In situ localisation and quantification of surfactins in a Bacillus subtilis swarming community by imaging mass spectrometry

Surfactins are a family of heptacyclopeptides in which the C-terminal carbonyl is linked with the beta-hydroxy group of a fatty acid acylating the N-terminal function of a glutamic acid residue. The fatty acyl chain is 12-16 carbon atoms long. These compounds, which are secreted by the Gram-positive bacterium Bacillus subtilis in stationary phase in liquid cultures, play an important role in swarming communities on the surface of agar media in the formation of dendritic patterns. TOF secondary ion MS (TOF-SIMS) imaging was used to map surfactins within 16-17 h swarming patterns, with a 2 mum spatial resolution. Surfactins were mainly located in the central mother colony (the site of initial inoculation), in a 'ring' surrounding the pattern and along the edges of the dendrites. In the mother colony and the interior of the dendrites, surfactins with shorter chain lengths are present, whereas in the ring surrounding the swarm community and between dendrites, surfactins with longer fatty acyl chain lengths were found. A quantitative analysis by MALDI-TOF MS showed a concentration gradient of surfactin from the mother colony to the periphery. The concentration of surfactin was approximately 400 pmol/mL in the mother colony and approximately 10 pmol/mL at the base of the dendrites, decreasing to 2 pmol/mL at their tips.     

Microbial remediation of NTO in aqueous industrial wastes

The bioremediation of aqueous wastes containing 5-nitro-1,2,4-triazol-3-one (NTO) was investigated. The microorganism used is a Bacillus licheniformis strain, isolated from the contaminated solutions by enrichment techniques. The biodegradation was carried out in the waste (15 g l-1 NTO) and proceeded through the nitroreduction of NTO, followed by the ring cleavage of the formed primary amine 5-amino-1,2,4-triazol-3-one (ATO). Both steps were optimized and according to the optimal conditions, the nitroreduction of NTO is total in 24 h, while the degradation of ATO requires 2 weeks of incubation. The end products of the biodegradation were carbon dioxide (40%), urea and a polar compound, assumed to be hydroxyurea. A mechanism of ATO ring cleavage was postulated in the light of experimental data, and led us to propose an overall degradation sequence for NTO.     

Phospholipid matrix as a target for sulfur mustard (HD): NMR study in model membrane systems

Although the interactions of sulfur mustard (HD) with nucleic acids and proteins have been well studied, the toxic interactions with the membrane matrix and specially the phospholipid bilayer have so far been poorly investigated. We have used several NMR techniques to study these interactions: ¹H NMR to observe the localization of HD in membranes of small unilamellar vesicles (SUV) of lecithin; ³¹P NMR to verify the hypothesis of pore formation in membranes of large unilamellar vesicles (LUV); and pseudo solid state ³¹P and ²H NMR to analyze the dynamic consequences of the presence of HD in multilayer dispersions of dimyristoylphosphatidylcholine (DMPC). Immediate and late modifications of the DMPC–HD complexes have been observed at the macroscopic and microscopic levels. After intoxication, HD is spontaneously incorporated into the membrane and locates at the level of the chain methylene groups. This incorporation occurs without formation of pores in the membrane. The presence of HD in the phospholipid dispersion differentially increases the membrane fluidity depending upon the level involved. Weak at the superficial level (phosphate group), this increase is dose-dependent on progression into the membrane. This increase is related to a lowering of transition temperature when measured at the chain level. Macroscopically, HD induces dose- and time-dependent modifications of the DMPC–HD complexes, leading to the formation of an optically transparent gel. This gel formation is confirmed at a microscopic level, where all structures disappear after intoxication. This revised version was published online in July 2006 with corrections to the Cover Date.     

Three dimensional structure and implications for the catalytic mechanism of 6-phosphogluconolactonase from Trypanosoma brucei

Enzymes from the pentose phosphate pathway (PPP) are potential drug targets for the development of new drugs against Trypanosoma brucei, the causative agent of African sleeping disease: for instance, the 6-phosphogluconate dehydrogenase is currently studied actively for such purposes. Structural and functional studies are necessary to better characterize the associated enzymes and compare them to their human homologues, in order to undertake structure-based drug design studies on such targets. In this context, the crystal structure of 6-phosphogluconolactonase (6PGL) from T. brucei, the second enzyme from PPP, was determined at 2.1 Angstroms resolution. Comparison of its sequence and structure to other related proteins in the 6PGL family with a known structure (Thermotoga maritima Tm6GPL 1PBT and Vibrio cholerae Vc6PGL (1Y89), which have not been discussed in print), or in the glucosamine-6-phosphate-deaminase family (hexameric Escherichia coli 1DEA and monomeric Bacillus subtilis 2BKV), allowed the identification of the 6PGL active site. In addition to the analysis of the crystal structure, 3D NMR interaction studies and docking experiments are reported here. Key residues involved in substrate binding or in catalysis were identified.     

Genetic differentiation of European anchovy (Engraulis encrasicolus) along the Moroccan coast reveals a phylogeographic break around the 25th parallel North

Seven microsatellite markers were used to investigate the population structure of the offshore ecotype of the European anchovy (Engraulis encrasicolus) by comparing 12 marine samples collected off the Moroccan coast with an inshore sample taken as a reference for the lagoonal ecotype. F-statistics, correspondence analysis and Bayesian assignment all concurred to cluster the European anchovy in this region into three groups: (i) one reference lagoonal sample, (ii) samples north of the 25°N latitude and (iii) samples south of it. Moreover, the Bayesian cluster analysis pointed toward the existence of an admixture between the group north of 25°N and the lagoonal ecotype, while this was not detectable with the group south of 25°N. Differential introgression between the two ecotypes could be one of the plausible explanations for the observed genetic structure and reveals the possible existence of a phylogeographic break around the 25th parallel North. Our study illustrates the fact that, for those species that encompass several incompletely isolated ecotypes, the level of gene flow among them may vary in space and serve as a tool for stock identification. This information may be useful to improve fishery management of this important harvested species along the Moroccan coast.     

Pathogenic Capacity of Helminthosporium spiciferum: Foliar Parasite of Rice in Morocco

In this study, artificially inoculation of six rice varieties widely cultured in the Gharb with Helminthosporium spiciferum showed different sensitivities against this isolate used. The foliar lesions are different from those noted on the plants of rice inoculated by Helminthosporium oryzae . Oryza sativa  = rice/ Helminthosporium spiciferum/ symptoms.     

Chloroplast DNA variation in the cultivated and wild olive taxa of the genus Olea L.

Polymorphism in the lengths of restriction fragments of the whole cpDNA molecule were studied in 15 taxa (species or subspecies) of the genus Olea. From restriction analysis using nine endonucleases, 28 site mutations and five length polymorphisms were identified, corresponding to 12 distinct chlorotypes. From a phenetic analysis based on a Nei’s dissimilarity matrix and a Dollo parsimony cladistic analysis using, as an outgroup, a species of the genus Phillyrea close to Olea, the ten taxa of section Olea were distinguished clearly from the five taxa of section Ligustroides which appear to posses more ancestral cpDNA variants. Within the section Ligustroides, the tropical species from central-western Africa, Olea hochtetteri, showed a chlorotype which differed substantially from those of the other four Olea taxa growing in southern Africa, supporting a previous assessment according to which O. hochtetteri may have been subjected to a long period of geographical isolation from the other Olea taxa. Within the Olea section, three phyla were identified corresponding to South and East Africa taxa, Asiatic taxa, and a group including Saharan, Macaronesian and Mediteranean taxa, respectively. On the basis of cpDNA variation, the closest Olea taxa to the single Mediterranean species, Olea europaea, represented by its very predominant chlorotype, observed in both wild and cultivated olive, were found to be Olea laperrinei (from the Sahara), Olea maroccana (from Maroccan High Atlas) and Olea cerasiformis (from Macaronesia). These three taxa, which all share the same chlorotype, may have a common maternal origin. Received: 5 December 1999 / Accepted: 30 December 1999     

Isolation and characterization of photoactive complexes of NADPH:protochlorophyllide oxidoreductase from wheat

A photoactive substrate-enzyme complex of the NADPH:protochlorophyllide oxidoreductase (POR; EC 1. 3. 1. 33) was purified from etiolated Triticum aestivum L. by gel chromatography after solubilization of prolamellar bodies by dodecyl-maltoside. Irradiation by a 1-ms flash induced the phototransformation of protocholorophyllide a (Pchlide) with −196 °C absorbance and emission maxima at 640 and 643 nm, respectively. The apparent molecular weight of this complex was 112 ± 24 kDa, which indicates aggregation of enzyme subunits. By lowering the detergent concentration in the elution buffer, a 1080 ± 250-kDa particle was obtained which displayed the spectral properties of the predominant form of photoactive Pchlide in vivo (−196 °C absorbance and fluorescence maxima at 650 and 653 nm). In this complex, POR was the dominant polypeptide. Gel chromatography in the same conditions of an irradiated sample of solubilized prolamellar bodies indicated rapid disaggregation of the complex after Pchlide phototransformation. High performance liquid chromatographic analysis of the POR complexes obtained using two detergent concentrations indicates a possible association of zeaxanthin and violaxanthin with the photoactive complex. Received: 25 February 1998 / Accepted: 8 June 1998