Fixation of [13N]N2 and transfer of fixed nitrogen in the Anthoceros-Nostoc symbiotic association

Abstract

The initial product of fixation of [(13)N]N2 by pure cultures of the reconstituted symbiotic association between Anthoceros punctatus L. and Nostoc sp. strain ac 7801 was ammonium; it accounted for 75% of the total radioactivity recovered in methanolic extracts after 0.5 min and 14% after 10 min of incubation. Glutamine and glutamate were the primary organic products synthesized from [(13)N]N2 after incubation times of 0.5-10 min. The kinetics of labeling of these two amino acids were characteristic of a precursor (glutamine) and product (glutamate) relationship. Results of inhibition experiments with methionine sulfoximine (MSX) and diazo-oxonorleucine were also consistent with the assimilation of N2-derived NH 4 (+) by Anthoceros-Nostoc through the sequential activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1), with little or no assimilation by glutamate dehydrogenase (EC 1.3.1.3). Isolated symbiotic Nostoc assimilated exogenous (13)NH 4 (+) into glutamine and glutamate and their formation was inhibited by MSX, indicating operation of the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway: However, relative to free-living cultures, isolated symbiotic Nostoc assimilated 80% less exogenous ammonium into glutamine and glutamate, implying that symbiotic Nostoc could assimilate only a fraction of N2-derived NH 4 (+) . This implication was tested by using Anthoceros associations reconstituted with wild-type or MSX-resistant strains of Nostoc incubated with [(13)N]N2 in the presence of MSX. The results of these experiments indicated that, in situ, symbiotic Nostoc assimilated about 10% of the N2-derived NH 4 (+) and that NH 4 (+) was made available to Anthoceros tissue where it was apparently assimilated by the GS-GOGAT pathway. Since less than 1% of the fixed N2 was lost to the suspension medium, it appears that transfer of NH 4 (+) from symbiont to host tissue was very efficient in this extracellular symbiotic association.

Functional analysis of the phosphoprotein PII (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942

“…In another set of experiments (Fig. 1B), cells grown in the presence of ammonium were transferred either to a medium lacking combined nitrogen or to a medium containing the inhibitors of glutamine synthetase and glutamate synthase, MSX (33, 38) and 6-diazo-5-oxo-L-norleucine (DON) (26,28,39), respectively. After 30 min and 1, 2, and 4 h, samples were removed and the phosphorylation state of P II was analyzed as mentioned above.…”

Regulation of Cellular Differentiation in Filamentous Cyanobacteria in Free-Living and Plant-Associated Symbiotic Growth States

“…Second, much of the nitrogen that is fixed is released and made available to the plant. The amounts of N 2 -derived NH 4 ϩ released by symbionts in associations grown under controlled conditions in the laboratory are 40% in Azolla (120), 80% in Anthoceros (118), and 90% in Gunnera (169). These values are in contrast to 6% (57) to 20% (178) of NH 4 ϩ and organic nitrogen combined by free-living N 2 -fixing cyanobacteria under variable conditions in the natural habitat.…”

“…Even 3 to 7 M NH 4 ϩ is sufficient to repress the appearance of heterocysts in Anabaena strain PCC 7120 (122). Since heterocysts persist in Nostoc within symbiotic cavities despite a level of ammonium that is much higher than this (35,36,118,120,169), it is imperative to understand the basis of the repression. The following discussion will not detail all genes now known to influence heterocyst formation and function; a more thorough listing can be found in references 211 and 212.…”

Evidence for plant-mediated regulation of nitrogenase expression in theAnthoceros-Nostoc symbiotic association

“…In situ 13N tracer experiments using mutants of Nostoc sp. strain UCD 7801, resistant to the glutamine synthetase inhibitor L-methionhe-DL-sub phoximine, established that symbiotic Nostoc releases 80 to 90% of its fixed nitrogen as ammonium to support growth of Anthoceros tissue (Meeks et al, 1985). Studies utilizing mutants of Nostoc UCD 7801, resistant to the E. L. Campbell and J .…”