Regulation of gene expression at the level of translation accounts for up to three orders of magnitude in its efficiency. We systematically compared the impact of several mRNA features on translation initiation at the first gene in an operon with those for the second gene. Experiments were done in a system with internal control based on dual cerulean and red (CER/RFP) fluorescent proteins. We demonstrated significant differences in the efficiency of Shine Dalgarno sequences acting at the leading gene and at the following genes in an operon. The majority of frequent intercistronic arrangements possess medium SD dependence, medium dependence on the preceding cistron translation and efficient stimulation by A/U-rich sequences. The second cistron starting immediately after preceding cistron stop codon displays unusually high dependence on the SD sequence.

SUMMARY We demonstrate that the antibiotic amicoumacin A (AMI) whose cellular target was unknown, is a potent inhibitor of protein synthesis. Resistance mutations in helix 24 of the 16S rRNA mapped the AMI binding site to the small ribosomal subunit. The crystal structure of bacterial ribosome in complex with AMI solved at 2.4 Å resolution revealed that the antibiotic makes contacts with universally conserved nucleotides of 16S rRNA in the E site and the mRNA backbone. Simultaneous interactions of AMI with 16S rRNA and mRNA and the in vivo experimental evidence suggest that it may inhibit the progression of the ribosome along mRNA. Consistent with this proposal, binding of AMI interferes with translocation in vitro. The inhibitory action of AMI can be partly compensated by mutations in the translation elongation factor G.

SignificanceAnalyzing complex microbial communities is the milestone of modern microbiology, calling for “deep functional profiling” techniques. While next generation sequencing revolutionized our understanding of microbiota communities, we still lack high-throughput technologies to precisely determine their functionality. Here we show how cultivation of individual bacteria inside droplets of microfluidic double water-in-oil-in-water emulsion enables us to isolate the clones with a desired activity. This approach allows us not only to select the potent antibiotic producer but also to discover a distinct mechanism of self-resistance as well as assess its efficiency on entire microbiomes. The outcome of this methodology shows that it could be effectively transferred to numerous applications in microbiology and biotechnology.

Yield of protein per translated mRNA may vary by four orders of magnitude. Many studies analyzed the influence of mRNA features on the translation yield. However, a detailed understanding of how mRNA sequence determines its propensity to be translated is still missing. Here, we constructed a set of reporter plasmid libraries encoding CER fluorescent protein preceded by randomized 5΄ untranslated regions (5΄-UTR) and Red fluorescent protein (RFP) used as an internal control. Each library was transformed into Escherchia coli cells, separated by efficiency of CER mRNA translation by a cell sorter and subjected to next generation sequencing. We tested efficiency of translation of the CER gene preceded by each of 48 natural 5΄-UTR sequences and introduced random and designed mutations into natural and artificially selected 5΄-UTRs. Several distinct properties could be ascribed to a group of 5΄-UTRs most efficient in translation. In addition to known ones, several previously unrecognized features that contribute to the translation enhancement were found, such as low proportion of cytidine residues, multiple SD sequences and AG repeats. The latter could be identified as translation enhancer, albeit less efficient than SD sequence in several natural 5΄-UTRs.

This review centers on the stationary phase of bacterial culture. The basic processes specific to the stationary phase, as well as the regulatory mechanisms that allow the bacteria to survive in conditions of stress, are described.

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

e In order to accelerate drug discovery, a simple, reliable, and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double-reporter system for not only antimicrobial activity detection but also simultaneous sorting of potential antimicrobials into those that cause ribosome stalling and those that induce the SOS response due to DNA damage. In this reporter system, the red fluorescent protein gene rfp was placed under the control of the SOS-inducible sulA promoter. The gene of the far-red fluorescent protein, katushka2S, was inserted downstream of the tryptophan attenuator in which two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to ribosome-stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need of enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals.T he spread of antibiotic resistance genes among pathogenic bacteria is leading to a gradual decrease in the efficiency of known antibiotics. Substantial efforts have been invested in platforms for new antibiotic development (1). High-throughput screening (HTS) is a major method for the discovery of new chemical scaffolds for drug discovery. However, in the search for new antibiotics, HTS demonstrated low efficiency (for a discussion, see references 2 and 3). Acceleration of the antibiotic development pipeline demands increased efficiency in the identification of mechanisms of action with both HTS of chemical libraries and screening of natural compounds. Ideally, the mechanism of action should be determined while screening for antibacterial activity. One of the major challenges in high-throughput screening is the development of a cost-effective procedure that could maximize information output while concomitantly minimizing the number of pipetting steps and the reagent costs.The most efficient way to reveal the mechanism of action is the application of reporter strains (for a recent review, see reference 4). However, the majority of the reporter strains developed thus far aim to identify a narrow group of chemically related compounds, such as tetracyclines (5), macrolides (6, 7), or ␤-lactams (8). Broader-spectrum reporters based on stress response promoters are also available (9-11). A combination of several reporter strains could help to classify more mechanisms of action, but that would require multiple experiments for a single substance being tested.The majority of antibiotics currently in clinical use target the cell wall, DNA, or protein biosynthesis. For the latter two mechanisms ...

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.