Abstract
Bacillus subtilis is a Gram-positive bacterium with a respiratory chain embedded in the cytoplasmic membrane. The respiratory chain is bifurcated after menaquinol into a cytochrome b6c + caa3 branch and a branch with up to three quinol oxidases. The complexes that generate the proton gradient are b6c, associated with caa3 and aa3 oxidase. The b6c and caa3 complexes form a supercomplex, and it is proposed to form respiratory strings in the membrane. There is still information missing about the quinol branch and if the primary oxidase quinol aa3 is associated with the electron donor complexes. It is unclear whether succinate quinone reductase (SQR) can form associations with the quinol branch or the cytochrome branch. In this paper, we show the separation of an almost pure b6c complex associated with cytochromes c550 and c551. We obtained a b6c + caa3 supercomplex of 600 kDa and SQR, aa3, and NADH dehydrogenase by dodecyl maltoside solubilization and separation of the respiratory chain components by ionic exchange chromatography. We found that aa3 does not associate with other complexes. SQR was associated with the b6c complex in a mutant lacking aa3. This association could facilitate electron transfer from SQR to menaquinone-7. The lack of associations between the abundant quinol oxidase aa3 and other complexes is a feature we cannot explain yet.
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Data availability statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
This work is the requirement to obtain the PhD degree in Sciences (Experimental Biology) for the student Gerardo Ignacio Picón Garrido in the Posgrado en Ciencias Biológicas, U.N.A.M. The work was supported by Grants DGAPA, PAPIIT IN219115 and IN219118. We are grateful to Biologist Bertha Pérez Gómez and Dr. Jesús Barrera Rojas for their technical assistance.
Funding
Funding for this research was obtained from Dirección General de Asuntos del Personal Académico (D.G.A.P.A.) (Universidad Nacional Autónoma de México, U.N.A.M.). Research Grants: IN219115 and IN912118.
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All authors contributed to the study conception and design.
GIPG: he grew Bacillus subtilis WT and mutants, prepared membranes for mutants, obtained the solubilization and isolation of respiratory complexes. Prepared the CNE and 2D SDS PAGE. APGG: also grew B. subtilis WT and assisted during the chromatographies, obtained the fractions and the visible spectroscopy analysis. AGL: prepared the samples and obtained the mass spectrometry analysis from the samples of CNE. LGV: contributed to the analysis of the results from mass spectrometry, selected the best ID values for all the proteins and assisted in the verification and analysis of all the mass spectrometry samples. CGL: donated material and chemicals, contributed to the discussion of the results and writing of the manuscript. EBGC: obtained funding and proposed the experiments, assisted in the analysis of results and discussion of the results and writing the manuscript.
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10863_2022_9951_MOESM1_ESM.png
Supplementary Material 1: Spectra of pools I-III obtained from the chromatography of solubilized membranes of WT (letters A through C) and solubilized membranes from mutant ∆Qox (LUH46) (letters D through F). Discontinuous line: ascorbate reduced membranes; continuous line: dithionite reduced membranes; dash-dotted line: the difference spectrum obtained by subtracting the dithionite-reduced minus ascorbate-added spectra.
10863_2022_9951_MOESM2_ESM.png
Supplementary Material 2: Spectra of membranes isolated from the mutants used in this work compared to the WT spectrum. A: dithionite-added spectrum of ∆Qox-aa3 membranes (dotted line) compared to the WT spectrum (continuous line). B: dithionite-added spectrum of ∆SQR membranes (dotted line) compared to the WT spectrum (continuous line). C: dithionite-added spectrum of ∆b6c membranes (dotted line) compared to the WT spectrum (continuous line)
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Picón Garrido, G.I., García García, A.P., González de la Vara, L. et al. Separation and analysis of Bacillus subtilis respiratory chain complexes. J Bioenerg Biomembr 54, 251–271 (2022). https://doi.org/10.1007/s10863-022-09951-6
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DOI: https://doi.org/10.1007/s10863-022-09951-6