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Lipopolysaccharides (LPS) constitute the outermost layer of Gram-negative bacteria and consequently play an important role in bacterial infections. In order to address public health issues posed by Gram-negative bacteria, it is necessary to elucidate the structure of the molecular actors at the forefront of infections. LPS virulence and toxicity are partially modulated by lipid A, a hydrophobic saccharolipid that anchors LPS to the bacterial outer membrane. Understanding the lipid A structure is inherently intertwined with understanding its role as an endotoxin. Accordingly, several successful strategies incorporating tandem mass spectrometry have been applied toward the structural analysis of lipid A. Herein, a shotgun HCD strategy was applied toward the characterization of the lipid A profile of Pseudomonas aeruginosa PAO1. This analysis was enhanced by the development of an LC-MS/MS approach to eliminate isomeric signals in the MS/MS spectra that confounded characterization. Importantly, combining reverse phase chromatography with HCD and ultraviolet photodissociation analyses of the lipid A profile revealed the presence of previously unreported lipid A acyl chain positional isomers. Altogether, these strategies provide the most in-depth structural and molecular characterization of PAO1 lipid A to date.

Lipopolysaccharides (LPS) constitute the outermost layer of Gram-negative bacteria and consequently play an important role in bacterial infections. In order to address public health issues posed by Gram-negative bacteria, it is necessary to elucidate the structure of the molecular actors at the forefront of infections. LPS virulence and toxicity are partially modulated by lipid A, a hydrophobic saccharolipid that anchors LPS to the bacterial outer membrane. Understanding the lipid A structure is inherently intertwined with understanding its role as an endotoxin. Accordingly, several successful strategies incorporating tandem mass spectrometry have been applied toward the structural analysis of lipid A. Herein, a shotgun HCD strategy was applied toward the characterization of the lipid A profile of Pseudomonas aeruginosa PAO1. This analysis was enhanced by the development of an LC–MS/MS approach to eliminate isomeric signals in the MS/MS spectra that confounded characterization. Importantly, combining reverse phase chromatography with HCD and ultraviolet photodissociation analyses of the lipid A profile revealed the presence of previously unreported lipid A acyl chain positional isomers. Altogether, these strategies provide the most in-depth structural and molecular characterization of PAO1 lipid A to date.

Biofilms have significance in medical, industrial, and environmental settings, and can cause important damage. As biofilms are tolerant to various stresses, including antibiotics, it is necessary to better understand their formation. For this reason, we characterized the phospholipidome of Pseudomonas aeruginosa, an opportunistic pathogen involved in numerous infections, during the first steps of the biofilm development. By a liquid chromatography-tandem mass spectrometry time-course analysis over a 24-h period, we compared the phospholipid (PL) composition of immobilized (attached) and planktonic (unattached) P. aeruginosa PAO1 cells. Our results showed that the PL content of P. aeruginosa PAO1 was mainly modulated by the incubation time, thus related to bacterial growth but also, more modestly, by the immobilization state. We observed that relative amounts of PL varied over time with two main profiles and that these profiles are correlated to its fatty acid composition, including the degree of unsaturation. A statistical analysis revealed that the PL contents of both attached and unattached PAO1 cells were significantly different mainly after 3 and 6 h of incubation and that the amounts of two PL presented a statistical difference between attached and unattached cells all along the 24-h period: PtdEtn 16:0_18:1 and PtdEtn 18:1_18:1.

Changes in leaf soluble proteome were explored in 3-month-old plants of metallicolous (M) and nonmetallicolous (NM) Agrostis capillaris L. populations exposed to increasing Cu concentrations (1-50 μM) to investigate molecular mechanisms underlying plant responses to Cu excess and tolerance of M plants. Plants were cultivated on perlite (CuSO4 spiked-nutrient solution). Soluble proteins, extracted by the trichloroacetic acid/acetone procedure, were separated with 2-DE (linear 4-7 pH gradient). Analysis of CCB-stained gels (PDQuest) reproducibly detected 214 spots, and 64 proteins differentially expressed were identified using LC-MS/MS. In both populations, Cu excess impacted both light-dependent (OEE, cytochrome b6-f complex, and chlorophyll a-b binding protein), and -independent (RuBisCO) photosynthesis reactions, more intensively in NM leaves (ferredoxin-NADP reductase and metalloprotease FTSH2). In both populations, upregulation of isocitrate dehydrogenase and cysteine/methionine synthases respectively suggested increased isocitrate oxidation and enhanced need for S-containing amino-acids, likely for chelation and detoxification. In NM leaves, an increasing need for energetic compounds was indicated by the stimulation of ATPases, glycolysis, pentose phosphate pathway, and Calvin cycle enzymes; impacts on protein metabolism and oxidative stress increase were respectively suggested by the rise of chaperones and redox enzymes. Overexpression of a HSP70 may be pivotal for M Cu tolerance by protecting protein metabolism. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD001930 (http//proteomecentral.proteomexchange.org/dataset/PXD001930).

Lipid A is a major compound of the outer membrane of gram-negative bacteria and is a key factor of bacterial virulence. As lipid A's structure differs among bacterial species and varies between strains of the same species, knowing its modifications is essential to understand its implications in the infectious process. To analyze these lipids, matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) is a well-suited method that is fast and efficient. However, there are limitations with the matrix and additives used, such as the suppression of signal or prompt fragmentations that could give a false overview of lipid A composition in biological samples. For a comprehensive analysis of the entire lipid A species present in a sample, we tested 16 matrices and 11 additives on two commercial lipids A. The first commercial one contains single phosphorylation group, and the second contains two phosphorylation and two ketodeoxyoctonic acid (KDO) groups. The lipid A containing KDO groups was essentially detected by the 3-hydroxypicolinic acid (3-HPA) matrix, whereas the monophosphorylated lipid A could be detected by 13 matrices out of the 16. We also demonstrated that the signal of diphosphorylated lipid A can be enhanced with the use of additives in the matrix. Our study indicated that the best conditions to obtain a clear signal of both lipids A without prompt fragmentation was the use of 3-HPA with 10mM trifluoroacetic acid (TFA).

EnterohaemorrhagicEscherichia colisuch as serotype O157:H7 are a leading cause of food-associated outbreaks. While the primary reservoir is associated with cattle, plant foods have been associated as sources of human infection.E. coliis able to grow in the tissue of food plants such as spinach. While fecal contamination is the primary suspect, soil has been underestimated as a potential reservoir. Persistence of bacterial populations in open systems is the product of growth, death, predation, and competition. Here we report thatE. coliO157:H7 can grow using the soluble compounds in soil, and characterize the effect of soil growth in the stationary phase proteome.E. coli933D (stxII-) was cultured in Soil Extracted Soluble Organic Matter (SESOM) and the culturable count determined for 24 d. The proteomes of exponential and stationary phase populations were characterized by 2D gel electrophoresis and protein spots were identified by MALDI-TOF mass spectrometry. While LB controls displayed a death phase, SESOM grown population remained culturable for 24 d, indicating an altered physiological state with superior longevity. This was not due to decreased cell density on entry to stationary phase as 24h SESOM populations concentrated 10-fold retained their longevity. Principal component analysis showed that stationary phase proteomes from SESOM and LB were different. Differences included proteins involved in stress response, motility, membrane and wall composition, nutrient uptake, translation and protein turnover, and anabolic and catabolic pathways, indicating an altered physiological state of soil-grown cells entering stationary phase. The results suggest thatE. colimay be a soil commensal that in absence of predation and competition maintains stable populations in soil.

Esca is one of the major diseases affecting vineyards with direct impact on product yield; nevertheless, scientific studies concerning its impact on grape quality are scarce. As an attempt to better understand the mechanisms behind "Esca proper" development in grapes, this work focused on the identification of proteins whose expression is altered by the disease. 2-DEs were performed on protein extracts from grape skins at different stages of maturity for two consecutive vintages. Grapes were collected in 2009 and in 2010 from plants that did not present signs of infection by Esca proper since the 2004 vintage and from plants that presented cast leaf symptoms at least once since 2004. For the first time, 13 proteins were shown to be influenced by Esca proper during the ripening process. Extensive bioinformatics analysis allowed the grouping of proteins involved in (i) stress tolerance and defense response, (ii) oxidative phosphorylation, (iii) oxidation-reduction processes in mitochondria, and (iv) oxidation-reduction processes in chloroplasts. Of these 13 proteins, cysteine synthase is the only one implicated in a metabolic pathway of oenological interest. This study shows how foliar symptoms of Esca proper may impact stress-related pathways in grapes, which are characterized by modifications in the chain of oxidative phosphorylation and redox scavenging.

Metamaterials have a number of interesting and potentially useful applications in a variety of fields, such as chemical and biological sensing, enhancement of spontaneous emission, nonlinear optics and as substrates for use in surface enhanced Raman spectroscopy (SERS). However, to date the low-wavelength cutoff for the majority of work at the higher frequency end of the spectrum has been determined by use of the coinage metals, which intrinsically prohibit their implementation below a vacuum wavelength of approximately 500nm for gold and 350nm for silver. Producing nanostructured plasmonic media that exhibit metamaterial functionalities in the ultraviolet will have a number of benefits. Not only will working in a new range of the electromagnetic spectrum allow for higher energy photons to be controlled, but a number of other benefits arise from the behaviour of different materials in the ultraviolet. For instance, many biological molecules, including DNA, exhibit fluorescence in the UV range, allowing for label-free detection and analysis of biological material; the intrinsic electronic absorption can be used to increase this label-free bio-sensitivity as well as enable the possibility of SE(R)RS, a process further enhanced by the frequency dependence on the efficiency of this scattering process. Here, we demonstrate the fabrication and characterisation of metamaterials operating in the deep-near UV. By using alternatives to the coinage metals, including aluminium and gallium, we have measured optical responses in the system down to approximately 200 nm. Sample preparation utilises a self-assembly method, allowing for the production of macroscopic-sized assemblies (> 1 cm2) of nanometric elements (radius ~ 25 nm, separation ~ 100 nm). Careful control of the fabrication conditions allows fine control of the structural parameters, which in turn allows tunability of the optical properties over a wide range of wavelengths (> 200 nm). The structures produced include gallium nanorods, oriented with their long axis perpendicular to the substrate and having a sub-wavelength interrod distance, fabricated via oxygen- and water-free electrodeposition into nanoporous anodised alumina (AAO), and aluminium nanohole arrays fabricated using AAO as a mask for ion milling, with elements at the same nanometric size scale. Both systems have been optically characterised across the UV and visible wavelength ranges and compared with numerical modelling in order to analyse and describe their behaviour.

Bacterial biofilms are complex cell communities found attached to surfaces and surrounded by an extracellular matrix composed of exopolysaccharides, DNA, and proteins. We investigated the whole-genome expression profile of Pseudomonas aeruginosa sessile cells (SCs) present in biofilms developed on a glass wool substratum. The transcriptome and proteome of SCs were compared with those of planktonic cell cultures. Principal component analysis revealed a biofilm-specific gene expression profile. Our study highlighted the overexpression of genes controlling the anthranilate degradation pathway in the SCs grown on glass wool for 24 h. In this condition, the metabolic pathway that uses anthranilate for Pseudomonas quinolone signal production was not activated, which suggested that anthranilate was primarily being consumed for energy metabolism. Transposon mutants defective for anthranilate degradation were analyzed in a simple assay of biofilm formation. The phenotypic analyses confirmed that P. aeruginosa biofilm formation partially depended on the activity of the anthranilate degradation pathway. This work points to a new feature concerning anthranilate metabolism in P. aeruginosa SCs.

Antibiotics such as chlortetracycline (CTC) have been used to promote growth of pigs for decades, but concerns over increased antibiotic-resistant infections in humans have prompted the development of alternative strategies. Developing alternatives to antibiotic growth promoters (AGPs) could be informed by information on the mechanisms of growth promotion, notably, how AGPs affect the microbial populations of the gastrointestinal tract. Pigs from three sows were aseptically delivered by cesarean section. Six piglets were distributed to each of two foster mothers until weaning, when piglets were fed a diet with or without 50 mg/kg CTC for 2 weeks. The ileal bacterial microbiota was characterized by using a cultivation-independent approach based on DNA extraction, PCR amplification, cloning, and sequencing of the 16S rRNA gene pool. The ileal and mucosal communities of these growing pigs were dominated by Lactobacillus bacteria, various members of the family Clostridiaceae , and members of the poorly known genus Turicibacter . Overall, CTC treatment resulted in three shifts: a decrease in L actobacillus johnsonii , an increase in L . amylovorus , and a decrease in Turicibacter phylotypes. The composition of the microbiota varied considerably between individual pigs, as revealed by shared operational taxonomic units (OTUs) and similarity (SONS) analysis (θ YC values). While the observed variation between untreated pigs obscured the possible effect of CTC, ∫-LIBSHUFF and SONS analyses of pooled libraries indicated a significant shift due to CTC in both the lumen and the mucosa, with some OTUs unique to either treated or control ileum. DOTUR analysis revealed little overlap between control and treated communities at the 3% difference level, indicating unique ileal communities in the presence of CTC.

Antibiotics have traditionally been used for growth promotion in the pork industry; however, their use in animal feed has recently been limited because of human health concerns. The intestinal microbiota plays an important role in mediating many physiological functions such as digestion and animal growth. It was hypothesized that use of antibiotics as growth promotants and subsequent variations in intestinal microbiota induce significant changes in the intestinal glycoconjugate composition, which ultimately affects animal growth and disease susceptibility. The aim of this study was to characterize the lectin binding profiles of the ileum of weanling pigs in response to the absence of intestinal microbiota and to the use of the antibiotic chlortetracycline as growth promotant. Eighteen half-sib piglets obtained by cesarean section were divided into 3 treatment groups (n = 6) and maintained as control, antibiotic-fed, and gnotobiotic piglets until 5 wk of age. The glycoconjugate composition of the ileal tissues was examined by lectin histochemistry. Lycopersicon esculentum lectin, Jacalin, Pisum sativum agglutinin, Lens culinaris agglutinin (LCA), and Sambucus nigra lectin showed significant differences (P < 0.05) in binding intensities on the dome and villous epithelium between the treatment groups. Griffonia simplicifolia lectin I, Glycine maxi agglutinin, and Arachis hypogea agglutinin exhibited differences (P < 0.05) between treatment groups in lectin binding on goblet cells. Triticum vulgaris agglutinin, Pisum sativum agglutinin, and LCA showed significant differences (P < 0.05) in binding intensities on dome, corona, and follicular regions of the ileum among treatment groups of animals. Overall, ileal tissues from gnotobiotic piglets expressed significantly weaker (P < 0.05) lectin binding for many lectins compared with control and antibiotic groups. This suggests that the intestinal microbiota plays an important role in the expression of sugar moieties in the intestine. Lectins LCA, Phaseolus vulgaris Leucoagglutinin, and Maackia amurensis lectin II showed significant differences (P < 0.05) in lectin bindings between control and antibiotic-fed piglets. This indicates that chlortetracycline as a growth promotant induces biologically relevant changes in the lectin binding profile of the ileum. These findings will help in further understanding the role of the gut microbiota and the mechanisms of action of antibiotics as growth promotants in pigs.

Bacillus cereus is believed to be a soil bacterium, but studied solely in laboratory culture media. The aim of this study was to assess the physiology of B. cereus growing on soil organic matter by a proteomic approach. Cells were cultured to mid-exponential phase in soil extracted solubilized organic matter (SESOM), which mimics the nutrient composition of soil, and in Luria-Bertani broth as control. Silver staining of the two-dimensional gels revealed 234 proteins spots up-regulated when cells were growing in SESOM, with 201 protein spots down-regulated. Forty-three of these differentially expressed proteins were detected by Colloidal Coomassie staining and identified by matrix assisted laser desorption ionization-time of flight MS of tryptic digests. These differentially expressed proteins covered a range of functions, primarily amino acid, lipid, carbohydrate and nucleic acid metabolism. These results suggested growth on soil-associated carbohydrates, fatty acids and/or amino acids, concomitant with shifts in cellular structure.

UNLABELLED Acquisition of Pseudomonas aeruginosa is known as a negative prognostic factor in patients with cystic fibrosis. We started a pilot study to evaluate Ps. aeruginosa gene expression directly from the sputum of infected patients. Total RNA was purified from 15 sputum samples collected from 10 patients, and the expression levels of five genes from Ps. aeruginosa were measured by RT-qPCR. Expression of algD, algR, antB, lasB and pqsA genes was determined in sputa that contained Ps. aeruginosa cells. The resultant data provided an overview of the expression of these genes in CF patients. Except for the correlation between algD expression and the mucoid phenotype, the gene expression profile could not be associated with the clinical status of patients. However, beyond the heterogeneity of the Ps. aeruginosa phenotype in sputum, we observed a correlation between the expression of antB and pqsA and a low level of lasB transcripts. SIGNIFICANCE AND IMPACT OF THE STUDY Pseudomonas aeruginosa infection leads to high morbidity and mortality in cystic fibrosis patients. The identification of Ps. aeruginosa-assigned factors is important to eradicate the colonization. We started a pilot study to evaluate the gene expression of Ps. aeruginosa directly from the sputum of infected patients. Preliminary results suggest that beyond the heterogeneity of the Ps. aeruginosa phenotype in sputum, we observe a correlation between the expression of antB and pqsA and a low level of lasB transcripts. This approach could shed some light on the behaviour of Ps. aeruginosa during pulmonary infection and may reveal some important elements for optimizing therapy.

Bacillus is commonly isolated from soils, with organisms of Bacillus cereus sensu lato being prevalent. Knowledge of the ecology of B. cereus and other Bacillus species in soil is far from complete. While the older literature favors a model of growth on soil-associated organic matter, the current paradigm is that B. cereus sensu lato germinates and grows in association with animals or plants, resulting in either symbiotic or pathogenic interactions. An in terra approach to study soil-associated bacteria is described, using filter-sterilized soil-extracted soluble organic matter (SESOM) and artificial soil microcosms (ASM) saturated with SESOM. B. cereus ATCC 14579 displayed a life cycle, with the ability to germinate, grow, and subsequently sporulate in both the liquid SESOM extract and in ASM inserted into wells in agar medium. Cells grew in liquid SESOM without separating, forming multicellular structures that coalesced to form clumps and encasing the ensuing spores in an extracellular matrix. Bacillus was able to translocate from the point of inoculation through soil microcosms as shown by the emergence of outgrowths on the surrounding agar surface. Microscopic inspection revealed bundles of parallel chains inside the soil. The motility inhibitor l -ethionine failed to suppress outgrowth, ruling out translocation by a flagellar-mediated mechanism such as swimming or swarming. Bacillus subtilis subsp. subtilis Marburg and four Bacillus isolates taken at random from soils also displayed a life cycle in SESOM and ASM and were all able to translocate through ASM, even in presence of l -ethionine. These data indicate that B. cereus is a saprophytic bacterium that is able to grow in soil and furthermore that it is adapted to translocate by employing a multicellular mode of growth.

Background Bacterial biofilms are predominant in natural ecosystems and constitute a public health threat because of their outstanding resistance to antibacterial treatments and especially to antibiotics. To date, several systems have been developed to grow bacterial biofilms in order to study their phenotypes and the physiology of sessile cells. Although relevant, such systems permit analysis of various aspects of the biofilm state but often after several hours of bacterial growth. Results Here we describe a simple and easy-to-use system for growing P. aeruginosa biofilm based on the medium adsorption onto glass wool fibers. This approach which promotes bacterial contact onto the support, makes it possible to obtain in a few minutes a large population of sessile bacteria. Using this growth system, we demonstrated the feasibility of exploring the early stages of biofilm formation by separating by electrophoresis proteins extracted directly from immobilized cells. Moreover, the involvement of protein synthesis in P. aeruginosa attachment is demonstrated. Conclusions Our system provides sufficient sessile biomass to perform biochemical and proteomic analyses from the early incubation period, thus paving the way for the molecular analysis of the early stages of colonization that were inaccessible to date. Electronic supplementary material The online version of this article (doi:10.1186/s12866-014-0253-z) contains supplementary material, which is available to authorized users.

International audience; The prerequisite to monitor gene expression is the selection of reference genes for normalization of RT-qPCR results. Using 13 sputum samples collected from 9 CF patients, we demonstrated that PA2875 and PA3340 are better reference genes than the previously used clpX and oprL genes. (C) 2013 Elsevier Ltd. All rights reserved.

Plasmonic technology relies on the coupling of light to surface electromagnetic modes on smooth or structured metal surfaces. While some applications utilise the resonant nature of surface polaritons, others require broadband characteristics. We demonstrate unidirectional and broadband plasmonic antennas with large acceptance angles based on chirped plasmonic gratings. Near-field optical measurements have been used to visualise the excitation of surface plasmon polaritons by such aperiodic structures. These weakly aperiodic plasmonic crystals allow the formation of a trapped rainbow-type effect in a two-dimensional geometry as surface polaritons of different frequencies are coherently excited in different locations over the plasmonic structure. Both the crystal's finite size and the finite lifetime of plasmonic states are crucial for the generation of broadband surface plasmon polaritons. This approach presents new opportunities for building unidirectional, broadband and broad-angle plasmonic couplers for sensing purposes, information processing, photovoltaic applications and shaping and manipulating ultrashort optical pulses.

It has been suggested that changes in diet at weaning in pigs induce intestinal inflammation which may be mediated through toll-like receptors (TLRs). We hypothesized that the use of antibiotics as growth promoters and subsequent changes in intestinal microbiota may mediate changes in the expression of TLRs in the intestine. Thus, this study was performed to assess the changes in intestinal TLRs in weanling pigs in response to the use of chlortetracycline as a growth promotant, and under gnotobiotic conditions. Eighteen cesarean-derived half-sib piglets were divided into three groups; antibiotic-fed, control (normal-fed) and gnotobiotic groups.TLR-2, -4, -5 and -9 gene expression and abundance of TLR-2 and -9 proteins in the ileum of pigs was assessed at 5 wk of age. No significant differences (p ?0.5) in TLR-2, -4, -5 and -9transcript levels and abundanceof TLR-2 and -9 proteins among three groups of pigswere observed.

Members of the genus Bacillus are commonly isolated from soils, with members of the Bacillus cereus group being prevalent. Our knowledge of the ecology of B. cereus and other aerobic spore-forming bacteria in soil is far from complete. We have developed an in terra approach to study soil-associated aerobes, using filter-sterilized soil extracted soluble organic matter (SESOM). B. cereus is able to germinate, grow and then sporulate when inoculated into SESOM, or in artificial soil microcosms (ASM). Furthermore, B. cereus switches from single-celled growth to form chains that coalesce to form clumps when cultured in SESOM. Data indicate that the switch to form bundles of chains in soil contributed to translocation of the population through soil by a process termed sliding. Methods for preparing SESOM and ASM are outlined, as are approaches to proteomic analysis and screening transposon mutant libraries for genes contributing to the multicellular phenotype.

The soil saprophyte Bacillus cereus forms biofilms at solid-liquid interfaces. The composition of the extracellular polymeric matrix is not known, but biofilms of other bacteria are encased in polysaccharides, protein, and also extracellular DNA (eDNA). A Tn 917 screen for strains impaired in biofilm formation at a solid-liquid interface yielded several mutants. Three mutants deficient in the purine biosynthesis genes purA , purC , and purL were biofilm impaired, but they grew planktonically like the wild type in Luria-Bertani broth. Biofilm populations had higher purA , purC , and purL transcript ratios than planktonic cultures, as measured by real-time PCR. Laser scanning confocal microscopy (LSCM) of BacLight-stained samples indicated that there were nucleic acids in the cell-associated matrix. This eDNA could be mobilized off the biofilm into an agarose gel matrix through electrophoresis, and it was a substrate for DNase. Glass surfaces exposed to exponentially growing populations acquired a DNA-containing conditioning film, as indicated by LSCM. Planktonic exponential-phase cells released DNA into an agarose gel matrix through electrophoresis, while stationary-phase populations did not do this. DNase treatment of planktonic exponential-phase populations rendered cells more susceptible than control populations to the DNA-interacting antibiotic actinomycin D. Exponential-phase purA cells did not contain detectable eDNA, nor did they convey a DNA-containing conditioning film to the glass surface. These results indicate that exponential-phase cells of B. cereus ATCC 14579 are decorated with eDNA and that biofilm formation requires DNA as part of the extracellular polymeric matrix.

International audience; Understanding the molecular basis of resistance to imatinib, a tyrosine kinase inhibitor used as front-line therapy in chronic myeloid leukemia, remains a challenge for successful treatment. In an attempt to identify new mechanisms of resistance, we performed a comparative proteomic analysis of an imatinib-resistant cell line generated from the erythroblastic cell line K562 (K562-r) for which no known mechanism of resistance has been detected. Bidimensional gel electrophoresis was carried out to compare the protein expression pattern of imatinib-sensitive and of imatinib-resistant K562 cells. Among the 400 matched spots on five pairs of gels, only 14 spots had a significantly increased or decreased expression leading to the identification of 24 proteins identified as scaffold proteins, metabolic enzymes, DNA translation and maturation, and chaperon proteins. Among the chaperon family, only Hsp70 and Hsc70 are overexpressed in K562-r, results confirmed by Western blotting. We recently reported the participation of Hsp70 overexpression in imatinib resistance whereas a role for Hsc70 has yet to be determined. Hsc70 is not involved in imatinib resistance as the inhibition of its expression by siRNA does not restore sensitivity to imatinib. In contrast, the induced decreased expression of Hsc70 was accompanied by a greater overexpression of Hsp70. This proteomic study therefore suggests opposing roles of Hsp70 and Hsc70 in imatinib resistance.

Bacillus cereus is believed to be a soil bacterium, but studied solely in laboratory culture media. The aim of this study was to assess the physiology of B. cereus growing on soil organic matter by a proteomic approach. Cells were cultured to mid-exponential phase in soil extracted solubilized organic matter (SESOM), which mimics the nutrient composition of soil, and in Luria-Bertani broth as control. Silver staining of the two-dimensional gels revealed 234 proteins spots up-regulated when cells were growing in SESOM, with 201 protein spots down-regulated. Forty-three of these differentially expressed proteins were detected by Colloidal Coomassie staining and identified by matrix assisted laser desorption ionization-time of flight MS of tryptic digests. These differentially expressed proteins covered a range of functions, primarily amino acid, lipid, carbohydrate and nucleic acid metabolism. These results suggested growth on soil-associated carbohydrates, fatty acids and/or amino acids, concomitant with shifts in cellular structure.

ObjectiveThe aim of this study was to analyze the interaction of surface free energy and roughness characteristics of different pyrolytic carbon heart valves with three bacterial species on biofilm formation.MethodsThree pyrolytic carbon heart valves (St Jude Medical [St Jude Medical Inc, Minneapolis, Minn], Sulzer Carbomedics [CarboMedics Inc, Austin, Tex], and MedicalCV [Medical Incorporated, Inver Grove Heights, Minn]) were tested. Roughness was measured by interferential microscopy and surface free energy by contact angle technique. To obtain a biofilm, prostheses were inserted into a bioreactor with Staphylococcus aureus P209, Staphylococcus epidermidis RP62A, or Pseudomonas aeruginosa PAO1. Adhesion was quantified by counting sessile bacteria. Morphologic characteristics of biofilms were evaluated with scanning electron microscopy.ResultsRoughness analysis revealed significant differences between the MedicalCV (35.18 ± 4.43 nm) valve and St Jude Medical (11.03 ± 3.11 nm; P < .0001) and Sulzer Carbomedics (8.80 ± 1.10 nm; P < .0001) valves. Analysis of surface free energy revealed a higher level for the MedicalCV valve (41.03 mJ · m−2) than for both the Sulzer Carbomedics (38.93 mJ · m−2) and St Jude Medical (31.51 mJ · m−2) models. These results showed a correlation between surface free energy and bacterial adhesion for S epidermidis and P aeruginosa species. Regardless of the support, we observed significant adhesion differences for the three bacterial species. S aureus was the most adherent species, S epidermidis was the least, and P aeruginosa was intermediate.ConclusionsOur results suggest that adhesion of S epidermidis and P aeruginosa are dependent on pyrolytic carbon surface free energy and roughness, although S aureus adhesion appears to be independent of these factors. Improvement of pyrolytic carbon physicochemical properties thus could lead to a reduction in valvular prosthetic infections.

Biofilm bacteria are widely held to exhibit a unique phenotype, typified by their increased resistance to antimicrobial agents. Numerous studies have been devoted to the identification of biofilm-specific genes, but surprisingly few have been reported to date. We compared the whole cell proteomes of 24 h old Bacillus cereus biofilms and the associated suspended population to exponential, transient and stationary phase planktonic cultures using the unbiased approach of principal component analysis, comparing the quantity variations of the 823 detected spots. The analyses support the hypothesis that biofilms of Gram positive bacteria have a unique pattern of gene expression. The data provides proteomic evidence for a new biofilm and surface influenced planktonic population which is distinct to both planktonic and biofilm cells.

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The protein maps of Pseudomonas aeruginosa cells from two natural (attached) and one artificial (gel-entrapped) immobilized-cell (IC) systems, together with their free (suspended) counterparts, were compared after incubation for 18 or 48 h in a minimal salt medium. Principal component analysis (PCA) was used to interpret the variations in protein spot densities that were observed on electropherogram obtained by two-dimensional electrophoresis (2-DE). PCA of the 2-DE data, a matrix of 933 rows (observations, i.e., spot density values) and 12 columns (variables, i.e., incubation conditions), in which observations were standardized horizontally, extracted four principal components (PCs) accounting for 78.75% of the variability in the protein expression profiles. PC1 opposed the two modes of growth (planktonic and immobilized) while PC2 discriminated between the incubation times of free cell cultures. The incubation conditions of ICs, including the immobilization procedure (entrapment vs attachment) and the nature of the biofilm substratum, were fairly separated in PC3 × PC4. The dependence of the protein patterns on the cell immobilization process was further illustrated by the identification of a number of peptides whose amount remained unchanged or was altered in ICs compared to free bacteria. These results reinforce the topical assertion that bacteria in the immobilized state display a specific physiological behavior but also question the existence of a unique IC phenotype.

International audience; The problems associated with biofilm infections in humans result from the distinct characteristics of biofilms, in particular their high level of resistance to antibiotics. One of the hypotheses that have been advanced to explain this resistance to antimicrobials is the phenotypic differentiation of biofilm cells. Although many studies on biofilms have highlighted physiological alterations following the attachment of bacteria to a surface, no studies have explicitly demonstrated a “biofilm” physiology. To contribute to this topical debate, we used principal component analysis to interpret spot quantity variations observed on electropherograms obtained by two-dimensional gel electrophoresis of crude protein extracts from planktonic and sessile Pseudomonas aeruginosa cells. These analyses showed that the proteome of attached P. aeruginosa cells differs from that of their planktonic counterparts. Furthermore, we found that the proteome of sessile P. aeruginosa is strongly dependent on the nature of the biofilm substratum.

Despite the sustained development of immobilized-cell (IC) technology over the last 20 years, our knowledge of IC physiology has remained for a long time in the shade of the technological advances involving IC systems. This review surveys the main physiological responses of artificially and naturally immobilized microorganisms and presents recent data on protein expression in ICs that support the existence of a specific metabolic behaviour of microbial cells in the immobilized state.

In a new area of postgenomics challenges, the optimization of protein identification has become a central goal in microbiochemistry. In this work, we demonstrate that the substitution of Coomassie Brilliant Blue for bromophenol blue in two-dimensional electrophoresis (2-DE) buffers improves the focusing of whole proteins from Pseudomonas aeruginosa. This improvement of focusing concerns more particularly basic proteins. This enhancement may be attributed to a better transfer from the first to the second dimension, which probably highlights an increase in the solubility of proteins in the IPG strips. Hence, the use of an efficient tracking dye in the 2-DE buffers may enlarge protein recovery on proteome maps.

Viable Escherichia coli cells were entrapped in agar gel layers and incubated in a phosphate-limited glucose medium. Immobilized bacteria displayed enhanced alkaline phosphatase activity and overexpressed the outer membrane protein PhoE as compared with free-floating organisms. These observations highlighted the existence of high phosphate deprivation within biofilm-like structures. In addition, the antimicrobial efficacy of latamoxef against immobilized bacteria was partly recovered in the presence of a high phosphate concentration. From these data, a possible role of phosphate deprivation in the high resistance of sessile-like organisms to antibiotics may be considered.