Ribosome heterogeneity in bacteria refers to variations in ribosome composition and function, but little is known about its physiological role. Many bacteria harbor multiple rpsU genes encoding bS21, one of the smallest ribosomal proteins. Due to their small size, some rpsU genes were missed in early annotations. Bradyrhizobium diazoefficiens and Sinorhizobium meliloti harbor rpsU1 and rpsU2 with unknown specific roles. In S. meliloti, rpsU1 is a part of a cold shock operon (1), and rpsU2 was annotated only in 2017. In a ribosome profiling (Ribo-seq) of S. meliloti, rpsU2 was identified as a translated small ORF, and bS21-2 but not bS21-1 was detected by mass spectrometry (2). Here, we aimed to study the ribosome heterogeneity based on rpsU1 and rpsU2 homologs in S. meliloti and B. diazoefficiens. We applied RNA-seq, qRT-PCR, immunoblot, and phenotypic assays to analyze wild type and mutant bacterial strains. We found that in exponentially growing S. meliloti 2011, the levels of rpsU1 mRNA are at least 30-fold lower than that of rpsU2. However, since according to Ribo-seq the translation efficiency (TE) of rpsU1 is approximately 10-fold higher than the TE of rpsU2, rpsU1 seems to be translated in exponential cultures, a suggestion supported by the failure to delete rpsU1 unless it was expressed ectopically. Depletion and overexpression of rpsU1 caused growth changes at 15°C. Upon cold shock, the level of rpsU1 mRNA level was strongly increased and that of rpsU2 mRNA was decreased till the differences were evened. Furthermore, we obtained a ∆ rpsU1 mutant of B. diazoefficiens, which is currently compared to the parental strain. To address the specific roles of the rpsU homologs in both species, we are analyzing changes in ribosome-associated mRNA pools upon deletion or manipulated expression of rpsU1 and rpsU2. Altogether, the results suggest that rpsU1 plays a role in the adaptation of S. meliloti to cold stress, but is also essential under standard conditions, when rpsU2 is predominantly used. By contrast, rpsU1 is not essential in B. diazoefficiens. The specific roles of the rpsU homologs in S. meliloti and B. diazoefficiens are still under investigation and most recent results will be shown.

References

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