Insertion sequencing (INSeq) analysis of Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) grown on glucose or succinate at both 21% and 1% O2 was used to understand how O2 concentration alters metabolism. Two transcriptional regulators were required for growth on glucose (pRL120207 (eryD) and RL0547 (phoB)), five on succinate (pRL100388, RL1641, RL1642, RL3427 and RL4524 (ecfL)) and three on 1% O2 (pRL110072, RL0545 (phoU) and RL4042). A novel toxin-antitoxin system was identified that could be important for generation of new plasmid-less rhizobial strains. Rlv3841 appears to use the methylglyoxal pathway alongside the ED pathway and TCA-cycle for optimal growth on glucose. Surprisingly the ED pathway was required for growth on succinate, suggesting sugars made by gluconeogenesis must undergo recycling. Altered amino acid metabolism was specifically needed for growth on glucose, including RL2082 (gatB) and pRL120419 (opaA, omega-amino acid:pyruvate transaminase). Growth on succinate specifically required enzymes of nucleobase synthesis including ribose-phosphate pyrophosphokinase (RL3468 (prs)) and a cytosine deaminase (pRL90208 (codA)). Succinate growth was particularly dependent on cell surface factors, including the PrsD-PrsE type I secretion system and UDP-galactose production. Only RL2393 (glnB, nitrogen regulatory protein PII) was specifically essential for growth on succinate at 1% O2, conditions similar to those experienced by N2-fixing bacteroids. Glutamate synthesis is constitutively activated in glnB mutants, suggesting consumption of 2-ketoglutarate may increase flux through the TCA-cycle, leading to excess reductant that cannot be reoxidized at 1% O2 and cell death.
