Identification of Proteins Associated with Polyhydroxybutyrate Granules from Herbaspirillum seropedicae SmR1 - Old Partners, New Players

Tirapelle, Evandro F.; Müller-Santos, Marcelo; Tadra-Sfeir, Michelle Z.; Kadowaki, Marco Antonio Seiki; Steffens, Maria; Monteiro, Rose A.; Souza, Emanuel Maltempi de; Pedrosa, Fábio O.; Chubatsu, Leda S.

doi:10.1371/journal.pone.0075066

Cited by 30 publications

(39 citation statements)

References 40 publications

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“…In addition, PhaR and PhaM raise the number of PGAPs to 20. The latter can also bind to DNA, in addition to PHB (19,20,22,30), and therefore must have multiple in vivo localizations, as described in Results. However, for most of the PhaZa PHB depolymerase isoenzymes, except for PhaZa1 and the two 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc), there is no or only poor evidence that they are bound to PHB granules in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…Proteome analysis has been used to study the composition of the surface layer of isolated PHB granules and to identify PGAPs. Recently, a Brazilian group determined the proteins of PHB granules isolated from Herbaspirillum seropedicae (30). Remarkably, a histone-like protein with some features similar to those of PhaM and PhaF (11,(22)(23)(24), two outer membrane proteins (OMPs), several hypothetical proteins, and also a protein of the tricarboxylic acid (TCA) cycle (aconitase) were identified, in addition to proteins with well-established functions in PHB metabolism (PHB synthase, the phasin PhaP, and the PHB depolymerase PhaZ).…”

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confidence: 99%

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Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

Sznajder

Pfeiffer

Jendrossek

2015

Appl Environ Microbiol

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Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative ␣/␤-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism. P olyhydroxybutyrate (PHB) and related polyhydroxyalkanoates (PHA) are storage compounds for carbon and energy and are widespread in prokaryotic species (1). PHB is deposited in the form of 200-to 500-nm particles (granules) when PHB-accumulating bacteria are cultivated in the presence of a surplus of a suitable carbon source. Ralstonia eutropha H16 (alternative designation, Cupriavidus necator) has become the model organism of PHB research, and the species is also used in industrial processes to produce PHB as a biodegradable polymer with plastic-like properties (2-4). The intensive research of the last few decades has led to a good understanding of the biochemical routes leading to PHB/PHA and of the biochemical properties of proteins with established functions in PHB/PHA metabolism. For example, the key enzymes of PHB synthesis, the PHB synthases (PhaCs), of many PHB-accumulating species have been purified; the coding genes were cloned; and the polymerization reaction has been studied (for overviews and recent results, see references 5 to 11). Meanwhile, much evidence has accumulated showing that PHB granules are not simply storage molecules but represent well-defined subcellular organelles that consist of a polymer core and a surface layer to which many proteins with specific functions are attached (12). Besides the aforementioned PHB synthase, small amphiphilic polypeptides, so-called phasin proteins (PhaP...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

Sznajder

Pfeiffer

Jendrossek

2015

Appl Environ Microbiol

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“…For reviews, see references 1, 5, 7, and 8. Other PHA-accumulating bacteria and Archaea are also known to have proteins specifically attached to the PHA surface (9)(10)(11)(12)(13)(14)(15). For an overview and more references, see Table 2 in reference 5.…”

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confidence: 99%

To Be or Not To Be a Poly(3-Hydroxybutyrate) (PHB) Depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha, Highly Active PHB Depolymerases with No Detectable Role in Mobilization of Accumulated PHB

Sznajder

Jendrossek

2014

Appl Environ Microbiol

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The putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, of Ralstonia eutropha H16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granules in vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ⌬phaP1 or ⌬phaP1-phaP5 mutant strains resulted in increased specific PHB depolymerase activity, especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB-permissive conditions in vivo. Expression of translational fusions of enhanced yellow fluorescent protein (EYFP) with PhaZd1 and PhaZd2 in which the active-site serines (S190 and Ser193) were replaced with alanine resulted in the colocalization of only PhaZd1 fusions with PHB granules. C-terminal fusions of inactive PhaZd2(S193A) with EYFP revealed the presence of spindlelike structures, and no colocalization with PHB granules was observed. Chromosomal deletion of phaZd1, phaZd2, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NBgluconate medium. Moreover, neither proteome analysis of purified native PHB granules nor lacZ fusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NBgluconate medium. In conclusion, PhaZd1 and PhaZd2 are two PHB depolymerases with a high capacity to degrade PHB when artificially expressed but are apparently not involved in PHB mobilization in the wild type. The true in vivo functions of PhaZd1 and PhaZd2 remain obscure. Ralstonia eutropha H16 is a chemolithoautotrophic betaproteobacterium that has become famous because of its ability to accumulate large amounts of poly(3-hydroxybutyrate) (PHB). R. eutropha is used for the commercial production of bioplastics (polyhydroxyalkanoates [PHAs] such as PHB and copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate) and is considered a model organism for PHA research (1-5). Investigation of the composition of the surface layer of PHB granules of R. eutropha H16 revealed an astonishingly high number of polypeptides that are predicted or have already been shown to be specifically attached to the PHB granule surface. These proteins include enzymes involved in biosynthesis (PHB synthase PhaC1) (6), in granule structure integrity (phasins PhaP1-PhaP7), in PHB mobilization (PhaZa1 to PhaZa5, PhaZb, PhaZc, PhaZd), and in other functions (PhaR, PhaM). For reviews, see references 1, 5, 7, and 8. Other PHA-accumulating bacteria and Archaea are also known to have proteins specifically attached to the PHA surface (9-15). For an overview and more references, see Table 2 in reference 5. Unfortunately, designation of PHB granule-associated proteins in R. eutropha is not uniformly used in literature: for alternative designations of PHB depolymerases and PHB oligomer hydrolases in R. eutropha, s...

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“…H. seropedicae is a diazotrophic ß-Proteobacterium found associated with important agricultural crops (Tirapelle et al, 2013). This bacterium produces polyhydroxybutyrate (PHB), aliphatic polyester, as a carbon storage and/or source of reducing equivalents (Catalan et al, 2007).…”

Section: Polyhydroxybutyratementioning

confidence: 99%

“…The PHB polymer is stored as intracellular insoluble granules coated mainly with proteins, some of which are directly involved in PHB synthesis, degradation and granule biogenesis. Tirapelle et al (2013) extracted the PHB granules from H. seropedicae and identified their associated-proteins by mass spectrometry. H. seropedicae cells were harvested by centrifugation and the cell pellet washed once with potassium phosphate buffer before being re-suspended and sonicated in the same buffer (Potter et al, 2004).…”

Section: Polyhydroxybutyratementioning

confidence: 99%

How to obtain the organelles of prokaryotic and microbial eukaryotic cells

Aderiye¹,

Oluwole²

2014

J. Cell Anim. Biol.

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An organelle is a specialized functional subunit within cells carrying out specific functions. These compartments which may or may not be enclosed in a lipid bilayer are found in microorganisms. While those found in eukaryotic cells are usually enclosed in lipid bilayer, those in prokaryotes don't. All microbes have compartments common to them like the nucleic acids, protein, ribosomes as well as unique intracellular structures found only in microbial subgroups. Such compartments include the mitochondria, endoplasmic reticulum, golgi apparatus amongst others unique to all eukaryotic cells only. Prokaryotes contain some micro-compartments unique to them including the carboxysomes, lipid bodies, polyhydroxybutyrate granules. The right choice of cell disruption methods that limit damage to the compartments is important in achieving successful compartment isolation and purification. Commonly applied methods include sonication, enzymatic lysis, detergent lysis, cavitation amongst others depending on the type of cells involved. Fractionation is the commonly utilized method for isolation and purification of organelles, utilizing ultracentrifugation and techniques that exploits size, density and surface charge variations of protoplasmic content. Such techniques include gradient centrifugation methods, use of beads, affinity purification chromatography methods and electrophoresis. Here, we review the compartments in microbial cells and the techniques employed to isolate and purify these intracellular components.

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Identification of Proteins Associated with Polyhydroxybutyrate Granules from Herbaspirillum seropedicae SmR1 - Old Partners, New Players

Cited by 30 publications

References 40 publications

Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

To Be or Not To Be a Poly(3-Hydroxybutyrate) (PHB) Depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha, Highly Active PHB Depolymerases with No Detectable Role in Mobilization of Accumulated PHB

How to obtain the organelles of prokaryotic and microbial eukaryotic cells

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