Bacterial endosymbionts of a nitrogen-fixing yeast Rhodotorula mucilaginosa JGTA-S1 – insights into a yet unknown micro-ecosystem†

Rhodotorula mucilaginosa JGTA-S1 is a yeast strain capable of fixing nitrogen and improving nitrogen nutrition in rice plants because of its nitrogen-fixing endobacteria, namely Stutzerimonas (Pseudomonas) stutzeri and Bradyrhizobium sp. To gain a deeper understanding of yeast endosymbionts, we conducted a whole-genome shotgun metagenomic analysis of JGTA-S1 cells grown under conditions of nitrogen sufficiency and deficiency. Our results showed that the endosymbiont population varied depending on the nitrogen regime. Upon mechanical disruption of yeast cells, we obtained endosymbionts in culturable form viz. Bacillus velezensis and Staphylococcus sp. under nitrogen-replete conditions and Lysinibacillus telephonicus., Brevibacillus sp., and Niallia circulans under nitrogen-depleted conditions. S. stutzeri and Bradyrhizobium sp. the previously reported endosymbionts remained unculturable. The culturable endosymbionts Staphylococcus sp. and Bacillus velezensis appear to possess genes for dissimilatory nitrate reduction (DNRA), an alternative pathway for ammonia synthesis. However, our findings suggest that these endosymbionts are facultative as they survive outside the host. The fitness of the yeast was not affected by curing of these microbes. Curing the yeast diazotrophic endosymbionts took a toll on its fitness. Our results also showed that the populations of S. stutzeri and B. velezensis increased significantly under nitrogen-depleted conditions compared to nitrogen-sufficient conditions. The importance of DNRA and nitrogen fixation is also reflected in the metagenomic reads of JGTA-S1.