| 1. |
- Pankratova, Galina, et al.
(författare)
-
Extracellular Electron Transfer by the Gram-positive Bacterium Enterococcus faecalis
- 2018
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 57, s. 4597-4603
-
Tidskriftsartikel (refereegranskat)abstract
- Extracellular electron transfer (EET) in microbial cells is essential for certain biotechnological applications and contributes to the biogeochemical cycling of elements and syntrophic microbial metabolism in complex natural environments. The Gram-positive lactic acid bacterium Enterococcus faecalis, an opportunistic human pathogen, is shown to be able to transfer electrons generated in fermentation metabolism to electrodes directly and indirectly via mediators. By exploiting E. faecalis wild-type and mutant cells it is demonstrated that reduced demethylmenaquinone in the respiratory chain in the bacterial cytoplasmic membrane is crucial for the EET. Heme-proteins are not involved and cytochrome bd oxidase activity was found to attenuate EET. These results are significant for the mechanistic understanding of EET in bacteria and for design of microbial electrochemical systems. The basic findings infer that in dense microbial communities, such as in biofilm and in the large intestine, metabolism in E. faecalis and similar Gram-positive lactic acid bacteria might be electrically connected to other microbes. Such an intercellular electron transfer might confer syntrophic metabolism that promote growth and other activities of bacteria in the microbiota of humans and animals.
|
|
| 2. |
- 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.
|
|
| 3. |
- 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.
|
|
| 4. |
- 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.
|
|
| 5. |
- 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.
|
|
| 6. |
- 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.
|
|
| 7. |
- 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.
|
|
| 8. |
- 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.
|
|
| 9. |
- 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.
|
|
| 10. |
- 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.
|
|
