Nucleoid‐associated PhaF phasin drives intracellular location and segregation of polyhydroxyalkanoate granules in <i>Pseudomonas putida</i> KT2442

Galán, Beatriz; Dinjaski, Nina; Maestro, Beatríz; Eugenio, Laura I. de; Escapa, Isabel F.; Sanz, Jesús M.; García, José Luis Rueda; Prieto, María Auxiliadora

doi:10.1111/j.1365-2958.2010.07450.x

Cited by 109 publications

(171 citation statements)

References 53 publications

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“…Recently, we identified a new PGAP, PhaM, that specifically binds to PHB and to DNA (31). We showed that PhaM is necessary for equal distribution of PHB granules to daughter cells during cell division, similar to PhaF in PHA MCL -accumulating Pseudomonas putida (9). In this study, we observed formation of the first visible granules with attached PhaC1-eYFP within only 5 min after transfer of PHB-free cells to conditions permissive for growth and PHB accumulation (Fig.…”

Section: Discussionsupporting

confidence: 61%

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

Pfeiffer

Jendrossek

2012

J Bacteriol

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Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granuleassociated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with "phasin 2 motifs." To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ⌬phaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins can have additional functions. A chromosomal deletion of phaP6 or phaP7 had no visible effect on formation of PHB granules. Survival of microorganisms during periods of starvation depends on their ability to accumulate reserve materials in times of surplus of nutrients. Many prokaryotes are able to accumulate reserve materials such as cyanophycin (CϩN reserve), polyhydroxyalkanoates (PHAs) (C reserve), and polyphosphate (phosphate reserve) in the form of inert osmotic inclusions. Poly(3-hydroxybutyrate) (PHB) is the most abundant PHA type among bacteria, and PHB contents of 90% of the cellular dry weight have been reported. Biosynthesis and biodegradation of PHB and related PHAs have been studied in several labs during the last 25 years (6,8,11,12,16,17,24,27,33,44,45). It turned out that PHB granules in vivo consist of an amorphous polymer core and a complexly organized proteinaceous surface layer. Meanwhile, at least five types of PHB granule-associated proteins (PGAPs) have been described for Ralstonia eutropha, most of which are present in multiple iso...

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Section: Discussionsupporting

confidence: 61%

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

Pfeiffer

Jendrossek

2012

J Bacteriol

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“…Interestingly, recent studies of granule formation in both R. eutropha (36) and Pseudomonas putida (13) have shown that a member of the phasin family in each organism (PhaM and PhaF, respectively) binds nonspecifically to DNA. Thus, in analogy to carboxysome segregation in Synechococcus elongatus (41), granules are equally segregated between daughter cells upon cell division (13), in this case utilizing nonspecific nucleoid binding by granules. Thus, nonspecific interaction of the phasin PhaM with the nucleoid in R. eutropha may explain why granules localized toward the center of the cell.…”

Section: Discussionmentioning

confidence: 99%

Growth and Localization of Polyhydroxybutyrate Granules in Ralstonia eutropha

Beeby

Cho

Stubbe

et al. 2011

Journal of Bacteriology

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The bacterium Ralstonia eutropha forms cytoplasmic granules of polyhydroxybutyrate that are a source of biodegradable thermoplastic. While much is known about the biochemistry of polyhydroxybutyrate production, the cell biology of granule formation and growth remains unclear. Previous studies have suggested that granules form either in the inner membrane, on a central scaffold, or in the cytoplasm. Here we used electron cryotomography to monitor granule genesis and development in 3 dimensions (3-D) in a near-native, "frozen-hydrated" state in intact Ralstonia eutropha cells. Neither nascent granules within the cell membrane nor scaffolds were seen. Instead, granules of all sizes resided toward the center of the cytoplasm along the length of the cell and exhibited a discontinuous surface layer more consistent with a partial protein coating than either a lipid mono-or bilayer. Putatively fusing granules were also seen, suggesting that small granules are continually generated and then grow and merge. Together, these observations support a model of biogenesis wherein granules form in the cytoplasm coated not by phospholipid but by protein. Previous thin-section electron microscopy (EM), fluorescence microscopy, and atomic force microscopy (AFM) results to the contrary may reflect both differences in nucleoid condensation and specimen preparation-induced artifacts.

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“…Furthermore, this phasin binds to DNA nonspecifically through a histone-like domain and plays a key role in intracellular localization and equal distribution of PHA granules to daughter cells during cell division (19,44). Last, PhaP from A. hydrophila (PhaP Ah ) was also observed to act as a positive regulator of the gene that encodes the PHA synthase in this organism, as the overexpression of phaP increased the expression of phaC (45).…”

Section: Role Of Phasins In Pha Metabolismmentioning

confidence: 99%

Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins

Mezzina

Pettinari

2016

Appl Environ Microbiol

102

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Phasins are the major polyhydroxyalkanoate (PHA) granule-associated proteins. They promote bacterial growth and PHA synthesis and affect the number, size, and distribution of the granules. These proteins can be classified in 4 families with distinctive characteristics. Low-resolution structural studies and in silico predictions were performed in order to elucidate the structure of different phasins. Most of these proteins share some common structural features, such as a preponderant ␣-helix composition, the presence of disordered regions that provide flexibility to the protein, and coiled-coil interacting regions that form oligomerization domains. Due to their amphiphilic nature, these proteins play an important structural function, forming an interphase between the hydrophobic content of PHA granules and the hydrophilic cytoplasm content. Phasins have been observed to affect both PHA accumulation and utilization. Apart from their role as granule structural proteins, phasins have a remarkable variety of additional functions. Different phasins have been determined to (i) activate PHA depolymerization, (ii) increase the expression and activity of PHA synthases, (iii) participate in PHA granule segregation, and (iv) have both in vivo and in vitro chaperone activities. These properties suggest that phasins might play an active role in PHA-related stress protection and fitness enhancement. Due to their granule binding capacity and structural flexibility, several biotechnological applications have been developed using different phasins, increasing the interest in the study of these remarkable proteins.

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Nucleoid‐associated PhaF phasin drives intracellular location and segregation of polyhydroxyalkanoate granules in Pseudomonas putida KT2442

Abstract: SummaryThe PhaF is a nucleoid-associated like protein of Pseudomonas putida KT2442 involved in the polyhydroxyalkanoate (

Cited by 109 publications

References 53 publications

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

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

Growth and Localization of Polyhydroxybutyrate Granules in Ralstonia eutropha

Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins

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