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Nonribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize important secondary metabolites such as antibiotics. NRPSs follow a modular synthetic logic whereby each successive amino-acid monomer is added to the peptide chain by successive multi-domain modules. The condensation domain catalyzes the central chemical event in the synthetic cycle, peptide-bond formation, and is present in every elongation module of the NRPS. Viomycin is an antituberculosis nonribosomal peptide that is synthesized by a series of four NRPS proteins and then modified by tailoring proteins. In order to study the mechanisms of peptide-bond formation in viomycin and in NRPSs in general, a structural study of the first condensation domain of the viomycin synthetase protein VioA (VioA-C1) was initiated. The gene for VioA-C1 was cloned from genomic DNA of Streptomyces vinaceus, expressed as an octahistidine-tagged construct and purified by column chromatography. VioA-C1 was crystallized using the sitting-drop vapor-diffusion method. X-ray diffraction data were collected on a rotating-anode source to 2.9 Å resolution. The data could be indexed in the orthorhombic space group P212121, with unit-cell parameters a = 46.165, b = 68.335, c = 146.423 Å. There is likely to be one monomer in the asymmetric unit, giving a solvent content of 49.2% and a Matthews coefficient ( VM) of 2.42 Å3 Da−1. Structural determination is in progress. [ABSTRACT FROM AUTHOR]

The binding site of the cyclic peptide antibiotics capreomycin and viomycin is located on the ribosomal subunit interface close to nucleotides C1409 in 16S rRNA and C1920 in 23S rRNA. In Mycobacterium tuberculosis, the 2′-hydroxyls of both nucleotides are methylated by the enzyme TlyA. Loss of these methylations through inactivation of TlyA confers resistance to capreomycin and viomycin. We report here that TlyA orthologues occur in diverse bacteria and fall into two distinct groups. One group, now termed TlyAI, has shorter N- and C-termini and methylates only C1920; the second group (now TlyAII) includes the mycobacterial enzyme, and these longer orthologues methylate at both C1409 and C1920. Ribosomal subunits are the preferred substrates for both groups of orthologues. Amino acid substitutions at the N-terminus of TlyAII reduce its ability to methylate these substrates. Growing pairs of recombinant TlyAII Escherichia coli strains in competition shows that even subtle changes in the level of rRNA methylation lead to significant differences in susceptibility to sub-inhibitory concentrations of capreomycin. The findings reveal that 2′- O-methyls at both C1409 and C1920 play a role in facilitating the inhibitory effects of capreomycin and viomycin on the bacterial ribosome. [ABSTRACT FROM AUTHOR]

We present evidence for the coexistence and coevolution of antibiotic resistance and biosynthesis genes in soil bacteria. The distribution of the streptomycin ( strA) and viomycin ( vph) resistance genes was examined in Streptomyces isolates. strA and vph were found either within a biosynthetic gene cluster or independently. Streptomyces griseus strains possessing the streptomycin cluster formed part of a clonal complex. All S. griseus strains possessing solely strA belonged to two clades; both were closely related to the streptomycin producers. Other more distantly related S. griseus strains did not contain strA. S. griseus strains with only vph also formed two clades, but they were more distantly related to the producers and to one another. The expression of the strA gene was constitutive in a resistance-only strain whereas streptomycin producers showed peak strA expression in late log phase that correlates with the switch on of streptomycin biosynthesis. While there is evidence that antibiotics have diverse roles in nature, our data clearly support the coevolution of resistance in the presence of antibiotic biosynthetic capability within closely related soil dwelling bacteria. This reinforces the view that, for some antibiotics at least, the primary role is one of antibiosis during competition in soil for resources. [ABSTRACT FROM AUTHOR]

The nonheme iron oxygenase VioC from Streptomyces vinaceus catalyzes Fe(II)-dependent and α-ketoglutarate-dependent Cβ-hydroxylation ofl-arginine during the biosynthesis of the tuberactinomycin antibiotic viomycin. Crystal structures of VioC were determined in complexes with the cofactor Fe(II), the substratel-arginine, the product (2 S,3 S)-hydroxyarginine and the coproduct succinate at 1.1–1.3 Å resolution. The overall structure reveals a β-helix core fold with two additional helical subdomains that are common to nonheme iron oxygenases of the clavaminic acid synthase-like superfamily. In contrast to other clavaminic acid synthase-like oxygenases, which catalyze the formation of threo diastereomers, VioC produces the erythro diastereomer of Cβ-hydroxylatedl-arginine. This unexpected stereospecificity is caused by conformational control of the bound substrate, which enforces a gauche(–) conformer for χ1 instead of the trans conformers observed for the asparagine oxygenase AsnO and other members of the clavaminic acid synthase-like superfamily. Additionally, the substrate specificity of VioC was investigated. The side chain of thel-arginine substrate projects outwards from the active site by undergoing interactions mainly with the C-terminal helical subdomain. Accordingly, VioC exerts broadened substrate specificity by accepting the analogsl-homoarginine andl-canavanine for Cβ-hydroxylation. [ABSTRACT FROM AUTHOR]

The cytotoxin colicin E3 targets the 30S subunit of bacterial ribosomes and specifically cleaves 16S rRNA at the decoding centre, thereby inhibiting translation. Although the cleavage site is well known, it is not clear which step of translation is inhibited. We studied the effects of colicin E3 cleavage on ribosome functions by analysing individual steps of protein synthesis. We find that the cleavage affects predominantly the elongation step. The inhibitory effect of colicin E3 cleavage originates from the accumulation of sequential impaired decoding events, each of which results in low occupancy of the A site and, consequently, decreasing yield of elongating peptide. The accumulation leads to an almost complete halt of translation after reading of a few codons. The cleavage of 16S rRNA does not impair monitoring of codon–anticodon complexes or GTPase activation during elongation-factor Tu-dependent binding of aminoacyl-tRNA, but decreases the stability of the codon–recognition complex and slows down aminoacyl-tRNA accommodation in the A site. The tRNA–mRNA translocation is faster on colicin E3-cleaved than on intact ribosomes and is less sensitive to inhibition by the antibiotic viomycin. [ABSTRACT FROM AUTHOR]

The basic peptide antibiotic viomycin rapidly inhibits polypeptide chain elongation when added to purified endogenous Escherichia colt polysomes actively engaged in polypeptide synthesis. After inhibition is established, the polysomal nascent peptide chains react only slowly with puromycin, while the chains of uninhibited polysomes or tetracycline-blocked polysomes react very rapidly. Since viomycin does not inhibit the peptidyl transferase reaction, these results indicate that the antibiotic confines peptidyl-tRNA to the ribosomal acceptor site and, consequently, that it blocks potypeptide chain elongation by inhibiting ribosomal translocation. Viomycin at concentrations strongly inhibitory for translocation (10 µM) does not impair the elongation-factor (EF)-G-plus-ribosome-dependent GTP hydrolysis, the formation of GDP- EF-G · ribosome- fusidic-acid complex and guanyl-5'-yl-methylene-diphosphonate · EF-G. ribosome complex. or the non-enzymic binding of AcPhe-tRNA to the ribosomal donor site. In contrast, viomycin strongly inhibits the non-enzyme translocation and the release of AcPhe-tRNA from the ribosomal acceptor site promoted by depletion of NH&4macr; ions. It is suggested that viomycin inhibits the movement of peptidyl-tRNA associated with translocation by interfering with its exit from the ribosomal acceptor site. Viomycin at high concentrations (10- 100 µM) partially inhibits the EF-G-plus-ribosome-dependent GTP hydrolysis and the turnover of the GDP · EF-G · ribosome · fusidic-acid complex. These effects, however, are probably unrelated to the inhibition of ribosomal translocation. The binding of viomycin to AcPhe-tRNA · ribosome - poly(U) complexes is readily reversible, since the antibiotic can be removed by filtration through molecular sieves. [ABSTRACT FROM AUTHOR]

The Streptomyces vinaceus viomycin phosphotransferase (vph) mRNA contains an untranslated leader with a conventional Shine-Dalgarno homology. The vph leader was removed by ligation of the vph coding sequence to the transcriptional start site of a Streptomyces or an Escherichia coli promoter, such that transcription would initiate at the first position of the vph start codon. Analysis of mRNA demonstrated that transcription initiated primarily at the A of the vph AUG translational start codon in both Streptomyces lividans and E. coli; cells expressing the unleadered vph mRNA were resistant to viomycin indicating that the Shine-Dalgarno sequence, or other features contained within the leader, was not necessary for vph translation. Addition of four nucleotides (5′-AUGC-3′) onto the 5′ end of the unleadered vph mRNA resulted in translation initiation from the vph start codon and the AUG triplet contained within the added sequence. Translational fusions of vph sequence to a Tn5 neo reporter gene indicated that the first 16 codons of vph coding sequence were sufficient to specify the translational start site and reading frame for expression of neomycin resistance in both E. coli and S. lividans. [ABSTRACT FROM AUTHOR]