BacA: a possible regulator that contributes to the biofilm formation of Pseudomonas aeruginosa.

Previously, we pointed out in P. aeruginosa PAO1 biofilm cells the accumulation of a hypothetical protein named PA3731 and showed that the deletion of the corresponding gene impacted its biofilm formation capacity. PA3731 belongs to a cluster of 4 genes (pa3732 to pa3729) that we named bac for "Biofilm Associated Cluster." The present study focuses on the PA14_16140 protein, i.e., the PA3732 (BacA) homolog in the PA14 strain. The role of BacA in rhamnolipid secretion, biofilm formation and virulence, was confirmed by phenotypic experiments with a bacA mutant. Additional investigations allow to advance that the bac system involves in fact 6 genes organized in operon, i.e., bacA to bacF. At a molecular level, quantitative proteomic studies revealed an accumulation of the BAC cognate partners by the bacA sessile mutant, suggesting a negative control of BacA toward the bac operon. Finally, a first crystallographic structure of BacA was obtained revealing a structure homologous to chaperones or/and regulatory proteins.

Detoxification Response of Pseudomonas fluorescens MFAF76a to Gaseous Pollutants NO2 and NO.

Bacteria are often exposed to nitrosative stress from their environment, from atmospheric pollution or from the defense mechanisms of other organisms. Reactive nitrogen species (RNS), which mediate nitrosative stress, are notably involved in the mammalian immune response through the production of nitric oxide (NO) by the inducible NO synthase iNOS. RNS are highly reactive and can alter various biomolecules such as lipids, proteins and DNA, making them toxic for biological organisms. Resistance to RNS is therefore important for the survival of bacteria in various environments, and notably to successfully infect their host. The fuel combustion processes used in industries and transports are responsible for the emission of important quantities of two major RNS, NO and the more toxic nitrogen dioxide (NO2). Human exposure to NO2 is notably linked to increases in lung infections. While the response of bacteria to NO in liquid medium is well-studied, few data are available on their exposure to gaseous NO and NO2. This study showed that NO2 is much more toxic than NO at similar concentrations for the airborne bacterial strain Pseudomonas fluorescens MFAF76a. The response to NO2 involves a wide array of effectors, while the response to NO seemingly focuses on the Hmp flavohemoprotein. Results showed that NO2 induces the production of other RNS, unlike NO, which could explain the differences between the effects of these two molecules.

Elicitation of Roots and AC-DC with PEP-13 Peptide Shows Differential Defense Responses in Multi-Omics.

The root extracellular trap (RET) has emerged as a specialized compartment consisting of root AC-DC and mucilage. However, the RET's contribution to plant defense is still poorly understood. While the roles of polysaccharides and glycoproteins secreted by root AC-DC have started to be elucidated, how the low-molecular-weight exudates of the RET contribute to root defense is poorly known. In order to better understand the RET and its defense response, the transcriptomes, proteomes and metabolomes of roots, root AC-DC and mucilage of soybean (Glycine max (L.) Merr, var. Castetis) upon elicitation with the peptide PEP-13 were investigated. This peptide is derived from the pathogenic oomycete Phytophthora sojae. In this study, the root and the RET responses to elicitation were dissected and sequenced using transcriptional, proteomic and metabolomic approaches. The major finding is increased synthesis and secretion of specialized metabolites upon induced defense activation following PEP-13 peptide elicitation. This study provides novel findings related to the pivotal role of the root extracellular trap in root defense.

Mitigation of monocyte driven thrombosis on cobalt chrome surfaces in contact with whole blood by thin film polar/hydrophobic/ionic polyurethane coatings.

Monocytes are active at the crossroads between inflammation and coagulation processes since they can secrete pro-inflammatory cytokines and express tissue factor (TF), a major initiator of coagulation. Cobalt-chrome (CoCr), a metal alloy, used as a biomaterial for vascular stents, has been shown to be potentially pro-thrombotic and pro-inflammatory. Research work with a polymer from a family of degradable-polar hydrophobic ionic polyurethanes (D-PHI), called HHHI, has been shown to exhibit anti-inflammatory responses from human monocytes. We have generated multifunctional polyurethane thin films (MPTF) based on the HHHI chemistry, as a thin coating for CoCr and have evaluated the reactivity of blood with MPTF-coated CoCr. The results showed that the coating of CoCr with MPTF derived from HHHI prevents thrombin generation, reduces coagulation activation, and suppresses fibrin formation in whole blood. Activation of monocytes was also suppressed at the surface of MPTF-coated CoCr and specifically the decrease in thrombin generation was accompanied by a significant decrease in TF and pro-inflammatory cytokine levels. Mass spectroscopy of the adsorbed proteins showed lower levels of fibrinogen, fibronectin and complement C3, C4, and C8 when compared to CoCr. We can conclude that MPTFs reduce the pro-thrombotic and pro-inflammatory phenotype of monocytes and macrophages on CoCr, and prevent clotting in whole blood.

Characterization of new outer membrane proteins of Pseudomonas aeruginosa using a combinatorial peptide ligand library.

Most often, the use of ProteoMiner beads has been restricted to human serum proteins for the normalization of major proteins, such as albumin. However, there are other situations of interest in which the presence of major proteins would quench the signals of low abundance polypeptides. We propose the use of these beads for investigating the envelope of the gram-negative bacterium Pseudomonas aeruginosa. Initially, we performed comparative 2D electrophoresis to qualitatively evaluate the incidence of the normalization stage. This demonstrated a significant reduction of the major membrane proteins. Thereafter, using shotgun analysis, the same protein extract was targeted by using combinatorial peptide ligand library capture. This treatment yielded 154 additional outer membrane proteins (OMPs) uncovered by the study of the crude sample.

Characterization of membrane lipidome changes in Pseudomonas aeruginosa during biofilm growth on glass wool.

Bacteria cells within biofilms are physiologically distinct from their planktonic counterparts. In particular they are more resistant to detrimental environmental conditions. In this study, we monitored the evolution of the phospholipid composition of the inner and outer membranes of P. aeruginosa during the biofilm formation (i.e., from 1-, 2-, to 6-day-old biofilm). Lipidome analyses were performed by electrospray ionization mass spectrometry. In addition to the lipidomic analysis, the fatty acid composition was analysed by gas chromatography/mass spectrometry. We found that the lipidome alterations of the inner and the outer membranes varied with the biofilm age. These alterations in phospholipid compositions reflect a higher diversity in sessile organisms than in planktonic counterparts. The diversity is characterized by the presence of PE 30∶1, PE 31∶0 and PG 31∶0 for the lower masses as well as PE 38∶1, 38∶2, 39∶1, 39∶2 and PG 38∶0, 38∶1, 38∶2, 39∶1, 39∶2 for the higher masses. However, this lipidomic feature tends to disappear with the biofilm age, in particular the high mass phospholipids tend to disappear. The amount of branched chains phospholipids mainly located in the outer membrane decreased with the biofilm age, whereas the proportion of cyclopropylated phospholipids increased in both membranes. In bacteria present in oldest biofilms, i.e., 6-day-old, the phospholipid distribution moved closer to that of planktonic bacteria.

Escherichia coli response to uranyl exposure at low pH and associated protein regulations.

Better understanding of uranyl toxicity in bacteria is necessary to optimize strains for bioremediation purposes or for using bacteria as biodetectors for bioavailable uranyl. In this study, after different steps of optimization, Escherichia coli cells were exposed to uranyl at low pH to minimize uranyl precipitation and to increase its bioavailability. Bacteria were adapted to mid acidic pH before exposure to 50 or 80 µM uranyl acetate for two hours at pH≈3. To evaluate the impact of uranium, growth in these conditions were compared and the same rates of cells survival were observed in control and uranyl exposed cultures. Additionally, this impact was analyzed by two-dimensional differential gel electrophoresis proteomics to discover protein actors specifically present or accumulated in contact with uranium.Exposure to uranium resulted in differential accumulation of proteins associated with oxidative stress and in the accumulation of the NADH/quinone oxidoreductase WrbA. This FMN dependent protein performs obligate two-electron reduction of quinones, and may be involved in cells response to oxidative stress. Interestingly, this WrbA protein presents similarities with the chromate reductase from E. coli, which was shown to reduce uranyl in vitro.