Draft genome sequence of broad-spectrum antibiotic sparsomycin-producing Streptomyces sparsogenes ATCC 25498 from the American Type Culture Collection.

OBJECTIVES: Sparsomycin, which exhibits rare broad-spectrum antibiotic and antitumour activity against bacteria, Archaea, Eucarya and various cancer cell lines, has been widely used as a powerful tool to study protein synthesis. Here we report the draft genome sequence of Streptomyces sparsogenes ATCC 25498 from the American Type Culture Collection (ATCC), which has become an organism of interest owing to its ability to produce sparsomycin. METHODS: The whole-genome sequence of S. sparsogenes ATCC 25498 was determined using a high-throughput Illumina HiSeq 2000 platform and genome assembly was performed using the SOAPdenovo method. RESULTS: Whole-genome sequencing analysis revealed a genome size of 10.0Mb. A total of 41 secondary metabolite biosynthetic gene clusters were identified. The gene cluster for the biosynthesis of sparsomycin was localised on scaffold 9. CONCLUSIONS: The genome sequence of S. sparsogenes ATCC 25498 will not only aid in understanding the regulatory mechanism of sparsomycin biosynthesis but will also reveal the ability of the isolate to produce novel bioactive secondary metabolites.

Sparsomycin Biosynthesis Highlights Unusual Module Architecture and Processing Mechanism in Non-ribosomal Peptide Synthetase.

Sparsomycin is a model protein synthesis inhibitor that blocks peptide bond formation by binding to the large ribosome subunit. It is a unique dipeptidyl alcohol, consisting of a uracil acrylic acid moiety and a monooxo-dithioacetal group. To elucidate the biosynthetic logic of sparsomycin, a biosynthetic gene cluster for sparsomycin was identified from the producer Streptomyces sparsogenes by genome mining, targeted gene mutations, and heterologous expression. Both the genetic and enzymatic studies revealed a minimum set of non-ribosomal peptide synthetases needed for generating the dipeptidyl alcohol scaffold of sparsomycin, featuring unusual mechanisms in dipeptidyl assembly and off-loading.

Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation.

In the absence of elongation factor EF-G, ribosomes undergo spontaneous, thermally driven fluctuation between the pre-translocation (classical) and intermediate (hybrid) states of translocation. These fluctuations do not result in productive mRNA translocation. Extending previous findings that the antibiotic sparsomycin induces translocation, we identify additional peptidyl transferase inhibitors that trigger productive mRNA translocation. We find that antibiotics that bind the peptidyl transferase A site induce mRNA translocation, whereas those that do not occupy the A site fail to induce translocation. Using single-molecule FRET, we show that translocation-inducing antibiotics do not accelerate intersubunit rotation, but act solely by converting the intrinsic, thermally driven dynamics of the ribosome into translocation. Our results support the idea that the ribosome is a Brownian ratchet machine, whose intrinsic dynamics can be rectified into unidirectional translocation by ligand binding.

Sparsomycin-linezolid conjugates can promote ribosomal translocation.

Sparsomycin is an antibiotic that targets the peptidyl transferase center of the ribosome and has the ability to promote ribosomal translocation in the absence of EF-G and GTP. Here we show that changes in the configurations at the two chiral centers of sparsomycin, especially at the chiral carbon, can greatly affect its capability to promote ribosomal translocation. More importantly, the incorporation of the pseudo-uracil moiety of sparsomycin into linezolid through a covalent linkage conferred on linezolid derivatives the ability to promote translocation, thus indicating the importance of interactions between this pseudo-uracil moiety, rRNA, and tRNA for promoting translocation. In addition, these translocation promoters can also effectively inhibit spontaneous reverse translocation; this suggests that they might promote forward translocation by trapping the ribosome in the post-translocation state and shifting the equilibrium between the pre- and post-translocation ribosome in the forward direction.

Absolute binding free energy calculations of sparsomycin analogs to the bacterial ribosome.

The interactions of the 50S subunit of bacterial ribosome with antibiotic sparsomycin (SPS) and five analogs (AN) are investigated through the calculation of the standard (absolute) binding free energy and the characterization of conformational dynamics. The standard binding free energies of the complexes are computed using free energy perturbation molecular dynamics (FEP/MD) simulations with explicit solvent. Restraining potentials are applied and then released during the simulation to efficiently sample the changes in translational, orientational, and conformational freedom of the ligand and receptor upon binding. The biasing effects of the restraining potentials are rigorously removed. The loss of conformational freedom of the ligand upon binding is determined by introducing a potential of mean force (PMF) as a function of the root-mean-square deviation (rmsd) of the ligand relative to its conformation in the bound state. To reduce the size of the simulated system, the binding pocket of the ribosome is simulated in the framework of the generalized solvent boundary potential (GSBP). The number of solvent molecules in the buried binding site is treated via grand canonical Monte Carlo (GCMC) during the FEP/MD simulations. The correlation coefficient between the calculated and measured binding free energies is 0.96, and the experimentally observed ranking order for the binding affinities of the six ligands is reproduced. However, while the calculated affinities of the strong binders agree well with the experimental values, those for the weak binders are underestimated.

Calculation of the standard binding free energy of sparsomycin to the ribosomal peptidyl-transferase P-site using molecular dynamics simulations with restraining potentials.

The standard (absolute) binding free energy of the antibiotic sparsomycin with the 50S bacteria ribosomal subunit is calculated using molecular dynamics (MD) free energy perturbation (FEP) simulations with restraining potentials developed by Wang et al. [Biophys. J. 91:2798-2814 (2006)]. In the simulation protocol, restraining potentials are activated for the orientational and translational movements of the ligand relative to the binding site when it is decoupled from the binding pocket, and then released once the ligand fully interacts with the rest of the system. A reduced system is simulated to decrease the computational cost of the FEP/MD calculations and the effects of the surrounding atoms are incorporated using the generalized solvent boundary potential (GSBP) method. The loss of conformational freedom of the ligand upon binding is characterized using the potential of mean force (PMF) as a function of the root-mean-square deviation (RMSD) relative to the bound conformation. The number of water molecules in the binding pocket is allowed to fluctuate dynamically in response to the ligand during the calculations by combining FEP/MD with grand canonical Monte Carlo (GCMC) simulations. The calculated binding free energy is about -6 kcal/mol, which is in reasonable agreement with the experimental value. The information gleaned from this study provides new insight on the recognition of ribosome by sparsomycin and highlights the challenges in calculations of absolute binding free energies in these systems.

23S rRNA nucleotides in the peptidyl transferase center are essential for tryptophanase operon induction.

Distinct features of the ribosomal peptide exit tunnel are known to be essential for recognition of specific amino acids of a nascent peptidyl-tRNA. Thus, a tryptophan residue at position 12 of the peptidyl-tRNA TnaC-tRNA(Pro) leads to the creation of a free tryptophan binding site within the ribosome at which bound tryptophan inhibits normal ribosome functions. The ribosomal processes that are inhibited are hydrolysis of TnaC-tRNA(Pro) by release factor 2 and peptidyl transfer of TnaC of TnaC-tRNA(Pro) to puromycin. These events are normally performed in the ribosomal peptidyl transferase center. In the present study, changes of 23S rRNA nucleotides in the 2585 region of the peptidyl transferase center, G2583A and U2584C, were observed to reduce maximum induction of tna operon expression by tryptophan in vivo without affecting the concentration of tryptophan necessary to obtain 50% induction. The growth rate of strains with ribosomes with either of these changes was not altered appreciably. In vitro analyses with mutant ribosomes with these changes showed that tryptophan was not as efficient in protecting TnaC-tRNA(Pro) from puromycin action as wild-type ribosomes. However, added tryptophan did prevent sparsomycin action as it normally does with wild-type ribosomes. These findings suggest that these two mutational changes act by reducing the ability of ribosome-bound tryptophan to inhibit peptidyl transferase activity rather than by reducing the ability of the ribosome to bind tryptophan. Thus, the present study identifies specific nucleotides within the ribosomal peptidyl transferase center that appear to be essential for effective tryptophan induction of tna operon expression.

Design at the atomic level: generation of novel hybrid biaryloxazolidinones as promising new antibiotics.

From the X-ray crystal structures of linezolid and the non-selective antibiotic sparsomycin, we have derived a new family of hybrid oxazolidinones. From this initial compound set we have developed a new biaryloxazolidinone scaffold that shows both potent antimicrobial activity as well as selective inhibition of ribosomal translation. The synthesis of these compounds is outlined.

Precise alignment of peptidyl tRNA by the decoding center is essential for EF-G-dependent translocation.

Translocation is an essential step in the elongation cycle of the protein synthesis that allows for the continual incorporation of new amino acids to the growing polypeptide. Movement of mRNA and tRNAs within the ribosome is catalyzed by EF-G binding and GTP hydrolysis. The 30S subunit decoding center is crucial for the selection of the cognate tRNA. However, it is not clear whether the decoding center participates in translocation. We disrupted the interactions in the decoding center by mutating the universally conserved 16S rRNA bases G530, A1492, and A1493, and the effects of these mutations on translocation were studied. Our results show that point mutation of any of these 16S rRNA bases inhibits EF-G-dependent translocation. Furthermore, the mutant ribosomes showed increased puromycin reactivity in the pretranslocation complexes, indicating that the dynamic equilibrium of the peptidyl tRNA between the classical and hybrid-state configurations is influenced by contacts in the decoding center.

Design at the atomic level: design of biaryloxazolidinones as potent orally active antibiotics.

We have developed a first generation of hybrid sparsomycin-linezolid compounds into a new family of orally bioavailable biaryloxazolidinones that have activity against both linezolid-susceptible and -resistant gram-positive bacteria as well as the fastidious gram-negative bacteria Haemophilus influenzae and Moraxella catarrahalis. The convergent synthesis of these new compounds is detailed.

Conserved residues Asp16 and Pro24 of TnaC-tRNAPro participate in tryptophan induction of Tna operon expression.

In Escherichia coli, interactions between the nascent TnaC-tRNA(Pro) peptidyl-tRNA and the translating ribosome create a tryptophan binding site in the ribosome where bound tryptophan inhibits TnaC-tRNA(Pro) cleavage. This inhibition delays ribosome release, thereby inhibiting Rho factor binding and action, resulting in increased tna operon transcription. Replacing Trp12 of TnaC with any other amino acid residue was previously shown to prevent tryptophan binding and induction of tna operon expression. Genome-wide comparisons of TnaC amino acid sequences identify Asp16 and Pro24, as well as Trp12, as highly conserved TnaC residues. Replacing these residues with other residues was previously shown to influence tryptophan induction of tna operon expression. In this study, in vitro analyses were performed to examine the potential roles of Asp16 and Pro24 in tna operon induction. Replacing Asp16 or Pro24 of TnaC of E. coli with other amino acids established that these residues are essential for free tryptophan binding and inhibition of TnaC-tRNA(Pro) cleavage at the peptidyl transferase center. Asp16 and Pro24 are in fact located in spatial positions corresponding to critical residues of AAP, another ribosome regulatory peptide. Sparsomycin-methylation protection studies further suggested that segments of 23S RNA were arranged differently in ribosomes bearing TnaCs with either the Asp16Ala or the Pro24Ala change. Thus, features of the amino acid sequence of TnaC of the nascent TnaC-tRNA(Pro) peptidyl-tRNA, in addition to the presence of Trp12, are necessary for the nascent peptide to create a tryptophan binding/inhibition site in the translating ribosome.

rRNA mutants in the yeast peptidyltransferase center reveal allosteric information networks and mechanisms of drug resistance.

To ensure accurate and rapid protein synthesis, nearby and distantly located functional regions of the ribosome must dynamically communicate and coordinate with one another through a series of information exchange networks. The ribosome is approximately 2/3 rRNA and information should pass mostly through this medium. Here, two viable mutants located in the peptidyltransferase center (PTC) of yeast ribosomes were created using a yeast genetic system that enables stable production of ribosomes containing only mutant rRNAs. The specific mutants were C2820U (Escherichia coli C2452) and Psi2922C (E. coli U2554). Biochemical and genetic analyses of these mutants suggest that they may trap the PTC in the 'open' or aa-tRNA bound conformation, decreasing peptidyl-tRNA binding. We suggest that these structural changes are manifested at the biological level by affecting large ribosomal subunit biogenesis, ribosomal subunit joining during initiation, susceptibility/resistance to peptidyltransferase inhibitors, and the ability of ribosomes to properly decode termination codons. These studies also add to our understanding of how information is transmitted both locally and over long distances through allosteric networks of rRNA-rRNA and rRNA-protein interactions.

Ribosomal features essential for tna operon induction: tryptophan binding at the peptidyl transferase center.

Features of the amino acid sequence of the TnaC nascent peptide are recognized by the translating ribosome. Recognition leads to tryptophan binding within the translating ribosome, inhibiting the termination of tnaC translation and preventing Rho-dependent transcription termination in the tna operon leader region. It was previously shown that inserting an adenine residue at position 751 or introducing the U2609C change in 23S rRNA or introducing the K90W replacement in ribosomal protein L22 prevented tryptophan induction of tna operon expression. It was also observed that an adenine at position 752 of 23S rRNA was required for induction. In the current study, the explanation for the lack of induction by these altered ribosomes was investigated. Using isolated TnaC-ribosome complexes, it was shown that although tryptophan inhibits puromycin cleavage of TnaC-tRNA(Pro) with wild-type ribosome complexes, it does not inhibit cleavage with the four mutant ribosome complexes examined. Similarly, tryptophan prevents sparsomycin inhibition of TnaC-tRNA(Pro) cleavage with wild-type ribosome complexes but not with these mutant ribosome complexes. Additionally, a nucleotide located close to the peptidyl transferase center, A2572, which was protected from methylation by tryptophan with wild-type ribosome complexes, was not protected with mutant ribosome complexes. These findings identify specific ribosomal residues located in the ribosome exit tunnel that recognize features of the TnaC peptide. This recognition creates a free tryptophan-binding site in the peptidyl transferase center, where bound tryptophan inhibits peptidyl transferase activity.

Rapid propagation of low-fitness drug-resistant mutants of human immunodeficiency virus type 1 by a streptococcal metabolite sparsomycin.

Here we report that sparsomycin, a streptococcal metabolite, enhances the replication of HIV-1 in multiple human T cell lines at a concentration of 400 nM. In addition to wild-type HIV-1, sparsomycin also accelerated the replication of low-fitness, drug-resistant mutants carrying either D30N or L90M within HIV-1 protease, which are frequently found mutations in HIV-1-infected patients on highly active antiretroviral therapy (HAART). Of particular interest was that replication enhancement appeared profound when HIV-1 such as the L90M-carrying mutant displayed relatively slower replication kinetics. The presence of sparsomycin did not immediately select the fast-replicating HIV-1 mutants in culture. In addition, sparsomycin did not alter the 50% inhibitory concentration (IC50) of antiretroviral drugs directed against HIV-1 including nucleoside reverse transcriptase inhibitors (lamivudine and stavudine), non-nucleoside reverse transcriptase inhibitor (nevirapine) and protease inhibitors (nelfinavir, amprenavir and indinavir). The IC50s of both zidovudine and lopinavir against multidrug resistant HIV-1 in the presence of sparsomycin were similar to those in the absence of sparsomycin. The frameshift reporter assay and Western blot analysis revealed that the replication-boosting effect was partly due to the sparsomycin's ability to increase the -1 frameshift efficiency required to produce the Gag-Pol transcript. In conclusion, the use of sparsomycin should be able to facilitate the drug resistance profiling of the clinical isolates and the study on the low-fitness viruses.

Regiospecificity of the peptidyl tRNA ester within the ribosomal P site.

The ribosomal peptidyl transferase center is expected to be regiospecific with regard to its tRNA substrates, yet the ester linkages between the tRNA and the amino acid or peptide are susceptible to isomerization between the O2' and O3' hydroxyls of the terminal A76 ribose sugar. To establish which isomer of the P site tRNA ester is utilized by the ribosome, we prepared two nonisomerizable transition state inhibitors with either an A76 O2' or O3' linkage. Strong preferential binding to the O3' regioisomer indicates that the peptidyl transferase proceeds through a transition state with an O3'-linked peptide in the P-site.

The hybrid state of tRNA binding is an authentic translation elongation intermediate.

The GTPase elongation factor (EF)-G is responsible for promoting the translocation of the messenger RNA-transfer RNA complex on the ribosome, thus opening up the A site for the next aminoacyl-tRNA. Chemical modification and cryo-EM studies have indicated that tRNAs can bind the ribosome in an alternative 'hybrid' state after peptidyl transfer and before translocation, though the relevance of this state during translation elongation has been a subject of debate. Here, using pre-steady-state kinetic approaches and mutant analysis, we show that translocation by EF-G is most efficient when tRNAs are bound in a hybrid state, supporting the argument that this state is an authentic intermediate during translation.

An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA.

The large ribosomal subunit catalyses the reaction between the alpha-amino group of the aminoacyl-tRNA bound to the A site and the ester carbon of the peptidyl-tRNA bound to the P site, while preventing the nucleophilic attack of water on the ester, which would lead to unprogrammed deacylation of the peptidyl-tRNA. Here we describe three new structures of the large ribosomal subunit of Haloarcula marismortui (Hma) complexed with peptidyl transferase substrate analogues that reveal an induced-fit mechanism in which substrates and active-site residues reposition to allow the peptidyl transferase reaction. Proper binding of an aminoacyl-tRNA analogue to the A site induces specific movements of 23S rRNA nucleotides 2618-2620 (Escherichia coli numbering 2583-2585) and 2541(2506), thereby reorienting the ester group of the peptidyl-tRNA and making it accessible for attack. In the absence of the appropriate A-site substrate, the peptidyl transferase centre positions the ester link of the peptidyl-tRNA in a conformation that precludes the catalysed nucleophilic attack by water. Protein release factors may also function, in part, by inducing an active-site rearrangement similar to that produced by the A-site aminoacyl-tRNA, allowing the carbonyl group and water to be positioned for hydrolysis.

Ribosomal protein L3: influence on ribosome structure and function.

Early studies demonstrated roles for ribosomal protein L3 in peptidyltransferase center formation and the ability of cells to propagate viruses. More recent studies have linked these two processes via the effects of mutants and drugs on programmed -1 ribosomal frameshifting. Here, we show that mutant forms of L3 result in ribosomes having increased affinities for both aminoacyl- and peptidyl-tRNAs. These defects potentiate the effects of sparsomycin, which promotes increased aminoalcyl-tRNA binding at the P-site, while antagonizing the effects anisomycin, a drug that promotes decreased peptidyl-tRNA binding at the A-site. The changes in ribosome affinities for tRNAs also correlate with decreased peptidyltransferase activities of mutant ribosomes, and with decreased rates of cell growth and protein synthesis. In vivo dimethylsulfate (DMS) protection studies reveal that small changes in L3 primary sequence also have significant effects on rRNA structure as far away as 100 A, supporting an allosteric model of ribosome function.

Structures of five antibiotics bound at the peptidyl transferase center of the large ribosomal subunit.

Structures of anisomycin, chloramphenicol, sparsomycin, blasticidin S, and virginiamycin M bound to the large ribosomal subunit of Haloarcula marismortui have been determined at 3.0A resolution. Most of these antibiotics bind to sites that overlap those of either peptidyl-tRNA or aminoacyl-tRNA, consistent with their functioning as competitive inhibitors of peptide bond formation. Two hydrophobic crevices, one at the peptidyl transferase center and the other at the entrance to the peptide exit tunnel play roles in binding these antibiotics. Midway between these crevices, nucleotide A2103 of H.marismortui (2062 Escherichia coli) varies in its conformation and thereby contacts antibiotics bound at either crevice. The aromatic ring of anisomycin binds to the active-site hydrophobic crevice, as does the aromatic ring of puromycin, while the aromatic ring of chloramphenicol binds to the exit tunnel hydrophobic crevice. Sparsomycin contacts primarily a P-site bound substrate, but also extends into the active-site hydrophobic crevice. Virginiamycin M occupies portions of both the A and P-site, and induces a conformational change in the ribosome. Blasticidin S base-pairs with the P-loop and thereby mimics C74 and C75 of a P-site bound tRNA.