Proline auxotrophy in Sinorhizobium meliloti results in a plant-specific symbiotic phenotype

Abstract

In order to effectively manipulate rhizobium-legume symbioses for our benefit, it is crucial to first gain a complete understanding of the underlying genetics and metabolism. Studies with rhizobium auxotrophs have provided insight into the requirement for amino acid biosynthesis during the symbiosis; however, a paucity of available L-proline auxotrophs has limited our understanding of the role of L-proline biosynthesis. Here, we examined the symbiotic phenotypes of a recently described Sinorhizobium meliloti L-proline auxotroph. Proline auxotrophy was observed to result in a host-plant-specific phenotype. The S. meliloti auxotroph displayed reduced symbiotic capability with alfalfa (Medicago sativa) due to a decrease in nodule mass formed and therefore a reduction in nitrogen fixed per plant. However, the proline auxotroph formed nodules on white sweet clover (Melilotus alba) that failed to fix nitrogen. The rate of white sweet clover nodulation by the auxotroph was slightly delayed, but the final number of nodules per plant was not impacted. Examination of white sweet clover nodules by confocal microscopy and transmission electron microscopy revealed the presence of the S. meliloti proline auxotroph cells within the host legume cells, but few differentiated bacteroids were identified compared with the bacteroid-filled plant cells of WT nodules. Overall, these results indicated that L-proline biosynthesis is a general requirement for a fully effective nitrogen-fixing symbiosis, likely due to a transient requirement during bacteroid differentiation.

Genomic resources for identification of the minimal N2‐fixing symbiotic genome

“…The lack of phenotypes suggest that aside from the classical symbiotic genes, pSymA‐encoded genes are generally not directly involved in nitrogen fixation. However, many are likely to contribute to the nodule occupancy competiveness of S. meliloti (Pobigaylo et al ., ) or provide host‐specific benefits not detected on alfalfa (Ardourel et al ., ; diCenzo et al ., ). This could potentially explain, in part, the high variability in the gene content of pSymA in various nodule isolates (Epstein et al ., ; Galardini et al ., ); most pSymA genes provide little global symbiotic advantage and so are gained or lost as necessary to better compete for nodule occupancy and to improve N 2 fixation in each individual environment.…”

“…Plant growth experiments were performed largely as described previously (Yarosh et al ., ; diCenzo et al ., ). Briefly, ∼ 5 × 10 8 colony‐forming units of S. meliloti were added to each of the sterile Magenta jars containing six to eight alfalfa ( M. sativa cv.…”

PhoU Allows Rapid Adaptation to High Phosphate Concentrations by Modulating PstSCAB Transport Rate in Sinorhizobium meliloti

“…Stationary-phase bacterial cultures were centrifuged at 3,901 ϫ g for 10 min, and the pellet was gently suspended in 2% (vol/vol) glutaraldehyde-0.1 M sodium phosphate buffer (pH 7.4). Preparation of the fixed samples for imaging and operation of the microscope were performed as described elsewhere (57).…”

A Virtual Nodule Environment (ViNE) for modelling the inter-kingdom metabolic integration during symbiotic nitrogen fixation

“…Nevertheless, as with all models, ViNE predictions were imperfect. However, as we often compared simulated phenotypes for M. truncatula with experimental data for M. sativa , and given that rhizobium mutant phenotypes are often plant specific [e.g., (114–116)], we cannot rule out that some of the inconsistencies are the result of plant-specific phenotypes. Going forward, we intend to continue to manually refine and update ViNE to maximize consistency with experimental observations.…”