Regulation of Polyhydroxybutyrate Synthesis in the Soil Bacterium Bradyrhizobium diazoefficiens

Polyhydroxybutyrate (PHB) is a carbon and energy reserve polymer in various prokaryotic species. We determined that, when grown with mannitol as the sole carbon source, Bradyrhizobium diazoefficiens produces a homopolymer composed only of 3-hydroxybutyrate units (PHB). Conditions of oxygen limitation (such as microoxia, oxic stationary phase and bacteroids inside legume nodules) were permissive for the synthesis of PHB, which was observed as cytoplasmic granules. To study the regulation of PHB synthesis, we generated mutants in the regulator gene phaR and the phasin genes phaP1 and phaP4. Under permissive conditions, mutation of phaR impaired PHB accumulation, and a phaP1/phaP4 double mutant produced more PHB than the wildtype, which was accumulated in a single, large cytoplasmic granule. Moreover, PhaR negatively regulated the expression of phaP1 and phaP4 as well as of phaA1, phaA2 (3-ketoacyl-CoA thiolase), phaC1, phaC2 (PHB synthases), and fixK2 (CRP/FNR-type transcription regulator of genes for microoxic lifestyle). In addition to the depressed PHB cycling, phaR mutants accumulated more extracellular polysaccharide and promoted higher plant shoot dry weight and competitiveness for nodulation than the wildtype, by contrast to phaC1 mutant strains, defective in PHB synthesis. These results suggest that phaR not only regulates PHB granules formation by controlling the expression of phasins and biosynthetic enzymes, but also acts as global regulator of excess carbon allocation and symbiosis by controlling fixK2.Fil: Quelas, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Mesa, S.. Consejo Superior de Investigaciones Científicas; EspañaFil: Mongiardini, Elias Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Jendrossek, D.. University of Stuttgart; AlemaniaFil: Lodeiro, Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Transesterification of PHA to Oligomers Covalently Bonded with (Bio)Active Compounds Containing Either Carboxyl or Hydroxyl Functionalities

© 2015 The Authors. Published by Public Library of Science. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.1371/journal.pone.0120149This manuscript presents the synthesis and structural characterisation of novel biodegradable polymeric controlled-release systems of pesticides with potentially higher resistance to weather conditions in comparison to conventional forms of pesticides. Two methods for the preparation of pesticide-oligomer conjugates using the transesterification reaction were developed. The first method of obtaining conjugates, which consist of bioactive compounds with the carboxyl group and polyhydroxyalkanoates (PHAs) oligomers, is "one-pot" transesterification. In the second method, conjugates of bioactive compounds with hydroxyl group and polyhydroxyalkanoates oligomers were obtained in two-step method, through cyclic poly(3-hydroxybutyrate) oligomers. The obtained pesticide-PHA conjugates were comprehensively characterised using GPC, 1H NMR and mass spectrometry techniques. The structural characterisation of the obtained products at the molecular level with the aid of mass spectrometry confirmed that both of the synthetic strategies employed led to the formation of conjugates in which selected pesticides were covalently bonded to PHA oligomers via a hydrolysable ester bond

Regulation of polyhydroxybutyrate synthesis in the soil bacterium <i>Bradyrhizobium diazoefficiens</i>

Polyhydroxybutyrate (PHB) is a carbon and energy reserve polymer in various prokaryotic species. We determined that, when grown with mannitol as the sole carbon source, Bradyrhizobium diazoefficiens produces a homopolymer composed only of 3-hydroxybutyrate units (PHB). Conditions of oxygen limitation (such as microoxia, oxic stationary phase, and bacteroids inside legume nodules) were permissive for the synthesis of PHB, which was observed as cytoplasmic granules. To study the regulation of PHB synthesis, we generated mutations in the regulator gene phaR and the phasin genes phaP1 and phaP4. Under permissive conditions, mutation of phaR impaired PHB accumulation, and a phaP1 phaP4 double mutant produced more PHB than the wild type, which was accumulated in a single, large cytoplasmic granule. Moreover, PhaR negatively regulated the expression of phaP1 and phaP4 as well as the expression of phaA1 and phaA2 (encoding a 3-ketoacyl coenzyme A [CoA] thiolases), phaC1 and phaC2 (encoding PHB synthases), and fixK2 (encoding a cyclic AMP receptor protein [CRP]/fumarate and nitrate reductase regulator [FNR]-type transcription factor of genes for microoxic lifestyle). In addition to the depressed PHB cycling, phaR mutants accumulated more extracellular polysaccharides and promoted higher plant shoot dry weight and competitiveness for nodulation than the wild type, in contrast to the phaC1 mutant strain, which is defective in PHB synthesis. These results suggest that phaR not only regulates PHB granule formation by controlling the expression of phasins and biosynthetic enzymes but also acts as a global regulator of excess carbon allocation and symbiosis by controlling fixK2.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Regulation of polyhydroxybutyrate synthesis in the soil bacterium <i>Bradyrhizobium diazoefficiens</i>

Polyhydroxybutyrate (PHB) is a carbon and energy reserve polymer in various prokaryotic species. We determined that, when grown with mannitol as the sole carbon source, Bradyrhizobium diazoefficiens produces a homopolymer composed only of 3-hydroxybutyrate units (PHB). Conditions of oxygen limitation (such as microoxia, oxic stationary phase, and bacteroids inside legume nodules) were permissive for the synthesis of PHB, which was observed as cytoplasmic granules. To study the regulation of PHB synthesis, we generated mutations in the regulator gene phaR and the phasin genes phaP1 and phaP4. Under permissive conditions, mutation of phaR impaired PHB accumulation, and a phaP1 phaP4 double mutant produced more PHB than the wild type, which was accumulated in a single, large cytoplasmic granule. Moreover, PhaR negatively regulated the expression of phaP1 and phaP4 as well as the expression of phaA1 and phaA2 (encoding a 3-ketoacyl coenzyme A [CoA] thiolases), phaC1 and phaC2 (encoding PHB synthases), and fixK2 (encoding a cyclic AMP receptor protein [CRP]/fumarate and nitrate reductase regulator [FNR]-type transcription factor of genes for microoxic lifestyle). In addition to the depressed PHB cycling, phaR mutants accumulated more extracellular polysaccharides and promoted higher plant shoot dry weight and competitiveness for nodulation than the wild type, in contrast to the phaC1 mutant strain, which is defective in PHB synthesis. These results suggest that phaR not only regulates PHB granule formation by controlling the expression of phasins and biosynthetic enzymes but also acts as a global regulator of excess carbon allocation and symbiosis by controlling fixK2.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Optimization of growth media components for polyhydroxyalkanoate (PHA) production from organic acids by Ralstonia eutropha

We employed systematic mixture analysis to determine optimal levels of acetate, propionate, and butyrate for cell growth and polyhydroxyalkanoate (PHA) production by Ralstonia eutropha H16. Butyrate was the preferred acid for robust cell growth and high PHA production. The 3-hydroxyvalerate content in the resulting PHA depended on the proportion of propionate initially present in the growth medium. The proportion of acetate dramatically affected the final pH of the growth medium. A model was constructed using our data that predicts the effects of these acids, individually and in combination, on cell dry weight (CDW), PHA content (%CDW), PHA production, 3HV in the polymer, and final culture pH. Cell growth and PHA production improved approximately 1.5-fold over initial conditions when the proportion of butyrate was increased. Optimization of the phosphate buffer content in medium containing higher amounts of butyrate improved cell growth and PHA production more than 4-fold. The validated organic acid mixture analysis model can be used to optimize R. eutropha culture conditions, in order to meet targets for PHA production and/or polymer HV content. By modifying the growth medium made from treated industrial waste, such as palm oil mill effluent, more PHA can be produced.Malaysia. Ministry of Science, Technology and Innovation (MOSTI

Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha

Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180 mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14 g/L branched-chain alcohols over the duration of 50 days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.United States. Dept. of EnergyUnited States. Advanced Research Projects Agency-Energ

Regulation of polyhydroxybutyrate synthesis in the soil bacterium <i>Bradyrhizobium diazoefficiens</i>

Polyhydroxybutyrate (PHB) is a carbon and energy reserve polymer in various prokaryotic species. We determined that, when grown with mannitol as the sole carbon source, Bradyrhizobium diazoefficiens produces a homopolymer composed only of 3-hydroxybutyrate units (PHB). Conditions of oxygen limitation (such as microoxia, oxic stationary phase, and bacteroids inside legume nodules) were permissive for the synthesis of PHB, which was observed as cytoplasmic granules. To study the regulation of PHB synthesis, we generated mutations in the regulator gene phaR and the phasin genes phaP1 and phaP4. Under permissive conditions, mutation of phaR impaired PHB accumulation, and a phaP1 phaP4 double mutant produced more PHB than the wild type, which was accumulated in a single, large cytoplasmic granule. Moreover, PhaR negatively regulated the expression of phaP1 and phaP4 as well as the expression of phaA1 and phaA2 (encoding a 3-ketoacyl coenzyme A [CoA] thiolases), phaC1 and phaC2 (encoding PHB synthases), and fixK2 (encoding a cyclic AMP receptor protein [CRP]/fumarate and nitrate reductase regulator [FNR]-type transcription factor of genes for microoxic lifestyle). In addition to the depressed PHB cycling, phaR mutants accumulated more extracellular polysaccharides and promoted higher plant shoot dry weight and competitiveness for nodulation than the wild type, in contrast to the phaC1 mutant strain, which is defective in PHB synthesis. These results suggest that phaR not only regulates PHB granule formation by controlling the expression of phasins and biosynthetic enzymes but also acts as a global regulator of excess carbon allocation and symbiosis by controlling fixK2.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Comparative and Functional Genomics of Rhodococcus opacus PD630 for Biofuels Development

The Actinomycetales bacteria Rhodococcus opacus PD630 and Rhodococcus jostii RHA1 bioconvert a diverse range of organic substrates through lipid biosynthesis into large quantities of energy-rich triacylglycerols (TAGs). To describe the genetic basis of the Rhodococcus oleaginous metabolism, we sequenced and performed comparative analysis of the 9.27 Mb R. opacus PD630 genome. Metabolic-reconstruction assigned 2017 enzymatic reactions to the 8632 R. opacus PD630 genes we identified. Of these, 261 genes were implicated in the R. opacus PD630 TAGs cycle by metabolic reconstruction and gene family analysis. Rhodococcus synthesizes uncommon straight-chain odd-carbon fatty acids in high abundance and stores them as TAGs. We have identified these to be pentadecanoic, heptadecanoic, and cis-heptadecenoic acids. To identify bioconversion pathways, we screened R. opacus PD630, R. jostii RHA1, Ralstonia eutropha H16, and C. glutamicum 13032 for growth on 190 compounds. The results of the catabolic screen, phylogenetic analysis of the TAGs cycle enzymes, and metabolic product characterizations were integrated into a working model of prokaryotic oleaginy.Cambridge-MIT InstituteMassachusetts Institute of Technology. (Seed Grant program)Shell Oil CompanyNational Institute of Allergy and Infectious Diseases (U.S.)United States. National Institutes of HealthNational Institutes of Health. Department of Health and Human Services (Contract No. HHSN272200900006C

Gene probing reveals the widespread distribution, diversity and abundance of isoprene-degrading bacteria in the environment

Background: Approximately 500 Tg of isoprene are emitted to the atmosphere annually, an amount similar to that of methane, and despite its significant effects on the climate, very little is known about the biological degradation of isoprene in the environment. Isolation and characterisation of isoprene degraders at the molecular level has allowed the development of probes targeting isoA encoding the α-subunit of the isoprene monooxygenase. This enzyme belongs to the soluble diiron centre monooxygenase family and catalyses the first step in the isoprene degradation pathway. The use of probes targeting key metabolic genes is a successful approach in molecular ecology to study specific groups of bacteria in complex environments. Here, we developed and tested a novel isoA PCR primer set to study the distribution, abundance, and diversity of isoprene degraders in a wide range of environments. Results: The new isoA probes specifically amplified isoA genes from taxonomically diverse isoprene-degrading bacteria including members of the genera Rhodococcus, Variovorax, and Sphingopyxis. There was no cross-reactivity with genes encoding related oxygenases from non-isoprene degraders. Sequencing of isoA amplicons from DNA extracted from environmental samples enriched with isoprene revealed that most environments tested harboured a considerable variety of isoA sequences, with poplar leaf enrichments containing more phylogenetically diverse isoA genes. Quantification by qPCR using these isoA probes revealed that isoprene degraders are widespread in the phyllosphere, terrestrial, freshwater and marine environments. Specifically, soils in the vicinity of high isoprene-emitting trees contained the highest number of isoprene-degrading bacteria. Conclusion: This study provides the molecular ecology tools to broaden our knowledge of the distribution, abundance and diversity of isoprene degraders in the environment, which is a fundamental step necessary to assess the impact that microbes have in mitigating the effects of this important climate-active gas

CD47 restricts antiviral function of alveolar macrophages during influenza virus infection

CD47 is an ubiquitously expressed surface molecule with significant impact on immune responses. However, its role for antiviral immunity is not fully understood. Here, we revealed that the expression of CD47 on immune cells seemed to disturb the antiviral immune response as CD47-deficient mice (CD47−/−) showed an augmented clearance of influenza A virus (IAV). Specifically, we have shown that enhanced viral clearance is mediated by alveolar macrophages (aMФ). Although aMФ displayed upregulation of CD47 expression during IAV infection in wildtype mice, depletion of aMФ in CD47−/− mice during IAV infection reversed the augmented viral clearance. We have also demonstrated that CD47 restricts hemoglobin (HB) expression in aMФ after IAV and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, with HB showing antiviral properties by enhancing the IFN-β response. Our study showed a negative role for CD47 during antiviral immune responses in the lung by confining HB expression in aMФ