Role of leucine zipper-like motifs in the oligomerization of Pseudomonas putida phasins

Background

Phasins are low molecular mass proteins that accumulate strongly in bacterial cells in response to the intracellular storage of polyhydroxyalkanoates (PHA). Although lacking catalytic activity, phasins are the major components of the surface of the PHA granules and could be potentially involved in the formation of a network-like protein layer surrounding the polyester inclusions. Structural models revealed phasins to possess coiled-coil regions that might be important in the establishment of protein-protein interactions. However, there is not experimental evidence of a coiled-coil mediated oligomerization in these proteins.

A Phasin with Many Faces: Structural Insights on PhaP from Azotobacter sp. FA8

Phasins are a group of proteins associated to granules of polyhydroxyalkanoates (PHAs). Apart from their structural role as part of the PHA granule cover, different structural and regulatory functions have been found associated to many of them, and several biotechnological applications have been developed using phasin protein fusions. Despite their remarkable functional diversity, the structure of these proteins has not been analyzed except in very few studies. PhaP from Azotobacter sp. FA8 (PhaP_Az) is a representative of the prevailing type in the multifunctional phasin protein family. Previous work performed in our laboratory using this protein have demonstrated that it has some very peculiar characteristics, such as its stress protecting effects in recombinant Escherichia coli, both in the presence and absence of PHA. The aim of the present work was to perform a structural characterization of this protein, to shed light on its properties. Its aminoacid composition revealed that it lacks clear hydrophobic domains, a characteristic that appears to be common to most phasins, despite their lipid granule binding capacity. The secondary structure of this protein, consisting of α-helices and disordered regions, has a remarkable capacity to change according to its environment. Several experimental data support that it is a tetramer, probably due to interactions between coiled-coil regions. These structural features have also been detected in other phasins, and may be related to their functional diversity.     

Identification of the haloarchaeal phasin (PhaP) that functions in polyhydroxyalkanoate accumulation and granule formation in Haloferax mediterranei

The polyhydroxyalkanoate (PHA) granule-associated proteins (PGAPs) are important for PHA synthesis and granule formation, but currently little is known about the haloarchaeal PGAPs. This study focused on the identification and functional analysis of the PGAPs in the haloarchaeon Haloferax mediterranei. These PGAPs were visualized with two-dimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF/TOF MS). The most abundant protein on the granules was identified as a hypothetical protein, designated PhaP. A genome-wide analysis revealed that the phaP gene is located upstream of the previously identified phaEC genes. Through an integrative approach of gene knockout/complementation and fermentation analyses, we demonstrated that this PhaP is involved in PHA accumulation. The ΔphaP mutant was defective in both PHA biosynthesis and cell growth compared to the wild-type strain. Additionally, transmission electron microscopy results indicated that the number of PHA granules in the ΔphaP mutant cells was significantly lower, and in most of the ΔphaP cells only a single large granule was observed. These results demonstrated that the H. mediterranei PhaP was the predominant structure protein (phasin) on the PHA granules involved in PHA accumulation and granule formation. In addition, BLASTp and phylogenetic results indicate that this type of PhaP is exclusively conserved in haloarchaea, implying that it is a representative of the haloarchaeal type PHA phasin.     

In Vivo Immobilization of Fusion Proteins on Bioplastics by the Novel Tag BioF

A new protein immobilization and purification system has been developed based on the use of polyhydroxyalkanoates (PHAs, or bioplastics), which are biodegradable polymers accumulated as reserve granules in the cytoplasm of certain bacteria. The N-terminal domain of the PhaF phasin (a PHA-granule-associated protein) from Pseudomonas putida GPo1 was used as a polypeptide tag (BioF) to anchor fusion proteins to PHAs. This tag provides a novel way to immobilize proteins in vivo by using bioplastics as supports. The granules carrying the BioF fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble BioF fusion protein can be obtained by a mild detergent treatment of the granule. The efficiency of this system has been demonstrated by constructing two BioF fusion products, including a functional BioF-β-galactosidase. This is the first example of an active bioplastic consisting of a biodegradable matrix carrying an active enzyme.     

Effects of Granule-Associated Protein PhaP on Glycerol-Dependent Growth and Polymer Production in Poly(3-Hydroxybutyrate)-Producing Escherichia coli

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular granules by many bacteria under unfavorable conditions, enhancing their fitness and stress resistance. Poly(3-hydroxybutyrate) (PHB) is the most widespread and best-known PHA. Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes for proteins involved in granule formation and/or with regulatory functions, such as phasins, that have been shown to affect polymer synthesis. This study evaluates the effect of PhaP, a phasin, on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of recombinant Escherichia coli carrying phaBAC from Azotobacter sp. strain FA8. Cells expressing phaP grew more, and accumulated more PHB, both using glucose and using glycerol as carbon sources. When cultures were grown in a bioreactor using glycerol, PhaP-bearing cells produced more polymer (2.6 times) and more biomass (1.9 times) than did those without the phasin. The effect of this protein on growth promotion and polymer accumulation is expected to be even greater in high-density cultures, such as those used in the industrial production of the polymer. The recombinant strain presented in this work has been successfully used for the production of PHB from glycerol in bioreactor studies, allowing the production of 7.9 g/liter of the polymer in a semisynthetic medium in 48-h batch cultures. The development of bacterial strains that can efficiently use this substrate can help to make the industrial production of PHAs economically feasible.     

Genetic and ultrastructural analysis of different mutants of Pseudomonas putida affected in the poly-3-hydroxy-n-alkanoate gene cluster

Functional analyses of the different proteins involved in the synthesis and accumulation of polyhydroxyalkanoates (PHAs) in P. putida U were performed using a mutant in which the pha locus had been deleted (PpUDeltapha). These studies showed that: (i) Pha enzymes cannot be replaced by other proteins in this bacterium, (ii) the transformation of PpDeltapha with a plasmid containing the locus pha fully restores the synthesis of bioplastics, (iii) the transformation of PpDeltapha with a plasmid harbouring the gene encoding the polymerase PhaC1 (pMCphaC1) permits the synthesis of polyesters (even in absence of phaC2ZDFI); however, in this strain (PpUDeltapha-pMCphaC1) the number of PHAs granules was higher than in the wild type, (iv) the expression of phaF in PpUDeltapha-pMCphaC1 restores the original phenotype, showing that PhaF is involved in the coalescence of the PHAs granules. Furthermore, the deletion of the phaDFI genes in P. putida U considerably decreases (> 70%) the biosynthesis of PHAs consisting of hydroxyalkanoates with aliphatic constituents, and completely prevents the synthesis of those ones containing aromatic monomers. Additional experiments revealed that the deletion of phaD in P. putida U strongly reduces the synthesis of PHA, this effect being restored by PhaF. Moreover, the overexpression of phaF in P. putida U, or in its DeltafadBA mutant, led to the collection of PHA over-producer strains.     

Role of phaD in Accumulation of Medium-Chain-Length Poly(3-Hydroxyalkanoates) in Pseudomonas oleovorans

Pseudomonas oleovorans is capable of producing poly(3-hydroxyalkanoates) (PHAs) as intracellular storage material. To analyze the possible involvement of phaD in medium-chain-length (MCL) PHA biosynthesis, we generated a phaD knockout mutant by homologous recombination. Upon disruption of the phaD gene, MCL PHA polymer accumulation was decreased. The PHA granule size was reduced, and the number of granules inside the cell was increased. Furthermore, mutant cells appeared to be smaller than wild-type cells. Investigation of MCL PHA granules revealed that the pattern of granule-associated proteins was changed and that the predominant protein PhaI was missing in the mutant. Complementation of the mutant with a phaD-harboring plasmid partially restored the wild-type characteristics of MCL PHA production and fully restored the granule and cell sizes. Furthermore, PhaI was attached to the granules of the complemented mutant. These results indicate that the phaD gene encodes a protein which plays an important role in MCL PHA biosynthesis. However, although its main effect seems to be the stabilization of MCL PHA granules, we found that the PhaD protein is not a major granule-associated protein and therefore might act by an unknown mechanism involving the PhaI protein.     

In vivo immobilization of fusion proteins on bioplastics by the novel tag BioF

A new protein immobilization and purification system has been developed based on the use of polyhydroxyalkanoates (PHAs, or bioplastics), which are biodegradable polymers accumulated as reserve granules in the cytoplasm of certain bacteria. The N-terminal domain of the PhaF phasin (a PHA-granule-associated protein) from Pseudomonas putida GPo1 was used as a polypeptide tag (BioF) to anchor fusion proteins to PHAs. This tag provides a novel way to immobilize proteins in vivo by using bioplastics as supports. The granules carrying the BioF fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble BioF fusion protein can be obtained by a mild detergent treatment of the granule. The efficiency of this system has been demonstrated by constructing two BioF fusion products, including a functional BioF-beta-galactosidase. This is the first example of an active bioplastic consisting of a biodegradable matrix carrying an active enzyme.     

The Molecular Basis of Iron-induced Oligomerization of Frataxin and the Role of the Ferroxidation Reaction in Oligomerization

The role of the mitochondrial protein frataxin in iron storage and detoxification, iron delivery to iron-sulfur cluster biosynthesis, heme biosynthesis, and aconitase repair has been extensively studied during the last decade. However, still no general consensus exists on the details of the mechanism of frataxin function and oligomerization. Here, using small-angle x-ray scattering and x-ray crystallography, we describe the solution structure of the oligomers formed during the iron-dependent assembly of yeast (Yfh1) and Escherichia coli (CyaY) frataxin. At an iron-to-protein ratio of 2, the initially monomeric Yfh1 is converted to a trimeric form in solution. The trimer in turn serves as the assembly unit for higher order oligomers induced at higher iron-to-protein ratios. The x-ray crystallographic structure obtained from iron-soaked crystals demonstrates that iron binds at the trimer-trimer interaction sites, presumably contributing to oligomer stabilization. For the ferroxidation-deficient D79A/D82A variant of Yfh1, iron-dependent oligomerization may still take place, although >50% of the protein is found in the monomeric state at the highest iron-to-protein ratio used. This demonstrates that the ferroxidation reaction controls frataxin assembly and presumably the iron chaperone function of frataxin and its interactions with target proteins. For E. coli CyaY, the assembly unit of higher order oligomers is a tetramer, which could be an effect of the much shorter N-terminal region of this protein. The results show that understanding of the mechanistic features of frataxin function requires detailed knowledge of the interplay between the ferroxidation reaction, iron-induced oligomerization, and the structure of oligomers formed during assembly.     

The role of EscD in supporting EscC polymerization in the type III secretion system of enteropathogenic Escherichia coli

The type III secretion system (T3SS) is a multi-protein complex that plays a central role in the virulence of many Gram-negative bacterial pathogens. In enteropathogenic Escherichia coli, a prevalent cause of diarrheal diseases, the needle complex base of the T3SS is formed by multi-rings: two concentric inner-membrane rings made by the two oligomerizing proteins (EscD and EscJ), and an outer ring made of a single oligomerizing protein (EscC). Although the oligomerization activity of these proteins is critical for their function and can, therefore, affect the virulence of the pathogen, the mechanisms underlying the oligomerization of these proteins have yet to be identified. In this study, we report that the proteins forming the inner-membrane T3SS rings, EscJ and EscD proteins, are crucial for the oligomerization of EscC. Moreover, we elucidate the oligomerization process of EscD and determine the contribution of individual regions of the protein to its self-oligomerization activity. We show that the oligomerization motif of EscD is located at its N-terminal portion and that its transmembrane domain can self-oligomerize, thus contributing to the self-oligomerization of the full-length EscD.     

Occurrence of polyhydroxyalkanoic acid granule-associated proteins related to the Alcaligenes eutrophus H16 GA24 protein in other bacteria

Abstract Fifty different polyhydroxyalkanoic acid (PHA)-accumulating bacterial strains were investigated for the occurrence of phasin proteins bound to PHA granules and related to the GA24 protein of Alcaligenes eutrophus H16, by isolating PHA granules and Western blot analysis of granule-associated proteins employing antibodies raised against the GA24 protein. It could be demonstrated that the PHA granules of many poly(3-hydroxybutyrate)-accumulating bacteria exhibited ja similar protein pattern, and a predominant protein of 24 ± 2 kDa occurred in the granules of A. eutrophus strains A7, CH34, JMP222, N9A and TF93 exhibiting N-terminal amino acid sequences identical to that of the GA24 protein. Proteins bound to the granules of A. latus, Burkholderia caryophvli B. cepacia B. solanacearum, Pseudomonas glathei. Rhodobacter sphaeroides and Telluria mixta also gave positive immunoreactions. Granule-associated proteins of small size also; occurred in various strains of the Gram-positive bacteria Bacillus megaterium and R. ruher as well as in the Gram-negative bacteria Azotohacter sp., Chromatium vinosum, Comamonas acidovorans, Methylobacterium sp., Mycoplana ruhra, Paracoccus denitrificans, Pseudomonas sp., Rhodospirillum ruhrum, Rubrivivax gelatinosus and Thiocystis violacea ; however, they gave no immunoreaction. This study clearly demonstrated that phasins are wide-spread if not essential in PHA-accümulating bacteria.     

Structure, Function, and Evolution of the Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein

Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging tick-borne virus of the Bunyaviridae family that is responsible for a fatal human disease for which preventative or therapeutic measures do not exist. We solved the crystal structure of the CCHFV strain Baghdad-12 nucleocapsid protein (N), a potential therapeutic target, at a resolution of 2.1 Å. N comprises a large globular domain composed of both N- and C-terminal sequences, likely involved in RNA binding, and a protruding arm domain with a conserved DEVD caspase-3 cleavage site at its apex. Alignment of our structure with that of the recently reported N protein from strain YL04057 shows a close correspondence of all folds but significant transposition of the arm through a rotation of 180 degrees and a translation of 40 Å. These observations suggest a structural flexibility that may provide the basis for switching between alternative N protein conformations during important functions such as RNA binding and oligomerization. Our structure reveals surfaces likely involved in RNA binding and oligomerization, and functionally critical residues within these domains were identified using a minigenome system able to recapitulate CCHFV-specific RNA synthesis in cells. Caspase-3 cleaves the polypeptide chain at the exposed DEVD motif; however, the cleaved N protein remains an intact unit, likely due to the intimate association of N- and C-terminal fragments in the globular domain. Structural alignment with existing N proteins reveals that the closest CCHFV relative is not another bunyavirus but the arenavirus Lassa virus instead, suggesting that current segmented negative-strand RNA virus taxonomy may need revision.     

Accumulation of the PhaP Phasin of Ralstonia eutropha Is Dependent on Production of Polyhydroxybutyrate in Cells

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of Ralstonia eutropha. R. eutropha phaC PHA synthase and phaP phasin gene replacement strains were constructed. The strains were engineered to express heterologous and/or mutant PHA synthase alleles and a phaP-gfp translational fusion in place of the wild-type alleles of phaC and phaP. The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of the phaP-gfp fusion. The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains. Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that R. eutropha has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.     

Metabolomic and proteomic insights into carbaryl catabolism by Burkholderia sp. C3 and degradation of ten N-methylcarbamates

Burkholderia sp. C3, an efficient polycyclic aromatic hydrocarbon degrader, can utilize nine of the ten N-methylcarbamate insecticides including carbaryl as a sole source of carbon. Rapid hydrolysis of carbaryl in C3 is followed by slow catabolism of the resulting 1-naphthol. This study focused on metabolomes and proteomes in C3 cells utilizing carbaryl in comparison to those using glucose or nutrient broth. Sixty of the 867 detected proteins were involved in primary metabolism, adaptive sensing and regulation, transport, stress response, and detoxification. Among the 41 proteins expressed in response to carbaryl were formate dehydrogenase, aldehyde-alcohol dehydrogenase and ethanolamine utilization protein involved in one carbon metabolism. Acetate kinase and phasin were 2 of the 19 proteins that were not detected in carbaryl-supported C3 cells, but detected in glucose-supported C3 cells. Down-production of phasin and polyhydroxyalkanoates in carbaryl-supported C3 cells suggests insufficient carbon sources and lower levels of primary metabolites to maintain an ordinary level of metabolism. Differential metabolomes (~196 identified polar metabolites) showed up-production of metabolites in pentose phosphate pathways and metabolisms of cysteine, cystine and some other amino acids, disaccharides and nicotinate, in contract to down-production of most of the other amino acids and hexoses. The proteomic and metabolomic analyses showed that carbaryl-supported C3 cells experienced strong toxic effects, oxidative stresses, DNA/RNA damages and carbon nutrient deficiency.     

Biochemical and Biophysical Characterisation of Higher Oligomeric Structure of Rat Nucleosome Assembly Protein 1

Nucleosome assembly protein 1 (NAP1) is a histone chaperone that exchanges histone H2A-H2B dimer from chromatin templates. Studies with yeast NAP1 (yNAP1) have revealed its existence as multiple oligomeric species in solution. Here, rat NAP1 (rNAP1), which is 98% identical to the human NAP1 (hNAP1) was used as a model to characterize the oligomeric structures of this protein in higher eukaryotes. Gel filtration chromatography and Dynamic light scattering of recombinant rNAP1 indicated that the protein exists as a complex mixture of multimeric species even at 500 mM ionic strength. The solution-state complexity remains unchanged even at higher ionic strengths. Equilibrium unfolding (ΔG 14.6 kcal mol^??1) shows that rNAP1, both dimeric and oligomeric, follow the two-state model of unfolding with no detectable intermediates. Homology modelling suggests that rat and yeast NAP1 share an overall similar structure with conserved domains. However, dissimilar substitutions like threonine and lysine with glycine in the β-hairpin involved in oligomerization, possibly leads to the observed differences in the oligomerization propensity of the two proteins. Molecular dynamic simulation (MDS) of the two structures also revealed that rNAP1 dimer is more stable owing to the extensive hydrogen bonding in comparison to yNAP1. Further, in vitro kinase assay showed that the phosphorylation of rNAP1 favors oligomerization with no effect on its histone binding capacity. Our results clearly suggest that there are differences in the in-solution behavior of rNAP1 compared to yNAP1 which may have in vivo functional implications for the regulation of these complexes during chromatin assembly and rearrangement.     

Polyhydroxyalkanoate (PHA) granule formation in<Emphasis Type="Italic"> Ralstonia eutropha</Emphasis> cells: a computer simulation

Computer simulation of polyhydroxyalkanoate (PHA) granule formation in vivo could help to design strategies to optimize the fermentation process and achieve higher yields of PHA. It could also suggest biotechnological approaches to control the granule size and molecular weight of the polymer. A computer program simulating the formation of PHA granules inside a Ralstonia eutropha cell was developed, based on published experimental data. The results are applicable to R. eutropha cells or other microorganisms and transgenic plants, where polyhydroxybutyrate production is made possible by heterologous expression systems. The simulation starts at the outset of the PHA accumulation phase when the cells are small and contain no PHA granules. In the presence of abundant glucose, the cell responds to phosphorus limitation by producing 3-hydroxybutyryl-CoA which undergoes polymerization on the few PHA synthase molecules present in the cytoplasm. The amphiphilic PHA synthase–PHA complex attracts additional PHA synthase molecules and granules begin to grow from these initiation sites. Phosphorus limitation and the appearance of PHA in the cytoplasm also stimulate production of phasin molecules that attach themselves to the growing granules. As the granules grow bigger, they begin to touch each other and move to optimize their packing. The phasin coat prevents the granules from coalescing. The size of the cell increases and its prolate ellipsoid shape becomes closer to spherical. The accumulation process stops either when the supply of glucose is exhausted or when the granules become tightly packed within the cell, so that access to their surface is limited. All important variables, such as cell dimensions, granule size, counts of granule-associated molecules, PHA yield, degree of polymerization of the PHA molecules, etc., are recorded in real time during the simulation. Examples of virtual experiments with the cell and their results are shown.     

Structure of the stress response protein DR1199 from Deinococcus radiodurans: a member of the DJ-1 superfamily

The expression level of protein DR1199 is observed to increase considerably in the radio-resistant bacterium Deinococcus radiodurans following irradiation. This protein belongs to the DJ-1 superfamily, which includes proteins with diverse functions, such as the archaeal proteases PhpI and PfpI, the bacterial chaperone Hsp31 and hyperosmotic stress protein YhbO, and the human Parkinson's disease-related protein DJ-1. All members of the superfamily are oligomeric, and the oligomerization interface varies from protein to protein. Although for many of these proteins, their function remains obscure, most of them are involved in cellular protection against environmental stresses. We have determined the structure of DR1199 to a resolution of 2.15 A, and we have tested its function and studied its role in the response to irradiation and more generally to oxidative stress in D. radiodurans. The protein is a dimer displaying an oligomerization interface similar to that observed for the YhbO and PhpI proteins. The cysteine in the catalytic triad (Cys 115) is oxidized in our structure, similar to modifications seen in the corresponding cysteine of the DJ-1 protein. The oxidation occurs spontaneously in DR1199 crystals. In solution, no proteolytic or chaperone activity was detected. On the basis of our results, we suggest that DR1199 might work as a general stress protein involved in the detoxification of the cell from oxygen reactive species, rather than as a peptidase in D. radiodurans.