both cysteines were found to be accessible to cysteine-specific alkylating reagents. In vivo studies of site-directed variants of StoA revealed that the active site cysteines are functionally important, as is Glu-71, which lies close to the active site and is conserved in many reducing extracytoplasmic TDORs. The structure and biophysical properties of StoA are very similar to those of ResA, a B. subtilis extracytoplasmic TDOR involved in cytochrome c maturation, raising important general questions about how these similar but non-redundant proteins achieve specificity. A detailed comparison of the two proteins demonstrates that relatively subtle differences, largely located around the active sites of the proteins, are sufficient to confer specificity.
|
|
| 34. |
- Degli Esposti, Mauro, et al.
(författare)
-
Respiratory heme A-containing oxidases originated in the ancestors of iron-oxidizing bacteria
- 2021
-
Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 12, s. 14-14
-
Tidskriftsartikel (refereegranskat)abstract
- Respiration is a major trait shaping the biology of many environments. Cytochrome oxidase containing heme A (COX) is a common terminal oxidase in aerobic bacteria and is the only one in mammalian mitochondria. The synthesis of heme A is catalyzed by heme A synthase (CtaA/Cox15), an enzyme that most likely coevolved with COX. The evolutionary origin of COX in bacteria has remained unknown. Using extensive sequence and phylogenetic analysis, we show that the ancestral type of heme A synthases is present in iron-oxidizing Proteobacteria such as Acidithiobacillus spp. These bacteria also contain a deep branching form of the major COX subunit (COX1) and an ancestral variant of CtaG, a protein that is specifically required for COX biogenesis. Our work thus suggests that the ancestors of extant iron-oxidizers were the first to evolve COX. Consistent with this conclusion, acidophilic iron-oxidizing prokaryotes lived on emerged land around the time for which there is the earliest geochemical evidence of aerobic respiration on earth. Hence, ecological niches of iron oxidation have apparently promoted the evolution of aerobic respiration.
|
|
| 35. |
- Erlendsson, Lydur, et al.
(författare)
-
Bacillus subtilis ResA is a thiol-disulfide oxidoreductase involved in cytochrome c synthesis
- 2003
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 278:20, s. 17852-17858
-
Tidskriftsartikel (refereegranskat)abstract
- Covalent attachment of heme to apocytochromes c in bacteria occurs on the outside of the cytoplasmic membrane and requires two reduced cysteinyls at the heme binding site. A constructed ResA-deficient Bacillus subtilis strain was found to lack c-type cytochromes. Cytochrome c synthesis was restored in the mutant by: (i) in trans expression of resA; (ii) deficiency in BdbD, a thioldisulfide oxidoreductase that catalyzes formation of an intramolecular disulfide bond in apocytochrome c after transfer of the polypeptide across the cytoplasmic membrane; or (iii) by addition of the reductant dithiothreitol to the growth medium. In vivo studies of ResA showed that it is membrane-associated with its thioredoxin-like domain on the outside of the cytoplasmic membrane. Analysis of a soluble form of the protein revealed two redox reactive cysteine residues with a midpoint potential of about -340 mV at pH 7. We conclude that ResA, probably together with another thiol-disulfide oxidoreductase, CcdA, is required for the reduction of the cysteinyls in the heme binding site of apocytochrome c.
|
|
| 36. |
- Erlendsson, Lydur, et al.
(författare)
-
Bacillus subtilis StoA is a thiol-disulfide oxidoreductase important for spore cortex synthesis
- 2004
-
Ingår i: Journal of Bacteriology. - 0021-9193. ; 186:18, s. 6230-6238
-
Tidskriftsartikel (refereegranskat)abstract
- Bacillus subtilis is an endospore-forming bacterium. There are indications that protein disulfide linkages occur in spores, but the role of thiol-disulfide chemistry in spore synthesis is not understood. Thiol-disulfide oxidoreductases catalyze formation or breakage of disulfide bonds in proteins. CcdA is the only B. subtilis thiol-disulfide oxidoreductase that has previously been shown to play some role in endospore biogenesis. In this work we show that lack of the StoA (YkvV) protein results in spores sensitive to heat, lysozyme, and chloroform. Compared to CcdA deficiency, StoA deficiency results in a 100-fold-stronger negative effect on sporulation efficiency. StoA is a membrane-bound protein with a predicted thioredoxin-like domain probably localized in the intermembrane space of the forespore. Electron microscopy of spores of CcdA- and StoA-deficient strains showed that the spore cortex is absent in both cases. The BdbD protein catalyzes formation of disulfide bonds in proteins on the outer side of the cytoplasmic membrane but is not required for sporulation. Inactivation of bdbD was found to suppress the sporulation defect of a strain deficient in StoA. Our results indicate that StoA is a thiol-disulfide oxidoreductase that is involved in breaking disulfide bonds in cortex components or in proteins important for cortex synthesis.
|
|
| 37. |
- Erlendsson, Lydur, et al.
(författare)
-
Mutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppress cytochrome c deficiency of CcdA-defective Bacillus subtilis cells
- 2002
-
Ingår i: Journal of Bacteriology. - 0021-9193. ; 184:5, s. 1423-1429
-
Tidskriftsartikel (refereegranskat)abstract
- Cytochromes of the c type in the gram-positive bacterium Bacillus subtilis are all membrane anchored, with their heme domains exposed on the outer side of the cytoplasmic membrane. They are distinguished from other cytochromes by having heme covalently attached by two thioether bonds. The cysteinyls in the heme-binding site (CXXCH) in apocytochrome c must be reduced in order for the covalent attachment of the heme to occur. It has been proposed that CcdA, a membrane protein, transfers reducing equivalents from thioredoxin in the cytoplasm to proteins on the outer side of the cytoplasmic membrane. Strains deficient in the CcdA protein are defective in cytochrome c and spore synthesis. We have discovered that mutations in the bdbC and bdbD genes can suppress the defects caused by lack of CcdA. BdbC and BdbD are thiol-disulfide oxidoreductases. Our experimental findings indicate that these B. subtilis proteins functionally correspond to the well-characterized Escherichia coli DsbB and DsbA proteins, which catalyze the formation of disulfide bonds in proteins in the periplasmic space.
|
|
| 38. |
- Frankenberg, Lena, et al.
(författare)
-
Enterococcus faecalis heme-dependent catalase
- 2002
-
Ingår i: Journal of Bacteriology. - 0021-9193. ; 184:22, s. 6351-6356
-
Tidskriftsartikel (refereegranskat)abstract
- Enterococcus faecalis cells cannot synthesize porphyrins and do not rely on heme for growth but can take up heme and use it to synthesize heme proteins. We recently described a cytochrome bd in E. faecalis strain V583 and here report the identification of a chromosomal gene, katA, encoding a heme-containing cytoplasmic catalase. The 54-kDa KatA polypeptide shows sequence similarity to members of the family of monofunctional catalases. A hexahistidyl-tagged version of the catalase was purified, and major characteristics of the enzyme were determined. It contains one protoheme IX group per KatA polypeptide. Catalase activity was detected only in E. faecalis cells grown in the presence of heme in the medium; about 2 and 10 muM hemin was required for half-maximal and maximal production of catalase, respectively. Our finding of a catalase whose synthesis is dependent on the acquisition of heme in the opportunistic pathogen E. faecalis might be of clinical importance. Studies of cellular heme transport and heme protein assembly and in vivo synthesis of metalloprotein analogs for biotechnological applications are impeded by the lack of experimental systems. We conclude that the E. faecalis cell potentially provides such a desired system.
|
|
| 39. |
- Fridén, H, et al.
(författare)
-
Cytochrome b558 of Bacillus subtilis
- 1988
-
Ingår i: Cytochrome Systems : Molecular Biology and Bioenergetics - Molecular Biology and Bioenergetics. - 9781461290780 - 9781461319412 ; , s. 641-647
-
Bokkapitel (refereegranskat)abstract
- The membranebound tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH) is associated with a b-type cytochrome in many organisms. 1,2 The cytochrome b is often found in stoichiometric amounts in isolated succinate-ubiquinone oxidoreductase (complex II) from bovine heart,3Neurospora crassa,4Ascaris suum5 and plant6 mitochondria as well as in SDH complexes isolated from both Gram-negative and Gram-positive8,9 bacteria whereas yeast (Saccharomyces cerevisiae) apparently lacks this type of cytochrome.10
|
|
| 40. |
- Fridén, H, et al.
(författare)
-
Low temperature EPR and MCD studies on cytochrome b-558 of the Bacillus subtilis succinate: quinone oxidoreductase indicate bis-histidine coordination of the heme iron
- 1990
-
Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - 0005-2728. ; 1041:2, s. 207-215
-
Tidskriftsartikel (refereegranskat)abstract
- Bacillus subtilis cytochrome b-558 was expressed in high amounts in Escherichia coli, solubilized from membranes with detergent and purified free from other hemoproteins. The cytochrome possibly contains two heme groups. To determine the axial ligands to the low-spin heme and the heme rhombicity, the cytochrome was analyzed using low-temperature electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopy. The combined results exclude bis-methionine, bis-lysine and histidine-methionine coordination. Bis-histidine coordination of the heme(s) with a near perpendicular orientation of the imidazole planes is strongly suggested by the highly axial low-spin EPR signals and the intense near infrared MCD spectrum (Δϵ=380 M−1·cm−1 at 4.2 K and 5 T) of the charge-transfer band at 1600 nm.
|
|
| 41. |
- Fridén, H, et al.
(författare)
-
Role of His residues in Bacillus subtilis cytochrome b558 for haem binding and assembly of succinate:quinone oxidoreductase (complex II)
- 1990
-
Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 4:6, s. 1045-1056
-
Tidskriftsartikel (refereegranskat)abstract
- Cytochrome 5558 in the cytoplasmic membrane ofBacilius subtiiis constitutes the anchor and electronacceptor to the flavoprotein (Fp) and iron-sulphurprotein (Ip) in succinate:quinone oxidoreductase, andseemingly contains two haem groups. EPR and MCDspectroscopic data indicate bis-imidazole ligation ofthe haem. Apo-cytochrome was found in the mem-brane fraction of haem-deficient B. subtilis, suggest-ing that during biogenesis of the oxidoreductase thecytochrome b558 polypeptide is embedded into themembrane prior to the incorporation of haem andsubsequent binding of Fp and Ip. The six His residuesin cytochrome b558 were individually changed to Tyrto attempt identification of residues serving as haemaxial ligands and to analyse the role of His residues forassembly and function of the oxidoreductase. Fromthe properties of the mutants, His-47 can be excludedas a haem ligand. The remaining His residues (atpositions 13,28,70,113 and 155) are located in or closeto four predicted transmembrane segments. TheTyr-28 and Tyr-70 mutant proteins appeared to lackone of the two haems. Only the Tyr-13 and Tyr-47mutant cytochromes were found to function asanchors for Fp and Ip, but the Tyr-13 mutant cyto-chrome assembles into an enzymatically defectivesuccinate:quinone oxidoreductase. It is concludedfrom a combination of the experimental findings,sequence comparisons and membrane topology datathat His-28, His-70 and His-155 are probably haemaxial ligands in a dihaem cytochrome 6558. His-70 andHis-155 may be tigands to the same haem.
|
|
| 42. |
- Geisler, Daniela, et al.
(författare)
-
Ca2+-binding and Ca2+-independent respiratory NADH and NADPH dehydrogenases of Arabidopsis thaliana.
- 2007
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 282:39, s. 28455-28464
-
Tidskriftsartikel (refereegranskat)abstract
- Type II NAD(P)H:quinone oxidoreductases are single polypeptide proteins widespread in the living world. They bypass the first site of respiratory energy conservation, constituted by the type I NADH dehydrogenases. To investigate substrate specificities and Ca2+ binding properties of seven predicted type II NAD(P)H dehydrogenases of Arabidopsis thaliana we have produced them as T7-tagged fusion proteins in Escherichia coli. The NDB1 and NDB2 enzymes were found to bind Ca2+, and a single amino acid substitution in the EF hand motif of NDB1 abolished the Ca2+ binding. NDB2 and NDB4 functionally complemented an E. coli mutant deficient in endogenous type I and type II NADH dehydrogenases. This demonstrates that these two plant enzymes can substitute for the NADH dehydrogenases in the bacterial respiratory chain. Three NDB-type enzymes displayed distinct catalytic profiles with substrate specificities and Ca2+ stimulation being considerably affected by changes in pH and substrate concentrations. Under physiologically relevant conditions, the NDB1 fusion protein acted as a Ca2+-dependent NADPH dehydrogenase. NDB2 and NDB4 fusion proteins were NADH-specific, and NDB2 was stimulated by Ca2+. The observed activity profiles of the NDB-type enzymes provide a fundament for understanding the mitochondrial system for direct oxidation of cytosolic NAD(P)H in plants. Our findings also suggest different modes of regulation and metabolic roles for the analyzed A. thaliana enzymes.
|
|
| 43. |
- Guan, Z, et al.
(författare)
-
Preparation and crystallization of a Bacillus subtilis arsenate reductase
- 2001
-
Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; D57, s. 1718-1721
-
Tidskriftsartikel (refereegranskat)abstract
- Arsenate reductase (AR) in B. subtilis is encoded by the chromosomal arsC gene. Together with arsB and arsR, arsC participates in detoxification processes for the arsenate and arsenite ions. Full-length arsenate reductase without any modification has been expressed in Escherichia coli and purified in a soluble form. The recombinant protein has been crystallized at 277 K using polyethyleneglycol (PEG) or poly(ethyleneglycol) methyl ether (PME) as the main precipitant. At least two forms of crystals large enough for data collection have been obtained from wild-type protein under different conditions. An orthorhombic crystal diffracted to beyond 2.2 Å with space group P212121 and unit-cell parameters a = 51.22, b = 91.62, c = 101.93 Å. A near-complete data set has been collected to 2.5 Å. The application of the flash-annealing technique was crucial for high resolution during the data collection. The SeMet-substituted AR has also been produced and crystallized under very similar conditions as the wild type, but the unit-cell parameters are very different. The crystals of the SeMet protein diffracted to higher resolution than those of the wild type.
|
|
| 44. |
- Hambraeus, Gustav, et al.
(författare)
-
Genome-wide survey of mRNA half-lives in Bacillus subtilis identifies extremely stable mRNAs
- 2003
-
Ingår i: Molecular Genetics and Genomics. - : Springer Science and Business Media LLC. - 1617-4615 .- 1617-4623. ; 269:5, s. 706-714
-
Tidskriftsartikel (refereegranskat)abstract
- We have used DNA microarrays to survey rates of mRNA decay on a genomic scale in early stationary-phase cultures of Bacillus subtilis. The decay rates for mRNAs corresponding to about 1500 genes could be estimated. About 80% of these mRNAs had a half-life of less than 7 min. More than 30 mRNAs, including both mono- and polycistronic transcripts, were found to be extremely stable, i.e. to have a half-life of greater than or equal to15 min. Only two such transcripts were known previously in B. subtilis. The results provide the first overview of mRNA decay rates in a gram-positive bacterium and help to identify polycistronic operons. We could find no obvious correlation between the stability of an mRNA and the function of the encoded protein. We have also not found any general features in the 5' regions of mRNAs that distinguish stable from unstable transcripts. The identified set of extremely stable mRNAs may be useful in the construction of stable recombinant genes for the overproduction of biomolecules in Bacillus species.
|
|
| 45. |
- Hansson, Mats, et al.
(författare)
-
Bacillus subtilis HemY is a peripheral membrane protein essential for protoheme IX synthesis which can oxidize coproporphyrinogen III and protoporphyrinogen IX
- 1994
-
Ingår i: Journal of Bacteriology. - 0021-9193. ; 176:19, s. 5962-5970
-
Tidskriftsartikel (refereegranskat)abstract
- The hemY gene of the Bacillus subtilis hemEHY operon is essential for protoheme IX biosynthesis. Two previously isolated hemY mutations were sequenced. Both mutations are deletions affecting the hemY reading frame, and they cause the accumulation of coproporphyrinogen III or coproporphyrin III in the growth medium and the accumulation of trace amounts of other porphyrinogens or porphyrins intracellularly. HemY was found to be a 53-kDa peripheral membrane-bound protein. In agreement with recent findings by Dailey et al. (J. Biol. Chem. 269:813-815, 1994) B. subtilis HemY protein synthesized in Escherichia coli oxidized coproporphyrinogen III and protoporphyrinogen IX to coproporphyrin and protoporphyrin, respectively. The protein is not a general porphyrinogen oxidase since it did not oxidize uroporphyrinogen III. The apparent specificity constant, kcat/Km, for HemY was found to be about 12-fold higher with coproporphyrinogen III as a substrate compared with protoporphyrinogen IX as a substrate. The protoporphyrinogen IX oxidase activity is consistent with the function of HemY in a late step of protoheme IX biosynthesis, i.e., HemY catalyzes the penultimate step of the pathway. However, the efficient coproporphyrinogen III to coproporphyrin oxidase activity is unexplained in the current view of protoheme IX biosynthesis.
|
|
| 46. |
- Hansson, Mats, et al.
(författare)
-
Cloning and characterization of the Bacillus subtilis hemEHY gene cluster, which encodes protoheme IX biosynthetic enzymes
- 1992
-
Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 174:24, s. 8081-8093
-
Tidskriftsartikel (refereegranskat)abstract
- Mutations that cause a block in a late step of the protoheme IX biosynthetic pathway, i.e., in a step after uroporphyrinogen III, map at 94 degrees on the Bacillus subtilis chromosomal genetic map. We have cloned and sequenced the hem genes at this location. The sequenced region contains six open reading frames: ponA, hemE, hemH, hemY, ORFA, and ORFB. The ponA gene product shows over 30% sequence identity to penicillin-binding proteins 1A of Escherichia coli, Streptococcus pneumoniae, and Streptococcus oralis and probably has a role in cell wall metabolism. The hemE gene was identified from amino acid sequence comparisons as encoding uroporphyrinogen III decarboxylase. The hemH gene was identified by enzyme activity analysis of the HemH protein expressed in E. coli. It encodes a water-soluble ferrochelatase which catalyzes the final step in protoheme IX synthesis, the insertion of ferrous iron into protoporphyrin IX. The function of the hemY gene product was not elucidated, but mutation analysis shows that it is required for a late step in protoheme IX synthesis. The hemY gene probably encodes an enzyme with coproporphyrinogen III oxidase or protoporphyrinogen IX oxidase activity or both of these activities. Inactivation of the ORFA and ORFB genes did not block protoheme IX synthesis. Preliminary evidence for a hemEHY mRNA was obtained, and a promoter region located in front of hemE was identified. From these combined results we conclude that the hemEHY gene cluster encodes enzymes for the synthesis of protoheme IX from uroporphyrinogen III and probably constitutes an operon.
|
|
| 47. |
- Hansson, Mats, et al.
(författare)
-
Isolated Bacillus subtilis HemY has coproporphyrinogen III to coproporphyrin III oxidase activity
- 1997
-
Ingår i: BBA - Protein Structure and Molecular Enzymology. - 0167-4838. ; 1340:1, s. 97-104
-
Tidskriftsartikel (refereegranskat)abstract
- Oxidation of coproporphyrinogen III to coproporphyrin III is found in extracts of Escherichia coli cells containing the Bacillus subtilis HemY protein (M. Hansson and L. Hederstedt, J. Bacteriol. 176, 5962-5970). We have analysed whether this activity is due to the heterologous expression system, since it in vivo would lead to disruption of the heme biosynthetic pathway. B. subtilis hemY was fused in its 3'-end to a polynucleotide encoding six histidine residues and expressed from plasmids in both E. coli and B. subtilis. The His6-tagged HemY protein extracted from membranes using non-ionic detergent was purified by Ni2+ affinity chromatography. Isolated HemY fusion protein synthesised in E. coli and B. subtilis oxidised coproporphyrinogen III to coproporphyrin III. No direct formation of protoporphyrin IX from coproporphyrinogen III could be detected. Our results suggest that the coproporphyrinogen III to coproporphyrin III activity of HemY is either avoided in B. subtilis in vivo or that coproporphyrin III is a heme biosynthetic intermediate in this bacterium.
|
|
| 48. |
- Hansson, Mats, et al.
(författare)
-
Purification and characterisation of a water-soluble ferrochelatase from Bacillus subtilis
- 1994
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 220:1, s. 201-208
-
Tidskriftsartikel (refereegranskat)abstract
- Bacillus subtilis ferrochelatase is encoded by the hemH gene of the hemEHY gene cluster and catalyses the incorporation of Fe2+ into protoporphyrin IX. B. subtilis ferrochelatase produced in Escherichia coli was purified. It was found to be a monomeric, water-soluble enzyme of molecular mass 35 kDa which in addition to Fe2+ can incorporate Zn2+ and Cu2+ into protoporphyrin IX. Chemical modification experiments indicated that the single cysteine residue in the ferrochelatase is required for enzyme activity although it is not a conserved residue compared to other ferrochelatases. In growing B. subtilis, the ferrochelatase constitutes approximately 0.05% (by mass) of the total cell protein, which corresponds to some 600 ferrochelatase molecules/cell. The turnover number of isolated ferrochelatase, 18-29 min-1, was found to be consistent with the rate of haem synthesis in exponentially growing cells (0.2 mol haem formed/min/mol enzyme). It is concluded that the B. subtilis ferrochelatase has enzymic properties which are similar to those of other characterised ferrochelatases of known primary structure, i.e. ferrochelatases of the mitochondrial inner membrane of yeast and mammalian cells. However, in contrast to these enzymes the B. subtilis enzyme is a water-soluble protein and should be more amenable to structural analysis.
|
|
| 49. |
- Hansson, Mats, et al.
(författare)
-
The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III
- 1991
-
Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 173, s. 2590-2599
-
Tidskriftsartikel (refereegranskat)abstract
- We have recently reported (M. Petricek, L. Rutberg, I. Schroder, and L. Hederstedt, J. Bacteriol. 172:2250-2258, 1990) the cloning and sequence of a Bacillus subtilis chromosomal DNA fragment containing hemA proposed to encode the NAD(P)H-dependent glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid (ALA) synthesis, hemX encoding a hydrophobic protein of unknown function, and hemC encoding hydroxymethylbilane synthase. In the present communication, we report the sequences and identities of three additional hem genes located immediately downstream of hemC, namely, hemD encoding uroporphyrinogen III synthase, hemB encoding porphobilinogen synthase, and hemL encoding glutamate-1-semialdehyde 2,1-aminotransferase. The six genes are proposed to constitute a hem operon encoding enzymes required for the synthesis of uroporphyrinogen III from glutamyl-tRNA. hemA, hemB, hemC, and hemD have all been shown to be essential for heme synthesis. However, deletion of an internal 427-bp fragment of hemL did not create a growth requirement for ALA or heme, indicating that formation of ALA from glutamate-1-semialdehyde can occur spontaneously in vivo or that this reaction may also be catalyzed by other enzymes. An analysis of B. subtilis carrying integrated plasmids or deletions-substitutions in or downstream of hemL indicates that no further genes in heme synthesis are part of the proposed hem operon.
|
|
| 50. |
- Hederstedt, Lars
(författare)
-
Complex II is complex too
- 2003
-
Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 299:5607, s. 671-672
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
|
|
