Recognition of tRNAIle with a UAU anticodon by isoleucyl-tRNA synthetase in lactic acid bacteria.

In almost all eubacteria, the AUA codon is translated by tRNAIle2 bearing lysidine at the wobble position. Lysidine is introduced by tRNAIle lysidine synthetase (TilS) via post-transcriptional modification of the cytidine of tRNAIle2 (CAU). Lactobacillus casei and Lactobacillus plantarum have tilS homologues and tRNAIle2 (CAU) genes. In addition, L. casei also has another tRNAIle2 gene with an UAU anticodon. L. plantarum has a tRNAIle (UAU)-like RNA. Here, we demonstrate that L. casei tRNAIle2 (UAU) is charged with isoleucine by L. casei isoleucyl-tRNA synthetase (IleRS) but not by L. plantarum IleRS, even though the amino acid identity of these two enzymes is over 60%. It has been reported that, in Mycoplasma mobile, which has its tRNAIle2 (UAU) but no tilS homologue, an Arg residue at position 865 of the IleRS is required for recognition of the UAU anticodon. This position is occupied by an Arg also in the IleRSs from both of the Lactobacillus species. Thus, other residues in L. casei, IleRS should also contribute to the recognition of tRNAIle2 (UAU). We found that a chimeric L. casei IleRS in which the N-terminal domain was replaced by the corresponding region of L. plantatarum IleRS has very low aminoacylation activity towards both tRNAIle2 (UAU) and tRNAIle1 (GAU). The A18G mutant had barely detectable aminoacylation activity towards either of the tRNAsIle . However, a double point mutant of A18G and G19N aminoacylated tRNAIle1 (GAU), but not tRNAIle2 (UAU). Our results suggest that, for L. casei IleRS, Ala18 and Gly19 also play a critical role in recognition of tRNAIle2 (UAU).

Fluorescent Aminoglycoside Antibiotics and Methods for Accurately Monitoring Uptake by Bacteria.

Characterizing how multidrug-resistant bacteria circumvent the action of clinically used or novel antibiotics requires a detailed understanding of how the antibiotics interact with and cross bacterial membranes to accumulate in the cells and exert their action. When monitoring the interactions of drugs with bacteria, it remains challenging to differentiate functionally relevant internalized drug levels from nonspecific binding. Fluorescence is a method of choice for observing dynamics of biomolecules. In order to facilitate studies involving aminoglycoside antibiotics, we have generated fluorescently labeled aminoglycoside derivatives with uptake and bactericidal activities similar, albeit with a moderate loss, to those of the parent drug. The method combines fluorescence microscopy with fluorescence-activated cell sorting (FACS) using neomycin coupled to nonpermeable cyanine dyes. Fluorescence imaging allowed membrane-bound antibiotic to be distinguished from molecules in the cytoplasm. Patterns of uptake were assigned to different populations in the FACS analysis. Our study illustrates how fluorescent derivatives of an aminoglycoside enable a robust characterization of the three components of uptake: membrane binding, EDPI, and EDPII. Because EDPI levels are weak compared to the two other types of accumulation and critical for the action of these drugs, the three components of uptake must be taken into account separately when drawing conclusions about aminoglycoside function.

Profiling of modified nucleosides from ribonucleic acid digestion by supercritical fluid chromatography coupled to high resolution mass spectrometry.

A method based on supercritical fluid chromatography coupled to high resolution mass spectrometry for the profiling of canonical and modified nucleosides was optimized, and compared to classical reverse-phase liquid chromatography in terms of separation, number of detected modified nucleosides and sensitivity. Limits of detection and quantification were measured using statistical method and quantifications of twelve nucleosides of a tRNA digest from E. coli are in good agreement with previously reported data. Results highlight the complementarity of both separation techniques to cover the largest view of nucleoside modifications for forthcoming epigenetic studies.

Characterization of redundant tRNAIles with CAU and UAU anticodons in Lactobacillus plantarum.

In most eubacteria, the minor AUA isoleucine codon is decoded by tRNAIle2, which has a lysidine (L) in the anticodon loop. The lysidine is introduced by tRNAIle-lysidine synthetase (TilS) through post-transcriptional modification of cytidine to yield an LAU anticodon. Some bacteria, Lactobacillus plantarum for example, possess two tRNAIle2(UAU) genes in addition to, two tRNAIle2(CAU) genes and the tilS gene. tRNA expression from all these genes would generate redundancy in a tRNA that decodes a rare AUA codon. In this study, we investigated the tRNA expression from these genes in L. plantarum and characterized the corresponding tRNAs. The tRNAIle2(CAU) gene products are modified by TilS to produce tRNAIle2(LAU), while tRNAIle2(UAU) lacks modification especially in the anticodon sequence. We found that tRNAIle2(LAU) is charged with isoleucine but tRNAIle2(UAU) is not. Our results suggest that the tRNAIle2 redundancy may be related to different roles of these tRNAs in the cell.

DNA-Guided Delivery of Single Molecules into Zero-Mode Waveguides.

Zero-mode waveguides (ZMWs) are powerful analytical tools corresponding to optical nanostructures fabricated in a thin metallic film capable of confining an excitation volume to the range of attoliters. This small volume of confinement allows single-molecule fluorescence experiments to be performed at physiologically relevant concentrations of fluorescently labeled biomolecules. Exactly one molecule to be studied must be attached at the floor of the ZMW for signal detection and analysis; however, the massive parallelism of these nanoarrays suffers from a Poissonian-limited distribution of these biomolecules. To date, there is no method available that provides full single-molecule occupancy of massively arrayed ZMWs. Here we report the performance of a DNA-guided method that uses steric exclusion properties of large DNA molecules to bias the Poissonian-limited delivery of single molecules. Non-Poissonian statistics were obtained with DNA molecules that contain a free-biotinylated extremity for efficient binding to the floor of the ZMW, which resulted in a decrease of accessibility for a second molecule. Both random-coiled and condensed DNA conformations drove non-Poissonian single-molecule delivery into ZMW arrays. The results suggest that an optimal balance between the rigidity and flexibility of the macromolecule is critical for favorable accessibility and single occupancy. The optimized method provides a means for full exploitation of these massively parallelized analytical tools.

Long and branched polyamines are required for maintenance of the ribosome, tRNAHis and tRNATyr in Thermus thermophilus cells at high temperatures.

Thermus thermophilus is an extremely thermophilic eubacterium that produces various polyamines. Aminopropylagmatine ureohydrolase (SpeB) and SAM decarboxylase-like protein 1 (SpeD1) are involved in the biosynthesis of spermidine from arginine. Because long and branched polyamines in T. thermophilus are synthesized from spermidine, the speB and speD1 gene-deleted strains (ΔspeB and ΔspeD1, respectively) cannot synthesize long and branched polyamines. Although neither strain grew at high temperatures (>75°C) in minimal medium, both strains survived at 80°C when they were cultured at 70°C until the mid-log phase and then shifted to 80°C. We therefore prepared the ΔspeB and ΔspeD1 cells using this culture method. Microscopic analysis showed that both strains can survive for 10 h after the temperature shift. Although the modification levels of 2'-O-methylguanosine at position 18, N7 -methylguanosine at position 46, 5-methyluridine at position 54 and N1 -methyladenosine at position 58 in the class I tRNA from both strains were normal, amounts of tRNATyr , tRNAHis , rRNAs and 70S ribosomes were decreased after the temperature shift. Furthermore, in vivo protein synthesis in both strains was completely lost 10 h after the temperature shift. Thus, long and branched polyamines are required for at least the maintenance of 70S ribosome and some tRNA species at high temperatures.

Use of Baby Spinach and Broccoli for imaging of structured cellular RNAs.

Fluorogenic RNA aptamers provide a powerful tool for study of RNA analogous to green fluorescent protein for the study of proteins. Spinach and Broccoli are RNAs selected in vitro or in vivo respectively to bind to an exogenous chromophore. They can be genetically inserted into an RNA of interest for live-cell imaging. Spinach aptamer has been altered to increase thermal stability and stabilize the desired folding. How well these fluorogenic RNA aptamers behave when inserted into structured cellular RNAs and how aptamer properties might be affected remains poorly characterized. Here, we report a study of the performance of distinct RNA Spinach and Broccoli aptamer sequences in isolation or inserted into the small subunit of the bacterial ribosome. We found that the ribosomal context helped maintaining the yield of the folded Baby Spinach aptamer; other versions of Spinach did not perform well in the context of ribosomes. In vivo, two aptamers clearly stood out. Baby Spinach and Broccoli aptamers yielded fluorescence levels markedly superior to all previous Spinach sequences including the super-folder tRNA scaffolded tSpinach2. Overall, the results suggest the use of Broccoli and Baby Spinach aptamers for live cell imaging of structured RNAs.

Folate-/FAD-dependent tRNA methyltransferase from Thermus thermophilus regulates other modifications in tRNA at low temperatures.

TrmFO is a N(5) , N(10) -methylenetetrahydrofolate (CH2 THF)-/FAD-dependent tRNA methyltransferase, which synthesizes 5-methyluridine at position 54 (m(5) U54) in tRNA. Thermus thermophilus is an extreme-thermophilic eubacterium, which grows in a wide range of temperatures (50-83 °C). In T. thermophilus, modified nucleosides in tRNA and modification enzymes form a network, in which one modification regulates the degrees of other modifications and controls the flexibility of tRNA. To clarify the role of m(5) U54 and TrmFO in the network, we constructed the trmFO gene disruptant (∆trmFO) strain of T. thermophilus. Although this strain did not show any growth retardation at 70 °C, it showed a slow-growth phenotype at 50 °C. Nucleoside analysis showed increase in 2'-O-methylguanosine at position 18 and decrease in N(1) -methyladenosine at position 58 in the tRNA mixture from the ∆trmFO strain at 50 °C. These in vivo results were reproduced by in vitro experiments with purified enzymes. Thus, we concluded that the m(5) U54 modification have effects on the other modifications in tRNA through the network at 50 °C. (35) S incorporations into proteins showed that the protein synthesis activity of ∆trmFO strain was inferior to the wild-type strain at 50 °C, suggesting that the growth delay at 50 °C was caused by the inferior protein synthesis activity.

Predicting the minimal translation apparatus: lessons from the reductive evolution of mollicutes.

Mollicutes is a class of parasitic bacteria that have evolved from a common Firmicutes ancestor mostly by massive genome reduction. With genomes under 1 Mbp in size, most Mollicutes species retain the capacity to replicate and grow autonomously. The major goal of this work was to identify the minimal set of proteins that can sustain ribosome biogenesis and translation of the genetic code in these bacteria. Using the experimentally validated genes from the model bacteria Escherichia coli and Bacillus subtilis as input, genes encoding proteins of the core translation machinery were predicted in 39 distinct Mollicutes species, 33 of which are culturable. The set of 260 input genes encodes proteins involved in ribosome biogenesis, tRNA maturation and aminoacylation, as well as proteins cofactors required for mRNA translation and RNA decay. A core set of 104 of these proteins is found in all species analyzed. Genes encoding proteins involved in post-translational modifications of ribosomal proteins and translation cofactors, post-transcriptional modifications of t+rRNA, in ribosome assembly and RNA degradation are the most frequently lost. As expected, genes coding for aminoacyl-tRNA synthetases, ribosomal proteins and initiation, elongation and termination factors are the most persistent (i.e. conserved in a majority of genomes). Enzymes introducing nucleotides modifications in the anticodon loop of tRNA, in helix 44 of 16S rRNA and in helices 69 and 80 of 23S rRNA, all essential for decoding and facilitating peptidyl transfer, are maintained in all species. Reconstruction of genome evolution in Mollicutes revealed that, beside many gene losses, occasional gains by horizontal gene transfer also occurred. This analysis not only showed that slightly different solutions for preserving a functional, albeit minimal, protein synthetizing machinery have emerged in these successive rounds of reductive evolution but also has broad implications in guiding the reconstruction of a minimal cell by synthetic biology approaches.

Ultra-high density protein spots achieved by on chip digitalized protein synthesis.

Current methodologies for arraying proteins using cell-free protein synthesis on a chip have spatial limitations that prevent reaching ultra-high density necessary for high throughput analysis. To circumvent this, we developed an on-chip method based on microcompartmentalization of protein synthesis. Proteins are synthesized in arrayed micrometer scale chambers from confined DNA template molecules. On-chip protein expression is highly efficient and the method can be used with a minimal amount of template i.e. single DNA molecules to perform digitalized cell-free protein synthesis (d-CFPS). A functionalized surface at the floor of the tightly sealed microchambers enables direct capture of expressed proteins. A density of 104 spots per mm² was achieved, which represents a gain by more than 3 orders of magnitude over conventional methods. This technique of forming such densely arrayed small protein spots is the first step towards the development of a general method that would allow fabrication of ultra-high density protein arrays for high-throughput analysis.

Enzymatic reaction in droplets manipulated with liquid dielectrophoresis.

Droplet generation and transportation for biological reactions are conducted with liquid dielectrophoresis (LDEP), forming two hundred picoliter droplets and aligning them in an open environment above the micro-machined electrodes. The generation of the dielectrophoresis signals was critically examined to actuate droplets in biological solutions without excessive Joule heating. Enzymatic reactions between ß-galactosidase and fluorescein di-ß-D-galactopyranoside were succeeded in manipulated droplets, which was confirmed by fluorescence imaging. These results allow us to propose the integration of LDEP actuation in high throughput biomolecular assays.

Protein-RNA footprinting: an evolving tool.

As more RNA molecules with important cellular functions are discovered, there is a strong need to characterize their structures, functions, and interactions. Chemical and enzymatic footprinting methods are used to map RNA secondary and tertiary structure, to monitor ligand interactions and conformational changes, and in the study of protein-RNA interactions. These methods provide data at single-nucleotide resolution that nicely complements the structural information available from X-ray diffraction, nuclear magnetic resonance spectroscopy (NMR), or cryo-electron microscopy. Footprinting methods also complement the dynamic information derived from single-molecule Förster resonance energy transfer. RNA footprinting tools have been used for decades, but we have recently seen spectacular advances, for instance, the use in combination with massive parallel sequencing techniques. Large libraries of RNA molecules (small or large in size) can now be probed in high-throughput manner when RNA footprinting methods are combined with fluorescent probe technologies and automation. In this article, after a brief historical overview, we summarize recent advances in RNA-protein footprinting methodologies that now integrate tools for massive parallel analysis.

Micro and nanotechnological tools for study of RNA.

Micro and nanotechnologies have originally contributed to engineering, especially in electronics. These technologies enable fabrication and assembly of materials at micrometer and nanometer scales and the manipulation of nano-objects. The power of these technologies has now been exploited in analyzes of biologically relevant molecules. In this review, the use of micro and nanotechnological tools in RNA research is described.

Complex transcriptional regulation of citrate metabolism in Clostridium perfringens.

A Gram-positive, spore-forming bacterium, Clostridium perfringens, possesses genes for citrate metabolism, which might play an important role in the utilization of citrate as a sole carbon source. In this study, we identified a chromosomal citCDEFX-mae-citS operon in C. perfringens strain 13, which is transcribed on three mRNAs of different sizes. Expression of the cit operon was significantly induced when 5 mM extracellular citrate was added to the growth medium. Most interestingly, three regulatory systems were found to be involved in the regulation of the expression of cit genes: 1) the two upstream divergent genes citG and citI; 2) two different two-component regulatory systems, CitA/CitB (TCS6 consisted of CPE0531/CPE0532) and TCS5 (CPE0518/CPE0519); and 3) the global two-component VirR/VirS-VR-RNA regulatory system known to regulate various genes for toxins and degradative enzymes. Our results suggest that in C. perfringens the citrate metabolism might be strictly controlled by a complex regulatory system.

Rapid detection of a cocaine-binding aptamer using biological nanopores on a chip.

This paper describes a methodology for the rapid and highly selective detection of cocaine using a membrane protein channel combined with a DNA aptamer. The DNA aptamer recognizes the cocaine molecule with high selectivity. We successfully detected a low concentration of cocaine (300 ng/mL, the drug test cutoff limit) within 60 s using a biological nanopore embedded in a microchip.

Lipid-coated microdroplet array for in vitro protein synthesis.

Monitoring complex biological assays such as in vitro protein synthesis over long periods in micrometer-sized cavities of poly(dimethyl siloxane) (PDMS) microfluidic devices requires a strategy that solves the adsorption and absorption problems on PDMS surfaces. In this study, we developed a technique that instantaneously arrays aqueous microdroplets coated with a phospholipid membrane within a single microfluidic device. The simple lipid bilayer coating effectively inhibits the adsorption of proteins and DNA, whereas the encapsulation of the droplet reduces the area in contact with the PDMS surface, resulting in decreased absorption in part. Although the size becomes smaller during the first few hours, a lipid-coated microdroplet array demonstrated a temporal stability of more than 20 h and a size uniformity of CV 3% in the device. Furthermore, we succeeded in expressing a green fluorescent protein by confining an in vitro translation system in the microdroplets, which was confirmed by scanning the fluorescence spectrum of the droplets, demonstrating that the lipid coat secured the synthetic reaction from the adsorption problem.

Identification of a two-component VirR/VirS regulon in Clostridium perfringens.

Clostridium perfringens, a Gram-positive anaerobic pathogen, is a causative agent of human gas gangrene that leads to severe rapid tissue destruction and can cause death within hours unless treated immediately. Production of several toxins is known to be controlled by the two-component VirR/VirS system involving a regulatory RNA (VR-RNA) in C. perfringens. To elucidate the precise regulatory network governed by VirR/VirS and VR-RNA, a series of microarray screening using VirR/VirS and VR-RNA-deficient mutants was performed. Finally, by qRT-PCR analysis, 147 genes (30 single genes and 21 putative operons) were confirmed to be under the control of the VirR/VirS-VR-RNA regulatory cascade. Several virulence-related genes for alpha-toxin, kappa-toxin, hyaluronidases, sialidase, and capsular polysaccharide synthesis were found. Furthermore, some genes for catalytic enzymes, various genes for transporters, and many genes for energy metabolism were also found to be controlled by the cascade. Our data indicate that the VirR/VirS-VR-RNA system is a global gene regulator that might control multiple cellular functions to survive and multiply in the host, which would turn out to be a lethal flesh-eating infection.

Interaction of the HIV-1 frameshift signal with the ribosome.

Ribosomal frameshifting on viral RNAs relies on the mechanical properties of structural elements, often pseudoknots and more rarely stem-loops, that are unfolded by the ribosome during translation. In human immunodeficiency virus (HIV)-1 type B a long hairpin containing a three-nucleotide bulge is responsible for efficient frameshifting. This three-nucleotide bulge separates the hairpin in two domains: an unstable lower stem followed by a GC-rich upper stem. Toeprinting and chemical probing assays suggest that a hairpin-like structure is retained when ribosomes, initially bound at the slippery sequence, were allowed multiple EF-G catalyzed translocation cycles. However, while the upper stem remains intact the lower stem readily melts. After the first, and single step of translocation of deacylated tRNA to the 30 S P site, movement of the mRNA stem-loop in the 5' direction is halted, which is consistent with the notion that the downstream secondary structure resists unfolding. Mechanical stretching of the hairpin using optical tweezers only allows clear identification of unfolding of the upper stem at a force of 12.8 +/- 1.0 pN. This suggests that the lower stem is unstable and may indeed readily unfold in the presence of a translocating ribosome.