IS1 transposition is enhanced by translation errors and by bacterial growth at extreme glucose levels

Transposition of insertion sequences (IS) is an enzyme-mediated process that only occurs in a minority of cells within a bacterial culture. Transposition is thus a rare event, but transposition frequency may vary depending on experimental conditions. For instance in a rich broth, IS elements are known to transpose during stationary phase but not during exponential growth. Using a reporter system which involves the activation of the cryptic bgl operon in Escherichia coli, we show that the frequency of IS1 transposition is a function of glucose concentration in the growth medium, it is increased by streptomycin amounts that are below minimum inhibitory concentration (sub-MIC) and is inhibited in an rpsL150 strain with high translation accuracy. Since starved cells are known to enhance ribosome frameshifting, our data suggests that growth conditions applied in this study could affect IS1 transposition by increasing translation infidelity.     

FERONIA and microtubules independently contribute to mechanical integrity in the Arabidopsis shoot

To survive, cells must constantly resist mechanical stress. In plants, this involves the reinforcement of cell walls, notably through microtubule-dependent cellulose deposition. How wall sensing might contribute to this response is unknown. Here, we tested whether the microtubule response to stress acts downstream of known wall sensors. Using a multistep screen with 11 mutant lines, we identify FERONIA (FER) as the primary candidate for the cell’s response to stress in the shoot. However, this does not imply that FER acts upstream of the microtubule response to stress. In fact, when performing mechanical perturbations, we instead show that the expected microtubule response to stress does not require FER. We reveal that the feronia phenotype can be partially rescued by reducing tensile stress levels. Conversely, in the absence of both microtubules and FER, cells appear to swell and burst. Altogether, this shows that the microtubule response to stress acts as an independent pathway to resist stress, in parallel to FER. We propose that both pathways are required to maintain the mechanical integrity of plant cells.

In all living organisms, cells must resist mechanical stress to survive. This study of the model plant Arabidopsis reveals that the candidate cell wall mechanoreceptor FERONIA and microtubules independently contribute to this mechanical feedback.     

Interactions between Head Blight Pathogens: Consequences for Disease Development and Toxin Production in Wheat Spikes

Head blight (HB) is one of the most damaging diseases on wheat, inducing significant yield losses and toxin accumulation in grains. Fungal pathogens responsible for HB include the genus Microdochium, with two species, and the toxin producer genus Fusarium, with several species. Field studies and surveys show that two or more species can coexist within a same field and coinfect the same plant or the same spike. In the current study, we investigated how the concomitant presence of F. graminearum and another of the HB complex species influences the spike colonization and the toxin production by the fungi. To study these interactions, 17 well-characterized isolates representing five species were inoculated alone or in pairs on wheat spikes in greenhouse and field experiments. The fungal DNA in the grains was estimated by quantitative PCR and toxin contents (deoxynivalenol and nivalenol) by ultraperformance liquid chromatography-UV detection-tandem mass spectrometry. The responses of the different isolates to the presence of a competitor were variable and isolate specific more than species specific. The development of the most aggressive isolates was either unchanged or a slightly increased, while the development of the less aggressive isolates was reduced. The main outcome of the study was that no trend of increased toxin production was observed in coinoculations compared to single inoculations. On the contrary, the amount of toxin produced was often lower than expected in coinoculations. We thus conclude against the hypothesis that the co-occurrence of several HB-causing species in the same field might aggravate the risk linked to fusarium toxins in wheat production.     

Molecular Composition of Staufen2-Containing Ribonucleoproteins in Embryonic Rat Brain

Messenger ribonucleoprotein particles (mRNPs) are used to transport mRNAs along neuronal dendrites to their site of translation. Numerous mRNA-binding and regulatory proteins within mRNPs finely regulate the fate of bound-mRNAs. Their specific combination defines different types of mRNPs that in turn are related to specific synaptic functions. One of these mRNA-binding proteins, Staufen2 (Stau2), was shown to transport dendritic mRNAs along microtubules. Its knockdown expression in neurons was shown to change spine morphology and synaptic functions. To further understand the molecular mechanisms by which Stau2 modulates synaptic function in neurons, it is important to identify and characterize protein co-factors that regulate the fate of Stau2-containing mRNPs. To this end, a proteomic approach was used to identify co-immunoprecipitated proteins in Staufen2-containing mRNPs isolated from embryonic rat brains. The proteomic approach identified mRNA-binding proteins (PABPC1, hnRNP H1, YB1 and hsc70), proteins of the cytoskeleton (α- and β-tubulin) and RUFY3 a poorly characterized protein. While PABPC1 and YB1 associate with Stau2-containing mRNPs through RNAs, hsc70 is directly bound to Stau2 and this interaction is regulated by ATP. PABPC1 and YB1 proteins formed puncta in dendrites of embryonic rat hippocampal neurons. However, they poorly co-localized with Stau2 in the large dendritic complexes suggesting that they are rather components of Stau2-containing mRNA particles. All together, these results represent a further step in the characterization of Stau2-containing mRNPs in neurons and provide new tools to study and understand how Stau2-containing mRNPs are transported, translationally silenced during transport and/or locally expressed according to cell needs.     

A systematic mutagenesis-driven strategy for site-resolved NMR studies of supramolecular assemblies

Obtaining sequence-specific assignments remains a major bottleneck in solution NMR investigations of supramolecular structure, dynamics and interactions. Here we demonstrate that resonance assignment of methyl probes in high molecular weight protein assemblies can be efficiently achieved by combining fast NMR experiments, residue-type-specific isotope-labeling and automated site-directed mutagenesis. The utility of this general and straightforward strategy is demonstrated through the characterization of intermolecular interactions involving a 468-kDa multimeric aminopeptidase, PhTET2.     

Primary culture and phenotyping of murine chondrocytes

The culture of chondrocytes is one of the most powerful tools for exploring the intracellular and molecular features of chondrocyte differentiation and activation. However, chondrocytes tend to dedifferentiate into fibroblasts when they are subcultured, which is a major problem. This protocol, involving primary cultures to limit dedifferentiation, describes two different methods for culturing chondrocytes of different anatomical origins (articular and costal chondrocytes, both of which represent hyaline cartilage) from mice. Mice are of particular interest for cellular and molecular studies, as many tools suitable for use in mice are available. In addition, rapid development of transgenic and gene-targeted mice provides powerful instruments for biological studies. The protocol can be divided into four stages: isolation of cartilage (15 min per animal), isolation of chondrocytes (2 h extended overnight), seeding of chondrocytes (1 h 30 min) and growth in culture (6 d). To obtain confluency of chondrocytes using this protocol takes 7 d. Methods for phenotyping chondrocytes are also provided.     

Secondary metabolites from plant-associated Pseudomonas are overproduced in biofilm

Plant rhizosphere soil houses complex microbial communities in which microorganisms are often involved in intraspecies as well as interspecies and inter-kingdom signalling networks. Some members of these networks can improve plant health thanks to an important diversity of bioactive secondary metabolites. In this competitive environment, the ability to form biofilms may provide major advantages to microorganisms. With the aim of highlighting the impact of bacterial lifestyle on secondary metabolites production, we performed a metabolomic analysis on four fluorescent Pseudomonas strains cultivated in planktonic and biofilm colony conditions. The untargeted metabolomic analysis led to the detection of hundreds of secondary metabolites in culture extracts. Comparison between biofilm and planktonic conditions showed that bacterial lifestyle is a key factor influencing Pseudomonas metabolome. More than 50% of the detected metabolites were differentially produced according to planktonic or biofilm lifestyles, with the four Pseudomonas strains overproducing several secondary metabolites in biofilm conditions. In parallel, metabolomic analysis associated with genomic prediction and a molecular networking approach enabled us to evaluate the impact of bacterial lifestyle on chemically identified secondary metabolites, more precisely involved in microbial interactions and plant-growth promotion. Notably, this work highlights the major effect of biofilm lifestyle on acyl-homoserine lactone and phenazine production in P. chlororaphis strains.     

Expression and purification of FtsW and RodA from Streptococcus pneumoniae,two membrane proteins involved in cell division and cell growth,respectively

FtsW and RodA are homologous integral membrane proteins involved in bacterial cell division and cell growth, respectively. Both proteins from Streptococcus pneumoniae were overexpressed in Escherichia coli as N-terminal His-tagged fusions. Their membrane addressing in E. coli was demonstrated by cell fractionation and was confirmed for FtsW by immunolocalization. Recombinant FtsW and RodA were solubilized from membranes using 3-(laurylamido)-N,N'-dimethylaminopropylamine oxide (LAPAO). The detergent was exchanged to polyoxyethylene 8 lauryl ether (C12E8) during one-step purification procedure by Co(2+)-affinity chromatography. This procedure yielded 50-150 microg protein per litre of culture. Both proteins are likely to be folded as they are resistant to trypsin digestion and could be incorporated into reconstituted lipid vesicles.     

Reduced Slow-Wave Rebound during Daytime Recovery Sleep in Middle-Aged Subjects

Cortical synchronization during NREM sleep, characterized by electroencephalographic slow waves (SW <4Hz and >75 μV), is strongly related to the number of hours of wakefulness prior to sleep and to the quality of the waking experience. Whether a similar increase in wakefulness length leads to a comparable enhancement in NREM sleep cortical synchronization in young and older subjects is still a matter of debate in the literature. Here we evaluated the impact of 25-hours of wakefulness on SW during a daytime recovery sleep episode in 29 young (27y ±5), and 34 middle-aged (51y ±5) subjects. We also assessed whether age-related changes in NREM sleep cortical synchronization predicts the ability to maintain sleep during daytime recovery sleep. Compared to baseline sleep, sleep efficiency was lower during daytime recovery sleep in both age-groups but the effect was more prominent in the middle-aged than in the young subjects. In both age groups, SW density, amplitude, and slope increased whereas SW positive and negative phase duration decreased during daytime recovery sleep compared to baseline sleep, particularly in anterior brain areas. Importantly, compared to young subjects, middle-aged participants showed lower SW density rebound and SW positive phase duration enhancement after sleep deprivation during daytime recovery sleep. Furthermore, middle-aged subjects showed lower SW amplitude and slope enhancements after sleep deprivation than young subjects in frontal and prefrontal derivations only. None of the SW characteristics at baseline were associated with daytime recovery sleep efficiency. Our results support the notion that anterior brain areas elicit and may necessitate more intense recovery and that aging reduces enhancement of cortical synchronization after sleep loss, particularly in these areas. Age-related changes in the quality of wake experience may underlie age-related reduction in markers of cortical synchronization enhancement after sustained wakefulness.