Bc17 And Cyt B150 A Fools Game

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An analysis of the current study of the bc1 complex

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Bc17 And Cyt B150 A Fools Game

  1. 1. The bc1 complex and cyt b150: The role of the bc1 complex in the light and oxygen regulation of the expression of the photosynthetic apparatus of R. sphaeroides<br />or<br />Why can strain BC-17 grow aerobically and not photosynthetically?<br />Why do ONLY some of the H217 Qi mutations turn green WHEN grown Anaerobically?<br />
  2. 2. R. sphaeroides strain BC-17<br />The bc1 complex is formed by four sub-units: cytF, cytB, and cytC as part of the fbc operon on chromosome I. <br />The fourth sub-unit cytQ, is encoded elsewhere on chromosome I. <br />R .sphaeroides strain BC17 is a kanamycin insertion mutant for the fbc operon<br />
  3. 3. Genome map of R. sphaeroides<br />cytQ mapped to ~ 2 o’clock on I <br />cyt FBC<br />From: http://mmg.uth.tmc.edu/sphaeroides/genome_map/phys_gen_map.pdf<br />
  4. 4. Role of the bc1 complex: 1994<br />The bc1 complex is thought to provide reduced cytochrome c to aa3<br />under aerobic conditions, and cbb3 under micro-aerophilic conditions<br />García-Horsman JA, et al., J Bact., 1994, 176 (18) 5587–5600.<br />
  5. 5. cbb3 oxidase: overview<br />Expressed maximally under micro-aerophilic or anaerobic conditions<br />Four non-identical sub-units CcoN, CcoO, CcoP (core), CcoQ (subunit IV)<br />Oxioreductase cbb3 is upstream of the prrA/prrB two – component regulatory system<br />cbb3 inversely regulates the PrrBA system via an unknown signal from the CcoQ subunit (RdxBH?)<br />The prrA/prrB regulates the expression of the bacteriochlorophyll synthesis loci in a positive manner<br />
  6. 6. Genome map of R. sphaeroides<br />ccoNOQP<br />From: http://mmg.uth.tmc.edu/sphaeroides/genome_map/phys_gen_map.pdf<br />
  7. 7. Much Mutational Work on ccb3 :1998<br />Aerobic<br />Double mutations of ccb3 with both the PrrAB system or the rdxBH gene show affect on photosynthetic expression as follows:<br />ccb3 deletion : spectral complexes expressed (CCOP1)<br />ccb3 deletion with the PrrAB deletion: lack of any photosynthetic complexes under any growth condition (PRRA2, PRRB1)<br />Filled bar: LHC II (850-800)<br />Open bar LHC I (875)<br />Anaerobic<br />O’Gara J, Eraso JM , S Kaplan J Bact., 1998, 180 (16) 4044–4050<br />
  8. 8. Role of the bc1 complex: 1999<br />The bc1 complex reduces cytochrome c2 providing reductant to cbb3<br />Aerobic conditions: O2 is the final electron acceptor<br />Anaerobic conditions the electron flow controls the ratio of the cartenoids SE (LHII) to SO (LHI)<br />Low light: SE SO <br />Dark + O2 : SE<br />+ DMSO: SE SO <br />SE – spheroidene (yellow)<br />SO – spheroidenone (red)<br />Yeliseev & Kaplan, J. Bact., 1996 ,178: 5877 – 5883; Oh & Kaplan, Biochemistry, 1999, 38: 2688-2696<br />
  9. 9. Role of the bc1 complex: 2000<br />Oh J & S Kaplan, The EMBO J, 2000, 19 (16) 4237 - 4247<br />
  10. 10. The bc1 complex is shown to provide reductant to either aa3 or cbb3 via cytochrome c2 or cy via mutational analysis (R. sphaeroides): 2001<br />Daldal, et.al., J Bact. 2001, 2013–2024. <br />
  11. 11. Anaerobic Growth Scheme for R. sphaeroides: 2005<br />Happ, et.al., Mol. Micro., 2005, 903 - 914 <br />
  12. 12. H217 Mutants of the QI site of the bc1 complex<br />Gray & Daldal (1994) H217D, R, and L in R. capsulatus<br />D and R grow photohetrotrophically with 60% of wild type bc1 levels, L is non-photosynthetically competent with 10% of wt bc1 levels<br />R. capsulatus was grown on MYPE media (LB for Rhodobacter genus) and authors noted some cases of reversion. <br /> Hacker & Gennis (1993) H217A, complex assembled, cyt bHoxidation blocked in R. sphaeroides strain BC-17<br />Grown on sistrom media (defined media), no mention of reversion published but personal communication indicates there was a difficulty in growing this mutation.<br />
  13. 13. DMSO ‘Rescues’ QI Site Mutations<br />Recent mutations constructed in R. sphaeroides consist of H217D, F, G, K, R, W, and Y<br />The D, G, K, R, and Y mutants are photosynthetically competent, yet will revert to H in the presence of oxygen or when grown photosynthetically on defined media (sistrom) with succinate as a carbon source.<br />The F and W mutations are photosynthetically incompetent when grown on defined media, and are lethal to the organism when oxygen is present.<br />The H217N and H217Q mutations both have been claimed to be highly detrimental to the organism (Hacker (Q), Padden (N). <br />Personal note: The H217N mutation would never stabilize in R. sphaeroides, initial selection after mating always yielded revertant or no colonies on the mating selection plate.<br />DMSO ‘rescues’ the 1st class of mutations, but the amount needed increases with the ‘severity’ of the mutation (R,K < D,Y <G <W <F <N,Q)<br />DMSO will also rescue W, but the organism remains photosynthetically incompetent and sickly.<br />DMSO has yet to be shown to be able to ‘rescue’ the H217F mutant<br />
  14. 14. DMSO decreases the amount of spectral complexes formed, 2000 <br />2.4.1: WT<br />CCOP1: ccoP mutant<br />CCOP1/FNRL:<br />ccoP and fnrL mutant<br />PPS1: ppsR mutant<br />Oh J & S Kaplan, The EMBO J, 2000, 19 (16) 4237 - 4247<br />
  15. 15. DorR represses cycA in the presence of DMSO<br />Tavano C., Comolli J., Donohue T., Microbio., 2004, 150 (6), 1893 - 1899 .<br />
  16. 16. Model of the DMSO Reductase System: 1998<br />Mouncey & Kaplan, J. Bact., 1998, 1951-1961<br />
  17. 17. Photosynthetic spectral complexes: 2007A: Aerobic; B. Anaerobic + DMSO<br />deletion<br />wildtype<br />mutant<br />mutant<br />Kim et. al., J. Bact., 2007, 5617-5625.<br />
  18. 18. Expression patterns of genes encoding components of the electron transport chain and other redox active proteins: 2004<br />Roh, J. H. et al. J. Biol. Chem. 2004, 9146-9155<br />
  19. 19. R. sphaeroides strain BC-17<br />Can not grow photosynthetically due to loss of reductant being passed to cbb3<br />Can grow aerobically using quinol oxidase (Qxt) in place of the bc1 complex<br />Null mutants for the cbb3 gene turn green in the presence of oxygen<br />
  20. 20. AppA/PpsR Regulatory System<br />PpsR is a repressor of many of the genes responsible for bacteriochlorophyll synthesis<br />AppA is an anti-repressor that block PpsR binding <br />AppA responds to the redox state of the quinone pool and blue light<br />
  21. 21. Zeilstra-Ryalls, J., et, al.,1998, J. Bact.,180:2801-2809.<br />
  22. 22. bc1complex and the b150 form<br />The bc1complex has three potentiometric midpoints observed during a redox titration: -90, 50 and 150 mV<br />Two of these three are ascribed to the two hemes present: -90 (bL) and 50 (bH) (see Fig I.)<br />The third midpoint b150 is proposed to be the ‘high-potential’ form of the bH cytochrome arising from a redox state between the quinone and bH at the Qi site.<br />The question is what is the nature of this redox state?<br />What is the contribution of each species?<br />How do the species interact with each other to form the 150 mv signal?<br />Does the Em of either species ‘tune’ to the conditions present?<br />Is the b150 form a product only of the QI:bH couple or are additional conditions necessary? <br />Redox poise, pH, mutations in the QI pocket<br />
  23. 23. R. capsulatus on MYPE <br />Gray & Dadal, Biochemistry, 1994, 723 - 733<br />
  24. 24. R. capsulatus H217 Mutant Data<br />Authors mention “Plots of the absorption change at various ambient redox potentials shows the spectrum of cytochrome b150 appears identical to that of ferro-cytochrome bH with a single maximum in the α band at 560 nm”<br />
  25. 25. A: 180 mV<br />B: 180 mV + 10 μM AA<br />C: 100 mV<br />D: 100 mV + 10 μM AA<br />cytochrome b red.<br />cytochrome c ox.<br />
  26. 26. The values from the Table in slide 24 were used in a program provided<br /> by Crofts which attempted to model the contribution of each species of the bc1 complex to the spectra observed under experimental conditions seen in slide 27.<br />Conclusion: The program successfully recreated the curves for the WT,<br />H217D and H217R mutants. However, the program failed to reproduce the curves measured for the H217L mutation. (slide 23)<br />
  27. 27. pMTS<br />H217R<br />H217L<br />H217D<br />
  28. 28. H217 mutations affect the formation of the chromatophore<br />Heavy fraction of WT<br /> chromatophore prep<br />Heavy fraction of H217x<br />chromatophore prep<br />The lack of ‘depth’ in the 542nm signal (seen below) can indicate either a lack of c2, or a lack of BRCs. When grown in the presence of DMSO the level of cytochrome c2 is decreased, and a irregular electron flow through the cbb3 oxidase will lead to a decrease in spectral complex formation by decreasing the amount of PrrA, a enhancer for photosynthetic gene expression. Both pathways lead to a decrease in spectral complex formation. From the image above it is clear the H217 mutation affects the expression of the genes responsible for the ‘greening’ of the chromatophore.<br />
  29. 29. Conclusion<br />The cyt b150 form has never been ‘uncoupled’ from the central physiology of the Rhodobacter species. The intact chromatophore is needed to study the cyt b150 form and the H217 mutations are difficult to study becausethe amino acid is essential. The cells will revert or die unless DMSO is provided. This has the effect of vastly reducing the spectral complexes of the chromatophore and the amount of cytochrome c2. For these reasons it is not clear if cyt b150 is exclusively due to the interaction of QIand bH, or does the surrounding physiology of the chromatophore play a role?<br />Any and all mutations in the bc1 complex will have the potential to affect the assembly of the chromatophore, by altering the activity of the PrrAB system. The chromatophore is the unit used to study the affect of the mutation, how can any conclusions be readily drawn if the unit of measurement fluxes along with the change? <br />To solve this problem the BC17 strain needs to be re-done in a DorR- / PpsR- background (Donohue 2004, Kaplan 2009). This combination of mutants will remove the repressor for photosynthetic gene expression and cytochrome c2 expression. This will eliminatethe stress on chromatophore formation for originating from the cbb3 complex under anaerobic conditions. This strain (BC 18?) will restore the full complement of photosynthetic complexes to the chromatophore and allow the H217 mutations, and all other bc1 complex mutations, to be studied uncoupled from the central Rhodobacterphysiology. <br />
  30. 30. H217D<br />
  31. 31. H217G<br />
  32. 32. H217R<br />
  33. 33. H217Y<br />

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