1887

Abstract

Polyhydroxybutyrate (PHB), a biodegradable polymer accumulated by bacteria is deposited intracellularly in the form of inclusion bodies often called granules. The granules are supramolecular complexes harbouring a varied number of proteins on their surface, which have specific but incompletely characterised functions. By comparison with other organisms that produce biodegradable polymers, only two phasins have been described to date for raising the possibility that more await discovery. Using a comparative proteomics strategy to compare the granules of wild-type with a PHB-negative mutant housing artificial PHB granules, we identified four potential PHB granules’ associated proteins. These were: Q2RSI4, an uncharacterised protein; Q2RWU9, annotated as an extracellular solute-binding protein; Q2RQL4, annotated as basic membrane lipoprotein; and Q2RQ51, annotated as glucose-6-phosphate isomerase. analysis revealed that Q2RSI4 harbours a family domain and shares low identity with a single-strand DNA-binding protein from . Fluorescence microscopy found that three proteins Q2RSI4, Q2EWU9 and Q2RQL4 co-localised with PHB granules. This work adds three potential new granule associated proteins to the repertoire of factors involved in bacterial storage granule formation, and confirms that proteomics screens are an effective strategy for discovery of novel granule associated proteins.

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2018-04-01
2020-01-22
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References

  1. KEGG 2015;www.genome.jp/dbget-bin/www_bfind?T00310 [acces- sed 2017]
  2. Jendrossek D, Pfeiffer D. New insights in the formation of polyhydroxyalkanoate granules (carbonosomes) and novel functions of poly(3-hydroxybutyrate). Environ Microbiol 2014;16:2357–2373 [CrossRef][PubMed]
    [Google Scholar]
  3. Grage K, Jahns AC, Parlane N, Palanisamy R, Rasiah IA et al. Bacterial polyhydroxyalkanoate granules: biogenesis, structure, and potential use as nano-/micro-beads in biotechnological and biomedical applications. Biomacromolecules 2009;10:660–669 [CrossRef][PubMed]
    [Google Scholar]
  4. Bresan S, Sznajder A, Hauf W, Forchhammer K, Pfeiffer D et al. Polyhydroxyalkanoate (PHA) granules have no phospholipids. Sci Rep 2016;6:26612 [CrossRef][PubMed]
    [Google Scholar]
  5. Boatman ES. Observations on the fine structure of spheroplasts of Rhodospirillum rubrum. J Cell Biol 1964;20:297–311 [CrossRef][PubMed]
    [Google Scholar]
  6. Jendrossek D. Polyhydroxyalkanoate granules are complex subcellular organelles (carbonosomes). J Bacteriol 2009;191:3195–3202 [CrossRef][PubMed]
    [Google Scholar]
  7. Wieczorek R, Pries A, Steinbüchel A, Mayer F. Analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in Alcaligenes eutrophus. J Bacteriol 1995;177:2425–2435 [CrossRef][PubMed]
    [Google Scholar]
  8. Pötter M, Müller H, Reinecke F, Wieczorek R, Fricke F et al. The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha. Microbiology 2004;150:2301–2311 [CrossRef][PubMed]
    [Google Scholar]
  9. Pfeiffer D, Jendrossek D. Interaction between poly(3-hydroxybutyrate) granule-associated proteins as revealed by two-hybrid analysis and identification of a new phasin in Ralstonia eutropha H16. Microbiology 2011;157:2795–2807 [CrossRef][PubMed]
    [Google Scholar]
  10. Pfeiffer D, Jendrossek D. Localization of poly(3-hydroxybutyrate) (PHB) granule-associated proteins during PHB granule formation and identification of two new phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16. J Bacteriol 2012;194:5909–5921 [CrossRef][PubMed]
    [Google Scholar]
  11. York GM, Stubbe J, Sinskey AJ. New insight into the role of the PhaP phasin of Ralstonia eutropha in promoting synthesis of polyhydroxybutyrate. J Bacteriol 2001;183:2394–2397 [CrossRef][PubMed]
    [Google Scholar]
  12. de Almeida A, Nikel PI, Giordano AM, Pettinari MJ. Effects of granule-associated protein PhaP on glycerol-dependent growth and polymer production in poly(3-hydroxybutyrate)-producing Escherichia coli. Appl Environ Microbiol 2007;73:7912–7916 [CrossRef][PubMed]
    [Google Scholar]
  13. Eggers J, Steinbüchel A. Impact of Ralstonia eutropha's poly(3-Hydroxybutyrate) (PHB) depolymerases and phasins on PHB storage in recombinant Escherichia coli. Appl Environ Microbiol 2014;80:7702–7709 [CrossRef][PubMed]
    [Google Scholar]
  14. Galán B, Dinjaski N, Maestro B, de Eugenio LI, Escapa IF et al. Nucleoid-associated PhaF phasin drives intracellular location and segregation of polyhydroxyalkanoate granules in Pseudomonas putida KT2442. Mol Microbiol 2011;79:402–418 [CrossRef][PubMed]
    [Google Scholar]
  15. Maestro B, Galán B, Alfonso C, Rivas G, Prieto MA et al. A new family of intrinsically disordered proteins: structural characterization of the major phasin PhaF from Pseudomonas putida KT2440. PLoS One 2013;8:e56904 [CrossRef][PubMed]
    [Google Scholar]
  16. Handrick R, Reinhardt S, Kimmig P, Jendrossek D. The "intracellular" poly(3-hydroxybutyrate) (PHB) depolymerase of Rhodospirillum rubrum is a periplasm-located protein with specificity for native PHB and with structural similarity to extracellular PHB depolymerases. J Bacteriol 2004;186:7243–7253 [CrossRef][PubMed]
    [Google Scholar]
  17. Handrick R, Reinhardt S, Schultheiss D, Reichart T, Schüler D et al. Unraveling the function of the Rhodospirillum rubrum activator of polyhydroxybutyrate (PHB) degradation: the activator is a PHB-granule-bound protein (phasin). J Bacteriol 2004;186:2466–2475 [CrossRef][PubMed]
    [Google Scholar]
  18. Maestro B, Sanz JM. Polyhydroxyalkanoate-associated phasins as phylogenetically heterogeneous, multipurpose proteins. Microb Biotechnol 2017;10:1323–1337 [CrossRef][PubMed]
    [Google Scholar]
  19. Horowitz DM, Sanders JKM. Amorphous, biomimetic granules of polyhydroxybutyrate: preparation, characterization, and biological implications. J Am Chem Soc 1994;116:2695–2702 [CrossRef]
    [Google Scholar]
  20. Koller M, Bona R, Hermann C, Horvat P, Martinz J et al. Biotechnological production of poly(3-hydroxybutyrate) with Wautersia eutropha by application of green grass juice and silage juice as additional complex substrates. Biocatal Biotransformation 2005;23:329–337 [CrossRef]
    [Google Scholar]
  21. Narancic T, Scollica E, Kenny ST, Gibbons H, Carr E et al. Understanding the physiological roles of polyhydroxybutyrate (PHB) in Rhodospirillum rubrum S1 under aerobic chemoheterotrophic conditions. Appl Microbiol Biotechnol 2016;100:8901–8912 [CrossRef][PubMed]
    [Google Scholar]
  22. Bose SK, Gest H, Ormerod JG. Light-activated hydrogenase activity in a photosynthetic bacterium – a permeability phenomenon. J Biol Chem 1961;236:PC13–PC14
    [Google Scholar]
  23. Handrick R, Reinhardt S, Jendrossek D. Mobilization of poly(3-hydroxybutyrate) in Ralstonia eutropha. J Bacteriol 2000;182:5916–5918 [CrossRef][PubMed]
    [Google Scholar]
  24. Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 2008;26:1367–1372 [CrossRef][PubMed]
    [Google Scholar]
  25. Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 2011;10:1794–1805 [CrossRef][PubMed]
    [Google Scholar]
  26. Pfeiffer D, Jendrossek D. Interaction between poly(3-hydroxybutyrate) granule-associated proteins as revealed by two-hybrid analysis and identification of a new phasin in Ralstonia eutropha H16. Microbiology 2011;157:2795–2807 [CrossRef][PubMed]
    [Google Scholar]
  27. Klask C, Raberg M, Heinrich D, Steinbüchel A, Klask C et al. Heterologous expression of various PHA synthase genes in Rhodospirillum rubrum. Chem Biochem Eng Q 2015;29:75–85 [CrossRef]
    [Google Scholar]
  28. Jendrossek D. Fluorescence microscopical investigation of poly(3-hydroxybutyrate) granule formation in bacteria. Biomacromolecules 2005;6:598–603 [CrossRef][PubMed]
    [Google Scholar]
  29. Uchino K, Saito T, Gebauer B, Jendrossek D. Isolated poly(3-hydroxybutyrate) (PHB) granules are complex bacterial organelles catalyzing formation of PHB from acetyl coenzyme A (CoA) and degradation of PHB to acetyl-CoA. J Bacteriol 2007;189:8250–8256 [CrossRef][PubMed]
    [Google Scholar]
  30. Wahl A, Schuth N, Pfeiffer D, Nussberger S, Jendrossek D. PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha. BMC Microbiol 2012;12:262 [CrossRef][PubMed]
    [Google Scholar]
  31. Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY et al. The perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 2016;13:731–740 [CrossRef][PubMed]
    [Google Scholar]
  32. Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL et al. Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 1999;112:531–552[PubMed]
    [Google Scholar]
  33. Käll L, Krogh A, Sonnhammer EL. Advantages of combined transmembrane topology and signal peptide prediction–the Phobius web server. Nucleic Acids Res 2007;35:W429–W432 [CrossRef][PubMed]
    [Google Scholar]
  34. Drozdetskiy A, Cole C, Procter J, Barton GJ. JPred4: a protein secondary structure prediction server. Nucleic Acids Res 2015;43:W389–W394 [CrossRef][PubMed]
    [Google Scholar]
  35. Gautier R, Douguet D, Antonny B, Drin G. HELIQUEST: a web server to screen sequences with specific alpha-helical properties. Bioinformatics 2008;24:2101–2102 [CrossRef][PubMed]
    [Google Scholar]
  36. Sznajder A, Pfeiffer D, Jendrossek D. Comparative proteome analysis reveals four novel polyhydroxybutyrate (PHB) granule-associated proteins in Ralstonia eutropha H16. Appl Environ Microbiol 2015;81:1847–1858 [CrossRef][PubMed]
    [Google Scholar]
  37. Munk AC, Copeland A, Lucas S, Lapidus A, del Rio TG et al. Complete genome sequence of Rhodospirillum rubrum type strain (S1T). Stand Genomic Sci 2011;4:293–302 [CrossRef][PubMed]
    [Google Scholar]
  38. Erb TJ, Fuchs G, Alber BE. (2S)-Methylsuccinyl-CoA dehydrogenase closes the ethylmalonyl-CoA pathway for acetyl-CoA assimilation. Mol Microbiol 2009;73:992–1008 [CrossRef][PubMed]
    [Google Scholar]
  39. Mande SC, Santosh Kumar CM, Sharma A. Evolution of bacterial Chaperonin 60 paralogues and moonlighting activity. In Hendersson B. (editor) Moonlighting Cell Stress Proteins in Microbial Infections Dordrecht: Springer; 2013
    [Google Scholar]
  40. Meyer RR, Laine PS. The single-stranded DNA-binding protein of Escherichia coli. Microbiol Rev 1990;54:342–380[PubMed]
    [Google Scholar]
  41. Pfeiffer D, Wahl A, Jendrossek D. Identification of a multifunctional protein, PhaM, that determines number, surface to volume ratio, subcellular localization and distribution to daughter cells of poly(3-hydroxybutyrate), PHB, granules in Ralstonia eutropha H16. Mol Microbiol 2011;82:936–951 [CrossRef][PubMed]
    [Google Scholar]
  42. Dinjaski N, Prieto MA. Smart polyhydroxyalkanoate nanobeads by protein based functionalization. Nanomedicine 2015;11:885–899 [CrossRef][PubMed]
    [Google Scholar]
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