Studies on the Influence of Phasins on Accumulation and Degradation of PHB and Nanostructure of PHB Granules inRalstoniaeutrophaH16

Abstract

Phasins play an important role in the formation of poly(3-hydroxybutyrate) [PHB] granules and affect their size and number in the cells. Recent studies on the PHB granule proteome and analysis of the complete genomic DNA sequence of Ralstonia eutropha H16 have identified three homologues of the phasin protein PhaP1. In this study, mutants of R. eutropha deficient in the expression of the phasin genes phaP1, phaP2, phaP3, phaP4, phaP12, phaP123, and phaP1234 were examined by gas chromatography. In addition, the nanostructures of the PHB granules of the wild-type and of the mutants were imaged by atomic force microscopy (AFM), and the molecular masses of the accumulated PHB were analyzed by gel permeation chromatography. For this, cells were cultivated under conditions permissive for accumulation of PHB and were then cultivated under conditions permissive for degradation of PHB. Mutants deficient in the expression of phaP2, phaP3, or phaP4 genes mobilized the stored PHB only slowly like the wild-type, whereas degradation occurred much earlier and faster in the phaP1 single mutant as well as in all multiple mutants defective in the phaP1 gene plus one or more other phasin genes. This indicated that the presence of the major phasin PhaP1 on the granule surface is important for PHB degradation and that this phasin is therefore of particular relevance for PHB accumulation. It was also shown that the molecular weights of the accumulated PHB were identical in all examined strains; phasins have therefore no influence on the molecular weight of PHB. The AFM images obtained in this study showed that the PHB granules of R. eutropha H16 form a single interconnected system inside the wild-type cells.

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

“…In some cases, the spreading was so pronounced that the network looked less like a network and more like the surface that is observed on a human brain, and the linear segments were similar to the nanostructures recently reported by Kuchta et al (16). For this reason, AFM probes that are very uniform and very sharp were utilized, with some of them having a radius of curvature as low as 2 nm.…”

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

“…Deletion of phaP1 results in formation of only one or two big PHB granules instead of 8 to 12 mediumsized granules in the wild type. Moreover, deletion of phaP1 enhanced degradation of PHB both in vivo (22) and in vitro (unpublished data). Deletion of other phasins (encoded by phaP2 to phaP7) or overexpression of phaP6 or phaP7 has no pronounced effect on size or number of accumulated PHB granules as determined by light microscopy (22,30; also this study).…”

“…Moreover, deletion of phaP1 enhanced degradation of PHB both in vivo (22) and in vitro (unpublished data). Deletion of other phasins (encoded by phaP2 to phaP7) or overexpression of phaP6 or phaP7 has no pronounced effect on size or number of accumulated PHB granules as determined by light microscopy (22,30; also this study). Interestingly, overexpression of PhaP5, PhaP6, or PhaP7 resulted in cell pole-localized PHB granules and indicated that these phasins can have additional properties compared to PhaP1 to PhaP4.…”

“…Colocalization of PhaP1 with PHB has been previously shown (7,26,51). Three other phasins (PhaP2, PhaP3, and PhaP4) have been postulated from the sequenced R. eutropha genome (32) on the basis of sequence similarity to PhaP1 (22,35), and recently, we identified a fifth phasin (PhaP5) via its interaction with PhaP2 in a two-hybrid approach (30). For most phasins, subcellular localization in vivo under defined culture conditions in the absence and presence of PHB has not yet been in- vestigated systematically.…”

A seawater-based open and continuous process for polyhydroxyalkanoates production by recombinant Halomonas campaniensis LS21 grown in mixed substrates

“…Yet this large single PHB granule is convenient for downstream PHB extraction as a large PHB granule size permits an easy centrifugation process due to its relative heavy weight compared with smaller and multiple PHB (PHA) granules commonly found in all other PHA accumulation bacteria [24,25].…”