Spatiotemporal kinetics of the SRP pathway in live E. coli cells.

Mechanistic details of the signal recognition particle (SRP)-mediated insertion of membrane proteins have been described from decades of in vitro biochemical studies. However, the dynamics of the pathway inside the living cell remain obscure. By combining in vivo single-molecule tracking with numerical modeling and simulated microscopy, we have constructed a quantitative reaction-diffusion model of the SRP cycle. Our results suggest that the SRP-ribosome complex finds its target, the membrane-bound translocon, through a combination of three-dimensional (3D) and 2D diffusional search, together taking on average 750 ms. During this time, the nascent peptide is expected to be elongated only 12 or 13 amino acids, which explains why, in Escherichia coli, no translation arrest is needed to prevent incorrect folding of the polypeptide in the cytosol. We also found that a remarkably high proportion (75%) of SRP bindings to ribosomes occur in the cytosol, suggesting that the majority of target ribosomes bind SRP before reaching the membrane. In combination with the average SRP cycling time, 2.2 s, this result further shows that the SRP pathway is capable of targeting all substrate ribosomes to translocons.

Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes.

DNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of 'poisons', namely natural product toxins (e.g. albicidin, microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We show that a PRP, QnrB1 provides specific protection against fluoroquinolones, which strictly requires ATP hydrolysis by gyrase. QnrB1 binds to the GyrB protein and stimulates ATPase activity of the isolated N-terminal ATPase domain of GyrB (GyrB43). We probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped the crosslinks to the GyrB43 protein. We propose a model in which QnrB1 binding allosterically promotes dissociation of the fluoroquinolone molecule from the cleavage complex.

Structure of ribosome-bound azole-modified peptide phazolicin rationalizes its species-specific mode of bacterial translation inhibition.

Ribosome-synthesized post-translationally modified peptides (RiPPs) represent a rapidly expanding class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) - an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants. The cryo-EM structure of the Escherichia coli 70S-PHZ complex reveals that the drug interacts with the 23S rRNA and uL4/uL22 proteins and obstructs ribosomal exit tunnel in a way that is distinct from other compounds. We show that the uL4 loop sequence determines the species-specificity of antibiotic action. PHZ expands the known diversity of LAPs and may be used in the future as biocontrol agent for agricultural needs.

Biosynthesis of Translation Inhibitor Klebsazolicin Proceeds through Heterocyclization and N-Terminal Amidine Formation Catalyzed by a Single YcaO Enzyme.

Klebsazolicin (KLB) is a recently discovered Klebsiella pneumonia peptide antibiotic targeting the exit tunnel of bacterial ribosome. KLB contains an N-terminal amidine ring and four azole heterocycles installed into a ribosomally synthesized precursor by dedicated maturation machinery. Using an in vitro system for KLB production, we show that the YcaO-domain KlpD maturation enzyme is a bifunctional cyclodehydratase required for the formation of both the core heterocycles and the N-terminal amidine ring. We further demonstrate that the amidine ring is formed concomitantly with proteolytic cleavage of azole-containing pro-KLB by a cellular protease TldD/E. Members of the YcaO family are diverse enzymes known to activate peptide carbonyls during natural product biosynthesis leading to the formation of azoline, macroamidine, and thioamide moieties. The ability of KlpD to simultaneously perform two distinct types of modifications is unprecedented for known YcaO proteins. The versatility of KlpD opens up possibilities for rational introduction of modifications into various peptide backbones.

Klebsazolicin inhibits 70S ribosome by obstructing the peptide exit tunnel.

Whereas screening of the small-molecule metabolites produced by most cultivatable microorganisms often results in the rediscovery of known compounds, genome-mining programs allow researchers to harness much greater chemical diversity, and result in the discovery of new molecular scaffolds. Here we report the genome-guided identification of a new antibiotic, klebsazolicin (KLB), from Klebsiella pneumoniae that inhibits the growth of sensitive cells by targeting ribosomes. A ribosomally synthesized post-translationally modified peptide (RiPP), KLB is characterized by the presence of a unique N-terminal amidine ring that is essential for its activity. Biochemical in vitro studies indicate that KLB inhibits ribosomes by interfering with translation elongation. Structural analysis of the ribosome-KLB complex showed that the compound binds in the peptide exit tunnel overlapping with the binding sites of macrolides or streptogramin-B. KLB adopts a compact conformation and largely obstructs the tunnel. Engineered KLB fragments were observed to retain in vitro activity, and thus have the potential to serve as a starting point for the development of new bioactive compounds.

Acinetodin and Klebsidin, RNA Polymerase Targeting Lasso Peptides Produced by Human Isolates of Acinetobacter gyllenbergii and Klebsiella pneumoniae.

We report the bioinformatic prediction and structural validation of two lasso peptides, acinetodin and klebsidin, encoded by the genomes of several human-associated strains of Acinetobacter and Klebsiella. Computation of the three-dimensional structures of these peptides using NMR NOESY constraints verifies that they contain a lasso motif. Despite the lack of sequence similarity to each other or to microcin J25, a prototypical lasso peptide and transcription inhibitor from Escherichia coli, acinetodin and klebsidin also inhibit transcript elongation by the E. coli RNA polymerase by binding to a common site. Yet, unlike microcin J25, acinetodin and klebsidin are unable to permeate wild type E. coli cells and inhibit their growth. We show that the E. coli cells become sensitive to klebsidin when expressing the outer membrane receptor FhuA homologue from Klebsiella pneumoniae. It thus appears that specificity to a common target, the RNA polymerase secondary channel, can be attained by a surprisingly diverse set of primary sequences folded into a common threaded-lasso fold. In contrast, transport into cells containing sensitive targets appears to be much more specific and must be the major determinant of the narrow range of bioactivity of known lasso peptides.

Structure, bioactivity, and resistance mechanism of streptomonomicin, an unusual lasso Peptide from an understudied halophilic actinomycete.

Natural products are the most historically significant source of compounds for drug development. However, unacceptably high rates of compound rediscovery associated with large-scale screening of common microbial producers have resulted in the abandonment of many natural product drug discovery efforts, despite the increasing prevalence of clinically problematic antibiotic resistance. Screening of underexplored taxa represents one strategy to avoid rediscovery. Herein we report the discovery, isolation, and structural elucidation of streptomonomicin (STM), an antibiotic lasso peptide from Streptomonospora alba, and report the genome for its producing organism. STM-resistant clones of Bacillus anthracis harbor mutations to walR, the gene encoding a response regulator for the only known widely distributed and essential two-component signal transduction system in Firmicutes. To the best of our knowledge, Streptomonospora had been hitherto biosynthetically and genetically uncharacterized, with STM being the first reported compound from the genus. Our results demonstrate that understudied microbes remain fruitful reservoirs for the rapid discovery of novel, bioactive natural products.