Determination of the thickness of the boundary layer surrounding bacterial PHA inclusion bodies, and implications for models describing the molecular architecture of this layer

Mayer, Frank; Hoppert, Michael

doi:10.1002/jobm.3620370108

Cited by 48 publications

(45 citation statements)

References 20 publications

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“…The second explanation is that in prior studies, the envelope was removed by the EM preparation procedure, leaving only the network and the crystalline lamellar layer, which would constitute an electron-dense layer that is approximately 4 nm thick. Therefore, when previous studies reported the boundary layer as being 4 nm thick (21), it may be that the network/lamellar shell was being measured. It is well established that EM preparatory procedures are deleterious to membranes and related attached structures (5).…”

Section: Discussionmentioning

confidence: 99%

“…Because of this, some researchers suggested that instead of an outer phospholipid layer, a protein layer, most logically composed of PhaP with embedded synthesis and depolymerizing enzymes, covers the inclusion (14,27). For a period of time, the difference between these two models seemed to be somewhat clarified by the determination by researchers that the outermost layer is 4 nm thick (21). This implies that it cannot be a lipid bilayer, which has been measured at 7 to 13 nm for C. necator H16 (21).…”

mentioning

confidence: 99%

“…For a period of time, the difference between these two models seemed to be somewhat clarified by the determination by researchers that the outermost layer is 4 nm thick (21). This implies that it cannot be a lipid bilayer, which has been measured at 7 to 13 nm for C. necator H16 (21). Similarly, it does not seem possible that it is a monolayer of PhaP, because quantitative Western blot analyses have determined that there is only enough PhaP present to cover 27 to 54% of the total inclusion surface (31,32).…”

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confidence: 99%

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PhaP Is Involved in the Formation of a Network on the Surface of Polyhydroxyalkanoate Inclusions in Cupriavidus necator H16

et al. 2008

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Polyhydroxyalkanoate (PHA) inclusions are polymeric storage inclusions formed in some bacterial species when carbon levels are high but levels of another essential nutrient, such as nitrogen, are low. Though much is known about PHA synthesis, little is known about inclusion structure. In this study, atomic force microscopy (AFM) was employed to elucidate the structure of PHA inclusions at the nanoscale level, including the characterization of different layers of structure. AFM data suggest that underneath the inclusion envelope, there is a 2-to 4-nm-thick network layer that resides on top of a harder layer that is likely to be a crystalline lamellar polymer. The network is comprised of ϳ20-nm-wide linear segments and junctions that are typically formed by the joining of three to four of the linear segments. In some cases, ϳ50-nm globular structures that are raised ϳ1 to 2 nm above the network are present at the junctions. These globular structures always have a central pore that is ϳ15 nm in diameter. To determine if the major surface protein of PHA inclusions, PhaP, is involved in the structure of this network, inclusions from Cupriavidus necator H16 ⌬phaP were examined. No network structure was detected. Instead, apparently random globular structures were found on the surfaces of the inclusions. When PhaP levels were reconstituted in this strain by the addition of phaP on a plasmid, the network was also reconstituted, albeit in a slightly different arrangement from that of the wild-type network. We conclude that PhaP participates in the formation of the inclusion network.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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PhaP Is Involved in the Formation of a Network on the Surface of Polyhydroxyalkanoate Inclusions in Cupriavidus necator H16

et al. 2008

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“…Phasins occur in any PHA SCLaccumulating bacterium, and are analogues of oleosins, which are bound to the surface of the oleosome in plants (Wieczorek et al, 1995;Steinbüchel et al, 1995;Wieczorek & Steinbüchel, 1996;Mayer & Hoppert, 1997;Hanley et al, 1999). Hitherto, only one phasin (PhaP1) has been known to occur in R. eutropha.…”

Section: Introductionmentioning

confidence: 99%

The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha

et al. 2004

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Analysis of the genome sequence of the polyhydroxyalkanoate-(PHA) accumulating bacterium Ralstonia eutropha strain H16 revealed three homologues (PhaP2, PhaP3 and PhaP4) of the phasin protein PhaP1. PhaP1 is known to constitute the major component of the layer at the surface of poly(3-hydroxybutyrate), poly(3HB), granules. PhaP2, PhaP3 and PhaP4 exhibited 42, 49 and 45 % identity or 61, 62 and 63 % similarity to PhaP1, respectively. The calculated molecular masses of PhaP1, PhaP2, PhaP3 and PhaP4 were 20?0, 20?2, 19?6 and 20?2 kDa, respectively. RT-PCR analysis showed that phaP2, phaP3 and phaP4 were transcribed under conditions permissive for accumulation of poly(3HB). 2D PAGE of the poly(3HB) granule proteome and analysis of the detected proteins by MALDI-TOF clearly demonstrated that PhaP1, PhaP3 and PhaP4 are bound to the poly(3HB) granules in the cells. PhaP3 was expressed at a significantly higher level in PhaP1-negative mutants. Occurrence of an unknown protein with an N-terminal amino-acid sequence identical to that of PhaP2 in crude cellular extracts of R. eutropha had previously been shown by others. Although PhaP2 could not be localized in vivo on poly(3HB) granules, in vitro experiments clearly demonstrated binding of PhaP2 to these granules. Further analysis of complete or partial genomes of other poly(3HB)-accumulating bacteria revealed the existence of multiple phasin homologues in Ralstonia solanacearum, Burkholderia fungorum and Azotobacter vinelandii. These new and unexpected findings should affect our current models of PHA-granule structure and may also have a considerable impact on the establishment of heterologous production systems for PHAs.

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“…eutropha H16 has not yet been identified. However, PHB granules, as well as TAG inclusions, possess a hydrophobic core of the polyester or lipid, respectively, which is thought to be surrounded by a monolayer of phospholipids (de Koning & Maxwell, 1993;Hocking & Marchessault, 1994;Mayer & Hoppert, 1997;Wältermann et al, 2005). This common structure allows the targeting of PhaP1 to PHB granules and also TAG inclusions, as demonstrated in this study.…”

Section: Discussionmentioning

confidence: 99%

The Ralstonia eutropha H16 phasin PhaP1 is targeted to intracellular triacylglycerol inclusions in Rhodococcus opacus PD630 and Mycobacterium smegmatis mc2155, and provides an anchor to target other proteins

et al. 2006

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The Ralstonia eutropha H16 phasin PhaP1 is targeted to intracellular triacylglycerol inclusions in Rhodococcus opacus PD630 and Mycobacterium smegmatis mc 2 155, and provides an anchor to target other proteins In Ralstonia eutropha, the H16 phasin PhaP1 represents the major phasin that binds to the surface of polyhydroxyalkanoate (PHA) inclusions. In this study, C-terminal fusions of PhaP1 with enhanced green fluorescent protein (eGFP) and with Escherichia coli b-galactosidase (LacZ) were expressed separately in the triacylglycerol (TAG)-accumulating actinomycetes Rhodococcus opacus PD630 and Mycobacterium smegmatis mc 2 155, employing the M. smegmatis acetamidase (ace) promoter of the Escherichia-Mycobacterium/Rhodococcus shuttle plasmid pJAM2. PhaP1 and the PhaP1 fusion proteins were expressed stably in the recombinant strains. Western blot analysis of cell fractions of Rh. opacus revealed that PhaP1 and the PhaP1-eGFP fusion protein were associated with the TAG inclusions, whereas no phasin or phasin fusion protein was detected in the soluble and membrane fractions. Additional electron microscopy/ immunocytochemistry studies demonstrated that PhaP1 was mainly located on the surface of intracellular TAG inclusions; in addition, some PhaP1 also occurred at the plasma membrane. Fluorescence microscopic investigations of the subcellular distribution of the PhaP1-eGFP fusion protein in vivo and on isolated TAG inclusions revealed that the fusion protein was bound to TAG inclusions at all stages of their formation, and to some extent at the cytoplasmic membrane. The PhaP1-LacZ fusion protein also bound to the TAG inclusions, and could be separated together with the inclusions from Rh. opacus crude extracts, thus demonstrating the immobilization of b-galactosidase activity on the inclusions. This is believed to be the first report demonstrating the ability of PhaP1 to bind to lipid inclusions in addition to PHA inclusions. Furthermore, it was demonstrated that this non-specificity of PhaP1 can be utilized to anchor enzymically active fusion proteins to a matrix of bacterial TAG inclusions.

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Determination of the thickness of the boundary layer surrounding bacterial PHA inclusion bodies, and implications for models describing the molecular architecture of this layer

Cited by 48 publications

References 20 publications

PhaP Is Involved in the Formation of a Network on the Surface of Polyhydroxyalkanoate Inclusions in Cupriavidus necator H16

PhaP Is Involved in the Formation of a Network on the Surface of Polyhydroxyalkanoate Inclusions in Cupriavidus necator H16

The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha

The Ralstonia eutropha H16 phasin PhaP1 is targeted to intracellular triacylglycerol inclusions in Rhodococcus opacus PD630 and Mycobacterium smegmatis mc2155, and provides an anchor to target other proteins

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