The nucleoid-associated protein Gbn binds to GATC sequences and affects sporulation and antibiotic production in Streptomyces

ABSTRACTBacterial chromosome structure is organized by a diverse group of proteins collectively called nucleoid-associated proteins (NAPs). Many NAPs have been studied in detail in Streptomyces, including Lsr2, HupA, HupS, and sIHF. Here, we show that SCO1839 represents a novel family of small NAPs unique to Actinobacteria and recognizes a consensus sequence consisting of GATC followed by (A/T)T. The protein was designated Gbn for GATC-binding NAP. Chromatin immunoprecipitation sequencing (ChIP-Seq) detected more than 2800 binding regions, encompassing some 3600 GATCWT motifs, which comprise 55% of all motifs in the S. coelicolor genome. DNA binding of Gbn in vitro increased DNA stiffness but not compaction, suggesting a role in regulation rather than chromosome organization. Despite the huge number of binding sites, the DNA binding profiles were nearly identical during vegetative and aerial growth. The exceptions were SCO1311 and SCOt32, for a tRNA editing enzyme and a tRNA that recognises the rare leucine codon CUA, respectively, which were nearly exclusively bound during vegetative growth. Deletion of gbn led to pleiotropic alterations in developmental timing, morphogenesis and antibiotic production. Taken together, our data show that Gbn is a highly pleiotropic NAP that impacts growth and development in streptomycetes.

Fluorothreonyl-tRNA deacylase prevents mistranslation in the organofluorine producerStreptomyces cattleya

Fluorine is an element with unusual properties that has found significant utility in the design of synthetic small molecules, ranging from therapeutics to materials. In contrast, only a few fluorinated compounds made by living organisms have been found to date, most of which derive from the fluoroacetate/fluorothreonine biosynthetic pathway first discovered inStreptomyces cattleya. While fluoroacetate has long been known to act as an inhibitor of the tricarboxylic acid cycle, the fate of the amino acid fluorothreonine is still not well understood. Here, we show that fluorothreonine can be misincorporated into protein in place of the proteinogenic amino acid threonine. We have identified two conserved proteins from the organofluorine biosynthetic locus, FthB and FthC, that are involved in managing fluorothreonine toxicity. Using a combination of biochemical, genetic, physiological, and proteomic studies, we show that FthB is atrans-acting transfer RNA (tRNA) editing protein, which hydrolyzes fluorothreonyl-tRNA 670-fold more efficiently than threonyl-RNA, and assign a role to FthC in fluorothreonine transport. Whiletrans-acting tRNA editing proteins have been found to counteract the misacylation of tRNA with commonly occurring near-cognate amino acids, their role has yet to be described in the context of secondary metabolism. In this regard, the recruitment of tRNA editing proteins to biosynthetic clusters may have enabled the evolution of pathways to produce specialized amino acids, thereby increasing the diversity of natural product structure while also attenuating the risk of mistranslation that would ensue.