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

ABSTRACTIdentification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated fromRalstonia eutrophabut 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 α/β-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 withphaB2(acetoacetyl-coenzyme A [CoA] reductase) andphaC2(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 confirmedin vivoby 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.

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Formation of Polyphosphate by Polyphosphate Kinases and Its Relationship to Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha Strain H16

Applied and Environmental Microbiology ◽

10.1128/aem.02279-15 ◽

2015 ◽

Vol 81 (24) ◽

pp. 8277-8293 ◽

Cited By ~ 11

Author(s):

Tony Tumlirsch ◽

Anna Sznajder ◽

Dieter Jendrossek

Keyword(s):

Fluorescent Protein ◽

Ralstonia Eutropha ◽

Yellow Fluorescent Protein ◽

Proteome Analysis ◽

Enhanced Yellow Fluorescent Protein ◽

Granule Formation ◽

Content Type ◽

Cell Extracts ◽

C And N

ABSTRACTA protein (PhaX) that interacted with poly(3-hydroxybutyrate) (PHB) depolymerase PhaZa1 and with PHB granule-associated phasin protein PhaP2 was identified by two-hybrid analysis. Deletion ofphaXresulted in an increase in the level of polyphosphate (polyP) granule formation and in impairment of PHB utilization in nutrient broth-gluconate cultures. A procedure for enrichment of polyP granules from cell extracts was developed. Twenty-seven proteins that were absent in other cell fractions were identified in the polyP granule fraction by proteome analysis. One protein (A2437) harbored motifs characteristic of type 1 polyphosphate kinases (PPK1s), and two proteins (A1212, A1271) had PPK2 motifs.In vivocolocalization with polyP granules was confirmed by expression of C- and N-terminal fusions of enhanced yellow fluorescent protein (eYFP) with the three polyphosphate kinases (PPKs). Screening of the genome DNA sequence for additional proteins with PPK motifs revealed one protein with PPK1 motifs and three proteins with PPK2 motifs. Construction and subsequent expression of C- and N-terminal fusions of the four new PPK candidates with eYFP showed that only A1979 (PPK2 motif) colocalized with polyP granules. The other three proteins formed fluorescent foci near the cell pole (apart from polyP) (A0997, B1019) or were soluble (A0226). Expression of theRalstonia eutropha ppk(ppkReu) genes in anEscherichia coliΔppkbackground and construction of a set of single and multiple chromosomal deletions revealed that both A2437 (PPK1a) and A1212 (PPK2c) contributed to polyP granule formation. Mutants with deletion of both genes were unable to produce polyP granules. The formation and utilization of PHB and polyP granules were investigated in different chromosomal backgrounds.

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Proteins with CHADs (Conserved Histidine α-Helical Domains) Are Attached to Polyphosphate Granules In Vivo and Constitute a Novel Family of Polyphosphate-Associated Proteins (Phosins)

Applied and Environmental Microbiology ◽

10.1128/aem.03399-16 ◽

2017 ◽

Vol 83 (7) ◽

Cited By ~ 11

Author(s):

Tony Tumlirsch ◽

Dieter Jendrossek

Keyword(s):

Fluorescent Protein ◽

Ralstonia Eutropha ◽

Yellow Fluorescent Protein ◽

Seed Plants ◽

Enhanced Yellow Fluorescent Protein ◽

Content Type ◽

Oil Seed ◽

Eukaryotic Microorganisms ◽

Specific Manner ◽

Associated Proteins

ABSTRACT On the basis of bioinformatic evidence, we suspected that proteins with a CYTH (CyaB thiamine triphosphatase) domain and/or a CHAD (conserved histidine α-helical domain) motif might represent polyphosphate (polyP) granule-associated proteins. We found no evidence of polyP targeting by proteins with CYTH domains. In contrast, two CHAD motif-containing proteins from Ralstonia eutropha H16 (A0104 and B1017) that were expressed as fusions with enhanced yellow fluorescent protein (eYFP) colocalized with polyP granules. While the expression of B1017 was not detectable, the A0104 protein was specifically identified in an isolated polyP granule fraction by proteome analysis. Moreover, eYFP fusions with the CHAD motif-containing proteins MGMSRV2-1987 from Magnetospirillum gryphiswaldense and PP2307 from Pseudomonas putida also colocalized with polyP granules in a transspecies-specific manner. These data indicated that CHAD-containing proteins are generally attached to polyP granules. Together with the findings from four previously polyP-attached proteins (polyP kinases), the results of this study raised the number of polyP-associated proteins in R. eutropha to six. We suggest designating polyP granule-bound proteins with CHAD motifs as phosins (phosphate), analogous to phasins and oleosins that are specifically bound to the surface of polyhydroxyalkanoate (PHA) granules in PHA-accumulating bacteria and to oil droplets in oil seed plants, respectively. IMPORTANCE The importance of polyphosphate (polyP) for life is evident from the ubiquitous presence of polyP in all species on earth. In unicellular eukaryotic microorganisms, polyP is located in specific membrane-enclosed organelles, called acidocalcisomes. However, in most prokaryotes, polyP is present as insoluble granules that have been designated previously as volutin granules. Almost nothing is known regarding the macromolecular composition of polyP granules. Particularly, the absence or presence of cellular compounds on the surface of polyP granules has not yet been investigated. In this study, we identified a novel class of proteins that are attached to the surface of polyP granules in three model species of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. These proteins are characterized by the presence of a CHAD (conserved histidine α-helical domain) motif that functions as a polyP granule-targeting signal. We suggest designating CHAD motif-containing proteins as phosins [analogous to phasins for poly(3-hydroxybutyrate)-associated proteins and to oleosins for oil droplet-associated proteins in oil seed plants]. The expression of phosins in different species confirmed their polyP-targeting function in a transspecies-specific manner. We postulate that polyP granules in prokaryotic species generally have a complex surface structure that consists of one to several polyP kinases and phosin proteins. We suggest differentiating polyP granules from acidocalcisomes by designating them as polyphosphatosomes.

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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

Applied and Environmental Microbiology ◽

10.1128/aem.01056-14 ◽

2014 ◽

Vol 80 (16) ◽

pp. 4936-4946 ◽

Cited By ~ 20

Author(s):

Anna Sznajder ◽

Dieter Jendrossek

Keyword(s):

Fluorescent Protein ◽

Ralstonia Eutropha ◽

Yellow Fluorescent Protein ◽

High Capacity ◽

Constitutive Expression ◽

Enhanced Yellow Fluorescent Protein ◽

Content Type ◽

Phb Depolymerase

ABSTRACTThe putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, ofRalstonia eutrophaH16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granulesin vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ΔphaP1or ΔphaP1-phaP5mutant 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 conditionsin 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 spindle-like structures, and no colocalization with PHB granules was observed. Chromosomal deletion ofphaZd1,phaZd2, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NB-gluconate medium. Moreover, neither proteome analysis of purified native PHB granules norlacZfusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NB-gluconate 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 truein vivofunctions of PhaZd1 and PhaZd2 remain obscure.

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Acidocalcisomes and Polyphosphate Granules Are Different Subcellular Structures in Agrobacterium tumefaciens

Applied and Environmental Microbiology ◽

10.1128/aem.02759-19 ◽

2020 ◽

Vol 86 (8) ◽

Cited By ~ 1

Author(s):

Celina Frank ◽

Dieter Jendrossek

Keyword(s):

Agrobacterium Tumefaciens ◽

Fluorescent Dyes ◽

Fluorescent Protein ◽

Ralstonia Eutropha ◽

Yellow Fluorescent Protein ◽

Eukaryotic Cells ◽

Enhanced Yellow Fluorescent Protein ◽

Content Type ◽

Polyphosphate Granules

ABSTRACT Acidocalcisomes are membrane-enclosed, polyphosphate-containing acidic organelles in lower Eukaryota but have also been described for Agrobacterium tumefaciens (M. Seufferheld, M. Vieira, A. Ruiz, C. O. Rodrigues, S. Moreno, and R. Docampo, J Biol Chem 278:29971–29978, 2003, https://doi.org/10.1074/jbc.M304548200). This study aimed at the characterization of polyphosphate-containing acidocalcisomes in this alphaproteobacterium. Unexpectedly, fluorescence microscopic investigation of A. tumefaciens cells using fluorescent dyes and localization of constructed fusions of polyphosphate kinases (PPKs) and of vacuolar H+-translocating pyrophosphatase (HppA) with enhanced yellow fluorescent protein (eYFP) suggested that acidocalcisomes and polyphosphate are different subcellular structures. Acidocalcisomes and polyphosphate granules were frequently located close together, near the cell poles. However, they never shared the same position. Mutant strains of A. tumefaciens with deletions of both ppk genes (Δppk1 Δppk2) were unable to form polyphosphate but still showed cell pole-located eYFP-HppA foci and could be stained with MitoTracker. In conclusion, A. tumefaciens forms polyP granules that are free of a surrounding membrane and thus resemble polyP granules of Ralstonia eutropha and other bacteria. The composition, contents, and function of the subcellular structures that are stainable with MitoTracker and harbor eYFP-HppA remain unclear. IMPORTANCE The uptake of alphaproteobacterium-like cells by ancestors of eukaryotic cells and subsequent conversion of these alphaproteobacterium-like cells to mitochondria are thought to be key steps in the evolution of the first eukaryotic cells. The identification of acidocalcisomes in two alphaproteobacterial species some years ago and the presence of homologs of the vacuolar proton-translocating pyrophosphatase HppA, a marker protein of the acidocalcisome membrane in eukaryotes, in virtually all species within the alphaproteobacteria suggest that eukaryotic acidocalcisomes might also originate from related structures in ancestors of alphaproteobacterial species. Accordingly, alphaproteobacterial acidocalcisomes and eukaryotic acidocalcisomes should have similar features. Since hardly any information is available on bacterial acidocalcisomes, this study aimed at the characterization of organelle-like structures in alphaproteobacterial cells, with A. tumefaciens as an example.

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C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽

10.1098/rsob.200010 ◽

2020 ◽

Vol 10 (5) ◽

pp. 200010

Author(s):

Navaneethan Palanisamy ◽

Mehmet Ali Öztürk ◽

Emir Bora Akmeriç ◽

Barbara Di Ventura

Keyword(s):

Escherichia Coli ◽

In Silico ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Time Lapse ◽

Enhanced Yellow Fluorescent Protein ◽

Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

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Enhancement of correct protein folding in vivo by a non-lytic baculovirus

Biochemical Journal ◽

10.1042/bj20040007 ◽

2004 ◽

Vol 382 (2) ◽

pp. 695-702 ◽

Cited By ~ 20

Author(s):

Yu HO ◽

Huei-Ru LO ◽

Tzu-Ching LEE ◽

Carol P. Y. WU ◽

Yu-Chan CHAO

Keyword(s):

Energy Transfer ◽

Fusion Protein ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Random Mutagenesis ◽

Resonance Energy ◽

Vector System ◽

Enhanced Yellow Fluorescent Protein

The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7% cell lysis, whereas L-BEVS showed 60% lysis of cells. The quality of protein expressed in both N- and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP–LUC–ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRET-based in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.

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Use of Degradation Tags To Control Protein Levels in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Applied and Environmental Microbiology ◽

10.1128/aem.03741-12 ◽

2013 ◽

Vol 79 (8) ◽

pp. 2833-2835 ◽

Cited By ~ 19

Author(s):

Brian P. Landry ◽

Jana Stöckel ◽

Himadri B. Pakrasi

Keyword(s):

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Terminal Protein ◽

Enhanced Yellow Fluorescent Protein ◽

Content Type ◽

Protein Levels ◽

Steady State Fluorescence ◽

Tunable Range ◽

Control Protein ◽

Synechocystis Sp

ABSTRACTWe generated a collection ofssrA-based C-terminal protein degradation tags with different degradation strengths. The steady-state fluorescence levels of different enhanced yellow fluorescent protein (eYFP) tag variants in aSynechocystissp. indicated a tunable range from 1% to 50% of untagged eYFP.

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In vivo visualization of uterine mast cells by two-photon microscopy

Reproduction ◽

10.1530/rep-13-0570 ◽

2014 ◽

Vol 147 (6) ◽

pp. 781-788 ◽

Cited By ~ 13

Author(s):

Franziska Schmerse ◽

Katja Woidacki ◽

Monika Riek-Burchardt ◽

Peter Reichardt ◽

Axel Roers ◽

...

Keyword(s):

Mast Cells ◽

Intravital Microscopy ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Specific Cell ◽

Enhanced Yellow Fluorescent Protein ◽

Two Photon Microscopy ◽

Two Photon ◽

Custom Made

Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for the study ofin vivobehavior of these cells. We have recently reported that uterine mast cells (uMCs) are important for implantation and placentation. However, theirin vivolocalization in uterus before and during pregnancy is unknown. Herein, we report the direct observation of uMCsin vivousing double-transgenic C57BL/6JMcpt5-Cre ROSA26-EYFPmice with high expression of enhanced yellow fluorescent protein in MC protease 5 (Cma1(Mcpt5))-expressing cells by intravital two-photon microscopy. We were able to monitor MCs livein uteroduring the murine estrous cycle and at different days of pregnancy. We demonstrated that uMCs accumulated during the receptive phase of the female (estrus) and persisted in large numbers at early pregnancy stages and around mid-gestation and declined in number in non-pregnant animals at diestrus. This intravital microscopy technique, including a custom-made microscope stage and the adaption of the surgical procedure, allowed the access of the uterus and implantations for imaging. The introduced application of intravital microscopy to C57BL/6J-Mcpt5-Cre ROSA26-EYFPmice offers a novel and powerfulin vivoapproach to further address the evident relevance of uMCs to reproductive processes with obvious clinical implications.

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BEM46 Shows Eisosomal Localization and Association with Tryptophan-Derived Auxin Pathway in Neurospora crassa

Eukaryotic Cell ◽

10.1128/ec.00061-14 ◽

2014 ◽

Vol 13 (8) ◽

pp. 1051-1063 ◽

Cited By ~ 9

Author(s):

K. Kollath-Leiß ◽

C. Bönniger ◽

P. Sardar ◽

F. Kempken

Keyword(s):

Neurospora Crassa ◽

Fluorescent Protein ◽

Developmental Stages ◽

Yellow Fluorescent Protein ◽

Content Type ◽

Bioinformatic Tools ◽

Ascospore Germination ◽

Alternatively Spliced ◽

Indole Production

ABSTRACTBEM46 proteins are evolutionarily conserved, but their functions remain elusive. We reported previously that the BEM46 protein inNeurospora crassais targeted to the endoplasmic reticulum (ER) and is essential for ascospore germination. In the present study, we established abem46knockout strain ofN. crassa. This Δbem46mutant exhibited a level of ascospore germination lower than that of the wild type but much higher than those of the previously characterizedbem46-overexpressing and RNA interference (RNAi) lines. Reinvestigation of the RNAi transformants revealed two types of alternatively splicedbem46mRNA; expression of either type led to a loss of ascospore germination. Our results indicated that the phenotype was not due tobem46mRNA downregulation or loss but was caused by the alternatively spliced mRNAs and the peptides they encoded. Using theN. crassaortholog of the eisosomal protein PILA fromAspergillus nidulans, we further demonstrated the colocalization of BEM46 with eisosomes. Employing the yeast two-hybrid system, we identified a single interaction partner: anthranilate synthase component II (encoded bytrp-1). This interaction was confirmedin vivoby a split-YFP (yellow fluorescent protein) approach. The Δtrp-1mutant showed reduced ascospore germination and increased indole production, and we used bioinformatic tools to identify a putative auxin biosynthetic pathway. The genes involved exhibited various levels of transcriptional regulation in the differentbem46transformant and mutant strains. We also investigated the indole production of the strains in different developmental stages. Our findings suggested that the regulation of indole biosynthesis genes was influenced bybem46overexpression. Furthermore, we uncovered evidence of colocalization of BEM46 with the neutral amino acid transporter MTR.

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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 ◽

10.1099/mic.0.051508-0 ◽

2011 ◽

Vol 157 (10) ◽

pp. 2795-2807 ◽

Cited By ~ 51

Author(s):

Daniel Pfeiffer ◽

Dieter Jendrossek

Keyword(s):

Gene Product ◽

Genomic Library ◽

Ralstonia Eutropha ◽

Hypothetical Protein ◽

Strong Interactions ◽

Granule Formation ◽

Associated Proteins ◽

Phb Synthase ◽

Two Hybrid

A large number of polypeptides are attached to poly(3-hydroxybutyrate) (PHB) granules of Ralstonia eutropha, such as PHB synthase (PhaC1), several PHB depolymerases (PhaZs) and phasins (PhaPs), the regulator protein PhaR Reu , and possibly others. In this study we used the bacterial adenylate cyclase-based two-hybrid assay to investigate interactions between known PHB granule-associated proteins (PGAPs) and to screen for new PGAPs. The utility of the system was tested by the in vivo verification of previously postulated interactions of the PHB synthase subunits of R. eutropha (PhaC1 homo-oligomerization) and of Bacillus megaterium (PhaC Bmeg –PhaR Bmeg hetero-oligomerization). Nine proteins (PhaA, PhaB1, PhaC1, PhaP1–PhaP4, PhaZ1 and PhaR), with established functions in PHB metabolism of R. eutropha, were tested for interaction in all combinations. While no significant interaction was detected between the PHB synthase PhaC1 and any of the other eight tested Pha proteins, strong interactions were found between all phasin proteins, in particular between PhaP2 and PhaP4. When PhaP2 was used as bait in a two-hybrid screening experiment with a genomic library of R. eutropha, the B1934 gene product was identified in 24 out of 53 isolated clones. B1934 encodes a hypothetical protein (15.7 kDa) with similarity to phasins of PHB-accumulating bacteria. A fusion protein of eYfp and the B1934 gene product colocalized with PHB granules, confirming that B1934 represents a new phasin (PhaP5). PhaP5 was not essential for PHB granule formation, but overexpression of PhaP5 increased the number of cells with PHB granules at the cell poles.

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