The structural basis for mRNA recognition and cleavage by the ribosome-dependent endonuclease RelE.

Translational control is widely used to adjust gene expression levels. During the stringent response in bacteria, mRNA is degraded on the ribosome by the ribosome-dependent endonuclease, RelE. The molecular basis for recognition of the ribosome and mRNA by RelE and the mechanism of cleavage are unknown. Here, we present crystal structures of E. coli RelE in isolation (2.5 A) and bound to programmed Thermus thermophilus 70S ribosomes before (3.3 A) and after (3.6 A) cleavage. RelE occupies the A site and causes cleavage of mRNA after the second nucleotide of the codon by reorienting and activating the mRNA for 2'-OH-induced hydrolysis. Stacking of A site codon bases with conserved residues in RelE and 16S rRNA explains the requirement for the ribosome in catalysis and the subtle sequence specificity of the reaction. These structures provide detailed insight into the translational regulation on the bacterial ribosome by mRNA cleavage.

Evidence for Enzymatic Backbone Methylation of the Main Membrane Lipids in the Archaeon Methanomassiliicoccus luminyensis.

Butanetriol and pentanetriol dibiphytanyl glycerol tetraethers (BDGTs and PDGTs, respectively) are recently identified classes of archaeal membrane lipids that are prominent constituents in anoxic subseafloor sediments. These lipids are intriguing, as they possess unusual backbones with four or five carbon atoms instead of the canonical three-carbon glycerol backbone. In this study, we examined the biosynthesis of BDGTs and PDGTs by the methanogen Methanomassiliicoccus luminyensis, the only available isolate known to produce these compounds, via stable isotope labeling with [methyl-13C]methionine followed by mass spectrometry analysis. We show that their biosynthesis proceeds from transfer(s) of the terminal methyl group of methionine to the more common archaeal membrane lipids, i.e., glycerol dibiphytanyl glycerol tetraethers (GDGTs). As this methylation targets a methylene group, a radical mechanism involving a radical S-adenosylmethionine (SAM) enzyme is probable. Over the course of the incubation, the abundance of PDGTs relative to BDGTs, expressed as backbone methylation index, increased, implying that backbone methylation may be related to the growth shift to stationary conditions, possibly due to limited energy and/or substrate availability. The increase of the backbone methylation index with increasing sediment age in a sample set from the Mediterranean Sea adds support for such a relationship. IMPORTANCE Butanetriol and pentanetriol dibiphytanyl glycerol tetraethers are membrane lipids recently discovered in anoxic environments. These lipids differ from typical membrane-spanning tetraether lipids because they possess a non-glycerol backbone. The biosynthetic pathway and physiological role of these unique lipids are currently unknown. Here, we show that in the strain Methanomassiliicoccus luminyensis, these lipids are the result of methyl transfer(s) from an S-adenosyl methionine (SAM) intermediate. We observed a relative increase of the doubly methylated compound, pentanetriol dibiphytanyl glycerol tetraether, in the stationary phase of M. luminyensis as well as in the subseafloor of the Mediterranean Sea and thus introduced a backbone methylation index, which could be used to further explore microbial activity in natural settings.

Exploring the Potential of Electrospray-Orbitrap for Stable Isotope Analysis Using Nitrate as a Model.

Widely used isotope ratio mass spectrometers have limited capabilities to measure metabolites, drugs, or small polyatomic ions without the loss of structural isotopic information. A new approach has recently been introduced that uses electrospray ionization Orbitrap to measure multidimensional isotope signatures of intact polar compounds. Using nitrate as a model compound, this study aims to establish performance metrics for comparisons with conventional IRMS at the natural abundance level. We present a framework on how to convert isotopolog intensities to δ values that are commonly used in the isotope geochemistry community. The quantification of seven nitrate isotopologs provides multiple pathways for obtaining the primary N and O δ values including non-mass-dependent O isotope variations, as well as opportunities to explore nonrandom isotopic distributions (i.e., clumping effects) within molecular nitrate. Using automation and the adaptation of measurement principles that are specific to isotope ratio analysis, nitrate δ15NAIR, δ18OVSMOW, and δ17OVSMOW were measured with a long-term precision of 0.4‰ or better for isotopic reference materials and purified nitrate from environmental samples. In addition, we demonstrate promising results for unpurified environmental samples in liquid form. With these new developments, this study connects the two largely disparate mass spectrometry fields of bioanalytical MS and isotope ratio MS, thus providing a route to measure new isotopic signatures in diverse organic and inorganic solutes.

Stable Isotope Analysis of Intact Oxyanions Using Electrospray Quadrupole-Orbitrap Mass Spectrometry.

The stable isotopes of sulfate, nitrate, and phosphate are frequently used to study geobiological processes of the atmosphere, ocean, as well as land. Conventionally, the isotopes of these and other oxyanions are measured by isotope-ratio sector mass spectrometers after conversion into gases. Such methods are prone to various limitations on sensitivity, sample throughput, or precision. In addition, there is no general tool that can analyze several oxyanions or all the chemical elements they contain. Here, we describe a new approach that can potentially overcome some of these limitations based on electrospray hyphenated with Quadrupole Orbitrap mass spectrometry. This technique yields an average accuracy of 1-2‰ for sulfate δ34S and δ18O and nitrate δ15N and δ18O, based on in-house and international standards. Less abundant variants such as δ17O, δ33S, and δ36S, and the 34S-18O "clumped" sulfate can be quantified simultaneously. The observed precision of isotope ratios is limited by the number of ions counted. The counting of rare ions can be accelerated by removing abundant ions with the quadrupole mass filter. Electrospray mass spectrometry (ESMS) exhibits high-throughput and sufficient sensitivity. For example, less than 1 nmol sulfate is required to determine 18O/34S ratios with 0.2‰ precision within minutes. A purification step is recommended for environmental samples as our proposed technique is susceptible to matrix effects. Building upon these initial provisions, new features of the isotopic anatomy of mineral ions can now be explored with ESMS instruments that are increasingly available to bioanalytical laboratories.

Refining the Application of Microbial Lipids as Tracers of Staphylococcus aureus Growth Rates in Cystic Fibrosis Sputum.

Chronic lung infections in cystic fibrosis (CF) could be treated more effectively if the effects of antimicrobials on pathogens in situ were known. Here, we compared changes in the microbial community composition and pathogen growth rates in longitudinal studies of seven pediatric CF patients undergoing intravenous antibiotic administration during pulmonary exacerbations. The microbial community composition was determined by counting rRNA with NanoString DNA analysis, and growth rates were obtained by incubating CF sputum with heavy water and tracing incorporation of deuterium into two branched-chain ("anteiso") fatty acids (a-C15:0 and a-C17:0) using gas chromatography-mass spectrometry (GC/MS). Prior to this study, both lipids were thought to be specific for Staphylococcaceae; hence, their isotopic enrichment was interpreted as a growth proxy for Staphylococcus aureus Our experiments revealed, however, that Prevotella is also a relevant microbial producer of a-C17:0 fatty acid in some CF patients; thus, deuterium incorporation into these lipids is better interpreted as a more general pathogen growth rate proxy. Even accounting for a small nonmicrobial background source detected in some patient samples, a-C15:0 fatty acid still appears to be a relatively robust proxy for CF pathogens, revealing a median generation time of ∼1.5 days, similar to prior observations. Contrary to our expectation, pathogen growth rates remained relatively stable throughout exacerbation treatment. We suggest two straightforward "best practices" for application of stable-isotope probing to CF sputum metabolites: (i) parallel determination of microbial community composition in CF sputum using culture-independent tools and (ii) assessing background levels of the diagnostic metabolite.IMPORTANCE In chronic lung infections, populations of microbial pathogens change and mature in ways that are often unknown, which makes it challenging to identify appropriate treatment options. A promising tool to better understand the physiology of microorganisms in a patient is stable-isotope probing, which we previously developed to estimate the growth rates of S. aureus in cystic fibrosis (CF) sputum. Here, we tracked microbial communities in a cohort of CF patients and found that anteiso fatty acids can also originate from other sources in CF sputum. This awareness led us to develop a new workflow for the application of stable-isotope probing in this context, improving our ability to estimate pathogen generation times in clinical samples.

Towards measuring growth rates of pathogens during infections by D2 O-labeling lipidomics.

RATIONALE: Microbial growth rate is an important physiological parameter that is challenging to measure in situ, partly because microbes grow slowly in many environments. Recently, it has been demonstrated that generation times of S. aureus in cystic fibrosis (CF) infections can be determined by D2 O-labeling of actively synthesized fatty acids. To improve species specificity and allow growth rate monitoring for a greater range of pathogens during the treatment of infections, it is desirable to accurately quantify trace incorporation of deuterium into phospholipids. METHODS: Lipid extracts of D2 O-treated E. coli cultures were measured on liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) instruments equipped with time-of-flight (TOF) and orbitrap mass analyzers, and used for comparison with the analysis of fatty acids by isotope-ratio gas chromatography (GC)/MS. We then developed an approach to enable tracking of lipid labeling, by following the transition from stationary into exponential growth in pure cultures. Lastly, we applied D2 O-labeling lipidomics to clinical samples from CF patients with chronic lung infections. RESULTS: Lipidomics facilitates deuterium quantification in lipids at levels that are useful for many labeling applications (>0.03 at% D). In the E. coli cultures, labeling dynamics of phospholipids depend largely on their acyl chains and between phospholipids we notice differences that are not obvious from absolute concentrations alone. For example, cyclopropyl-containing lipids reflect the regulation of cyclopropane fatty acid synthase, which is predominantly expressed at the beginning of stationary phase. The deuterium incorporation into a lipid that is specific for S. aureus in CF sputum indicates an average generation time of the pathogen on the order of one cell doubling per day. CONCLUSIONS: This study demonstrates how trace level measurement of stable isotopes in intact lipids can be used to quantify lipid metabolism in pure cultures and provides guidelines that enable growth rate measurements in microbiome samples after incubation with a low percentage of D2 O.

A guide to precise measurements of isotope abundance by ESI-Orbitrap MS.

Stable isotopes of carbon, hydrogen, nitrogen, oxygen and sulfur are widespread in nature. Nevertheless, their relative abundance is not the same everywhere. This is due to kinetic isotope effects in enzymes and other physical principles such as equilibrium thermodynamics. Variations in isotope ratios offer unique insights into environmental pollution, trophic relationships in ecology, metabolic disorders and Earth history including climate history. Although classical isotope ratio mass spectrometry (IRMS) techniques still struggle to access intramolecular information like site-specific isotope abundance, electrospray ionization-Orbitrap mass spectrometry can be used to achieve precise and accurate intramolecular quantification of isotopically substituted molecules ('isotopocules'). This protocol describes two procedures. In the first one, we provide a step-by-step beginner's guide for performing multi-elemental, intramolecular and site-specific stable isotope analysis in unlabeled polar solutes by direct infusion. Using a widely available calibration solution, isotopocules of trifluoroacetic acid and immonium ions from the model peptide MRFA are quantified. In the second approach, nitrate is used as a simple model for a flow injection routine that enables access to a diverse range of naturally occurring isotopic signatures in inorganic oxyanions. Each procedure takes 2-3 h to complete and requires expertise only in general mass spectrometry. The workflows use optimized Orbitrap IRMS data-extraction and -processing software and are transferable to various analytes amenable to soft ionization, including metabolites, peptides, drugs and environmental pollutants. Optimized mass spectrometry systems will enable intramolecular isotope research in many areas of biology.

Discovering Nature's Fingerprints: Isotope Ratio Analysis on Bioanalytical Mass Spectrometers.

For a generation or more, the mass spectrometry that developed at the frontier of molecular biology was worlds apart from isotope ratio mass spectrometry, a label-free approach done on optimized gas-source magnetic sector instruments. Recent studies show that electrospray-ionization Orbitraps and other mass spectrometers widely used in the life sciences can be fine-tuned for high-precision isotope ratio analysis. Since isotope patterns form everywhere in nature based on well-understood principles, intramolecular isotope measurements allow unique insights into a fascinating range of research topics. This Perspective introduces a wider readership to current topics in stable isotope research with the aim of discussing how soft-ionization mass spectrometry coupled with ultrahigh mass resolution can enable long-envisioned progress. We highlight novel prospects of observing isotopes in intact polar compounds and speculate on future directions of this adventure into the overlapping realms of biology, chemistry, and geology.

Microbial Denitrification: Active Site and Reaction Path Models Predict New Isotopic Fingerprints.

The study of isotopic fingerprints in nitrate (δ15N, δ18O, Δ17O) has enabled pivotal insights into the global nitrogen cycle and revealed new knowledge gaps. Measuring populations of isotopic homologs of intact NO3 - ions (isotopologues) shows promise to advance the understanding of nitrogen cycling processes; however, we need new theory and predictions to guide laboratory experiments and field studies. We investigated the hypothesis that the isotopic composition of the residual nitrate pool is controlled by the N-O bond-breaking step in Nar dissimilatory nitrate reductase using molecular models of the enzyme active sites and associated kinetic isotope effects (KIEs). We integrated the molecular model results into reaction path models representing the reduction of nitrate under either closed-system or steady-state conditions. The predicted intrinsic KIE (15ε and 18ε) of the Nar active site matches observed fractionations in both culture and environmental studies. This is what would be expected if the isotopic composition of marine nitrate were controlled by dissimilatory nitrate reduction by Nar. For a closed system, the molecular models predict a pronounced negative 15N-18O clumping anomaly in residual nitrate. This signal could encode information about the amount of nitrate consumed in a closed system and thus constrain initial nitrate concentration and its isotopic composition. Similar clumped isotope anomalies can potentially be used to distinguish whether a system is open or closed to new nitrate addition. These mechanistic predictions can be tested and refined in combination with emerging ESI-Orbitrap measurements.

A planetary health perspective on synthetic methionine.

Methionine is an amino acid that humans and farm animals must derive from food. This metabolite, a tightly regulated resource in ecosystems, has become a mass commodity in the global economy, with well over 1 million tons being produced annually from petroleum to fortify livestock feed. Viewed from the standpoint of planetary health, anthropogenic methionine synthesis is an important enabler of low-cost animal protein production, with interdependent but unexamined effects on human health and ecosystems. At a time when agrochemical engineering is shifting the way sulphur is assimilated and moves up our food chain, research suggests that dietary methionine restriction alone captures many healthspan benefits noted with calorie restriction. As such, methionine synthesis is an excellent exemplar of planetary scale anthropogenic activity that manifests at the molecular scale of cellular metabolism, with potential systemic effects on human health. In this Personal View we establish the scale and historical trajectory of the methionine industry and provide a preliminary model for tracing this amino acid through the food supply into the human body. We draw together insights across disparate publications of applied animal agriculture, human nutrition, and biomedical research to call for cross-disciplinary dialogue on responsible use of methionine-augmentation technologies.

The structural basis for specific decoding of AUA by isoleucine tRNA on the ribosome.

Decoding of the AUA isoleucine codon in bacteria and archaea requires modification of a C in the anticodon wobble position of the isoleucine tRNA. Here, we report the crystal structure of the archaeal tRNA2(Ile), which contains the modification agmatidine in its anticodon, in complex with the AUA codon on the 70S ribosome. The structure illustrates how agmatidine confers codon specificity for AUA over AUG.

Decoding in the absence of a codon by tmRNA and SmpB in the ribosome.

In bacteria, ribosomes stalled at the end of truncated messages are rescued by transfer-messenger RNA (tmRNA), a bifunctional molecule that acts as both a transfer RNA (tRNA) and a messenger RNA (mRNA), and SmpB, a small protein that works in concert with tmRNA. Here, we present the crystal structure of a tmRNA fragment, SmpB and elongation factor Tu bound to the ribosome at 3.2 angstroms resolution. The structure shows how SmpB plays the role of both the anticodon loop of tRNA and portions of mRNA to facilitate decoding in the absence of an mRNA codon in the A site of the ribosome and explains why the tmRNA-SmpB system does not interfere with normal translation.

Insights into translational termination from the structure of RF2 bound to the ribosome.

The termination of protein synthesis occurs through the specific recognition of a stop codon in the A site of the ribosome by a release factor (RF), which then catalyzes the hydrolysis of the nascent protein chain from the P-site transfer RNA. Here we present, at a resolution of 3.5 angstroms, the crystal structure of RF2 in complex with its cognate UGA stop codon in the 70S ribosome. The structure provides insight into how RF2 specifically recognizes the stop codon; it also suggests a model for the role of a universally conserved GGQ motif in the catalysis of peptide release.

Structure refinement of cyclosporin A in chloroform by using RDCs measured in a stretched PDMS-gel.

New developments concerning alignment media for apolar solvents like chloroform make it possible to measure anisotropic parameters such as residual dipolar couplings (RDCs) at relatively low concentrations and natural isotopic abundance. As RDCs provide structural restraints with respect to an external coordinate system, long-range structural arrangements of the time-averaged structure can be determined with high precision. The method is demonstrated on the well-studied cyclo-undecapeptide Cyclosporin A (CsA), for which crystal and conventionally derived NMR structures are available. Neither crystal nor NMR structure are consistent with heteronuclear D(CH) RDCs measured in a stretched poly(dimethylsiloxane) gel, and refinement by using the anisotropic parameter results in a highly defined structure with a slightly changed backbone conformation. The applied methods and interpretation of the structural model are discussed.