| Two general types of phosphotransferase system (PTS) have been identified in bacteria:the sugar PTS, dedicated to carbohydrate transport, and the nitrogen PTS, which exerts regulatory functions. Nitrogen phosphotransferase system (PTSNtr) consists of EⅠNtr, NPr and EⅡANtr. The active phosphate moiety derived from phosphoenolpyruvate is transferred through EⅠNtr and NPr to EIIANtr. In the previous study explored by this laboratory, soybean plants inoculated with EINtr mutant of Sinorhizobium fredii CCBAU45436 showed a typical nitrogen starvation phenotype. This will further investigate the role of nitrogen phosphotransferase system components in S. fredii CCBAU45436 symbioses with its legume hosts.S. fredii can establish nitrogen-fixing symbiosis with legume crops soybean (determinate nodules) and pigeonpea (indeterminate nodules). In this study, S. fredii mutants of ptsP and ptsO (encoding EⅠNtr and NPr, respectively) formed ineffective nodules on soybean and pigeonpea, while the ptsN (encoding EⅡANtr) mutant is not defective in symbiosis with soybean and pigeonpea. A notable reduction in the number of bacteroids within each symbiosome and poly-β-hydroxybutyrate (PHB) granules in bacteroids was observed in nodules infected by the ptsP or ptsO mutants but not in those infected with the ptsN mutant. However these defects of ptsP and ptsO mutants were recovered in the double mutants ptsPptsN or ptsOptsN, implying a negative role of unphosphorylated EⅡANtr in symbiosis. These results imply that EⅡANtr might not be essential for symbiosis but the non-phosphorylated EⅡANtr in the ptsP or ptsO mutants could play unidentified negative regulation roles. However, a potential unknown positive regulation role of EⅡANtr might also be involved in optimizing symbiosis. To test these possibilities, nodulation occupancy experiments were carried out by equally mixing representative strains in inoculants. As expected, the ptsP and ptsO mutants occupied a very low number of nodules, when inoculated equally with CCBAU45436. The ptsN mutant showed a smaller but also significant reduction in nodule occupancy compared to CCBAU45436, indicating a positive role of EⅡANtr in symbiosis. Moreover, ptsPN occupied 5% and 8% of nodules when equally inoculated with CCBAU45436 and the AptsN mutant in soybean, respectively, implying an essential role of cellular processes other than the transfer of phosphoryl group to EⅡANtr.In order to illustrate a more detailed picture of symbiotic defects associated with ptsP and ptsO mutants, dynamic changes in the growth of host plants, and the development of nodules and bacteroids were recorded. On soybean plants, both the ptsP and ptsO mutants formed normal pink nodules at early stages, but these nodules exhibited premature senescence compared with those induced by wild-type strain. In the further test of the ptsP mutant on pigeonpea plants, symbiotic defects at 28 dpi (days post inoculation) regarding leaf color and the development of PHB granules in bacteroids, were partially and fully recovered at 45 dpi and 67 dpi, sequentially. Therefore, it can be speculated that the weakness of symbiotic ability of the ptsP and ptsO mutants might be related to carbon source supply. Thus, ptsⅠ encoding sugar PTS’El was deleted in the background of wild-type strain and the ptsP mutant. The resultant strains were inoculated on soybean and pigeonpea plants. The results showed that the ptsⅠ mutant was not defective in symbiosis with soybean and pigeonpea, while the double mutant ptsPⅠ formed ineffective nodules similar to the ptsP mutant. These findings imply that the function of PTSNtr in symbiosis might not depend on the sugar PTS. Moreover, Western blotting experiments using the ptsP mutant and wild type strain uncovered that EINtr may be the main source of EIIANtr phosphate group. |
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