Role of two RpoN in Bradyrhizobium sp. strain DOA9 in symbiosis and free-living growth

Abstract

RpoN is an alternative sigma factor (sigma 54) that recruits the core RNA polymerase to promoters of genes. In bacteria, RpoN has diverse physiological functions. In rhizobia, RpoN plays a key role in the transcription of nitrogen fixation (nif) genes. The Bradyrhizobium sp. DOA9 strain contains a chromosomal (c) and plasmid (p) encoded RpoN protein. We used single and double rpoN mutants and reporter strains to investigate the role of the two RpoN proteins under free-living and symbiotic conditions. We observed that the inactivation of rpoNc or rpoNp severely impacts the physiology of the bacteria under free-living conditions, such as the bacterial motility, carbon and nitrogen utilization profiles, exopolysaccharide (EPS) production, and biofilm formation. However, free-living nitrogen fixation appears to be under the primary control of RpoNc. Interestingly, drastic effects of rpoNc and rpoNp mutations were also observed during symbiosis with Aeschynomene americana. Indeed, inoculation with rpoNp, rpoNc, and double rpoN mutant strains resulted in decreases of 39, 64, and 82% in the number of nodules, respectively, as well as a reduction in nitrogen fixation efficiency and a loss of the bacterium’s ability to survive intracellularly. Taken together, the results show that the chromosomal and plasmid encoded RpoN proteins in the DOA9 strain both play a pleiotropic role during free-living and symbiotic states.

Exploring the cellular surface polysaccharide and root nodule symbiosis characteristics of the rpoN mutants of Bradyrhizobium sp. DOA9 using synchrotron-based Fourier transform infrared microspectroscopy in conjunction with X-ray absorption spectroscopy

“…In the previous work, we demonstrated that the regulatory RpoN is required to express other nitrogen-fixing genes under free-living and symbiotic conditions. Moreover, the study on the deletion of rpoN genes in DOA9 suggested an alternative sigma factor affecting colony morphology characteristics, leading to differences in its performance in nitrogen-fixing nodule phenotypes, as described by Wongdee et al ( 13 ).…”

“…To confirm the effect of the RpoN mutation on the colony size of strain DOA9 ( 13 ), single colonies from both the DOA9WT and DOA9∆ rpoN mutant strains were examined on a yeast extract-mannitol (YEM) agar plate. The mutant strains were smaller ( Fig.…”

“…Genome analysis revealed that DOA9 contains one chromosome (c) and one symbiotic plasmid (p), both of which carry nif genes, including the nitrogenase structural genes, nifH , nifD , and nifK , and nitrogenase regulatory genes, nifA and rpoN ( 12 ). Our recent study demonstrated that deletion mutations of the nitrogenase regulatory rpoN genes in DOA9 cause a reduction in CSP production under free-living conditions ( 13 ). All rpoN mutant strains exhibited smaller colonies with different CSP characteristics than the wild type (WT), which is related to their lesser effectiveness in producing root nodules and plant growth phenotypes.…”

“…Interestingly, a reduction in nitrogen fixation activity with the appearance of small and less red-colored nodules was found in A. americana inoculated with rpoN mutant strains. Although the CSP production and symbiotic nitrogen fixation efficiency of these mutants have been investigated by analyzing the weight of dried CSP, nodule appearance under microscopes, and nitrogenase activity assays, respectively ( 13 ), these methods could only represent the physiological function of the two RpoN in DOA9 that affected CSP production, nitrogen fixation, and nodule phenotypes. Therefore, a deeper understanding of the functional components in the CSP structure of cell growth under free-living conditions and the biochemical changes in nodules formed by the rpoN mutants requires advanced analysis technology.…”

Identifying functional multi-host shuttle plasmids to advance synthetic biology applications inMesorhizobiumandBradyrhizobium

“…Additionally, M. japonicum exhibits a broad legume host range, whereas some other mesorhizobia, as is typical of Mesorhizobium huakuii strains, have a narrow host range. Within the family Bradyrhizobiaceae , one broad host range strain that has been well characterized in recent years is of particular interest to us (Teamtisong et al ., 2014; Songwattana et al ., 2017; Wongdee et al ., 2023). Bradyrhizobium sp.…”