| 1. |
- Pagels, Martin, et al.
(författare)
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Redox sensing by a Rex-family repressor is involved in the regulation of anaerobic gene expression in Staphylococcus aureus
- 2010
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 76:5, s. 1142-1161
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Tidskriftsartikel (refereegranskat)abstract
- P>An alignment of upstream regions of anaerobically induced genes in Staphylococcus aureus revealed the presence of an inverted repeat, corresponding to Rex binding sites in Streptomyces coelicolor. Gel shift experiments of selected upstream regions demonstrated that the redox-sensing regulator Rex of S. aureus binds to this inverted repeat. The binding sequence - TTGTGAAW(4)TTCACAA - is highly conserved in S. aureus. Rex binding to this sequence leads to the repression of genes located downstream. The binding activity of Rex is enhanced by NAD+ while NADH, which competes with NAD+ for Rex binding, decreases the activity of Rex. The impact of Rex on global protein synthesis and on the activity of fermentation pathways under aerobic and anaerobic conditions was analysed by using a rex-deficient strain. A direct regulatory effect of Rex on the expression of pathways that lead to anaerobic NAD+ regeneration, such as lactate, formate and ethanol formation, nitrate respiration, and ATP synthesis, is verified. Rex can be considered a central regulator of anaerobic metabolism in S. aureus. Since the activity of lactate dehydrogenase enables S. aureus to resist NO stress and thus the innate immune response, our data suggest that deactivation of Rex is a prerequisite for this phenomenon.
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| 2. |
- Aggarwal, Swati, et al.
(författare)
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A protocol for production of perdeuterated OmpF porin for neutron crystallography
- 2021
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928. ; 188
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Tidskriftsartikel (refereegranskat)abstract
- Hydrogen atoms are at the limit of visibility in X-ray structures even at high resolution. Neutron macromolecular crystallography (NMX) is an unambiguous method to locate hydrogens and study the significance of hydrogen bonding interactions in biological systems. Since NMX requires very large crystals, very few neutron structures of proteins have been determined yet. In addition, the most common hydrogen isotope 1H gives rise to significant background due to its large incoherent scattering cross-section. Therefore, it is advantageous to substitute as many hydrogens as possible with the heavier isotope 2H (deuterium) to reduce the sample volume requirement. While the solvent exchangeable hydrogens can be substituted by dissolving the protein in heavy water, complete deuterium labelling – perdeuteration – requires the protein to be expressed in heavy water with a deuterated carbon source. In this work, we developed an optimized method for large scale production of deuterium-labelled bacterial outer membrane protein F (OmpF) for NMX. OmpF was produced using deuterated media with different carbon sources. Mass spectrometry verified the integrity and level of deuteration of purified OmpF. Perdeuterated OmpF crystals diffracted X-rays to a resolution of 1.9 Å. This work lays the foundation for structural studies of membrane protein by neutron diffraction in future.
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| 3. |
- Al-Eryani, Yusra, et al.
(författare)
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Exploring structure and interactions of the bacterial adaptor protein YjbH by crosslinking mass spectrometry
- 2016
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Ingår i: Proteins: Structure, Function and Genetics. - : Wiley. - 0887-3585. ; 84:9, s. 1234-1245
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Tidskriftsartikel (refereegranskat)abstract
- Adaptor proteins assist proteases in degrading specific proteins under appropriate conditions. The adaptor protein YjbH promotes the degradation of an important global transcriptional regulator Spx, which controls the expression of hundreds of genes and operons in response to thiol-specific oxidative stress in Bacillus subtilis. Under normal growth conditions, the transcription factor is bound to the adaptor protein and therefore degraded by the AAA+ protease ClpXP. If this binding is alleviated during stress, the transcription factor accumulates and turns on genes encoding stress-alleviating proteins. The adaptor protein YjbH is thus a key player involved in these interactions but its structure is unknown. To gain insight into its structure and interactions we have used chemical crosslinking mass spectrometry. Distance constraints obtained from the crosslinked monomer were used to select and validate a structure model of YjbH and then to probe its interactions with other proteins. The core structure of YjbH is reminiscent of DsbA family proteins. One lysine residue in YjbH (K177), located in one of the α-helices outside the thioredoxin fold, crosslinked to both Spx K99 and Spx K117, thereby suggesting one side of the YjbH for the interaction with Spx. Another lysine residue that crosslinked to Spx was YjbH K5, located in the long and presumably very flexible N-terminal arm of YjbH. Our crosslinking data lend support to a model proposed based on site-directed mutagenesis where the YjbH interaction with Spx can stabilize and present the C-terminal region of Spx for protease recognition and proteolysis. Proteins 2016; 84:1234–1245.
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| 4. |
- Alferov, Sergey, et al.
(författare)
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Electrical communication of cytochrome enriched Escherichia coli JM109 cells with graphite electrodes
- 2009
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Ingår i: Electrochimica Acta. - : Elsevier BV. - 0013-4686 .- 1873-3859. ; 54:22, s. 4979-4984
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Konferensbidrag (refereegranskat)abstract
- In the present study three different strains of Escherichia coli (JM 109 - a native "wild type" strain, JM 109/pBSD 1300 - a strain overproducing the membrane anchor domain of Bacillus subtilis succinate-quinone reductase, SQR, a protein that contains two transmembraneously arranged heme groups and JM109/pLUV 1900 - a strain overproducing cytochrome c(550) from B. subtilis, a protein where the cytochrome domain is anchored to the membrane with a transmembrane helix) were immobilised on the surface of a spectrographic graphite electrode and tested for electrical communication using mediators. Such compounds as ferricyanide, 2,6-dichlorophenolindophenol (DCPIP) and ubiquinone (Q(0)) were used as soluble mediators and two flexible osmium redox polymers; poly(1-vinylimidazole)(12)-[Os-(4,4'-dimethyl-2,2'-di'pyridyl)(2)Cl-2](2 +/+) (osmium redox polymer I) and poly(vinylpyridine)-[Os-(N,N'-methylated-2,2'-biimidazole)(3)](2+/3+) (osmium redox polymer II) were co-immobilised with the bacterial cells onto the electrode surface. The effects of applied potential, buffer pH and different substrates were compared for the different combinations bacterial strains - mediators. Through the introduction of the cytochromes in the bacterial membrane it was established that it had great effect on the ability of the bacterial cells to effectively communicate with artificial mediators. The introduction of the transmembraneously arranged heme groups of B. subtilis made it possible for this strain to communicate with the Os-polymers, whereas the introduction of the cytochrome c(550) had an effect especially increasing ability of Q(0) to act as an efficient e(-) acceptor. (C) 2009 Elsevier Ltd. All rights reserved.
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| 5. |
- Awad, Wael, et al.
(författare)
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Structural Basis for YjbH Adaptor-Mediated Recognition of Transcription Factor Spx
- 2019
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Ingår i: Structure. - : Elsevier BV. - 0969-2126. ; 27:6, s. 6-936
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Tidskriftsartikel (refereegranskat)abstract
- YjbH is a bacterial adaptor protein required for efficient proteolysis of the RNA polymerase-binding transcription factor Spx by the ClpXP protease. We report the structure of YjbH in complex with Spx. YjbH comprises a DsbA-like thioredoxin domain connected via a linker to a C-terminal domain reminiscent of the winged helix-turn-helix fold. The interaction between YjbH and Spx involves a large surface area. Binding to YjbH stabilizes the C-terminal ClpX recognition region of Spx. We show that mutation of critical YjbH contact residues abrogates Spx recognition. Small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry analyses determined the existence of a stable heterodimeric complex in solution and provide evidence that binding of Spx to YjbH reduces the overall conformational flexibility of Spx. Our findings provide insights into the molecular basis for Spx recognition and suggest a model for how YjbH stabilizes Spx and displays the C terminus of Spx for engagement by ClpXP. Awad et al. determined the crystal structure of the ClpXP adaptor protein YjbH in complex with the transcription factor Spx. Structural dynamics of the complex were investigated by hydrogen-deuterium exchange mass spectrometry. The insights provided in this work add molecular details to the recognition of Spx by YjbH.
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| 6. |
- Azarkina, Natalia, et al.
(författare)
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A bb´-type quinol oxidase in Bacillus subtilis strain 168
- 1999
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 1083-351X .- 0021-9258. ; 274, s. 32810-32817
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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'.
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| 7. |
- Belitsky, B R, et al.
(författare)
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An lrp-like gene of Bacillus subtilis involved in branched-chain amino acid transport
- 1997
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Ingår i: Journal of Bacteriology. - 0021-9193. ; 179:17, s. 5448-5457
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Tidskriftsartikel (refereegranskat)abstract
- The azlB locus of Bacillus subtilis was defined previously by a mutation conferring resistance to a leucine analog, 4-azaleucine (J. B. Ward, Jr., and S. A. Zahler, J. Bacteriol. 116:727-735, 1973). In this report, azlB is shown to be the first gene of an operon apparently involved in branched-chain amino acid transport. The product of the azlB gene is an Lrp-like protein that negatively regulates expression of the azlBCDEF operon. Resistance to 4-azaleucine in azlB mutants is due to overproduction of AzlC and AzlD, two novel hydrophobic proteins.
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| 8. |
- Bengtsson, Jenny, et al.
(författare)
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CtaG is required for formation of active cytochrome C oxidase in Bacillus subtilis
- 2004
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Ingår i: Microbiology. - : Microbiology Society. - 1465-2080 .- 1350-0872. ; 150, s. 415-425
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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.
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| 9. |
- Coman, Vasile, et al.
(författare)
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Electrical wiring of live, metabolically enhanced Bacillus subtilis cells with flexible osmium-redox polymers.
- 2009
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 131:44, s. 16171-16176
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Tidskriftsartikel (refereegranskat)abstract
- The present study explores genetic engineering of the respiratory chain and the application of two different flexible osmium redox polymers to achieve efficient electric communication between the gram-positive organism Bacillus subtilis and an electrode. Poly(1-vinylimidazole)(12)-[Os-(4,4'-dimethyl-2,2'-bipyridyl)(2)Cl(2)](+/2+) (osmium redox polymer I) and poly(vinylpyridine)-[Os-(N,N'-methylated-2,2'-biimidazole)(3)](2+/3+) (osmium redox polymer II) were investigated for efficient electrical "wiring" of viable gram-positive bacterial cells to electrodes. Using a B. subtilis strain that overproduces succinate/quinone oxidoreductase (respiratory complex II), we were able to improve the current response several fold using succinate as substrate, in both batch and flow analysis modes, and using gold and graphite electrodes. The efficiency of the osmium redox polymer, working as electron transfer mediator between the cells and the electrode, was compared with that of a soluble mediator (hexacyanoferrate). The results demonstrated that mediators did not have to pass the cytosolic membrane to bring about an efficient electronic communication between bacterial cells with a thick cell wall and electrodes.
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| 10. |
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