New insights into the functioning and structure of the PE and PP plastispheres from the Mediterranean Sea.

Plastic debris are accumulating in the marine environment and aggregate microorganisms that form a new ecosystem called the plastisphere. Better understanding the plastisphere is crucial as it has self-sufficient organization and carries pathogens or organisms that may be involved in the pollutant adsorption and/or plastic degradation. To date, the plastisphere is mainly described at the taxonomic level and the functioning of its microbial communities still remains poorly documented. In this work, metagenomic and metaproteomic analyzes were performed on the plastisphere of polypropylene and polyethylene plastic debris sampled on a pebble beach from the Mediterranean Sea. Our results confirmed that the plastisphere was organized as self-sufficient ecosystems containing highly active primary producers, heterotrophs and predators such as nematode. Interestingly, the chemical composition of the polymer did not impact the structure of the microbial communities but rather influenced the functions expressed. Despite the fact that the presence of hydrocarbon-degrading bacteria was observed in the metagenomes, polymer degradation metabolisms were not detected at the protein level. Finally, hydrocarbon degrader (i.e., Alcanivorax) and pathogenic bacteria (i.e., Vibrionaceae) were observed in the plastispheres but were not very active as no proteins involved in polymer degradation or pathogeny were detected. This work brings new insights into the functioning of the microbial plastisphere developed on plastic marine debris.

Evaluation of gold layer configuration for plasmonic fiber grating biosensors.

Gold-coated fiber Bragg gratings (FBGs) are nowadays a mature technology for lab-on-fiber sensing based on surface plasmon resonance (SPR) excitation. Tilted FBGs bring valuable assets such as easy light injection, remote operation in very small volumes of analytes and immunity to temperature fluctuations. Different gold configurations have been reported to date, without considering their relative performances in terms of biochemical sensing. In this work, we experimentally study the impact of the gold coating on the cladding mode distribution in the tilted FBG amplitude spectrum and subsequently on its sensitivity to cytokeratins used as biomarkers for cancer diagnosis. Some relevant configurations of gold coatings are produced and tested, relying on both the sputtering and electroless plating (ELP) processes. The obtained results confirm that the coating thickness and its roughness drive the biosensing performances. The experimental limit of detection for cytokeratins 17 sensing reaches 14 fM for the most sensitive configurations.

New insight into the photoheterotrophic growth of the isocytrate lyase-lacking purple bacterium Rhodospirillum rubrum on acetate.

Purple non-sulfur bacteria are well known for their metabolic versatility. One of these bacteria, Rhodospirillum rubrum S1H, has been selected by the European Space Agency to ensure the photoheterotrophic assimilation of volatile fatty acids in its regenerative life support system, MELiSSA. Here, we combined proteomic analysis with bacterial growth analysis and enzymatic activity assays in order to better understand acetate photoassimilation. In this isocitrate lyase-lacking organism, the assimilation of two-carbon compounds cannot occur through the glyoxylate shunt, and the citramalate cycle has been proposed to fill this role, while, in Rhodobacter sphaeroides, the ethylmalonyl-CoA pathway is used for acetate assimilation. Using proteomic analysis, we were able to identify and quantify more than 1700 unique proteins, representing almost one-half of the theoretical proteome of the strain. Our data reveal that a pyruvate : ferredoxin oxidoreductase (NifJ) could be used for the direct assimilation of acetyl-CoA through pyruvate, potentially representing a new redox-balancing reaction. We additionally propose that the ethylmalonyl-CoA pathway could also be involved in acetate assimilation by the examined strain, since specific enzymes of this pathway were all upregulated and activity of crotonyl-CoA reductase/carboxylase was increased in acetate conditions. Surprisingly, we also observed marked upregulation of glutaryl-CoA dehydrogenase, which could be a component of a new pathway for acetate photoassimilation. Finally, our data suggest that citramalate could be an intermediate of the branched-chain amino acid biosynthesis pathway, which is activated during acetate assimilation, rather than a metabolite of the so-called citramalate cycle.

HylA, an alternative hydrolase for initiation of catabolism of the phenylurea herbicide linuron in Variovorax sp. strains.

Variovorax sp. strain WDL1, which mineralizes the phenylurea herbicide linuron, expresses a novel linuron-hydrolyzing enzyme, HylA, that converts linuron to 3,4-dichloroaniline (DCA). The enzyme is distinct from the linuron hydrolase LibA enzyme recently identified in other linuron-mineralizing Variovorax strains and from phenylurea-hydrolyzing enzymes (PuhA, PuhB) found in Gram-positive bacteria. The dimeric enzyme belongs to a separate family of hydrolases and differs in Km, temperature optimum, and phenylurea herbicide substrate range. Within the metal-dependent amidohydrolase superfamily, HylA and PuhA/PuhB belong to two distinct protein families, while LibA is a member of the unrelated amidase signature family. The hylA gene was identified in a draft genome sequence of strain WDL1. The involvement of hylA in linuron degradation by strain WDL1 is inferred from its absence in spontaneous WDL1 mutants defective in linuron hydrolysis and its presence in linuron-degrading Variovorax strains that lack libA. In strain WDL1, the hylA gene is combined with catabolic gene modules encoding the downstream pathways for DCA degradation, which are very similar to those present in Variovorax sp. SRS16, which contains libA. Our results show that the expansion of a DCA catabolic pathway toward linuron degradation in Variovorax can involve different but isofunctional linuron hydrolysis genes encoding proteins that belong to evolutionary unrelated hydrolase families. This may be explained by divergent evolution and the independent acquisition of the corresponding genetic modules.

Genome sequence of the edible cyanobacterium Arthrospira sp. PCC 8005.

We determined the genome sequence of Arthrospira sp. PCC 8005, a cyanobacterial strain of great interest to the European Space Agency for its nutritive value and oxygenic properties in the Micro-Ecological Life Support System Alternative (MELiSSA) biological life support system for long-term manned missions into space.

The genome of cyprinid herpesvirus 3 encodes 40 proteins incorporated in mature virions.

Koi herpesvirus, also known as cyprinid herpesvirus 3 (CyHV-3), is the aetiological agent of an emerging and mortal disease in common and koi carp. CyHV-3 virions present the characteristic morphology of other members of the order Herpesvirales, being composed of an envelope, a capsid containing the genome and a tegument. This study identified CyHV-3 structural proteins and the corresponding encoding genes using liquid chromatography tandem mass spectrometry-based proteomic approaches. In addition, exponentially modified protein abundance index analyses were used to estimate the relative abundance of the identified proteins in CyHV-3 virions. These analyses resulted in the identification of 40 structural proteins, which were classified based on bioinformatic analyses as capsid (three), envelope (13), tegument (two) and unclassified (22) structural proteins. Finally, a search for host proteins in purified CyHV-3 virions indicated the potential incorporation of up to 18 distinct cellular proteins. The identification of the proteins incorporated into CyHV-3 virions and determination of the viral genes encoding these proteins are key milestones for further fundamental and applied research on this virus.

Use of Mycobacterium avium subsp. paratuberculosis specific coding sequences for serodiagnosis of bovine paratuberculosis.

In this study, the finished complete genome of Mycobacterium avium subsp. paratuberculosis (Map) was screened for specific coding sequences that could be very valuable in the design of a sensitive and specific Map detection serological assay. Eighty-seven Map-specific sequences were retained. Among these, three candidate antigens have been analysed for their serodiagnostic potential. These antigens were selected on the basis of their putative immunogenicity as predicted by in silico analysis. The antigens were cloned in Escherichia coli, expressed, and purified before testing in an antibody detection ELISA test, using a well characterized panel of 18 and 48 sera from Map infected and uninfected cattle, respectively. Two of these antigens, antigen 6 and MAP1637c, yielded in our conditions a sensitivity of 72% and 82%, respectively, for a specificity of 98%. It is particularly noticeable that, when probed with the same serum panel, the most widely used European paratuberculosis commercial seroassay (Pourquier test) yielded a sensitivity of 72% for a specificity of only 92%.

Thimet oligopeptidase (EC 3.4.24.15) activates CPI-0004Na, an extracellularly tumour-activated prodrug of doxorubicin.

CPI-0004Na is a tetrapeptidic extracellularly tumour-activated prodrug of doxorubicin. The tetrapeptide structure ensures blood stability and selective cleavage by unidentified peptidase(s) released by tumour cells. The purpose of this work was to identify the enzyme responsible for the first rate-limiting step of CPI-0004Na activation, initially attributed to a 70 kDa acidic (pI=5.2) metallopeptidase active at neutral pH that was subsequently purified from HeLa cell homogenates. Two electrophoretic bands were isolated and identified by matrix-assisted laser desorption ionisation-time of flight (MALDI-tof) and electrospray ionisation-quadrupole-time of flight (ESI-Q-tof) mass spectrometry as thimet oligopeptidase (TOP). The identity of the CPI-0004Na activating enzyme and TOP was further supported by the similar substrate specificity of the purified enzyme and recombinant TOP, by thiol stimulation of CPI-0004Na cleavage by cancer cell conditioned media (unique characteristic of TOP) and by the inhibition of CPI-0004Na activation by specific inhibitors or immunoprecipitation. Although other enzymes can be involved, TOP clearly appears to be a likely candidate for extracellular activation of the CPI-0004Na prodrug.

Recombinant interferon-gamma secreted by Chinese hamster ovary-320 cells cultivated in suspension in protein-free media is protected against extracellular proteolysis by the expression of natural protease inhibitors and by the addition of plant protein hydrolysates to the culture medium.

Biosafety requirements increasingly restrict the cultivation of mammalian cells producing therapeutic glycoproteins to conditions that are devoid of any compound of animal origin. On cultivation in serum-free media, the proteases inhibitors, usually found in serum, cannot protect secreted recombinant proteins against unwanted endogenous proteolysis. Chinese hamster ovary (CHO) cells, secreting recombinant human interferon-gamma (CHO-320 cell line) and cultivated in suspension in an original protein-free medium, expressed at least two members of the matrix metalloproteinases (MMP), either at the cell surface (proMMP-14 and MMP-14) or secreted (proMMP-9). In addition, tissue- and urinary-type plasminogen activators were also secreted in such culture conditions. At the cell surface, dipeptidyl peptidase IV and tripeptidyl peptidase II (TPPII) activities were also detected, and their activities decreased during time course of batch cultures. The proteolytic activities of these proteins were counterbalanced by (1) their expression as zymogens (proMMP-9, proMMP-14), (2) the expression of their natural inhibitors, tissue inhibitors of metalloproteinases-1 and -2 and plasminogen activator inhibitor-1 (PAI-1), or (3) the addition of plant protein hydrolysates to the culture medium, acting as a nonspecific source of TPPII inhibitors. This study points out that, even in protein-free media, recombinant proteins secreted by CHO cells are actively protected against physiological and unwanted extracellular proteolysis either by endogenous or by exogenous inhibitors.

Proteomics of bronchoalveolar lavage fluid.

Lung diseases are essentially multi-factorial diseases that require a global analysis, and thus, cannot be understood through the sole analysis of individual or small numbers of genes. Proteome analysis has rapidly developed in the post-genome era and is now widely accepted as the obligated complementary technology for genetic profiling. It has been shown to be a powerful tool for the study of human diseases and for identifying novel prognostic, diagnostic and therapeutic markers. During last years, proteomic approaches applied to lung diseases are centred on the analysis of proteins in samples, such as cell cultures, biopsies and physiological fluids like serum and, especially, bronchoalveolar lavage fluid (BALF). BALF is presently the most common way of sampling the components of the epithelial lining fluid (ELF) and the most faithful reflect of the protein composition of the pulmonary airways. This review focuses on the state of the investigations of BALF proteome and its powerful contribution both to a better knowledge of the lung structure at the molecular level and to the study of lung disorders at the clinical level.

Purification, characterization, gene cloning and preliminary X-ray data of the exo-inulinase from Aspergillus awamori.

Extracellular exo-inulinase has been isolated from a solid-phase culture of the filamentous fungus Aspergillus awamori var. 2250. The apparent molecular mass of the monomer enzyme was 69 +/- kDa, with a pI of 4.4 and a pH optimum of 4.5. The enzyme hydrolysed the beta-(2-->1)-fructan (inulin) and beta-(2-->6)-fructan (levan) via exo-cleavage, releasing fructose. The values for the Michaelis constants K(m) and V(max) in the hydrolysis of inulin were 0.003 +/- 0.0001 mM and 175 +/- 5 micromol.min(-1).mg(-1). The same parameters in the hydrolysis of levan were 2.08 +/- 0.04 mg/ml and 1.2 +/- 0.02 micromol/min per mg, respectively. The gene and cDNA encoding the A. awamori exo-inulinase were cloned and sequenced. The amino acid sequence indicated that the protein belongs to glycoside hydrolase family 32. A surprisingly high similarity was found to fructosyltransferase from Aspergillus foetidus (90.7% on the level of the amino acid sequence), despite the fact that the latter enzyme is unable to hydrolyse inulin and levan. Crystals of the native exo-inulinase were obtained and found to belong to the orthorhombic space group P2(1)2(1)2(1) with cell parameters a=64.726 A (1A=0.1 nm), b=82.041 A and c=136.075 A. Crystals diffracted beyond 1.54 A, and useful X-ray data were collected to a resolution of 1.73 A.

Modifier effect of ENOS in autosomal dominant polycystic kidney disease.

A significant phenotypical variability is observed in autosomal dominant polycystic kidney disease (ADPKD). ADPKD is associated with altered endothelial-dependent vasodilation and decreased vascular production of nitric oxide (NO). Thus, ENOS, the gene coding for the endothelial nitric oxide synthase (eNOS), could have a modifier effect in ADPKD. In order to test this hypothesis, we genotyped 173 unrelated ADPKD patients from Belgium and the north of France for the Glu298Asp, intron 4 VNTR and T-786C polymorphisms of ENOS and looked for their influence on the age at end-stage renal disease (ESRD). In males (n = 93), the Glu298Asp polymorphism was associated with a lower age at ESRD (Glu/Asp + Asp/Asp: 49.0 +/- 1.2 years, n = 53; Glu/Glu: 53.5 +/- 1.5 years, n = 40; simple regression, P = 0.02; multiple regression, P = 0.006). This effect was confirmed in a subset of males linked to PKD1 and reaching ESRD before age 45, and by a cumulative renal survival analysis in PKD1-linked families. Further studies demonstrated that NO synthase (NOS) activity was decreased in renal artery samples from ADPKD males harbouring the Asp298 allele, in association with post-translational modifications and partial cleavage of eNOS. No significant effect of the other polymorphisms was found in males, and no polymorphism influenced the age at ESRD in females. In conclusion, the frequent Glu298Asp polymorphism of ENOS is associated with a 5 year lower mean age at ESRD in this subset of ADPKD males. This effect could be due to a decreased NOS activity and a partial cleavage of eNOS, leading to a further decrease in the vascular production of NO.

Structural modifications in the membrane-bound regions of the gastric H+/K+-ATPase upon ligand binding.

Extensive trypsin proteolysis was used to examine the accessibility of membrane bound segments of the gastric H+/K+-ATPase under different experimental conditions known to induce either the E1 or the E2 conformation. Membrane-anchored peptides were isolated after trypsinolysis and identified by sequencing. We show that several membrane bound segments are involved in the conformational change. In the N-terminal region, a M1-M2 peptide (12 kDa) was found to be associated with the membrane fraction after digestion in the presence of K+ or in the presence of vanadate (12 kDa and 15 kDa). In the M3 and M4 region, no difference was observed for the peptide obtained in E1 or E2-K conformations, but the peptide generated in the presence of vanadate begins 12 amino-acid residues earlier in the sequence. Cytoplasmic loop region: we show here that a peptide beginning at Asp574 and predicted to end at Arg693 is associated with the membrane for a vanadate-induced conformation. In the M5-M6 region, the membrane-anchored peptide obtained on E1 is 39 amino acids shorter than the E2 peptide. In the M7-M8 region, the same peptide encompassing the M7 and M8 transmembrane segments was produced for E1 and E2 conformations.

Structure and dynamics of the membrane-embedded domain of LmrAinvestigated by coupling polarized ATR-FTIR spectroscopy and (1)H/(2)H exchange.

Bacterial LmrA, an integral membrane protein of Lactococcus lactis, confers multidrug resistance by mediating active extrusion of a wide variety of structurally unrelated compounds. Similar to its eucaryotic homologue P-gp, this protein is a member of the ATP-binding cassette (ABC) superfamily. Different predictive models, based on hydropathy profiles, have been proposed to describe the structure of the ABC transporters in general and of LmrA in particular. We used polarized attenuated total reflection infrared spectroscopy, combined with limited proteolysis, to investigate the secondary structure and the orientation of the transmembrane segments of LmrA. We bring the first experimental evidence that the membrane-embedded domain of LmrA is composed of transmembrane-oriented alpha-helices. Furthermore, a new approach was developed in order to provide information about membrane domain dynamics. Monitoring the infrared linear dichroism spectra in the course of (1)H/(2)H exchange allowed to focus the recording of exchange rates on the membrane-embedded region of the protein only. This approach revealed an unusual structural dynamics, indicating high flexibility in this antibiotic binding and transport region.

Maternal tobacco smoking and lung epithelium-specific proteins in amniotic fluid.

The bronchiolar 16 kD Clara cell secretory protein (CC16) and the alveolar surfactant-associated protein A (SP-A) are secreted in the amniotic fluid (AF), where they reflect the growth and the maturity of the fetal lung. To evaluate the possible effects of in utero tobacco smoke exposure upon infant bronchoalveolar epithelium function and maturity, CC16 and SP-A levels were determined in AF obtained at term (36-41 wk) from 28 nonsmoking, 18 smoke-exposed, and 28 smoking mothers with uncomplicated pregnancies. Tobacco smoke exposure was assessed by questionnaire and the assay in AF and maternal urine of cotinine, a stable nicotine metabolite. The specificity of the changes of CC16 and SP-A concentrations in AF was assessed by comparison with nonpulmonary proteins of high- (albumin and transferrin) or low-molecular weight (beta2-microglobulin, retinol binding protein, cystatin-C). Pulmonary and nonpulmonary AF proteins were also compared by two-dimensional gel electrophoresis between smoking and nonsmoking mothers. The levels of CC16 and SP-A as well as low- and high-molecular-weight proteins were not significantly different between the three smoking categories. The protein pattern of AF, established by two-dimensional gel electrophoresis, did not reveal any quantitative or qualitative difference between nonsmoking (n = 10), smoke-exposed (n = 5), and smoking mothers (n = 5). By multiple regression analysis of possible determinants, tobacco smoke did not emerge as a significant predictor of CC16 and SP-A concentrations in AF. SP-A level was dependent only on gestational age at birth (r2 = 0.1, p = 0.001), whereas CC16 correlated only with the levels of low-molecular weight proteins (r2 = 0.2, p = 0.0001). The latter correlation suggests that CC16 enters AF not only as a result of its secretion at the surface of the respiratory tract but also partly following its elimination by the fetal kidney. This study suggests that maternal smoking during pregnancy is not associated with alterations of the secretory functions of the epithelium of the distal airways and the alveoli at term.

Proteomics as the tool to search for lung disease markers in bronchoalveolar lavage.

Most lung disorders are known to be associated to considerable modifications of surfactant composition. Numerous of these abnormalities have been exploited in the past to diagnose lung diseases, allowing proper treatment and follow-up. Diagnosis was then based on phospholipid content, surface tension and cytological features of the epithelial lining fluid (ELF), sampled by bronchoalveolar lavage (BAL) during fiberoscopic bronchoscopy. Today, it appears that the protein content of ELF displays a remarkably high complexity, not only due to the wide variety of the proteins it contains but also because of the great diversity of their cellular origins. The significance of the use of proteome analysis of BAL fluid for the search for new lung disease marker proteins and for their simultaneous display and analysis in patients suffering from lung disorders has been examined.

Lentil seed aquaporins form a hetero-oligomer which is phosphorylated by a Mg(2+)-dependent and Ca(2+)-regulated kinase.

In plants, aquaporins regulate the water flow through membranes during growth, development and stress responses. We have isolated two isoforms of the aquaporin family from the protein-storage vacuoles of lentil (Lens culinaris Med.) seeds. Chemical cross-linking experiments showed that both isoforms belong to the same oligomer in the membrane and are phosphorylated by a membrane-bound protein kinase. We assigned the kinase activity to a 52 kDa protein that is magnesium-dependent and calcium-regulated.

Purification and characterization of two voltage-dependent anion channel isoforms from plant seeds.

Mitochondria were isolated from imbibed seeds of lentil (Lens culinaris) and Phaseolus vulgaris. We copurified two voltage-dependent anion channel from detergent solubilized mitochondria in a single purification step using hydroxyapatite. The two isoforms from P. vulgaris were separated by chromatofocusing chromatography in 4 M urea without any loss of channel activity. Channel activity of each isoform was characterized upon reconstitution into diphytanoyl phosphatidylcholine planar lipid bilayers. Both isoforms form large conductance channels that are slightly anion selective and display cation selective substates.

Membrane topology of VacA cytotoxin from H. pylori.

The interaction of VacA with membranes involves: (i) a low pH activation that induces VacA monomerization in solution, (ii) binding of the monomers to the membrane, (iii) oligomerization and (iv) channel formation. To better understand the structure-activity relationship of VacA, we determined its topology in a lipid membrane by a combination of proteolytic, structural and fluorescence techniques. Residues 40-66, 111-169, 205-266, 548-574 and 723-767 were protected from proteolysis because of their interaction with the membrane. This last peptide was shown to most probably adopt a surface orientation. Both alpha-helices and beta-sheets were found in the structure of the protected peptides.

Characterization of the interaction of IpaB and IpaD, proteins required for entry of Shigella flexneri into epithelial cells, with a lipid membrane.

Entry of Shigella flexneri into epithelial cells and lysis of the phagosome involve the IpaB, IpaC, and IpaD proteins, which are secreted by type III secretion machinery. We report here the purification of IpaB and IpaD and the characterization of their lipid-binding properties as a function of pH. The interaction of IpaB with the membrane was quite independent of the pH whereas that of IpaD took place only at low pH. To support the data obtained with the purified proteins, we designed a system in which protein secretion by live bacteria was induced in the presence of liposomes, thereby allowing interaction of proteins with lipids directly after secretion and bypassing any purification step. In these conditions, both IpaB and IpaC, as well as minor amounts of IpaA and IpgD, were associated with the membrane and the ratio of IpaB to IpaC was modulated by the pH. The relevance of these results with respect to the dual roles of IpaB, IpaC and IpaD in induction of membrane ruffles and lysis of the endosomal membrane is discussed.