In this work we have established a new experimental model for studying the mechanisms and processes which change expression of certain gene groups via manipulations of translational machinery components. Streptomyces albus SAM2 was chosen as a model organism.
Using SAM2, we studied influence of merodiploid state of rpsL gene (encodes S12 ribosomal protein) on Streptomyces metabolism. In particular, presence of two different alleles of rpsL in one genome confers the strains to ability to produce heterogeneous population of ribosomes carrying either wild type or mutated S12 proteins. The impact of such mixed ribosomal population on antibiotic production is unclear, as well as molecular mechanisms linking the former to a variety of phenotypes. Merodiploids showed altered phenotypes depending on the type of S12 aminoacid substitution encoded by additional rpsL alleles. Strain L90K_ex showed the highest increases in antibiotic titers. Therefore, merodiploids are of practical interest as a platform for strain improvement.
The other important contribution of this work is studying the role of posttranscriptional tRNA modifications (PTTM) in Streptomyces. First of all, we were interested in PTTMs that contribute to proper function of tRNALeuUAA, the only one for translation of the rarest Streptomyces codon UUA. This tRNA is a “switch” to trigger morphogenesis and secondary metabolism. Without it, translation of UUA-containing mRNAs would be abortive. Our working hypothesis was that PTTMs may determine the occurence of mature (translationally competent) tRNALeuUAA.
The generation of bldA gene knock-out was necessary part of this work, since the properties of bldA mutant for S. albus were not described. The ΔbldA strain did not reveal classic “Bald colony” phenotype and was able to form aerial mycelium and spores on most solid mediums. However, strain did not produce metabolites directed by gene clusters carrying TTA-containing (TTA+) genes. The ΔbldA did not express ТТА+ reporter gene sco3479 (lacZ). Yet, we were able to detect low level of adpA-gusA reporter fusion expression. This could be the evidence for UUA mistranslation in adpA-mRNA in the absence of tRNALeuUAA. It is possible that adpA expression in S. albus is not under strict translational control of tRNALeuUAA and low amount of AdpA in ΔbldA is enough to escape morphological arrest. bldA knock-out resulted in candicidin overproduction. Hence, depletion of tRNALeuUAA could be an interesting strategy for overexpression of TTA-independent antibiotics. The detailed mechanisms of such activation of secondary metabolism require future studies.
For S. albus J1074 strain a large group of genes was bioinformatically identified that possibly control formation of different tRNA modifications. It was decided to study two of those genes, XNR_1074 and XNR_1078, encoding possible MiaA and MiaB orthologues, respectively. It is known that Mia-proteins take part in ms2i6A37 formation for most tRNAХХA. tRNALeuUAA could be one of those. We generated XNR_1074 and XNR_1078 knockouts for deeper study of mentioned genes role in SAM2. The final product of Mia-dependent modifications in wild type is ms2io6A37 derivative. Sequential action of proteins МіаА (XNR_1074), МіаВ (XNR_1078) and МіаЕ yields the final hypermodified nucleoside. We were not able to unambigiously identify MiaE homolog in SAM2.
We studied the properties of ΔmiaA and ΔmiaB strains. Knock-out of miaB practically does not affect morphology of native secondary metabolites production, but reduces the accumulation of bldA-dependent aranciamycin and moenomycin. At the same time, the miaA knock-out leads to considerable delay in morphological differentiation (as compred to SAM2), and practically abolished production of TTA-containing antibiotics. Interestingly, ΔmiaA overpoduced different forms of antimycins. Strains ΔmiaA and ΔbldA exhibited similar phenotypes. Besides, expression of TTA-containing reporter genes was affected in ΔmiaA. Lower level of adpA expression and delay of ТТА+-gusA expression was detected for ΔmiaA. Considering partial complementation of ΔmiaA phenotype with TTA-free adpA, we assume that delayed and complicated expression of TTA-containing genes could be the component of defective ΔmiaA phenotype. It is possible that the absence of ms2io6A37 in tRNALeuUAA complicates UUA decoding.
The Mia-proteins modify not only tRNALeuUAA but a wider set of other tRNAXXA, which also contribute to normal metabolism of SAM2. The double mutant S. albus Δmia/bldA::aac(3)IV-oriT phenotype support such a conjecture, since it exhibited morphogenetic defects more severe than that of single mutants ΔmiaA or ΔbldA. In summary, tRNA modifications are important for Streptomyces metabolism and their absence results in specific phenotypes. The mechanisms of such phenotypes formation and role of tRNA modifications in expression of particular gene groups deserve further studies for fundamental and practical reasons.
