Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates.

A comprehensive report of the acetylene reduction assay for measurement of N2 fixation is presented. The objective is to facilitate the effective use and identify some potential limitations of the method. The report is based on more than 200 accounts of the use of this technique in 15 countries during the last 5 years. Methods of measurement of N2 fixation are compared. Nomenclature, e.g., N2[C2H2] fixed, is introduced to identify values of N2 fixation determined by C2H2-C2H2 assay. The biochemical basis of the assay is described along with relevant characteristics including Km, C2H2/N2 conversion factor, and specific N2[C2H2]-fixing activities obtained with various systems. Effects of combined nitrogen, temperature, light, pO2, N2, pC2H2 and water on activity are summarized. Available methods for sample preparation, assay chamber, gas phase, assay condition, termination of reaction, C2H4 analysis and expression of results are compared. The many uses of the C2H2-C2H4 assay for investigations of the biochemistry of nitrogenase and physiology of N2-fixing organisms, definition of N2-fixing organisms and measurement of field N2 fixation by legume, non-legume, soil, marine, rhizosphere, phylloplane and mammalian samples are tabulated.

Applications of the acetylene-ethylene assay for measurement of nitrogen fixation

Publisher Summary This chapter focuses on the polymeric reserve materials and thus excludes the consideration of lipids other than poly-β-hydroxybutyrate, and the disaccharide trehalose, which plays an important role in the economy of the yeast cell, but displays certain features that are at variance with the normally accepted behavior of storage compounds. The chapter considers the evidence for the energy-storage roles of glycogen, polyphosphates and poly-p-hydroxybutyrate, and the current state of knowledge concerning the regulation of their biosynthesis and degradation in the microbial cell. The intracellular location of the enzymes concerned with polymer synthesis and degradation is an intriguing problem. The most detailed work has been carried out with PHB granules, which have PHB synthetase and a factor involved in depolymerase activity associated with the membrane that bounds the granule. The depolymerase system is a complex one that also involves a soluble fraction, underlining the importance of reactions at liquid-solid interfaces in the process and emphasizing that relatively little work has been done to isolate polyphosphate or glycogen granules in their native state in order to examine the possibility of regulation by a membranous system.

The Role and Regulation of Energy Reserve Polymers in Micro-organisms

INTRODUCfION Techniques for Studying the Identity of Transported Solutes of Nitrogen . Nitrogen in Phloem and Xylem Fluids . Uptake. Assimilation. and Transport of Nitrogen by Roots . Transport of Nitrogen from Nodulated Roots of Nr/ixing Plants .. Initial Distribution of Xylem-Borne Nitrogen in Shoots .. Export of Nitrogen from Leaves in Phloem . Export of Nitrogen from Stem Tissue in the Phloem . RelationshipS Between Transport and Storage of Nitrogen .. Transport of Nitrogen to Deve/oping Fruits and Seeds Loading of Nitrogenous Solutes onto Xylem and Phloem .. Relationships Between Nitrogen Transport and the Production and Partitioning of Photosynthate .. CONCLUSIONS ..

Transport and Partitioning of Nitrogenous Solutes

Ion-exclusion high-pressure liquid chromatography (HPLC) was used to measure poly-β-hydroxybutyrate (PHB) in Rhizobium japonicum bacteroids. The products in the acid digest of PHB-containing material were fractionated by HPLC on Aminex HPX-87H ion-exclusion resin for organic acid analysis. Crotonic acid formed from PHB during acid digestion was detected by its intense absorbance at 210 nm. The Aminex-HPLC method provides a rapid and simple chromatographic technique for routine analysis of organic acids. Results of PHB analysis by Aminex-HPLC were confirmed by gas chromatography and spectrophotometric analysis.

/pdf/analysis-of-poly-b-hydroxybutyrate-in-rhizobium-japonicum-38vk3j5jqv.pdf

Analysis of Poly-β-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection

Soybean (Glycine max) nodule bacteroids contain high concentrations of poly-β-hydroxybutyrate and possess a depolymerase system that catalyzes the hydrolysis of the polymer. Changes in poly-β-hydroxybutyrate content and in activities of nitrogenase, β-hydroxybutyrate dehydrogenase, and isocitrate lyase in nodule bacteroids were investigated under conditions in which the supply of carbohydrate from the soybean plants was interrupted. The poly-β-hydroxybutyrate content of bacteroids did not decrease appreciably until the carbohydrate supply from the host plants was limited by incubation of excised nodules, incubation of plants in the dark, or by senescence of the host plant. Isocitrate lyase activity in bacteroids was not detected until poly-β-hydroxybutyrate utilization appeared to begin. The presence of a supply of poly-β-hydroxybutyrate in nodule bacteroids was not sufficient for maintenance of high nitrogenase activity under conditions of limited carbohydrate supply from the host plant. An unusually high activity of β-hydroxybutyrate dehydrogenase was observed in bacteroid extracts but no significant change in the activity of this enzyme was observed as a result of apparent utilization of poly-β-hydroxybutyrate by nodule bacteroids.

http://www.plantphysiol.org/content/plantphysiol/47/6/750.full.pdf

Poly-β-hydroxybutyrate Utilization by Soybean (Glycine max Merr.) Nodules and Assessment of Its Role in Maintenance of Nitrogenase Activity

A non-haem iron protein was isolated from an extract of soya-bean nodule bacteroids by a procedure including protamine sulphate and heat precipitation followed by chromatography on DEAE-cellulose. The purified protein contains non-haem iron and acid-labile sulphur and exhibits a spectrum with a rather broad absorption shoulder in the region 380–440nm and a more prominent peak at 280nm. From sedimentation-velocity measurements an apparent s20,w value of 1.3S was calculated. The protein functions as an electron carrier between the reducing system of illuminated chloroplast fragments and nitrogenase from nodule bacteroids, but it failed to function as a cofactor for the photochemical reduction of NADP in the presence of spinach chloroplasts. Also, it is inactive as a cofactor in the enzymic degradation of pyruvate to acetyl phosphate and CO2 in the presence of a ferredoxin-free extract of Clostridium pasteurianum. Repeated freezing, storage and thawing of the non-haem iron protein resulted in a marked loss of activity in the photochemical acetylene-reduction assay. A major portion of the activity that was lost was restored as a result of treatment with sodium sulphide, mercaptoethanol and ferrous ammonium sulphate.

/pdf/purification-and-some-properties-of-a-non-haem-iron-protein-2iibe1861f.pdf

Purification and some properties of a non-haem iron protein from the bacteroids of soya-bean (Glycine max Merr) nodules.

A series of investigations were conducted with the objective of elucidating natural pathways of electron transport from respiratory processes to the site of N2 fixation in nodule bacteroids. A survey of dehydrogenase activities in a crude extract of soybean nodule bacteroids revealed relatively high activities of NAD-specific β-hydroxybutyrate and glyceraldehyde-3-phosphate dehydrogenases. Moderate activities of NADP-specific isocitrate and glucose-6-phosphate dehydrogenases were observed. By use of the ATP-dependent acetylene reduction reaction catalyzed by soybean bacteroid nitrogenase, and enzymes and cofactors from bacteroids and other sources, the following sequences of electron transport to bacteroid nitrogenase were demonstrated: (1) H2 to bacteroid nitrogenase in presence of a nitrogenase-free extract ofC. pasteurianum; (2) β-hydroxybutyrate to bacteroid nitrogenase in a reaction containing β-hydroxybutyrate dehydrogenase, NADH dehydrogenase, NAD and benzyl viologen; (3) β-hydroxybutyrate dehydrogenase, to nitrogenase in reaction containing NADH dehydrogenase, NAD and either FMN or FAD; (4) light-dependent transfer of electrons from ascorbate to bacteroid nitrogenase in a reaction containing photosystem I from spinach chloroplasts, 2,6-dichlorophenolindophenol, and either azotoflavin from Azotobacter or non-heme iron protein from bacteroids; (5) glucose-6-phosphate to bacteroid nitrogenase in a system that included glucose-6-phosphate dehydrogenase, NADP, NADP-ferredoxin reductase from spinach, azotoflavin from Azotobacter and bacteroid non-heme iron protein. The electron transport factors, azotoflavin and bacteroid non-heme iron protein, failed to function in the transfer of electrons from an NADH-generating system to bacteroid nitrogenase. When FMN or FAD were added to systems containing azotoflavin and bacteroid non-heme iron protein, electrons apparently were transferred to the flavin-nucleotides and then nitrogenase without involvement of azotoflavin and bacteroid non-heme iron protein.

Investigations into the pathway of electron transport to the nitrogenase from nodule bacteroids

Ceratonova shasta is a myxozoan parasite of salmon and trout transmitted by waterborne actinospores. Based on DNA sequence data and host specificity, 4 distinct parasite genotypes are recognized. Genotypes I and II are common in the lower reaches of the Klamath River, Oregon–California, but only infection by genotype I causes mortality in Chinook salmon. We conducted sentinel fish exposures and determined genotype composition in river water during exposure, and in fish gills, intestine, and tank water post-exposure to determine whether: (1) transmission of parasites having different genotypes is host-specific and (2) all transmitted genotypes persist in the host through to release as waterborne stages. Initial parasite transmission to the fish host appears indiscriminant, since we detected both genotypes I and II in 83.6% of the fish gills sampled. However, only genotype I was detected in fish that succumbed to infection, while both genotypes persisted in fish that survived. Persistence was lik...

Transmission and Persistence of Ceratonova shasta Genotypes in Chinook Salmon

/pdf/transmission-and-persistence-of-ceratomyxa-shasta-399ggdeq0s.pdf

Transmission and Persistence of Ceratomyxa shasta

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