Genetic analysis of Streptomyces albus J1074 mia mutants suggests complex relationships between post-transcriptional tRNAXXA modifications and physiological traits

Abstract

Proteins MiaA and MiaB catalyze sequential isopentenylation and methylthiolation, respectively, of adenosine residue in 37th position of tRNA XXA . The mia mutations were recently shown by us to affect secondary metabolism and morphology of Streptomyces. However, it remained unknown as to whether both or one of the aforementioned modifications is critical for colony development and antibiotic production. Here, we addressed this issue through analysis of Streptomyces albus J1074 strains carrying double miaAmiaB knockout or extra copy of miaB gene. The double mutant differed from wild-type and miaAminus strains in severity of morphological defects, growth dynamics, and secondary metabolism. Introduction of extra copy of miaB gene into miaA mutant restored aerial mycelium formation to the latter on certain solid media. Hence, miaB gene might be involved in tRNA thiomethylation in the absence of miaA; either MiaA-or MiaB-mediated modification appears to be enough to support normal metabolic and morphological processes in Streptomyces.

The Origins and Roles of Methylthiolated Cytokinins: Evidence From Among Life Kingdoms

“…Despite the availability of mass spectrometry methods for the analysis of 2MeS-CKs (Tarkowski et al, 2010;Kisiala et al, 2019) and emerging number of reports of their presence in different biological systems (including mammals, plants, pathogen-host associations, bacteria, and algae), the functional significance of 2MeS-CKs continues to remain poorly understood (Esberg et al, 1999;Morrison et al, 2015a;Kambhampati et al, 2017;Daudu et al, 2019;Koshla et al, 2019). Most knowledge about the role of 2MeS-CKs stems from their occurrence in post translational tRNA modifications which have a role in translational efficiency, fidelity, and specificity of codon/anticodon interactions (Persson and Björk, 1993;Golovko et al, 2000;Koshla et al, 2020). Deletion of key genes in the methylthiolation pathway can result in aberrant phenotypes most often related to impacts on translation (Koshla et al, 2019).…”

“…However, the impact of the released, methylthiolated CK compounds on the physiology of organisms has yet to be fully understood. Koshla et al (2019Koshla et al ( , 2020 examined the impact of miaA, B and AB null mutants in Streptomyces albus showing that miaA and B deficiency caused significant changes in morphology of Streptomyces and its secondary metabolome profile. Likely caused by less efficient translation, the miaAB mutant was devoid of endogenous antibacterial activity while the parent miaA mutant still displayed some antibacterial activity (Koshla et al, 2019).…”

“…Likely caused by less efficient translation, the miaAB mutant was devoid of endogenous antibacterial activity while the parent miaA mutant still displayed some antibacterial activity (Koshla et al, 2019). miaB mutants had delayed biomass production and miaAB mutants produced sparse aerial hyphae without spore chains compared to the parental mutant miaA (Koshla et al, 2020), suggesting that, even in the absence of miaA, miaB functions to maintain cell cycle and morphology. Phenotypic discrimination between the mutated bacterial lines was highly conditional and only possible on specific media during early stages of their lifecycle; indicating that the impact of methylthiolation may be specific to certain stages of the growth cycle under defined nutrient conditions (Koshla et al, 2020).…”

“…miaB mutants had delayed biomass production and miaAB mutants produced sparse aerial hyphae without spore chains compared to the parental mutant miaA (Koshla et al, 2020), suggesting that, even in the absence of miaA, miaB functions to maintain cell cycle and morphology. Phenotypic discrimination between the mutated bacterial lines was highly conditional and only possible on specific media during early stages of their lifecycle; indicating that the impact of methylthiolation may be specific to certain stages of the growth cycle under defined nutrient conditions (Koshla et al, 2020). Although the levels of unbound 2MeS-CKs were not analyzed, this study suggests that the removal of genes responsible for thiolation impacts total free 2MeS-CKs and thereby also has an impact on the overall development of the bacteria (Koshla et al, 2020).…”

“…While most studies focus on the impact of gene deletion and the resulting effects which might then be rescued through gene complementation (Koshla et al, 2020), few have added exogenous 2MeS-CKs to rescue deletion phenotypes or applied exogenous methylthiol CK derivatives to examine their impact on a biological system.…”