Abstract
The aim of this study was to investigate the effect of the synthesis and degradation of the nitrogen-rich polymer cyanophycin in rhizobia on symbiotic nitrogen fixation and crop yield in legumes. For this, cyanophycin synthetase from Anabaena sp. PCC7120 was expressed in the bacteroids of the symbiont S. meliloti 1021 either alone or together with an intracellular cyanophycinase from the same bacterium, either in the wild type or in a polyhydroxybutyratenegative (PHB-) mutant, and the effect on the growth of alfalfa host plants was studied. All strains induced the formation of nitrogen-fixing nodules in the host, but clear differences in various parameters were noticeable. Alfalfa plants infected with the wild type expressing only cyanophycin synthetase showed significantly lower shoot nitrogen contents and higher nitrogen fixation rates than plants inoculated with wild type, but the wild type phenotype was exceeded in S. meliloti 1021 expressing cyanophycinase in addition to cyanophycin synthetase. Growth of plants infected with the PHB- mutant expressing only cyanophycin synthetase was severely impaired in comparison to growth of plants infected with the PHB- mutant expressing both or neither of the two proteins. Transmission electron micrographs of sections of nodules induced by wild type S. meliloti 1021 producing cyanophycin synthetase and cyanophycinase showed that the rough endoplasmic reticulum and Golgi membranes were extended further in comparison to the nodules induced by the wild type, the PHB- mutant, or all other recombinant S. meliloti strains, indicating higher metabolic activities in these nodules.
Highlights
Cyanophycin was discovered over one hundred years ago during light microscopic examinations of cyanobacteria [1]
Alfalfa plants inoculated with the phbC-negative mutant Rm4775, which is unable to synthesize PHB, demonstrated an insignificantly [P>0.01] higher acetylene reduction activity 55 days after inoculation in comparison to the acetylene reduction activity levels of plants inoculated with the wild type (Figure 2)
The plants inoculated with the recombinant phbC-negative mutant harbouring cphA1 showed significantly (P
Summary
Cyanophycin (multi-L-arginyl-poly-L-aspartic acid, cyanophycin granule polypeptide or CGP) was discovered over one hundred years ago during light microscopic examinations of cyanobacteria [1]. CGP is synthesized in the cytoplasm of most cyanobacteria [3,4,5] and of several non-photosynthetic bacteria [6,7] and deposited in the cells as water-insoluble membrane-less granules. CGP is usually insoluble in water at physiological pH, but is soluble under acidic (pH9) conditions [8]. A water-soluble form of CGP was observed in cells of a recombinant Escherichia coli strain [7]. Intracellular synthesis of CGP is catalyzed by cyanophycin synthetase, which is encoded by cphA [7]. It was shown that the cyanophycin synthetase of the first cluster (CphA1) contributed more to the synthesis and accumulation of CGP than the second cyanophycin synthetase (CphA2) [12]
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