Cuckoo sRNAs in Agrobacterium tumefaciens

Small regulatory RNAs (sRNAs) are essential for bacterial adaptation to environmental changes. Since they play a significant role in regulating both mRNA translation and stability, sRNAs are involved in controlling various cell processes, including stress responses. sRNAs of the αr14 family appear to play a role in stress responses and are characterized by a CCUCCUCC anti-Shine Dalgarno sequence in each loop, which led Reinkensmeier and Giegerich to refer to them as "cuckoo" sRNAs (1). The soil bacterium and plant pathogen Agrobacterium tumefaciens contains three cuckoo sRNAs, which are dependent on the RNA chaperone Hfq and are positively regulated by the LysR-type transcriptional regulator LsrB. Deletion of one cuckoo sRNA resulted in minor effects, raising the question of whether the paralogs complement each other. Therefore, we constructed a mutant lacking all three cuckoo sRNAs and analyzed the transcriptome and proteome of the triple deletion mutant to better understand the function of these sRNAs.

The transcriptome and proteome analysis of the triple deletion mutant showed an altered expression of numerous ABC transporter substrate-binding proteins. The most upregulated target was afuA, coding for an iron(III) ABC transporter substrate-binding protein. The increased afuA mRNA may explain the growth defect observed in the triple deletion mutant under elevated iron concentrations. Despite the increased iron uptake, the oxidative stress response remained unaffected. Additionally, several ABC transporter substrate-binding proteins, that bind sugars, were downregulated in the transcriptome of the cuckoo sRNAs deletion strain. Oxidation of sucrose in the periplasm leads to ketosucrose, which can be transported into the cytoplasm or released into the cultivation medium. We found an increased ketosucrose concentration in the medium of the cuckoo sRNAs deletion mutant compared to the wild type. This suggests that one of the downregulated ABC transporter substrate-binding proteins is involved in ketosucrose uptake. Both phenotypes can be compensated by a single cuckoo sRNA suggesting that the three cuckoo sRNAs have redundant functions and are able to complement each other. In summary, the cuckoo sRNAs of A. tumefaciens seem to be involved in the regulation of the uptake of iron and ketosucrose.

(1) Reinkensmeier & Giegerich. 2015. RNA Biology