Proteomic Analysis of the Developing Intracytoplasmic Membrane in Rhodobacter sphaeroides During Adaptation to Low Light Intensity

  • Kamil Woronowicz
  • Robert A. NiedermanEmail author
Conference paper
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 675)


Although the primary photochemical events in the facultative photoheterotrophic purple bacterium Rhodobacter sphaeroides are now well understood, comparatively little is known about how their photosynthetic apparatus is assembled. Here we present a proteomic analysis of the intracytoplasmic membrane (ICM) assembly process during adaptation to lowered light intensity, in which the size of the photosynthetic units is greatly expanded by addition of the light-harvesting 2 (LH2) peripheral antenna complex. When the isolated ICM-derived chromatophore vesicles were subjected to clear native gel electrophoresis (CNE), four pigmented bands appeared; the top and bottom bands contained the reaction center – light-harvesting 1 (RC–LH1) core complex and LH2 peripheral antenna, respectively, while the two bands of intermediate migration contained associations of the LH2 and core complexes. Proteomic analysis revealed a large array of other proteins associated with the CNE gel bands – in particular, several F1FO-ATP synthase subunits gave unexpectedly high spectral counts, given the inability to detect this coupling factor, as well as the more abundant cytochrome bc 1 complex, by atomic force microscopy. Significant levels of general membrane assembly factors were also found, as well as numerous proteins of unknown function including high counts for RSP6124 that were correlated with LH2 levels. When combined with further AFM and spectroscopic studies, these proteomic approaches are expected to provide a much-improved understanding of the overall assembly process.


Spectral Count Reaction Center Complex Peripheral Antenna Lithium Dodecyl Sulfate Bacterium Rhodobacter Sphaeroides 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was supported by Supported by US Department of Energy Grant No. DE-FG02-08ER15957 from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science. We thank Oluwatobi B. Olubanjo for assistance with membrane purification procedures and Prof. Peter Lobel and Dr. Haiyan Zheng of the Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey, for conducting the proteomics analysis.


  1. Bahatyrova S, Frese RN, Siebert CA, van der Werf KO, van Grondelle R, Niederman RA, Bullough PA, Otto C, Olsen JD, Hunter CN (2004) The native architecture of a photosynthetic membrane. Nature 430:1058–1062PubMedCrossRefGoogle Scholar
  2. Broglie RM, Hunter CN, Delepelaire P, Niederman RA, Chua N-H, Clayton RK (1980) Isolation and characterization of pigment-protein complexes of Rhodopseudomonas sphaeroides by lithium dodecyl sulfate/polyacrylamide gel electrophoresis. Proc Natl Acad Sci USA 77:87–91PubMedCrossRefGoogle Scholar
  3. Feniouk BA, Cherepanov DA, Voskoboynikova NE, Mulkidjanian AY, Junge W (2002) Chromatophore vesicles of Rhodobacter capsulatus contain on average one FOF1-ATP synthase each. Biophys J 82:1115–1122PubMedCrossRefGoogle Scholar
  4. Gubellini F, Francia F, Turina P, Lévy D, Venturoli G, Melandri BA (2007) Heterogeneity of photosynthetic membranes from Rhodobacter capsulatus: Size dispersion and ATP synthase distribution. Biochim Biophys Acta 1767:1340–1352PubMedCrossRefGoogle Scholar
  5. Holmes NG, Hunter CN, Niederman RA, Crofts AR (1980) Identification of the pigment pool responsible for the flash-induced carotenoid band shift in Rhodopseudomonas sphaeroides. FEBS Lett 115:43–48CrossRefGoogle Scholar
  6. Hunter CN, Daldal F, Thurnauer MC, Beatty JT (eds) (2008) The purple phototrophic bacteria. Advances in photosynthesis and respiration, vol 28. Springer, DordrechtGoogle Scholar
  7. Hunter CN, Pennoyer JD, Sturgis JN, Farrelly D, Niederman RA (1988) Oligomerization states and associations of light-harvesting pigment-protein complexes of Rhodobacter sphaeroides as analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemistry 27:3459–3467CrossRefGoogle Scholar
  8. Hunter CN, Tucker JD, Niederman RA (2005) The assembly and organisation of photosynthetic membranes in Rhodobacter sphaeroides. Photochem Photobiol Sci 4:1023–1027PubMedCrossRefGoogle Scholar
  9. Koblízek M, Shih JD, Breitbart SI, Ratcliffe EC, Kolber ZS, Hunter CN, Niederman RA (2005) Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence. Biochim Biophys Acta 1706:220–231PubMedCrossRefGoogle Scholar
  10. Mackenzie C, Choudhary M, Larimer FW, Predki PF, Stilwagen S, Armitage JP, Barber RD, Donohue TJ, Hosler JP, Newman JE, Shapleigh JP, Sockett RE, Zeilstra-Ryalls J, Kaplan S (2001) The home stretch, a first analysis of the nearly completed genome of Rhodobacter sphaeroides 2.4.1. Photosynth Res 70:19–41PubMedCrossRefGoogle Scholar
  11. Niederman RA, Mallon DE, Parks LC (1979) Membranes of Rhodopseudomonas sphaeroides. VI. Isolation of a fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes. Biochim Biophys Acta 555:210–220PubMedCrossRefGoogle Scholar
  12. Niederman RA (2006) Structure, function and formation of bacterial intracytoplasmic membranes. In: Shively JM (ed) Complex intracellular structures in prokaryotes, microbiology monographs, vol 2. Springer, Berlin, Heidelberg, pp 193–227CrossRefGoogle Scholar
  13. Roh JH, Smith WE, Kaplan S (2004) Effects of oxygen and light intensity on transcriptome expression in Rhodobacter sphaeroides 2.4.1. Redox active gene expression profile. J Biol Chem 279:9146–9155PubMedCrossRefGoogle Scholar
  14. Schägger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199:223–231PubMedCrossRefGoogle Scholar
  15. Sturgis JN, Hunter CN, Niederman RA (1988) Spectra and extinction coefficients of near-infrared absorption bands in membranes of Rhodobacter sphaeroides mutants lacking light-harvesting and reaction center complexes. Photochem Photobio l 48:243–247CrossRefGoogle Scholar
  16. Sturgis JN, Tucker JD, Olsen JD, Hunter CN, Niederman RA (2009) Atomic force microscopy studies of native photosynthetic membranes. Biochemistry 48:3679–3698PubMedCrossRefGoogle Scholar
  17. Tatusov RL, Koonin EV, Lipman DJ (1997) A genomic perspective on protein families. Science 278:631–637PubMedCrossRefGoogle Scholar
  18. Westerhuis WHJ, Sturgis JN, Ratcliffe EC, Hunter CN, Niederman RA (2002) Isolation, size estimates and spectral heterogeneity of an oligomeric series of light-harvesting 1 complexes from Rhodobacter sphaeroides. Biochemistry 41:8698–8707PubMedCrossRefGoogle Scholar
  19. Wittig I, Karas M, Schägger H (2007) High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol Cell Proteomics 6:1215–1225PubMedCrossRefGoogle Scholar
  20. Young CS, Beatty JT (2003) Multi-level regulation of purple bacterial light-harvesting complexes. In: Green BR, Parson WW (ed) Light-harvesting antennas in photosynthesis advances in photosynthesis and respiration, vol 13. Kluwer Academic, Dordrecht, pp 449–470Google Scholar
  21. Zeng X, Choudhary M, Kaplan S (2003) A second and unusual pucBAoperon of Rhodobacter sphaeroides 2.4.1: Genetics and function of the encoded polypeptides. J Bacteriol 185:6171–6184PubMedCrossRefGoogle Scholar
  22. Zeng X, Roh JH, Callister SJ, Tavano CL, Donohue TJ, Lipton MS, Kaplan S (2007) Proteomic characterization of the Rhodobacter sphaeroides 2.4.1 photosynthetic membrane: identification of new proteins. J Bacteriol 189:7464–7474PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of Molecular Biology and BiochemistryRutgers UniversityPiscatawayUSA

Personalised recommendations