1.Overexpression of the thiostrepton-resistance gene from Streptomyces azureus in Escherichia coli and characterization of recognition sites of the 23S rRNA A1067 2'-methyltransferase in the guanosine triphosphatase center of 23S ribosomal RNA.
Bechthold A1, Floss HG. Eur J Biochem. 1994 Sep 1;224(2):431-7.
The thiostrepton-resistance gene encoding the 23S rRNA A1067 methyltransferase from Streptomyces azureus has been overexpressed in Escherichia coli using a T7-RNA-polymerase-dependent expression vector. The protein was efficiently expressed at levels up to 20% of total soluble protein and purified to near homogeneity. Kinetic parameters for S-adenosyl-L-methionine (Km = 0.1 mM) and an RNA fragment containing nucleotides 1029-1122 of the 23S ribosomal RNA from E. coli (Km = 0.001 mM) were determined. S-Adenosyl-L-homocysteine showed competitive product inhibition (Ki = 0.013 mM). Binding of either thiostrepton or protein L11 inhibited methylation. RNA sequence variants of the RNA fragment with mutations in nucleotides 1051-1108 were tested as substrates for the methylase. The experimental data indicate that methylation is dependent on the secondary structure of the hairpin including nucleotide A1067 and the exact sequence U(1066)-A(1067)-G(1068)-A(1069)-A(1070) of the single strand.
2.Binding induced RNA conformational changes control substrate recognition and catalysis by the thiostrepton resistance methyltransferase (Tsr).
Kuiper EG1, Conn GL2. J Biol Chem. 2014 Sep 19;289(38):26189-200. doi: 10.1074/jbc.M114.574780. Epub 2014 Aug 1.
Ribosomal RNA (rRNA) post-transcriptional modifications are essential for ribosome maturation, translational fidelity, and are one mechanism used by both antibiotic-producing and pathogenic bacteria to resist the effects of antibiotics that target the ribosome. The thiostrepton producer Streptomyces azureus prevents self-intoxication by expressing the thiostrepton-resistance methyltransferase (Tsr), which methylates the 2'-hydroxyl of 23 S rRNA nucleotide adenosine 1067 within the thiostrepton binding site. Tsr is a homodimer with each protomer containing an L30e-like amino-terminal domain (NTD) and a SPOUT methyltransferase family catalytic carboxyl-terminal domain (CTD). We show that both enzyme domains are required for high affinity RNA substrate binding. The Tsr-CTD has intrinsic, weak RNA affinity that is necessary to direct the specific high-affinity Tsr-RNA interaction via NTDs, which have no detectable RNA affinity in isolation. RNA structure probing experiments identify the Tsr footprint on the RNA and structural changes in the substrate, induced specifically upon NTD binding, which are necessary for catalysis by the CTD.
3.Draft Genome Sequence of Thiostrepton-Producing Streptomyces azureus ATCC 14921.
Sakihara K1, Maeda J1, Tashiro K2, Fujino Y3, Kuhara S2, Ohshima T4, Ogata S1, Doi K5. Genome Announc. 2015 Oct 22;3(5). pii: e01183-15. doi: 10.1128/genomeA.01183-15.
Streptomyces azureus ATCC 14921 belongs to the Streptomyces cyaneus cluster and is known to be a thiostrepton producer. Here, we report a draft genome sequence for this strain, consisting of 350 contigs containing a total of 8,790,525 bp, 8,164 predicted coding sequences, and a G+C content of 70.9%.
4.Characterization of an 8.7-kilobase thiostrepton resistance-encoding plasmid (pGIF3) of Streptomyces incarnatus.
Malina H1, Robert-Gero M. Appl Environ Microbiol. 1992 Mar;58(3):895-9.
The low-copy-number 8.7-kb plasmid pGIF3 of Streptomyces incarnatus was studied after cloning in the Escherichia coli vector pBR322; a restriction map was constructed. Southern blot analysis showed that pGIF3 in S. incarnatus occurs predominantly as integrated in a larger replicon. The plasmid carries a gene for thiostrepton resistance having no homology with the known thiostrepton resistance gene from Streptomyces azureus.
