Ornithine cycle in Nostoc PCC 73102. Occurrence and localization of ornithine carbamoyl transferase, and the effects of external carbon and ornithine on nitrogenase activity and citrulline synthesis

Abstract

Lindblad, P. 1992. Ornilhine cycle in Nostoc PCC 73102. Occurrence and localization of ornithine carbamoyl transferase. and the effects of external carbon and ornithine on nitrogenase activity and citruiline synthesis. -Physiol. Plant. 84: 275-282.Cells of free-living nitrogen-fixing Nostoc PCC 73102, a filamentous hcterocystous cyanobacterium originally isolated from coralloid roots of the cycad Macrozamia, were examined for the presence of ornilhsne carbamoyl transferase (OCT) by native-PAGE/in situ activity stain, and SDS-PAGE/Weslcrn immunoblots. Transmission electron microscopy and immunocytologicai labeling were used to study the cellular and subceilular distribution of OCT in the Nosioc cells. Moreover, the effects of photoautotrophic and dark helerotrophic growth metabolism on growth, nitrogenase activity and in vivo citrulline synthesis were investigated. PAGE in combination with in situ activity staining demonstrated an in vitro active OCT with a molecular weight of approximately 80 kDa. SDS-PAGE/Western immunoHoIs revealed that a polypeptide with a molecular weight of approximately 38 kDa was immunologically related to OCT purified from pea (Fisum sativum L. cv. Alaska). Immunolocalization demonstrated that the OCT protein was located both in vegetative cells and heterocysts. Using the particle analysis of an image processor, the labeling associated with the photosynthetic vegetative cells was calculated lo be 7.5.6 (± .S.5) gold particles iim"' compared with 62.0 (±7.5) in the nitrogen-fixing heterocysts. Glucose and fructose stimulated both cyanobacteriai growth and nitrogenase activity in light and darkness. Addition of exogenous ornithine decreased nitrogenase activity. In light grown cells, additions of glucose and fructose in combination with ornithine not only stimulated growth and nitrogenase activity but also in vivo citrulline synthesis, measured as "COj-fixation into ['*C]-citrulline. In darkness no stimulation was observed on in vivo citrulline synthesis. The substantial stimulation of nitrogenase activity by additions of external glucose and fructose, both in the light and in darkness, was not followed by a simultaneous stimulation of in vivo citrulline synthesis.

Root-based N2-fixing Symbioses: Legumes, Actinorhizal Plants, Parasponia sp. and Cycads

“…Nostoc, as opposed to many other cyanobacteria, can easily switch from growing photoautotrophically to growing heterotrophically (Rippka et al, 1979) which probably is a very important part of their symbiotic competence. Evidence of the heterotrophic nature of cycad cyanobionts includes the fact that externally added fructose and glucose to cycad isolates grown in darkness stimulated their nitrogenase activity (Lindblad, 1992;Martel et al, 1993). The carbon supplied by the host is still unknown, and whether the mucilage filling the extracellular space of the symbiotic coralloid roots has a role in the carbon supply to the cyanobiont, or whether it is of cyanobacterial origin, is also an open question.…”

Hydrogen uptake in Nostoc sp. strain PCC 73102. Cloning and characterization of a hupSL homologue

“…strain PCC 73102 has been proposed as the type strain of the species Nostoc punctiforme in the PCC classification. Axenic, N 2 -fixing, and non-N 2 -fixing cultures were grown in BG11 0 and BG11 media, respectively (Stanier et al 1971), in continuous light (Thorn Polylux 4000 and Osram Warmtone Warm White 400-700 nm; 40 µmol photons m -2 s -1 ), at 26°C with the use of a magnetic stirrer to obtain a homogeneous cell suspension (Lindblad 1992). The plasmid used in the ligation steps in this study was pBluescript II SK (+) (Stratagene), and the bacterial strain Escherichia coli XL1-Blue (Stratagene; La Jolla, Calif., USA) was used in the transformations.…”

Plant-type and bacterial-type ferredoxins in a nitrogen-fixing cyanobacterium: Nostoc sp. strain PCC 73102

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed, including controls, as described earlier [16,17]. strain PCC 73102 were sedimented by centrifugation (13 000 × g, 10 min) and solubilization buffer (1 : 1, v/v; 10 mM Tris-HCI, pH 8.0, containing 1 mM EDTA, 5% /3mercaptoethanol, 2.5% SDS and 0.5% Triton X-100) was added before the samples were boiled, at 100°C, for 5 rain.…”

“…Fixation, embedding, sectioning and immunogold labelling were all performed as described previously [16], except for the present cells being embedded in LRWhite (TAAB, Reading, UK). Polyclonal rabbit-anti-major plant-type-ferredoxin antiserum (1 : 1000 dilution [18]), or polyclonal rabbit-anti-bacterial-type-ferredoxin antiserum (1:500 dilution [18]), and secondary IgG (goatanti-rabbit IgG (BioCell, Cardiff, UK), 1 : 20 dilution) conjugated to 5 nm colloidal gold particles were used.…”