Induction of systemic resistance to Botrytis cinerea in tomato by Pseudomonas aeruginosa 7NSK2 : role of salicylic acid, pyochelin, and pyocyanin

The rhizobacterium Pseudomonas aeruginosa 7NSK2 produces secondary metabolites such as pyochelin (Pch), its precursor salicylic acid (SA), and the phenazine compound pyocyanin. Both 7NSK2 and mutant KMPCH (Pch-negative, SA-positive) induced resistance to Botrytis cinerea in wild-type but not in transgenic NahG tomato. SA-negative mutants of both strains lost the capacity to induce resistance. On tomato roots, KMPCH produced SA and induced phenylalanine ammonia lyase activity, while this was not the case for 7NSK2. In 7NSK2, SA is probably very efficiently converted to Pch. However, Pch alone appeared not to be sufficient to induce resistance. In mammalian cells, Fe-Pch and pyocyanin can act synergistically to generate highly reactive hydroxyl radicals that cause cell damage. Reactive oxygen species are known to play an important role in plant defense. To study the role of pyocyanin in induced resistance, a pyocyanin-negative mutant of 7NSK2, PHZ1, was generated. PHZ1 is mutated in the phzM gene encoding an O-methyltransferase. PHZ1 was unable to induce resistance to B. cinerea, whereas complementation for pyocyanin production or co-inoculation with mutant 7NSK2-562 (Pch-negative, SA-negative, pyocyanin-positive) restored induced resistance. These results suggest that pyocyanin and Pch, rather than SA, are the determinants for induced resistance in wild-type P aeruginosa 7NSK2

The insect pathogen Serratia marcescens Db10 uses a hybrid non-ribosomal peptide synthetase-polyketide synthase to produce the antibiotic althiomycin

There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1-alb6. Bioinformatic analysis of the proteins encoded by alb1-6 predicted a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line (Alb4/5/6), tailoring enzymes (Alb2/3) and an export/resistance protein (Alb1), and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2-Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism

Effect of shear stress on Pseudomonas aeruginosa isolated from the cystic fibrosis lung

Chronic colonization of the lungs by Pseudomonas aeruginosa is one of the major causes of morbidity and mortality in cystic fibrosis (CF) patients. To gain insights into the characteristic biofilm phenotype of P. aeruginosa in the CF lungs, mimicking the CF lung environment is critical. We previously showed that growth of the non-CF-adapted P. aeruginosa PAO1 strain in a rotating wall vessel, a device that simulates the low fluid shear (LS) conditions present in the CF lung, leads to the formation of in-suspension, self-aggregating biofilms. In the present study, we determined the phenotypic and transcriptomic changes associated with the growth of a highly adapted, transmissible P. aeruginosa CF strain in artificial sputum medium under LS conditions. Robust self-aggregating biofilms were observed only under LS conditions. Growth under LS conditions resulted in the upregulation of genes involved in stress response, alginate biosynthesis, denitrification, glycine betaine biosynthesis, glycerol metabolism, and cell shape maintenance, while genes involved in phenazine biosynthesis, type VI secretion, and multidrug efflux were downregulated. In addition, a number of small RNAs appeared to be involved in the response to shear stress. Finally, quorum sensing was found to be slightly but significantly affected by shear stress, resulting in higher production of autoinducer molecules during growth under high fluid shear (HS) conditions. In summary, our study revealed a way to modulate the behavior of a highly adapted P. aeruginosa CF strain by means of introducing shear stress, driving it from a biofilm lifestyle to a more planktonic lifestyle. IMPORTANCE: Biofilm formation by Pseudomonas aeruginosa is one of the hallmarks of chronic cystic fibrosis (CF) lung infections. The biofilm matrix protects this bacterium from antibiotics as well as from the immune system. Hence, the prevention or reversion of biofilm formation is believed to have a great impact on treatment of chronic P. aeruginosa CF lung infections. In the present study, we showed that it is possible to modulate the behavior of a highly adapted transmissible P. aeruginosa CF isolate at both the transcriptomic and phenotypic levels by introducing shear stress in a CF-like environment, driving it from a biofilm to a planktonic lifestyle. Consequently, the results obtained in this study are of great importance with regard to therapeutic applications that introduce shear stress in the lungs of CF patients

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Microradiographic and histological evaluation of the bone-screw and bone-plate interface of orthodontic miniplates in patients

SummaryObjectives: To describe the tissue reactions at the bone-titanium interface of orthodontic miniplates in humans. Materials and methods: Forty-two samples, consisting of tissue fragments attached or not to miniplates or their fixation screws, were collected from 24 orthodontic patients treated with miniplate anchorage, at the time of removal of their miniplates. The samples were embedded in methylmethacrylate and cut into undecalcified sections which were submitted to microradiographic analysis. The sections were also stained and examined under ordinary light. Results: Three types of reactions were observed both on the histological sections and on the microradiographs. 1. The majority of the stable miniplates were easy to remove (34/42). The tissue samples collected consisted mainly in mature lamellar bone with some medullary spaces containing blood vessels, 2. two screws were highly osseointegrated and required the surgeon to remove them by trephining (2/42). They were surrounded by bone tissue which extended to the miniplate. The histological features were similar to the previous group, though the bone-screw contact was higher, and 3. in six samples obtained after unstable miniplate removal during the treatment, we observed either some woven bone trabeculae or loose connective tissue, without any histological sign of inflammation. Limitations and Conclusion: For evident ethical reasons, our data were limited by the size of the tissue fragments and the limited number of patients and variety of clinical presentations. The healing reactions consisted mainly in mature lamellar bone tissue sparsely in contact with the screw or the miniplate, with signs of a moderate remodelling activit

Using remotely sensed solar radiation data for reference evapotranspiration estimation at a daily time step

International audienceSolar radiation is an important climatic variable for assessing reference evapotranspiration (E0), but it is seldom available in weather station records. Meteosat satellite images processed with the Heliosat-2 method provide the HelioClim-1 database, which displays spatialized solar radiation data at a daily time step for Europe and Africa. The aim of the present work was to investigate the interest of satellite-sensed solar radiation for E0 calculation, where air temperature is the sole local weather data available. There were two study areas in Southern France. One (Southwest, SW) is characterized by Oceanic climate and the other (Southeast, SE) by Mediterranean climate. A data set of daily values for 19 weather stations spanning five years (2000–2004) was used. First, a sensitivity analysis of the Penman–Monteith formula to climate input variables was performed, using the Sobol' method. It shows that E0 is mainly governed by solar radiation during summer, and by wind speed during winter. Uncertainties of HelioClim-1 solar radiation data and their repercussions on E0 formulae were evaluated, using the FAO-56 Penman–Monteith formulae (PM) and radiation-based methods (Turc, TU; Priestley–Taylor, PT and Hargreaves-Radiation, HR). It was shown that HelioClim-1 data slightly underestimate solar radiation and provide relative RMSE (root mean square error) of 20% of the mean annual value for SW and 14% for SE. The propagation of HelioClim-1 data uncertainties is small in PM but considerable in radiation methods. Four estimation methods were then compared to PM data: the 1985 Hargreaves formula (HT) based on air temperature only; TU, PT and HR, based on air temperature and satellite-sensed solar radiation. Radiation methods were more precise and more accurate than HT, with RMSE ranging from 0.52 mm to 0.86 mm against 0.67–0.96 mm. These results suggest that using satellite-sensed solar radiation may improve E0 estimates for areas where air temperature is the only available record at ground level

Low emittance lattice for LEP

In order to obtain the largest luminosity with LEP2, it is attractive to make the beam emittance as small as possible because the beam-beam effect is not a limitation at the energy of E~90 GeV for the obtained bunch currents. This can be achieved with a high tune lattice. Two possible candidates are lattices with a horizontal phase advance of 108o or 135o per cell. Both have a vertical phase advance of 60o. These lattice were developed during 1994 and the results are presented. tests to reach high intensity for the 108o lattice were performed and the bunch current achieved is compared with expectations. For this lattice the detuning v.s. intensity and several optics parameters were measured as well

Terrestrial laser scanning as a key element in the integrated monitoring of tidal influences on a twin-tube concrete tunnel

In 2012, for the first time in Belgium, integrated monitoring was carried out to assess the deformations of a twin-tube tunnel below the River Scheldt in Antwerp under the influence of estuarine tides. Levelling measurements during a tide cycle showed a variation in the height of levelling bolts of up to 10mm between low and high tide. Simultaneous strain-gauge measurements also showed a significant difference in strains. A deformation of the tunnel section during each twice-daily tide cycle, resulting in an eccentric egg or pumpkin shape, could hold large risks for the strength and durability of the tunnel structure. However, laser scanning results showed that there were no significant deformations of the tunnel's shape during the tide cycle. The laser scanning measurements resulted in a more thorough view of the deformation pattern which was much needed and improved the long-term risk assessment of the tunnel

Ferrous Iron Is a Significant Component of Bioavailable Iron in Cystic Fibrosis Airways

Chronic, biofilm-like infections by the opportunistic pathogen Pseudomonas aeruginosa are a major cause of mortality in cystic fibrosis (CF) patients. While much is known about P. aeruginosa from laboratory studies, far less is understood about what it experiences in vivo. Iron is an important environmental parameter thought to play a central role in the development and maintenance of P. aeruginosa infections, for both anabolic and signaling purposes. Previous studies have focused on ferric iron [Fe(III)] as a target for antimicrobial therapies; however, here we show that ferrous iron [Fe(II)] is abundant in the CF lung (~39 µM on average for severely sick patients) and significantly correlates with disease severity (ρ = −0.56, P = 0.004), whereas ferric iron does not (ρ = −0.28, P = 0.179). Expression of the P. aeruginosa genes bqsRS, whose transcription is upregulated in response to Fe(II), was high in the majority of patients tested, suggesting that increased Fe(II) is bioavailable to the infectious bacterial population. Because limiting Fe(III) acquisition inhibits biofilm formation by P. aeruginosa in various oxic in vitro systems, we also tested whether interfering with Fe(II) acquisition would improve biofilm control under anoxic conditions; concurrent sequestration of both iron oxidation states resulted in a 58% reduction in biofilm accumulation and 28% increase in biofilm dissolution, a significant improvement over Fe(III) chelation treatment alone. This study demonstrates that the chemistry of infected host environments coevolves with the microbial community as infections progress, which should be considered in the design of effective treatment strategies at different stages of disease. IMPORTANCE: Iron is an important environmental parameter that helps pathogens thrive in sites of infection, including those of cystic fibrosis (CF) patients. Ferric iron chelation therapy has been proposed as a novel therapeutic strategy for CF lung infections, yet until now, the iron oxidation state has not been measured in the host. In studying mucus from the infected lungs of multiple CF patients from Europe and the United States, we found that ferric and ferrous iron change in concentration and relative proportion as infections progress; over time, ferrous iron comes to dominate the iron pool. This information is relevant to the design of novel CF therapeutics and, more broadly, to developing accurate models of chronic CF infections