Natural and engineered hydroxyectoine production based on the Pseudomonas stutzeri ectABCD-ask gene cluster.

We report on the presence of a functional hydroxyectoine biosynthesis gene cluster, ectABCD-ask, in Pseudomonas stutzeri DSM5190(T) and evaluate the suitability of P. stutzeri DSM5190(T) for hydroxyectoine production. Furthermore, we present information on heterologous de novo production of the compatible solute hydroxyectoine in Escherichia coli. In this host, the P. stutzeri gene cluster remained under the control of its salt-induced native promoters. We also noted the absence of trehalose when hydroxyectoine genes were expressed, as well as a remarkable inhibitory effect of externally applied betaine on hydroxyectoine synthesis. The specific heterologous production rate in E. coli under the conditions employed exceeded that of the natural producer Pseudomonas stutzeri and, for the first time, enabled effective hydroxyectoine production at low salinity (2%), with the added advantage of simple product processing due to the absence of other cosolutes.

Structural and mechanistic basis for translation inhibition by macrolide and ketolide antibiotics.

Macrolides and ketolides comprise a family of clinically important antibiotics that inhibit protein synthesis by binding within the exit tunnel of the bacterial ribosome. While these antibiotics are known to interrupt translation at specific sequence motifs, with ketolides predominantly stalling at Arg/Lys-X-Arg/Lys motifs and macrolides displaying a broader specificity, a structural basis for their context-specific action has been lacking. Here, we present structures of ribosomes arrested during the synthesis of an Arg-Leu-Arg sequence by the macrolide erythromycin (ERY) and the ketolide telithromycin (TEL). Together with deep mutagenesis and molecular dynamics simulations, the structures reveal how ERY and TEL interplay with the Arg-Leu-Arg motif to induce translational arrest and illuminate the basis for the less stringent sequence-specific action of ERY over TEL. Because programmed stalling at the Arg/Lys-X-Arg/Lys motifs is used to activate expression of antibiotic resistance genes, our study also provides important insights for future development of improved macrolide antibiotics.

Genome-driven investigation of compatible solute biosynthesis pathways of Pseudomonas syringae pv. syringae and their contribution to water stress tolerance.

The foliar pathogen Pseudomonas syringae pv. syringae exhibits an exceptional ability to survive on asymptomatic plants as an epiphyte. Intermittent wetting events on plants lead to osmotic and matric stresses which must be tolerated for survival as an epiphyte. In this study, we have applied bioinformatic, genetic, and biochemical approaches to address water stress tolerance in P. syringae pv. syringae strain B728a, for which a complete genome sequence is available. P. syringae pv. syringae B728a is able to produce the compatible solutes betaine, ectoine, N-acetylglutaminylglutamine amide (NAGGN), and trehalose. Analysis of osmolyte profiles of P. syringae pv. syringae B728a under a variety of in vitro and in planta conditions reveals that the osmolytes differentially contribute to water stress tolerance in this species and that they interact at the level of transcription to yield a hierarchy of expression. While the interruption of a putative gene cluster coding for NAGGN biosynthesis provided the first experimental evidence of the NAGGN biosynthetic pathway, application of this knockout strain and also a gfp reporter gene fusion strain demonstrated the small contribution of NAGGN to cell survival and desiccation tolerance of P. syringae pv. syringae B728a under in planta conditions. Additionally, detailed investigation of ectC, an orphan of the ectoine cluster (lacking the ectA and ectB homologs), revealed its functionality and that ectoine production could be detected in NaCl-amended cultures of P. syringae pv. syringae B728a to which sterilized leaves of Syringa vulgaris had been added.

Ornithine capture by a translating ribosome controls bacterial polyamine synthesis.

Polyamines are essential metabolites that play an important role in cell growth, stress adaptation and microbial virulence1-3. To survive and multiply within a human host, pathogenic bacteria adjust the expression and activity of polyamine biosynthetic enzymes in response to different environmental stresses and metabolic cues2. Here, we show that ornithine capture by the ribosome and the nascent peptide SpeFL controls polyamine synthesis in γ-proteobacteria by inducing the expression of the ornithine decarboxylase SpeF4, via a mechanism involving ribosome stalling and transcription antitermination. In addition, we present the cryogenic electron microscopy structure of an Escherichia coli ribosome stalled during translation of speFL in the presence of ornithine. The structure shows how the ribosome and the SpeFL sensor domain form a highly selective binding pocket that accommodates a single ornithine molecule but excludes near-cognate ligands. Ornithine pre-associates with the ribosome and is then held in place by the sensor domain, leading to the compaction of the SpeFL effector domain and blocking the action of release factor 1. Thus, our study not only reveals basic strategies by which nascent peptides assist the ribosome in detecting a specific metabolite, but also provides a framework for assessing how ornithine promotes virulence in several human pathogens.

Publisher Correction: Ornithine capture by a translating ribosome controls bacterial polyamine synthesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ribosomal stalling landscapes revealed by high-throughput inverse toeprinting of mRNA libraries.

Although it is known that the amino acid sequence of a nascent polypeptide can impact its rate of translation, dedicated tools to systematically investigate this process are lacking. Here, we present high-throughput inverse toeprinting, a method to identify peptide-encoding transcripts that induce ribosomal stalling in vitro. Unlike ribosome profiling, inverse toeprinting protects the entire coding region upstream of a stalled ribosome, making it possible to work with random or focused transcript libraries that efficiently sample the sequence space. We used inverse toeprinting to characterize the stalling landscapes of free and drug-bound Escherichia coli ribosomes, obtaining a comprehensive list of arrest motifs that were validated in vivo, along with a quantitative measure of their pause strength. Thanks to the modest sequencing depth and small amounts of material required, inverse toeprinting provides a highly scalable and versatile tool to study sequence-dependent translational processes.

How Widespread is Metabolite Sensing by Ribosome-Arresting Nascent Peptides?

In order to colonize a niche and compete for scarce resources, microorganisms have evolved means to adjust the expression levels of their biosynthetic enzymes in response to the changing levels of metabolites available to them. To do so, they often rely on transcription factors or structured RNAs that directly sense the concentration of metabolites and turn genes on or off accordingly. In some instances, however, a metabolite can be sensed by an actively translating ribosome bearing a nascent polypeptide whose specific amino acid sequence interferes with translation. These "arrest peptides" lead to the formation of stalled ribosome nascent chain complexes on the mRNA that can regulate the expression of downstream genes through transcriptional or translational mechanisms. Although this process was discovered over three and a half decades ago, the extent to which arrest peptides regulate gene expression in response to cell metabolites is unknown. Here, we examine the physical constraints imposed by the ribosome on peptide-mediated ligand sensing and review attempts to assess the diversity of arrest peptides to date. In addition, we outline a possible way forward to establish how pervasive metabolite sensing by arrest peptides is in nature.