Cleavage of natural rubber by rubber oxygenases in Gram-negative bacteria.

Bacterial degradation of natural rubber (NR) in an oxic environment is initiated by oxidative cleavage of double bonds in the NR-carbon backbone and is catalyzed by extracellular haem-containing rubber oxygenases. NR-cleavage products of sufficiently low molecular mass are taken up by the cells and metabolized for energy and biomass formation. Gram-negative and Gram-positive NR-degrading bacteria (usually) employ different types of rubber oxygenases such as RoxA and/or RoxB (most Gram-negative NR-degraders) or latex clearing protein Lcp (most Gram-positive NR-degraders). In order to find novel orthologues of Rox proteins, we have revisited databases and provide an update of Rox-like proteins. We describe the putative evolution of rubber oxygenases and confirm the presence of a third subgroup of Rox-related proteins (RoxCs), the biological function of which remains, however, unclear. We summarize the knowledge on the taxonomic position of Steroidobacter cummioxidans 35Y and related species. Comparison of genomic and biochemical features of strain 35Y with other species of the genus Steroidobacter suggests that strain 35Y represents a species of a novel genus for which the designation Aurantibaculum gen. nov. is proposed. A short summary on the capabilities of NR-degrading consortia, that could be superior in biotechnological applications compared to pure cultures, is also provided. KEY POINTS: • Three types of rubber oxygenases exist predominantly in Gram-negative microbes • S. cummioxidans 35Y contains RoxA and RoxB which are superior in activity • S. cummioxidans 35Y represents a species of a novel genus.

Polyphosphate Kinases Phosphorylate Thiamine Phosphates.

Polyphosphate kinases (PPKs) catalyze the reversible transfer of the γ-phosphate moiety of ATP (or of another nucleoside triphosphate) to a growing chain of polyphosphate (polyP). In this study we describe that PPKs of various sources are additionally able to phosphorylate thiamine diphosphate (ThP2) to produce thiamine triphosphate (ThP3) and even thiamine tetraphosphate (ThP4) in vitro. Furthermore, all tested PPK2s, but not PPK1s, were able to phosphorylate thiamine monophosphate (ThP1) to ThP2 and ThP3 although at low efficiency. The predicted masses and identities of the mono- and oligo-phosphorylated thiamine metabolites were identified by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Moreover, the biological activity of ThP2, that was synthesized by phosphorylation of ThP1 with polyP and PPK, as a cofactor of ThP2-dependent enzymes (here transketolase TktA from Escherichia coli) was confirmed in a coupled enzyme assay. In conclusion, our study shows that PPKs are promiscuous enzymes that are presumably involved in the formation of a variety of phosphorylated metabolites in vivo.

Migration of Polyphosphate Granules in Agrobacterium tumefaciens.

Agrobacterium tumefaciens has two polyphosphate (polyP) kinases, one of which (PPK1AT) is responsible for the formation of polyP granules, while the other (PPK2AT) is used for replenishing the NTP pools by using polyP as a phosphate donor to phosphorylate nucleoside diphosphates. Fusions of eYFP with PPK2AT or of the polyP granule-associated phosin PptA from Ralstonia eutropha always co-localized with polyP granules in A. tumefaciens and allowed the tracking of polyP granules in time-lapse microscopy experiments without the necessity to label the cells with the toxic dye DAPI. Fusions of PPK1AT with mCherry formed fluorescent signals often attached to, but not completely co-localizing with, polyP granules in wild-type cells. Time-lapse microscopy revealed that polyP granules in about one-third of a cell population migrated from the old pole to the new cell pole shortly before or during cell division. Many cells de novo formed a second (nonmigrating) polyP granule at the opposite cell pole before cell division was completed, resulting in two daughter cells each having a polyP granule at the old pole after septum formation. Migration of polyP granules was disordered in mitomycin C-treated or in PopZ-depleted cells, suggesting that polyP granules can associate with DNA or with other molecules that are segregated during the cell cycle.

The Multiple Roles of Polyphosphate in Ralstonia eutropha and Other Bacteria.

An astonishing variety of functions has been attributed to polyphosphate (polyP) in prokaryotes. Besides being a reservoir of phosphorus, functions in exopolysaccharide formation, motility, virulence and in surviving various forms of stresses such as exposure to heat, extreme pH, oxidative agents, high osmolarity, heavy metals and others have been ascribed to polyP. In this contribution, we will provide a historical overview on polyP, will then describe the key proteins of polyP synthesis, the polyP kinases, before we will critically assess of the underlying data on the multiple functions of polyP and provide evidence that - with the exception of a P-storage-function - most other functions of polyP are not relevant for survival of Ralstonia eutropha, a biotechnologically important beta-proteobacterial species.

The protective role of PHB and its degradation products against stress situations in bacteria.

Many bacteria produce storage biopolymers that are mobilized under conditions of metabolic adaptation, for example, low nutrient availability and cellular stress. Polyhydroxyalkanoates are often found as carbon storage in Bacteria or Archaea, and of these polyhydroxybutyrate (PHB) is the most frequently occurring PHA type. Bacteria usually produce PHB upon availability of a carbon source and limitation of another essential nutrient. Therefore, it is widely believed that the function of PHB is to serve as a mobilizable carbon repository when bacteria face carbon limitation, supporting their survival. However, recent findings indicate that bacteria switch from PHB synthesis to mobilization under stress conditions such as thermal and oxidative shock. The mobilization products, 3-hydroxybutyrate and its oligomers, show a protective effect against protein aggregation and cellular damage caused by reactive oxygen species and heat shock. Thus, bacteria should have an environmental monitoring mechanism directly connected to the regulation of the PHB metabolism. Here, we review the current knowledge on PHB physiology together with a summary of recent findings on novel functions of PHB in stress resistance. Potential applications of these new functions are also presented.

Characterization of Agrobacterium tumefaciens PPKs reveals the formation of oligophosphorylated products up to nucleoside nona-phosphates.

Agrobacterium tumefaciens synthesizes polyphosphate (polyP) in the form of one or two polyP granules per cell during growth. The A. tumefaciens genome codes for two polyphosphate kinase genes, ppk1AT and ppk2AT, of which only ppk1AT is essential for polyP granule formation in vivo. Biochemical characterization of the purified PPK1AT and PPK2AT proteins revealed a higher substrate specificity of PPK1AT (in particular for adenine nucleotides) than for PPK2AT. In contrast, PPK2AT accepted all nucleotides at comparable rates. Most interestingly, PPK2AT catalyzed also the formation of tetra-, penta-, hexa-, hepta-, and octa-phosphorylated nucleosides from guanine, cytosine, desoxy-thymidine, and uridine nucleotides and even nona-phosphorylated adenosine. Our data-in combination with in vivo results-suggest that PPK1AT is important for the formation of polyP whereas PPK2AT has the function to replenish nucleoside triphosphate pools during times of enhanced demand. The potential physiological function(s) of the detected oligophosphorylated nucleotides await clarification. KEY POINTS: •PPK1AT and PPK2AT have different substrate specificities, •PPK2AT is a subgroup 1 member of PPK2s, •PPK2AT catalyzes the formation of polyphosphorylated nucleosides.

A universal polyphosphate kinase: PPK2c of Ralstonia eutropha accepts purine and pyrimidine nucleotides including uridine diphosphate.

Polyphosphosphate kinases (PPKs) catalyse the reversible transfer of the γ-phosphate group of a nucleoside-triphosphate to a growing chain of polyphosphate. Most known PPKs are specific for ATP, but some can also use GTP as a phosphate donor. In this study, we describe the properties of a PPK2-type PPK of the ß-proteobacterium Ralstonia eutropha. The purified enzyme (PPK2c) is highly unspecific and accepts purine nucleotides as well as the pyridine nucleotides including UTP as substrates. The presence of a polyP primer is not necessary for activity. The corresponding nucleoside diphosphates and microscopically detectable polyphosphate granules were identified as reaction products. PPK2c also catalyses the formation of ATP, GTP, CTP, dTTP and UTP from the corresponding nucleoside diphosphates, if polyP is present as a phosphate donor. Remarkably, the nucleoside-tetraphosphates AT(4)P, GT(4)P, CT(4)P, dTT(4)P and UT(4)P were also detected in substantial amounts. The low nucleotide specificity of PPK2c predestines this enzyme in combination with polyP to become a powerful tool for the regeneration of ATP and other nucleotides in biotechnological applications. As an example, PPK2c and polyP were used to replace ATP and to fuel the hexokinase-catalysed phosphorylation of glucose with only catalytic amounts of ADP. KEY POINTS: • PPK2c of R. eutropha can be used for regeneration of any NTP or dNTP. • PPK2c is highly unspecific and accepts all purine and pyrimidine nucleotides. • PPK2c forms polyphosphate granules in vitro from any NTP.

Response surface method for polyhydroxybutyrate (PHB) bioplastic accumulation in Bacillus drentensis BP17 using pineapple peel.

Polyhydroxybutyrate (PHB) is a biodegradable biopolymer which is useful for various applications including packing, medical and coating materials. An endospore-forming bacterium (strain BP17) was isolated from composted soil and evaluated for PHB production. Strain BP17, taxonomically identified as Bacillus drentensis, showed enhanced PHB accumulation and was selected for further studies. To achieve maximum PHB production, the culture conditions for B. drentensis BP17 were optimized through response surface methodology (RSM) employing central composite rotatable design (CCRD). The final optimum fermentation conditions included: pineapple peel solution, 11.5% (v/v); tryptic soy broth (TSB), 60 g/L; pH, 6.0; inoculum size, 10% (v/v) and temperature, 28°C for 36 h. This optimization yielded 5.55 g/L of PHB compared to the non-optimized condition (0.17 g/L). PHB accumulated by B. drentensis BP17 had a polydispersity value of 1.59 and an average molecular weight of 1.15x105 Da. Thermal analyses revealed that PHB existed as a thermally stable semi-crystalline polymer, exhibiting a thermal degradation temperature of 228°C, a melting temperature of 172°C and an apparent melting enthalpy of fusion of 83.69 J/g. It is evident that B. drentensis strain BP17 is a promising bacterium candidate for PHB production using agricultural waste, such as pineapple peel as a low-cost alternative carbon source for PHB production.

Acidocalcisomes and Polyphosphate Granules Are Different Subcellular Structures in Agrobacterium tumefaciens.

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.

Towards the understanding of the enzymatic cleavage of polyisoprene by the dihaem-dioxygenase RoxA.

Utilization of polyisoprene (natural rubber) as a carbon source by Steroidobacter cummioxidans 35Y (previously Xanthomonas sp. strain 35Y) depends on the formation and secretion of rubber oxygenase A (RoxA). RoxA is a dioxygenase that cleaves polyisoprene to 12-oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD), a suitable growth substrate for S. cummioxidans. RoxA harbours two non-equivalent, spectroscopically distinguishable haem centres. A dioxygen molecule is bound to the N-terminal haem of RoxA and identifies this haem as the active site. In this study, we provide insights into the nature of this unusually stable dioxygen-haem coordination of RoxA by a re-evaluation of previously published together with newly obtained biophysical data on the cleavage of polyisoprene by RoxA. In combination with the meanwhile available structure of RoxA we are now able to explain several uncommon and previously not fully understood features of RoxA, the prototype of rubber oxygenases in Gram-negative rubber-degrading bacteria.

Solimonas fluminis has an active latex-clearing protein.

The utilization of rubber (poly (cis-1,4-isoprene)) by rubber-degrading bacteria depends on the synthesis of rubber oxygenases that cleave the polymer extracellularly to low molecular weight products that can be taken up and used as a carbon source. All so far described Gram-negative rubber-degrading species use two related ≈ 70 kDa rubber oxygenases (RoxA and RoxB) for the primary attack of rubber while all described Gram-positive rubber-degrading strains use RoxA/RoxB-unrelated latex-clearing proteins (Lcps, ≈ 40 kDa) as rubber oxygenase(s). In this study, we identified an lcp orthologue in a Gram-negative species (Solimonas fluminis). We cloned and heterologously expressed the lcp gene of S. fluminis HR-BB, purified the corresponding Lcp protein (LcpHR-BB) from recombinant Escherichia coli, and biochemically characterised the LcpHR-BB activity. LcpHR-BB cleaved polyisoprene to a mixture of C20 and higher oligoisoprenoids at a specific activity of 1.5 U/mg. Furthermore, spectroscopic investigation identified LcpHR-BB as a b-haem-containing protein with an oxidised, fivefold coordinated (open) haem centre. To the best of our knowledge, this is the first report that Gram-negative bacteria can have an active rubber oxygenase of the Lcp type.

Formation of an Organic-Inorganic Biopolymer: Polyhydroxybutyrate-Polyphosphate.

A considerable variety of different biopolymers is formed by the entirety of organisms present on earth. Most of these compounds are organic polymers such as polysaccharides, polyamino acids, polynucleotides, polyisoprenes or polyhydroxyalkanoates (PHAs), but some biopolymers can consist of solely inorganic monomers such as phosphate in polyphosphates (polyPs). In this contribution, we describe the formation of an organic-inorganic block copolymer consisting of poly(3-hydroxybutyrate) (PHB) and polyP. This was achieved by the expression of a fusion of the polyP kinase gene (ppk2c) with the PHB synthase gene (phaC) of Ralstonia eutropha in a polyP-free and PHB-free mutant background of R. eutropha. The fusion protein catalyzed both the formation of polyP by its polyP kinase domain and the formation of PHB by its PHB synthase domain. It was also possible to synthesize the polyP-PHB polymer in vitro with purified Ppk2c-PhaC, if the monomers, adenosine triphosphate (ATP) and 3-hydroxybutyryl-CoA (3HB-CoA), were provided. Most likely, the formed block copolymer (polyP-protein-PHB) turns into a blend of polyP and PHB after release from the enzyme.

Rubber oxygenases.

Natural rubber (NR), poly(cis-1,4-isoprene), is used in an industrial scale for more than 100 years. Most of the NR-derived materials are released to the environment as waste or by abrasion of small particles from our tires. Furthermore, compounds with isoprene units in their molecular structures are part of many biomolecules such as terpenoids and carotenoids. Therefore, it is not surprising that NR-degrading bacteria are widespread in nature. NR has one carbon-carbon double bond per isoprene unit and this functional group is the primary target of NR-cleaving enzymes, so-called rubber oxygenases. Rubber oxygenases are secreted by rubber-degrading bacteria to initiate the break-down of the polymer and to use the generated cleavage products as a carbon source. Three main types of rubber oxygenases have been described so far. One is rubber oxygenase RoxA that was first isolated from Xanthomonas sp. 35Y but was later also identified in other Gram-negative rubber-degrading species. The second type of rubber oxygenase is the latex clearing protein (Lcp) that has been regularly found in Gram-positive rubber degraders. Recently, a third type of rubber oxygenase (RoxB) with distant relationship to RoxAs was identified in Gram-negative bacteria. All rubber oxygenases described so far are haem-containing enzymes and oxidatively cleave polyisoprene to low molecular weight oligoisoprenoids with terminal CHO and CO-CH3 functions between a variable number of intact isoprene units, depending on the type of rubber oxygenase. This contribution summarises the properties of RoxAs, RoxBs and Lcps.

Rhizobacter gummiphilus NS21 has two rubber oxygenases (RoxA and RoxB) acting synergistically in rubber utilisation.

Biodegradation of poly(cis-1,4-isoprene) (rubber) by Gram-negative bacteria has been investigated on the enzymatic level only in Steroidobacter cummioxidans 35Y (previously Xanthomonas sp. 35Y). This species produces two kinds of rubber oxygenases, RoxA35Y and RoxB35Y, one of which (RoxB35Y) cleaves polyisoprene to a mixture of C20- and higher oligoisoprenoids while the other (RoxA35Y) cleaves polyisoprene and RoxB35Y-derived oligoisoprenoids to the C15-oligoisoprenoid 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). ODTD can be taken up by S. cummioxidans and used as a carbon source. Gram-positive rubber-degrading bacteria employ another type of rubber oxygenase, latex clearing protein (Lcp), for the initial oxidative attack of the polyisoprene molecule. In this contribution, we examined which type of rubber oxygenase is present in the only other well-documented Gram-negative rubber-degrading species, Rhizobacter gummiphilus NS21. No homologue for an Lcp protein but homologues for a putative RoxA and a RoxB protein (the latter identical to a previously postulated LatA-denominated rubber cleaving enzyme) were identified in the genome of strain NS21. The roxANS21 and roxBNS21 genes were separately expressed in a ∆roxA35Y/∆roxB35Y background of S. cummioxidans 35Y and restored the ability of the mutant to produce oligoisoprenoids. The RoxANS21 and RoxBNS21 proteins were each purified and biochemically characterised. The results-in combination with in silico analysis of databases-indicate that Gram-negative rubber-degrading bacteria generally utilise two synergistically acting rubber oxygenases (RoxA/RoxB) for efficient cleavage of polyisoprene to ODTD.

Correction: Metabolic and taxonomic insights into the Gram-negative natural rubber degrading bacterium Steroidobacter cummioxidans sp. nov., strain 35Y.

[This corrects the article DOI: 10.1371/journal.pone.0197448.].

Metabolic and taxonomic insights into the Gram-negative natural rubber degrading bacterium Steroidobacter cummioxidans sp. nov., strain 35Y.

The pathway of rubber (poly [cis-1,4-isoprene]) catabolism is well documented for Gram-positive rubber degraders but only little information exists for Gram-negative species. The first documented potent rubber degrading Gram-negative strain is Xanthomonas sp. strain 35Y that uses extracellular rubber oxygenases for the initial cleavage of the polyisoprene molecule. However, neither the exact phylogenetic position of Xanthomonas sp. strain 35Y nor the catabolic pathway of the primary polyisoprene cleavage products have been investigated. In this contribution, we started to address both these issues by a comprehensive taxonomic characterization and by the analysis of the draft genome sequence of strain 35Y. Evaluation of the 16S rRNA gene sequence pointed to a borderline taxonomic position of strain 35Y as a novel species of the genus Steroidobacter. Further, substantial differences in the genotypic properties of strain 35Y and the members of the genus Steroidobacter, including average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH), resolved the taxonomic position of strain 35Y and suggested its positioning as a novel species of the genus Steroidobacter. This was further confirmed by comparative analysis of physiological and biochemical features of strain 35Y with other members of the genus Steroidobacter. Thus, we conclude that strain 35Y represents a novel species of the genus Steroidobacter, for which we propose the designation Steroidobacter cummioxidans sp. nov., strain 35YT. A comprehensive analysis of the draft genome of S. cummioxidans strain 35Y revealed similarities but also substantial differences to rubber degrading Gram-positive counterparts. In particular, the putative transporters for the uptake of polyisoprene cleavage products differ from Gram-positive rubber degrading species. The draft genome sequence of S. cummioxidans strain 35Y will be useful for researchers to experimentally verify the predicted similarities and differences in the pathways of polyisoprene catabolism in Gram-positive and Gram-negative rubber degrading species.

Poly(3-Hydroxybutyrate) (PHB) Polymerase PhaC1 and PHB Depolymerase PhaZa1 of Ralstonia eutropha Are Phosphorylated In Vivo.

In this study, we screened poly(3-hydroxybutyrate) (PHB) synthase PhaC1 and PHB depolymerase PhaZa1 of Ralstonia eutropha for the presence of phosphorylated residues during the PHB accumulation and PHB degradation phases. Thr373 of PHB synthase PhaC1 was phosphorylated during the stationary growth phase but was not modified during the exponential and PHB accumulation phases. Ser35 of PHB depolymerase PhaZa1 was identified in the phosphorylated form during both the exponential and stationary growth phases. Additional phosphosites were identified for both proteins in sample-dependent forms. Site-directed mutagenesis of the codon for Thr373 and other phosphosites of PhaC1 revealed a strong negative impact on PHB synthase activity. Modifications of Thr26 and Ser35 of PhaZa1 reduced the ability of R. eutropha to mobilize PHB in the stationary growth phase. Our results show that phosphorylation of PhaC1 and PhaZa1 can be important for the modulation of the activities of PHB synthase and PHB depolymerase.IMPORTANCE Poly(3-hydroxybutyrate) (PHB) and related polyhydroxyalkanoates (PHAs) are important intracellular carbon and energy storage compounds in many prokaryotes. The accumulation of PHB or PHAs increases the fitness of cells during periods of starvation and under other stress conditions. The simultaneous presence of PHB synthase (PhaC1) and PHB depolymerase (PhaZa1) on synthesized PHB granules in Ralstonia eutropha (alternative designation, Cupriavidus necator) was previously shown in several laboratories. These findings imply that the activities of PHB synthase and PHB depolymerase should be regulated to avoid a futile cycle of simultaneous synthesis and degradation of PHB. Here, we addressed this question by identifying the phosphorylation sites on PhaC1 and PhaZa1 and by site-directed mutagenesis of the identified residues. Furthermore, we conducted in vitro and in vivo analyses of PHB synthase activity and PHB contents.

Inactivation of an intracellular poly-3-hydroxybutyrate depolymerase of Azotobacter vinelandii allows to obtain a polymer of uniform high molecular mass.

A novel poly-3-hydroxybutyrate depolymerase was identified in Azotobacter vinelandii. This enzyme, now designated PhbZ1, is associated to the poly-3-hydroxybutyrate (PHB) granules and when expressed in Escherichia coli, it showed in vitro PHB depolymerizing activity on native or artificial PHB granules, but not on crystalline PHB. Native PHB (nPHB) granules isolated from a PhbZ1 mutant had a diminished endogenous in vitro hydrolysis of the polyester, when compared to the granules of the wild-type strain. This in vitro degradation was also tested in the presence of free coenzyme A. Thiolytic degradation of the polymer was observed in the nPHB granules of the wild type, resulting in the formation of 3-hydroxybutyryl-CoA, but was absent in the granules of the mutant. It was previously reported that cultures of A. vinelandii OP grown in a bioreactor showed a decrease in the weight average molecular weight (Mw) of the PHB after 20 h of culture, with an increase in the fraction of polymers of lower molecular weight. This decrease was correlated with an increase in the PHB depolymerase activity during the culture. Here, we show that in the phbZ1 mutant, neither the decrease in the Mw nor the appearance of a low molecular weight polymers occurred. In addition, a higher PHB accumulation was observed in the cultures of the phbZ1 mutant. These results suggest that PhbZ1 has a role in the degradation of PHB in cultures in bioreactors and its inactivation allows the production of a polymer of a uniform high molecular weight.

Determination of Polyhydroxybutyrate (PHB) Content in Ralstonia eutropha Using Gas Chromatography and Nile Red Staining.

Ralstonia eutropha H16 produces and mobilizes (re-utilizes) intracellular polyhydroxybutyrate (PHB) granules during growth. This protocol describes the visualization of intracellular Nile red stained PHB granules and the quantification of PHB by gas chromatography. Our first method describes how to analyze PHB granules by fluorescence microscopy qualitatively. Our second approach enables the conversion of PHB to volatile hydroxycarboxylic acid methyl esters by acidic methanolysis and their quantification by gas chromatography. Through this method, it is possible to obtain an absolute quantification of PHB, e.g., per cell dry weight.

Production of functionalized oligo-isoprenoids by enzymatic cleavage of rubber.

In this study, we show the proof of concept for the production of defined oligo-isoprenoids with terminal functional groups that can be used as starting materials for various purposes including the synthesis of isoprenoid-based plastics. To this end, we used three types of rubber oxygenases for the enzymatic cleavage of rubber [poly(cis-1,4-isoprene)]. Two enzymes, rubber oxygenase RoxAXsp and rubber oxygenase RoxBXsp , originate from Xanthomonas sp. 35Y; the third rubber oxygenase, latex-clearing protein (LcpK30 ), is derived from Gram-positive rubber degraders such as Streptomyces sp. K30. Emulsions of polyisoprene (latex) were treated with RoxAXsp , RoxBXsp , LcpK30 or with combinations of the three proteins. The cleavage products were purified by solvent extraction and FPLC separation. All products had the same general structure with terminal functions (CHO-CH2 - and -CH2 -COCH3 ) but differed in the number of intact isoprene units in between. The composition and m/z values of oligo-isoprenoid products were determined by HPLC-MS analysis. Our results provide a method for the preparation of reactive oligo-isoprenoids that can likely be used to convert polyisoprene latex or rubber waste materials into value-added molecules, biofuels, polyurethanes or other polymers.