Characterization of PHB1 and Its Role in Mitochondrial Maturation and Yolk Platelet Degradation during Development of Artemia Embryos

Background

To cope with harsh environments, crustaceans such as Artemia produce diapause gastrula embryos (cysts) with suppressed metabolism. Metabolism and development resume during post-diapause development, but the mechanism behind these cellular events remains largely unknown.

Principal Finding

Our study investigated the role of prohibitin 1 (PHB1) in metabolic reinitiation during post-diapause development. We found that PHB1 was developmentally regulated via changes in phosphorylation status and localization. Results from RNA interference experiments demonstrated PHB1 to be critical for mitochondrial maturation and yolk degradation during development. In addition, PHB1 was present in yolk platelets, and it underwent ubiquitin-mediated degradation during the proteolysis of yolk protein.

Conclusions/Significance

PHB1 has an indispensable role in coordinating mitochondrial maturation and yolk platelet degradation during development in Artemia. This novel function of PHB1 provides new clues to comprehend the roles of PHB1 in metabolism and development.     

Poly-3-hydroxybutyrate metabolism in the type II methanotroph Methylocystis parvus OBBP

Differences in carbon assimilation pathways and reducing power requirements among organisms are likely to affect the role of the storage polymer poly-3-hydroxybutyrate (PHB). Previous researchers have demonstrated that PHB functions as a sole growth substrate in aerobic cultures enriched on acetate during periods of carbon deficiency, but it is uncertain how C(1) metabolism affects the role of PHB. In the present study, the type II methanotroph Methylocystis parvus OBBP did not replicate using stored PHB in the absence of methane, even when all other nutrients were provided in excess. When PHB-rich cultures of M. parvus OBBP were deprived of carbon and nitrogen for 48 h, they did not utilize significant amounts of stored PHB, and neither cell concentrations nor concentrations of total suspended solids changed significantly. When methane and nitrogen both were present, PHB and methane were consumed simultaneously. Cells with PHB had significantly higher specific growth rates than cells lacking PHB. The addition of formate (a source of reducing power) to PHB-rich cells delayed PHB consumption, but the addition of glyoxylate (a source of C(2) units) did not. This and results from other researchers suggest that methanotrophic PHB metabolism is linked to the supply of reducing power as opposed to the supply of C(2) units for synthesis.     

Prohibitin deficiency causes opposing lipid metabolism between 3T3-L1 adipocytes and Clone 9 hepatocytes

Prohibitin (PHB) is a highly conserved protein in eukaryotic cells that are present in multiple cellular compartments and has potential roles as a tumor suppressor, an anti-proliferative protein, a regulator of cell-cycle progression and in apoptosis. In the present study, we generated PHB-deficient 3T3-L1 adipocytes and Clone 9 (C9) hepatocytes by oligonucleotide siRNA and investigated whether PHB affect lipid metabolism. It was revealed that PHB deficiency caused opposing lipid metabolism between the two cell models. PHB deficiency increased expression of adipogenic, lipogenic, and other lipid metabolic proteins in 3T3-L1 adipocytes, whereas significantly decreased the levels of those proteins in C9 cells. Collectively, PHB deficiency promoted lipid metabolism in 3T3-L1 adipocytes while it aggravated lipid metabolism in C9 hepatocytes.     

Stoichiometry and kinetics of poly-beta-hydroxybutyrate metabolism in aerobic, slow growing, activated sludge cultures

This paper discusses the poly-beta-hydroxybutyrate (PHB) metabolism in aerobic, slow growing, activated sludge cultures, based on experimental data and on a metabolic model. The dynamic conditions which occur in activated sludge processes were simulated in a 2-L sequencing batch reactor (SBR) by subjecting a mixed microbial population to successive periods of external substrate availability (feast period) and no external substrate availability (famine period). Under these conditions intracellular storage and consumption of PHB was observed. It appeared that in the feast period, 66% to almost 100% of the substrate consumed is used for storage of PHB, the remainder is used for growth and maintenance processes. Furthermore, it appeared that at high sludge retention time (SRT) the growth rate in the feast and famine periods was the same. With decreasing SRT the growth rate in the feast period increased relative to the growth rate in the famine period. Acetate consumption and PHB production in the feast period both proceeded with a zero-order rate in acetate and PHB concentration respectively. PHB consumption in the famine period could best be described kinetically with a nth-order degradation equation in PHB concentration. The obtained results are discussed in the context of the general activated sludge models.     

Recovery of Poly-β-Hydroxybutyrate from Estuarine Microflora

Poly-β-hydroxybutyrate (PHB) is a uniquely procaryotic endogenous storage polymer whose metabolism has been shown to reflect environmental perturbations in laboratory monocultures. When hydrolyzed for 45 min in 5% sodium hypochlorite, PHB can be isolated from estuarine detrital microflora in high yield and purified free from non-PHB microbial components. Lyophilization of frozen estuarine samples shortens the exposure time to NaOCl necessary for maximal recovery. Lyophilized samples of hardwood leaves, Vallisneria, and the aerobic upper millimeter of estuarine muds yielded PHB. The efficiency of incorporation of sodium [1-¹⁴C]acetate into PHB is very high and is stimulated by aeration. PHB was not recovered from the anaerobic portions of sediments unless they were aerated for a short time. Levels of PHB in the detrital microbial community do not correlate with the microbial biomass as measured by the extractible lipid phosphate, suggesting that PHB-like eucaryotic endogenous storage materials may more accurately reflect the metabolic status of the population than its biomass.     

Recovery of Poly-beta-Hydroxybutyrate from Estuarine Microflora

Poly-beta-hydroxybutyrate (PHB) is a uniquely procaryotic endogenous storage polymer whose metabolism has been shown to reflect environmental perturbations in laboratory monocultures. When hydrolyzed for 45 min in 5% sodium hypochlorite, PHB can be isolated from estuarine detrital microflora in high yield and purified free from non-PHB microbial components. Lyophilization of frozen estuarine samples shortens the exposure time to NaOCl necessary for maximal recovery. Lyophilized samples of hardwood leaves, Vallisneria, and the aerobic upper millimeter of estuarine muds yielded PHB. The efficiency of incorporation of sodium [1-C]acetate into PHB is very high and is stimulated by aeration. PHB was not recovered from the anaerobic portions of sediments unless they were aerated for a short time. Levels of PHB in the detrital microbial community do not correlate with the microbial biomass as measured by the extractible lipid phosphate, suggesting that PHB-like eucaryotic endogenous storage materials may more accurately reflect the metabolic status of the population than its biomass.     

Fermentative and aerobic metabolism in Rhizobium etli.

Strains of Rhizobium etli, Rhizobium meliloti, and Rhizobium tropici decreased their capacity to grow after successive subcultures in minimal medium, with a pattern characteristic for each species. During the growth of R. etli CE 3 in minimal medium (MM), a fermentation-like response was apparent: the O2 content was reduced and, simultaneously, organic acids and amino acids were excreted and poly-beta-hydroxybutyrate (PHB) was accumulated. Some of the organic acids excreted into the medium were tricarboxylic acid (TCA) cycle intermediates, and, concomitantly, the activities of several TCA cycle and auxiliary enzymes decreased substantially or became undetectable. Optimal and sustained growth and a low PHB content were found in R. etli CE 3 when it was grown in MM inoculated at a low cell density with O2 maintained at 20% or with the addition of supplements that have an effect on the supply of substrates for the TCA cycle. In the presence of supplements such as biotin or thiamine, no amino acids were excreted and the organic acids already excreted into the medium were later reutilized. Levels of enzyme activities in cells from supplemented cultures indicated that carbon flux through the TCA cycle was maintained, which did not happen in MM. It is proposed that the fermentative state in Rhizobium species is triggered by a cell density signal that results in the regulation of some of the enzymes responsible for the flux of carbon through the TCA cycle and that this in turn determines how much carbon is available for the synthesis and accumulation of PHB. The fermentative state of free-living Rhizobium species may be closely related to the metabolism that these bacteria express during symbiosis.     

Measuring the fitness of symbiotic rhizobia

The legume-rhizobia symbiosis is an important model system for research on the evolution of cooperation and conflict. A key strength of this system is that the fitness consequences of greater or lesser investment in cooperative behaviors can be measured for each partner. Most empirical studies have characterized the fitness of symbiotic rhizobia exclusively by their numbers within nodules, often estimated using nodule size as a proxy. Here we show that the relationship between nodule size and rhizobial numbers can differ drastically between strains of the same species. We further show that differences in accumulation of the storage polyester poly-3-hydroxybutyrate (PHB), which can support future reproduction, can be large enough that even direct measurements of rhizobial numbers alone can lead to qualitatively incorrect conclusions. Both results come from a comparison of strains differing in production of the ethylene-inhibitor rhizobitoxine (Rtx). A broader study (using three legume-rhizobia species pairs) showed that PHB/cell cannot be reliably estimated from its correlation with rhizobia/nodule or nodule size. Differences in PHB between strains or treatments will not always make major contributions to differences in fitness, but situation-specific data are needed before PHB can safely be neglected.     

Poly(hydroxyalkanoate) in cyanobacteria: an overview

Abstract In this paper an overview is given on the occurrence of poly(hydroxyalkanoate) (PHA) in cyanobacteria and its possible role as a putative reserve compound. Comparisons are made with the function of other storage compounds that occur in cyanobacteria. For the cyanobacteria Oscillatoria limosa and Gloeothece sp. PCC 6909, some experimental data on the accumulation and mobilization of PHA are presented. O. limosa presumably contains poly(hydroxyvalerate) (PHV), whereas in Gloeothece poly(hydroxybutyrate) (PHB) was detected. Both species accumulated PHA to 6–9% of the dry weight. In Gloeothece PHB accumulation was stimulated by the addition of acetate but in O. limosa this was not the case. PHA was not involved in dark metabolism in either of the strains.     

Identification and characterization of the intracellular poly-3-hydroxybutyrate depolymerase enzyme PhaZ of Sinorhizobium meliloti

Background  

S. meliloti forms indeterminate nodules on the roots of its host plant alfalfa (Medicago sativa). Bacteroids of indeterminate nodules are terminally differentiated and, unlike their non-terminally differentiated counterparts in determinate nodules, do not accumulate large quantities of Poly-3-hydroxybutyrate (PHB) during symbiosis. PhaZ is in intracellular PHB depolymerase; it represents the first enzyme in the degradative arm of the PHB cycle in S. meliloti and is the only enzyme in this half of the PHB cycle that remains uncharacterized.     

On-line monitoring of PHB production by mixed microbial cultures using respirometry,titrimetry and chemometric modelling

This work describes a method for on-line monitoring of biomass production, acetate consumption and intracellular polyhydroxybutyrate (PHB) storage by mixed microbial cultures (MMC). The method is based on reliable and easily available on-line measurements, namely pH, dissolved oxygen, dissolved carbon dioxide, on-line respirometry and on-line titrimetric analysis. Biomass production refers to active biomass growth and also to the synthesis of extracellular polymeric substances (EPS). The composition and kinetics of EPS synthesis has high variability depending on the culture enrichment protocol. Since the metabolism for EPS production is rather difficult to define, it was not possible to develop a reliable estimation model based on metabolic principles only. Instead, projection of latent structures (PLS) linear regression constrained by steady state carbon balance was employed. PHB concentration and biomass production rate were directly estimated by the PLS model, whereas acetate concentration was indirectly estimated through the carbon balance. The method was validated experimentally with data of four experiments carried out in a SBR. Accurate on-line estimations were obtained with regression coefficients (r²) of 0.986 and 0.980 for biomass concentration, 0.976 and 0.999 for PHB and 0.992 and 0.999 for acetate concentration in calibration and validation, respectively. These results confirm the ability of the proposed methodology for on-line monitoring of the state variables in PHB production process by MMC.     

Peroxisomal polyhydroxyalkanoate biosynthesis is a promising strategy for bioplastic production in high biomass crops

Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers with diverse plastic‐like properties. PHA biosynthesis in transgenic plants is being developed as a way to reduce the cost and increase the sustainability of industrial PHA production. The homopolymer polyhydroxybutyrate (PHB) is the simplest form of these biodegradable polyesters. Plant peroxisomes contain the substrate molecules and necessary reducing power for PHB biosynthesis, but peroxisomal PHB production has not been explored in whole soil‐grown transgenic plants to date. We generated transgenic sugarcane (Saccharum sp.) with the three‐enzyme Ralstonia eutropha PHA biosynthetic pathway targeted to peroxisomes. We also introduced the pathway into Arabidopsis thaliana, as a model system for studying and manipulating peroxisomal PHB production. PHB, at levels up to 1.6%–1.8% dry weight, accumulated in sugarcane leaves and A. thaliana seedlings, respectively. In sugarcane, PHB accumulated throughout most leaf cell types in both peroxisomes and vacuoles. A small percentage of total polymer was also identified as the copolymer poly (3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) in both plant species. No obvious deleterious effect was observed on plant growth because of peroxisomal PHA biosynthesis at these levels. This study highlights how using peroxisomal metabolism for PHA biosynthesis could significantly contribute to reaching commercial production levels of PHAs in crop plants.     

Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate.

Rhizobium etli accumulates poly-beta-hydroxybutyrate (PHB) in symbiosis and in free life. PHB is a reserve material that serves as a carbon and/or electron sink when optimal growth conditions are not met. It has been suggested that in symbiosis PHB can prolong nitrogen fixation until the last stages of seed development, but experiments to test this proposition have not been done until now. To address these questions in a direct way, we constructed an R. etli PHB-negative mutant by the insertion of an Omega-Km interposon within the PHB synthase structural gene (phaC). The identification and sequence of the R. etli phaC gene are also reported here. Physiological studies showed that the PHB-negative mutant strain was unable to synthesize PHB and excreted more lactate, acetate, pyruvate, beta-hydroxybutyrate, fumarate, and malate than the wild-type strain. The NAD+/NADH ratio in the mutant strain was lower than that in the parent strain. The oxidative capacity of the PHB-negative mutant was reduced. Accordingly, the ability to grow in minimal medium supplemented with glucose or pyruvate was severely diminished in the mutant strain. We propose that in free life PHB synthesis sequesters reductive power, allowing the tricarboxylic acid cycle to proceed under conditions in which oxygen is a limiting factor. In symbiosis with Phaseolus vulgaris, the PHB-negative mutant induced nodules that prolonged the capacity to fix nitrogen.     

Nuclear Magnetic Resonance Studies of Poly(3-Hydroxybutyrate) and Polyphosphate Metabolism in Alcaligenes eutrophus

The metabolic pathways of poly(3-hydroxybutyrate) (PHB) and polyphosphate in the microorganism Alcaligenes eutrophus H16 were studied by ¹H, ¹³C, and ³¹P nuclear magnetic resonance (NMR) spectroscopy and by conventional analytical techniques. A. eutrophus cells accumulated two storage polymers of PHB and polyphosphate in the presence of carbon and phosphate sources under aerobic conditions after exhaustion of nitrogen sources. The solid-state cross-polarization/magic-angle spinning ¹³C NMR spectroscopy was used to study the biosynthetic pathways of PHB and other cellular biomass components from ¹³C-labeled acetate. The solid-state ¹³C NMR analysis of lyophilized intact cells grown on [1-¹³C]acetate indicated that the carbonyl carbon of acetate was selectively incorporated both into the carbonyl and methine carbons of PHB and into the carbonyl carbons of proteins. The ³¹P NMR analysis of A. eutrophus cells in suspension showed that the synthesis of intracellular polyphosphate was closely related to the synthesis of PHB. The roles of PHB and polyphosphate in the cells were studied under conditions of carbon, phosphorus, and nitrogen source starvation. Under both aerobic and anaerobic conditions PHB was degraded, whereas little polyphosphate was degraded. The rate of PHB degradation under anaerobic conditions was faster than that under aerobic conditions. Under anaerobic conditions, acetate and 3-hydroxybutyrate were produced as the major extracellular metabolites. The implications of this observation are discussed in connection with the regulation of PHB and polyphosphate metabolism in A. eutrophus.     

New insights in the formation of polyhydroxyalkanoate granules (carbonosomes) and novel functions of poly(3‐hydroxybutyrate)

The metabolism of polyhydroxybutyrate (PHB) and related polyhydroxyalkanoates (PHAs) has been investigated by many groups for about three decades, and good progress was obtained in understanding the mechanisms of biosynthesis and biodegradation of this class of storage molecules. However, the molecular events that happen at the onset of PHB synthesis and the details of the initiation of PHB/PHA granule formation, as well as the complex composition of the proteinaceous surface layer of PHB/PHA granules, have only recently come into the focus of research and were not reviewed yet. In this contribution, we summarize the progress in understanding the initiation and formation of the PHA granule complex at the example of Ralstonia eutropha H16 (model organism of PHB‐accumulating bacteria). Where appropriate, we include information on PHA granules of Pseudomonas putida as a representative species for medium‐chain‐length PHA‐accumulating bacteria. We suggest to replace the previous micelle mode of PHB granule formation by the Scaffold Model in which the PHB synthase initiation complex is bound to the bacterial nucleoid. In the second part, we highlight data on other forms of PHB: oligo‐PHB with ≈100 to 200 3‐hydroxybutyrate (3HB) units and covalently bound PHB (cPHB) are unrelated in function to storage PHB but are presumably present in all living organisms, and therefore must be of fundamental importance.     

Identification of the pivotal role of glutamate in enhancing insect cell growth using factor analysis

Although the insect cell/baculovirus system is an important expression platform for recombinant protein production, our understanding of insect cell metabolism with respect to enhancing cell growth capability and productivity is still limited. Moreover, different host insect cell lines may have different growth characteristics associated with diverse product yields, which further hampers the elucidation of insect cell metabolism. To address this issue, the growth behaviors and utilization profiles of common metabolites among five cultured insect cell lines (derived from two insect hosts, Spodoptera frugiperda and Spodoptera exigua) were investigated in an attempt to establish a metabolic framework that can interpret the different cell growth behaviors. To analyze the complicated metabolic dataset, factor analysis was introduced to differentiate the crucial metabolic variations among these cells. Factor analysis was used to decompose the metabolic data to obtain the underlying factors with biological meaning that identify glutamate (a metabolic intermediate involved in glutaminolysis) as a key metabolite for insect cell growth. Notably, glutamate was consumed in significant amounts by fast-growing insect cell lines, but it was produced by slow-growing lines. A comparative experiment using cells grown in culture media with and without glutamine (the starting metabolite in glutaminolysis) was conducted to further confirm the pivotal role of glutamate. The factor analysis strategy allowed us to elucidate the underlying structure and inter-correlation between insect cell growth and metabolite utilization to provide some insights into insect cell growth and metabolism, and this strategy can be further extended to large-scale metabolomic analysis.     

Metabolic pathway analysis of a recombinant yeast for rational strain development

Elementary mode analysis has been used to study a metabolic pathway model of a recombinant Saccharomyces cerevisiae system that was genetically engineered to produce the bacterial storage compound poly-beta-hydroxybutyrate (PHB). The model includes biochemical reactions from the intermediary metabolism and takes into account cellular compartmentalization as well as the reversibility/irreversibility of the reactions. The reaction network connects the production and/or consumption of eight external metabolites including glucose, acetate, glycerol, ethanol, PHB, CO(2), succinate, and adenosine triphosphate (ATP). Elementary mode analysis of the wild-type S. cerevisiae system reveals 241 unique reaction combinations that balance the eight external metabolites. When the recombinant PHB pathway is included, and when the reaction model is altered to simulate the experimental conditions when PHB accumulates, the analysis reveals 20 unique elementary modes. Of these 20 modes, 7 produce PHB with the optimal mode having a theoretical PHB carbon yield of 0.67. Elementary mode analysis was also used to analyze the possible effects of biochemical network modifications and altered culturing conditions. When the natively absent ATP citrate-lyase activity is added to the recombinant reaction network, the number of unique modes increases from 20 to 496, with 314 of these modes producing PHB. With this topological modification, the maximum theoretical PHB carbon yield increases from 0.67 to 0.83. Adding a transhydrogenase reaction to the model also improves the theoretical conversion of substrate into PHB. The recombinant system with the transhydrogenase reaction but without the ATP citrate-lyase reaction has an increase in PHB carbon yield from 0.67 to 0.71. When the model includes both the ATP citrate-lyase reaction and the transhydrogenase reaction, the maximum theoretical carbon yield increases to 0.84. The reaction model was also used to explore the possibility of producing PHB under anaerobic conditions. In the absence of oxygen, the recombinant reaction network possesses two elementary modes capable of producing PHB. Interestingly, both modes also produce ethanol. Elementary mode analysis provides a means of deconstructing complex metabolic networks into their basic functional units. This information can be used for analyzing existing pathways and for the rational design of further modifications that could improve the system's conversion of substrate into product.