The RNase YbeY Is Vital for Ribosome Maturation, Stress Resistance, and Virulence of the Natural Genetic Engineer Agrobacterium tumefaciens.

Riboregulation involving regulatory RNAs, RNA chaperones, and ribonucleases is fundamental for the rapid adaptation of gene expression to changing environmental conditions. The gene coding for the RNase YbeY belongs to the minimal prokaryotic genome set and has a profound impact on physiology in a wide range of bacteria. Here, we show that the Agrobacterium tumefaciensybeY gene is not essential. Deletion of the gene in the plant pathogen reduced growth, motility, and stress tolerance. Most interestingly, YbeY is crucial for A. tumefaciens-mediated T-DNA transfer and tumor formation. Comparative proteomics by using isobaric tags for relative and absolute quantitation (iTRAQ) revealed dysregulation of 59 proteins, many of which have previously been found to be dependent on the RNA chaperone Hfq. YbeY and Hfq have opposing effects on production of these proteins. Accumulation of a 16S rRNA precursor in the ybeY mutant suggests that A. tumefaciens YbeY is involved in rRNA processing. RNA coimmunoprecipitation-sequencing (RIP-Seq) showed binding of YbeY to the region immediately upstream of the 16S rRNA. Purified YbeY is an oligomer with RNase activity. It does not physically interact with Hfq and thus plays a partially overlapping but distinct role in the riboregulatory network of the plant pathogen.IMPORTANCE Although ybeY gene belongs to the universal bacterial core genome, its biological function is incompletely understood. Here, we show that YbeY is critical for fitness and host-microbe interaction in the plant pathogen Agrobacterium tumefaciens Consistent with the reported endoribonuclease activity of YbeY, A. tumefaciens YbeY acts as a RNase involved in maturation of 16S rRNA. This report adds a worldwide plant pathogen and natural genetic engineer of plants to the growing list of bacteria that require the conserved YbeY protein for host-microbe interaction.

Linear programming model can explain respiration of fermentation products.

Many differentiated cells rely primarily on mitochondrial oxidative phosphorylation for generating energy in the form of ATP needed for cellular metabolism. In contrast most tumor cells instead rely on aerobic glycolysis leading to lactate to about the same extent as on respiration. Warburg found that cancer cells to support oxidative phosphorylation, tend to ferment glucose or other energy source into lactate even in the presence of sufficient oxygen, which is an inefficient way to generate ATP. This effect also occurs in striated muscle cells, activated lymphocytes and microglia, endothelial cells and several mammalian cell types, a phenomenon termed the "Warburg effect". The effect is paradoxical at first glance because the ATP production rate of aerobic glycolysis is much slower than that of respiration and the energy demands are better to be met by pure oxidative phosphorylation. We tackle this question by building a minimal model including three combined reactions. The new aspect in extension to earlier models is that we take into account the possible uptake and oxidation of the fermentation products. We examine the case where the cell can allocate protein on several enzymes in a varying distribution and model this by a linear programming problem in which the objective is to maximize the ATP production rate under different combinations of constraints on enzymes. Depending on the cost of reactions and limitation of the substrates, this leads to pure respiration, pure fermentation, and a mixture of respiration and fermentation. The model predicts that fermentation products are only oxidized when glucose is scarce or its uptake is severely limited.

Mathematical models for explaining the Warburg effect: a review focussed on ATP and biomass production.

For producing ATP, tumour cells rely on glycolysis leading to lactate to about the same extent as on respiration. Thus, the ATP synthesis flux from glycolysis is considerably higher than in the corresponding healthy cells. This is known as the Warburg effect (named after German biochemist Otto H. Warburg) and also applies to striated muscle cells, activated lymphocytes, microglia, endothelial cells and several other cell types. For similar phenomena in several yeasts and many bacteria, the terms Crabtree effect and overflow metabolism respectively, are used. The Warburg effect is paradoxical at first sight because the molar ATP yield of glycolysis is much lower than that of respiration. Although a straightforward explanation is that glycolysis allows a higher ATP production rate, the question arises why cells do not re-allocate protein to the high-yield pathway of respiration. Mathematical modelling can help explain this phenomenon. Here, we review several models at various scales proposed in the literature for explaining the Warburg effect. These models support the hypothesis that glycolysis allows for a higher proliferation rate due to increased ATP production and precursor supply rates.

Profound impact of Hfq on nutrient acquisition, metabolism and motility in the plant pathogen Agrobacterium tumefaciens.

As matchmaker between mRNA and sRNA interactions, the RNA chaperone Hfq plays a key role in riboregulation of many bacteria. Often, the global influence of Hfq on the transcriptome is reflected by substantially altered proteomes and pleiotropic phenotypes in hfq mutants. Using quantitative proteomics and co-immunoprecipitation combined with RNA-sequencing (RIP-seq) of Hfq-bound RNAs, we demonstrate the pervasive role of Hfq in nutrient acquisition, metabolism and motility of the plant pathogen Agrobacterium tumefaciens. 136 of 2544 proteins identified by iTRAQ (isobaric tags for relative and absolute quantitation) were affected in the absence of Hfq. Most of them were associated with ABC transporters, general metabolism and motility. RIP-seq of chromosomally encoded Hfq3xFlag revealed 1697 mRNAs and 209 non-coding RNAs (ncRNAs) associated with Hfq. 56 ncRNAs were previously undescribed. Interestingly, 55% of the Hfq-bound ncRNAs were encoded antisense (as) to a protein-coding sequence suggesting that A. tumefaciens Hfq plays an important role in asRNA-target interactions. The exclusive enrichment of 296 mRNAs and 31 ncRNAs under virulence conditions further indicates a role for post-transcriptional regulation in A. tumefaciens-mediated plant infection. On the basis of the iTRAQ and RIP-seq data, we assembled a comprehensive model of the Hfq core regulon in A. tumefaciens.

Membrane lipids in Agrobacterium tumefaciens: biosynthetic pathways and importance for pathogenesis.

Many cellular processes critically depend on the membrane composition. In this review, we focus on the biosynthesis and physiological roles of membrane lipids in the plant pathogen Agrobacterium tumefaciens. The major components of A. tumefaciens membranes are the phospholipids (PLs), phosphatidylethanolamine (PE), phosphatidylglycerol, phosphatidylcholine (PC) and cardiolipin, and ornithine lipids (OLs). Under phosphate-limited conditions, the membrane composition shifts to phosphate-free lipids like glycolipids, OLs and a betaine lipid. Remarkably, PC and OLs have opposing effects on virulence of A. tumefaciens. OL-lacking A. tumefaciens mutants form tumors on the host plant earlier than the wild type suggesting a reduced host defense response in the absence of OLs. In contrast, A. tumefaciens is compromised in tumor formation in the absence of PC. In general, PC is a rare component of bacterial membranes but amount to ~22% of all PLs in A. tumefaciens. PC biosynthesis occurs via two pathways. The phospholipid N-methyltransferase PmtA methylates PE via the intermediates monomethyl-PE and dimethyl-PE to PC. In the second pathway, the membrane-integral enzyme PC synthase (Pcs) condenses choline with CDP-diacylglycerol to PC. Apart from the virulence defect, PC-deficient A. tumefaciens pmtA and pcs double mutants show reduced motility, enhanced biofilm formation and increased sensitivity towards detergent and thermal stress. In summary, there is cumulative evidence that the membrane lipid composition of A. tumefaciens is critical for agrobacterial physiology and tumor formation.

Hfq influences multiple transport systems and virulence in the plant pathogen Agrobacterium tumefaciens.

The Hfq protein mediates gene regulation by small RNAs (sRNAs) in about 50% of all bacteria. Depending on the species, phenotypic defects of an hfq mutant range from mild to severe. Here, we document that the purified Hfq protein of the plant pathogen and natural genetic engineer Agrobacterium tumefaciens binds to the previously described sRNA AbcR1 and its target mRNA atu2422, which codes for the substrate binding protein of an ABC transporter taking up proline and γ-aminobutyric acid (GABA). Several other ABC transporter components were overproduced in an hfq mutant compared to their levels in the parental strain, suggesting that Hfq plays a major role in controlling the uptake systems and metabolic versatility of A. tumefaciens. The hfq mutant showed delayed growth, altered cell morphology, and reduced motility. Although the DNA-transferring type IV secretion system was produced, tumor formation by the mutant strain was attenuated, demonstrating an important contribution of Hfq to plant transformation by A. tumefaciens.

Analgesic efficacy of quick-release versus standard lornoxicam for pain after third molar surgery: a randomized, double-blind, placebo-controlled, single-dose trial.

OBJECTIVE: The aim of this study was to evaluate the analgesic efficacy and time to onset of effect of the lornoxicam quick-release (LNX-QR) tablet compared with the standard-release tablet (LNX-ST). METHODS: In this randomized, double-blind, single-dose trial, 200 patients with moderate pain after surgical removal of an impacted third molar were randomized to treatment with an LNX-QR 8 mg tablet (80 patients), an LNX-ST 8 mg tablet (80 patients) or placebo (40 patients). Pain intensity (PI) and pain relief (PAR) were assessed (numerical and verbal rating scales) for 6 hours, and time to onset of PAR was recorded. The cumulated sum of PI differences (SPID) and PAR (TOTPAR) were calculated. Tolerability was evaluated by occurrence of adverse events. RESULTS: Kaplan-Meier analysis of time to onset of analgesic efficacy demonstrated a significantly faster onset with LNX-QR than placebo or LNX-ST (p < 0.0001). Median time of onset was 32 minutes (range 29-37) for LNX-QR and 46 minutes (range 37-59) for LNX-ST. The analgesic efficacy of LNX-QR and LNX-ST were superior to that of placebo, whereas paired comparisons of TOTPAR and SPID showed LNX-QR to be superior to LNX-ST (p < 0.05). CONCLUSION: LNX-QR provided a faster onset and superior analgesic effect against pain following third molar surgery than LNX-ST.

Intravenous acetaminophen (paracetamol): comparable analgesic efficacy, but better local safety than its prodrug, propacetamol, for postoperative pain after third molar surgery.

We compared an acetaminophen (paracetamol) 1 g (n = 51) formulation for infusion with propacetamol 2 g (n = 51) and placebo (n = 50) in a randomized, controlled, double-blind, parallel group trial in patients with moderate-to-severe pain after third molar surgery. Treatment efficacy was assessed in house for 6 h after starting the 15-min infusion. Significant effects versus placebo (P < 0.01) were obtained with both active treatments on pain relief, pain intensity difference on a 100-mm visual analog scale, and on a categorical scale (except for propacetamol at 6 h). No significant differences were noted between active groups except at 1 h. Six-hour weighted sums of primary assessments showed significantly better efficacy than placebo (P < 0.0001) and no difference between active treatments. Median stopwatch time to onset of pain relief for active treatment was 6-8 min after infusion start. Active treatments showed comparable efficacy with a significantly longer duration of analgesia and better patients' global evaluation compared with placebo. The incidence of patients reporting local pain at the infusion site was significantly less frequent after IV acetaminophen or placebo (0%) in comparison with propacetamol (49%). In conclusion, acetaminophen 1 g and propacetamol 2 g were superior to placebo regarding analgesic efficacy, with a more frequent incidence of local pain at the infusion site for propacetamol.