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

Cells of free-living nitrogen-fixing Nostoc PCC 73102, a filamentous heterocystous cyanobacterium originally isolated from coralloid roots of the cycad Macrozamia. were examined for the presence of ornithine carbamoyl transferase (OCT) by native-PAGE/in situ activity stain, and SDS-PAGE/Western immunoblots. Transmission electron microscopy and immunocytological labeling were used to study the cellular and subcellular distribution of OCT in the Nostoc cells. Moreover, the effects of photoautotrophic and dark heterotrophic 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 immunoblots revealed that a polypeptide with a molecular weight of approximately 38 kDa was immunologically related to OCT purified from pea (Pisum 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 to be 75.6 (± 5.5) gold particles μm^?2 compared with 62.0 (± 7.5) in the nitrogen-fixing heterocysts. Glucose and fructose stimulated both cyanobacterial 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 ¹⁴CO₂-fixation into ¹⁴C]-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.