Rapid coupling of Surface Plasmon Resonance (SPR and SPRi) and ProteinChip™ based mass spectrometry for the identification of proteins in nucleoprotein interactions

We compared coupling approaches of SPR to LC-MS and ProteinChip™-based mass spectrometry (SELDI™) as a means of identifying proteins captured on DNA surfaces. The approach we outline has the potential to allow multiple, quantitative analysis of macromolecular interactions followed by rapid mass spectrometry identification of retained material

The absence of SigX may result in a nutritional stress response in Pseudomonas aeruginosa

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Expression of the translocator protein (TSPO) from Pseudomonas fluorescens Pf0-1 requires the stress regulatory sigma factors AlgU and RpoH

International audienceThe translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is an evolutionary conserved protein that is found in many Eukarya, Archae and Bacteria, in which it plays several important functions including membrane biogenesis, signaling and stress response. A tspo homologue gene has been identified in several members of the Pseudomonas genus, among which the soil bacterium P. fluorescens Pf0-1. In this bacterium, the tspo gene is located in the vicinity of a putative hybrid histidine kinase-encoding gene. Since tspo has been involved in water stress related response in plants, we explored the effects of hyperosmolarity and temperature on P. fluorescens Pf0-1 tspo expression using a strategy based on lux-reporter fusions. (B) Co-transcription of Pfl01_2810 and tspo by RT-PCR assay. RT-PCR was assayed on tspo (1), Pfl01_2810 (2) and on the putative operonic structure Pfl01_2810-tspo (3), using primers located into tspo (1) or Pfl01_2810 (2) ORFs, or into both tspo and Pfl01_2810 ORFs (3). RT-PCR achieved on total RNA did not lead to a PCR fragment (line 4, negative control). L: Ladder 1kb+ (Biorad ®), (C) Growth curves in microtitre wells in LB medium at 28 C and relative luminescence (RL) activity of pTSPO and pHK-TSPO in P. fluorescens Pf0-1. The tspo gene (Pfl01_2811) of P. fluorescens Pf0-1 forms an operonic structure with Pfl01_2810 and is expressed transiently during the bacterial growth. (A) Schematic representation of the genomic environment of tspo and of transcriptional fusions pTSPO and pHK-TSPO. Grey bars represent the beginning of the luxCDABE reporter cassette of the promoterless pAB133 vector. The position of the studied promoter region is indicated relatively to the translational initiation start of each ORF. Pfl01_ 2809 lux CDABE pHK-TSPO-253 bp lux CDABE pTSPO-227 bp P. fluorescens Pf0-1 chromosome lux transcriptional fusions Pfl01_2810 tsp

In vitro assessment of the probiotic properties and bacteriocinogenic potential of Pediococcus pentosaceus MZF16, isolated from artisanal Tunisian meat « Dried Ossban »

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Evaluation of probiotic and bacteriocinogenic potential of Pediococcus pentosaceus MZF16 isolated from artisanal Tunisian meat "Dried Ossban"

International audiencePediococcus pentosaceus MZF16 has been isolated from artisanal Tunisian meat so called “Dried Ossban”, an original ecological niche, and identified by MALDI-TOF mass spectrometry and 16S rDNA sequencing. This bacterium showed a high tolerance to gastric stress conditions, and toward bile salts. P. pentosaceus MZF16 also demonstrated a hydrophobic surface profile (high adhesion to xylene), autoaggregation, and adhesive abilities to the human intestinal Caco-2/TC7 cell line. These properties may help the bacterium colonizing the gut. Furthermore, MZF16 was found to be resistant to gentamycin and chloramphenicol but did not harbor any transferable resistance determinants and/or virulence genes. The data also demonstrated absence of cytotoxicity of this strain. Conversely, P. pentosaceus MZF16 can slightly stimulate the immune system and enhance the intestinal epithelial barrier function. Moreover, this bacterium has been shown to be highly active against Listeria spp. due to bacteriocin production. Characterization of the bacteriocin by PCR amplification, sequencing and bioinformatic analyses revealed that MZF16 produces a bacteriocin 100% identical to coagulin, a pediocin-like inhibitory substance produced by Bacillus coagulans. To our knowledge, this is the first report that highlights the production of a pediocin 100% identical to coagulin in a Pediococcus strain. As coagulin, pediocin MZF16 has the consensus sequence YYGNGVXCXXXXCXVXXXXA (X denotes any amino acid), which confirms its belonging to class IIa bacteriocins, and its suitability to preserve foods from Listeria monocytogenes development. According to these results, P. pentosaceus MZF16 can be proposed as a probiotic and bioprotective agent for fermented foods, including Tunisian dry meat and sausages. Further investigations will aim to study the behavior of this strain in meat products as a component of functional food

High-affinity DNA binding sites for H-NS provide a molecular basis for selective silencing within proteobacterial genomes

The global transcriptional regulator H-NS selectively silences bacterial genes associated with pathogenicity and responses to environmental insults. Although there is ample evidence that H-NS binds preferentially to DNA containing curved regions, we show here that a major basis for this selectivity is the presence of a conserved sequence motif in H-NS target transcriptons. We further show that there is a strong tendency for the H-NS binding sites to be clustered, both within operons and in genes contained in the pathogenicity-associated islands. In accordance with previously published findings, we show that these motifs occur in AT-rich regions of DNA. On the basis of these observations, we propose that H-NS silences extensive regions of the bacterial chromosome by binding first to nucleating high-affinity sites and then spreading along AT-rich DNA. This spreading would be reinforced by the frequent occurrence of the motif in such regions. Our findings suggest that such an organization enables the silencing of extensive regions of the genetic material, thereby providing a coherent framework that unifies studies on the H-NS protein and a concrete molecular basis for the genetic control of H-NS transcriptional silencing

The Temperature-Regulation of Pseudomonas aeruginosa cmaX-cfrX-cmpX Operon Reveals an Intriguing Molecular Network Involving the Sigma Factors AlgU and SigX

Pseudomonas aeruginosa is a highly adaptable Gram-negative opportunistic pathogen, notably due to its large number of transcription regulators. The extracytoplasmic sigma factor (ECFσ) AlgU, responsible for alginate biosynthesis, is also involved in responses to cell wall stress and heat shock via the RpoH alternative σ factor. The SigX ECFσ emerged as a major regulator involved in the envelope stress response via membrane remodeling, virulence and biofilm formation. However, their functional interactions to coordinate the envelope homeostasis in response to environmental variations remain to be determined. The regulation of the putative cmaX-cfrX-cmpX operon located directly upstream sigX was investigated by applying sudden temperature shifts from 37°C. We identified a SigX- and an AlgU- dependent promoter region upstream of cfrX and cmaX, respectively. We show that cmaX expression is increased upon heat shock through an AlgU-dependent but RpoH independent mechanism. In addition, the ECFσ SigX is activated in response to valinomycin, an agent altering the membrane structure, and up-regulates cfrX-cmpX transcription in response to cold shock. Altogether, these data provide new insights into the regulation exerted by SigX and networks that are involved in maintaining envelope homeostasis

Effect of epinephrine on Pseudomonas aeruginosa

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Effect of epinephrine on Pseudomonas aeruginosa

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