Association of Hemolytic Activity of Pseudomonas entomophila,a Versatile Soil Bacterium,with Cyclic Lipopeptide Production

Pseudomonas entomophila is an entomopathogenic bacterium that is able to infect and kill Drosophila melanogaster upon ingestion. Its genome sequence suggests that it is a versatile soil bacterium closely related to Pseudomonas putida. The GacS/GacA two-component system plays a key role in P. entomophila pathogenicity, controlling many putative virulence factors and AprA, a secreted protease important to escape the fly immune response. P. entomophila secretes a strong diffusible hemolytic activity. Here, we showed that this activity is linked to the production of a new cyclic lipopeptide containing 14 amino acids and a 3-C₁₀OH fatty acid that we called entolysin. Three nonribosomal peptide synthetases (EtlA, EtlB, EtlC) were identified as responsible for entolysin biosynthesis. Two additional components (EtlR, MacAB) are necessary for its production and secretion. The P. entomophila GacS/GacA two-component system regulates entolysin production, and we demonstrated that its functioning requires two small RNAs and two RsmA-like proteins. Finally, entolysin is required for swarming motility, as described for other lipopeptides, but it does not participate in the virulence of P. entomophila for Drosophila. While investigating the physiological role of entolysin, we also uncovered new phenotypes associated with P. entomophila, including strong biocontrol abilities.Pseudomonas entomophila is a recently isolated Pseudomonas species that is closely related to the saprophytic soil bacterium Pseudomonas putida. It was initially characterized as a natural pathogen of Drosophila (63). Indeed, P. entomophila was first isolated from flies sampled in Guadeloupe, and it is highly pathogenic for Drosophila larvae and adults. P. entomophila can also effectively kill members of other insect orders (e.g., Bombyx mori, Anopheles gambiae), which makes it a new entomopathogenic bacterium. Its ability to infect and kill Drosophila melanogaster very efficiently after ingestion makes it an appropriate model for the study of host-pathogen interactions (38, 62, 63).In order to unravel features contributing to the entomopathogenic properties of P. entomophila, its genome was sequenced. The results suggest that this strain is a ubiquitous, metabolically versatile bacterium that may colonize diverse habitats, including soil, rhizosphere, and aquatic systems, as shown for P. putida KT2440 (62). However, in contrast to the P. putida genome, the P. entomophila genome contains many genes that are predicted to be important for virulence toward insects. Notably, P. entomophila could secrete many degradative enzymes (proteases and lipases), putative toxins, and secondary metabolites (62). Similar factors have been shown to play a key role in the virulence of other entomopathogenic bacteria like Photorhabdus and Xenorhabdus sp. (27, 29).Insertional mutagenesis allowed the identification of several P. entomophila genes required to infect and/or kill Drosophila. This analysis demonstrated that P. entomophila virulence is under the control of the GacS/GacA two-component system (62, 63), a global regulatory system which is known to control secondary metabolite production, protein secretion, and pathogenic abilities in gammaproteobacteria (37, 65). Another study indicates that P. entomophila can counteract the Drosophila gut immune response as a result of the secretion of an abundant protease, AprA, which degrades antimicrobial peptides produced by gut epithelia and thereby promotes bacterial persistence (38). However, an AprA-deficient mutant remains virulent to some extent, indicating that P. entomophila virulence is multifactorial, AprA being one virulence factor among others.The secretion of virulence factors is a common mechanism employed by pathogens to compromise host defenses. Several entomopathogenic bacteria (e.g., Photorhabdus luminescens) secrete toxins that allow them to impair host function (8). The starting point of this study was the observation that, in contrast to several other Pseudomonas strains, P. entomophila secretes a strong diffusible hemolytic activity (which is also controlled by the Gac system). This raises the possibility of a link between this hemolytic activity and the pathogenicity of P. entomophila for Drosophila. Indeed, bacterial hemolysins are exotoxins that attack blood cell membranes and cause cell rupture by poorly defined mechanisms. It was conceivable that this hemolytic activity could be a readout for the ability of P. entomophila to damage the epithelial cells of the Drosophila gut, which plays a crucial role in its virulence (10, 33, 63).In this study, the P. entomophila hemolytic factor was identified as a cyclic lipopeptide (CLP) whose structure was elucidated. CLPs are versatile molecules with antimicrobial, cytotoxic, and surfactant properties that are produced by members of the genera Bacillus, Serratia, Burkholderia, and Pseudomonas (31, 41, 43, 50). They are produced by a ribosome-independent mechanism that utilizes multifunctional enzymes called nonribosomal peptide synthetases (NRPSs) (42, 59). These NRPSs are composed of repeated amino acid activation modules containing domains for condensation, aminoacyl adenylation, and thiolation. Modules are responsible for activation and incorporation of amino acids into the growing peptide. A large number of prokaryotic and some eukaryotic organisms synthesize peptide metabolites via this nonribosomal mechanism of biosynthesis (42, 47).Several genes involved in P. entomophila lipopeptide production were identified, three of them encoding NRPSs. The physiological role of this lipopeptide was also investigated, and it does not seem to play a role in the process of virulence towards Drosophila and Dictyostelium or in the P. entomophila biocontrol activity that was uncovered by this study. This suggests that the lifestyle of this newly identified bacterium is probably quite versatile and that lipopeptide production could be required only under specific circumstances.     

Knock-down of the COX3 and COX17 gene expression of cytochrome c oxidase in the unicellular green alga Chlamydomonas reinhardtii

The COX3 gene encodes a core subunit of mitochondrial cytochrome c oxidase (complex IV) whereas the COX17 gene encodes a chaperone delivering copper to the enzyme. Mutants of these two genes were isolated by RNA interference in the microalga Chlamydomonas. The COX3 mRNA was completely lacking in the cox3-RNAi mutant and no activity and assembly of complex IV were detected. The cox17-RNAi mutant presented a reduced level of COX17 mRNA, a reduced activity of the cytochrome c oxidase but no modification of its amount. The cox3-RNAi mutant had only 40% of the wild-type rate of dark respiration which was cyanide-insensitive. The mutant presented a 60% decrease of H₂O₂ production in the dark compared to wild type, which probably accounts for a reduced electron leakage by respiratory complexes III and IV. In contrast, the cox17-RNAi mutant showed no modification of respiration and of H₂O₂ production in the dark but a two to threefold increase of H₂O₂ in the light compared to wild type and the cox3-RNAi mutant. The cox17-RNAi mutant was more sensitive to cadmium than the wild-type and cox3-RNAi strains. This suggested that besides its role in complex IV assembly, Cox17 could have additional functions in the cell such as metal detoxification or Reactive Oxygen Species protection or signaling. Concerning Cox3, its role in Chlamydomonas complex IV is similar to that of other eukaryotes although this subunit is encoded in the nuclear genome in the alga contrary to the situation found in all other organisms.     

Has the final countdown to wildlife extinction in Northern Central African Republic begun?

The wildlife populations of Northern Central African Republic experienced precipitous declines during the 1970s and 1980s. While anecdotes coming out of the region indicate that the wildlife populations remain under serious threat, little is known about their status. An aerial sample count was carried out in the Northern Central African Republic at the end of the dry season in June 2005 and covered an 85,000 km² complex landscape containing national parks, hunting reserves and community hunting areas. Results show a dramatic decline of wildlife since the previous survey in 1985. In 20 years, large mammals’ numbers decreased by 65%, probably because of poaching and diseases brought by illegal cattle transhumance. Elephant (Loxodonta africana) and Buffon kob (Kobus kob) populations showed the greatest decline (over 80% each), while buffalo (Syncerus caffer), roan antelope (Hippotragus equinus) and Giant Lord’s Derby Eland (Taurotragus derbianus) populations seem stable or increasing over these last 20 years. The analysis of the wildlife population distribution by status of the different types of protected areas (national parks, hunting areas) showed that individual encounter rates of elephant and buffalo were lower in national parks than in neighbouring hunting areas, while those for roan, giraffe (Giraffa camelopardalis) and Buffon kob were higher in the national parks.     

Altered SK3/KCa2.3-mediated migration in adenomatous polyposis coli (Apc) mutated mouse colon epithelial cells

Lost of adenomatous polyposis coli gene (Apc) disturbs the migration of intestinal epithelial cells but the mechanisms have not been fully characterized. Since we have demonstrated that SK3/KCa2.3 channel promotes cancer cell migration, we hypothesized that Apc mutation may affect SK3/KCa2.3 channel-mediated colon epithelial cell motility. We report evidence that SK3/KCa2.3 channel promotes colon epithelial cells motility. Following Apc mutation SK3/KCa2.3 expression is largely reduced leading to a suppression of the SK3/KCa2.3 channel mediated-cell migration. Our findings reveal a previously unknown function of the SK3/KCa2.3 channel in epithelial colonic cells, and suggest that Apc is a powerful regulator SK3/KCa2.3 channel.     

A Major Determinant for Binding and Aminoacylation of tRNA in Cytoplasmic Alanyl-tRNA Synthetase Is Mutated in Dominant Axonal Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth disease (CMT) is the most common cause of inherited peripheral neuropathy, with an estimated frequency of 1/2500. We studied a large family with 17 patients affected by the axonal form of CMT (CMT2). Analysis of the 15 genes or loci known to date was negative. Genome-wide genotyping identified a CMT2 locus in 16q21-q23 between D16S3050 and D16S3106. The maximum two-point LOD score was 4.77 at θ = 0 for marker D16S3050. Sequencing of candidate genes identified a unique mutation, c.986G>A (p.Arg329His), affecting a totally conserved amino acid in the helical domain of cytoplasmic alanyl-tRNA synthetase (AlaRS). A second family with the same mutation and a different founder was then identified in a cohort of 91 CMT2 families. Although mislocation of mutant Arg329His-AlaRS in axons remains to be evaluated, experimental data point mostly to a quantitative reduction in tRNA^Ala aminoacylation. Aminoacylation and editing functions closely cooperate in AlaRS, and Arg329His mutation could also lead to qualitative errors participating in neurodegeneration. Our report documents in 18 patients the deleterious impact of a mutation in human cytoplasmic AlaRS and broadens the spectrum of defects found in tRNA synthetases. Patients present with sensory-motor distal degeneration secondary to predominant axonal neuropathy, slight demyelination, and no atypical or additional CNS features.     

Complex patterns of hybridization between exotic and native North American poplar species

? Premise of the study: Poplars and their hybrids are seen as important candidates for bioenergy initiatives. However, many concerns have been raised about large-scale plantations of new poplar cultivars. The deployment of such plants with novel traits brings the risk of potential spread of novel genome regions (including exotic genes, transgenes, or other heritable modifications) into natural populations of related species. The possibility of introgression is especially high in poplars because reproductive barriers between species are weak. Knowledge of the frequency of hybridization between cultivated trees and natural populations is one important step in the risk-assessment process. ? Methods: We studied the rate of spontaneous hybridization from two sexually mature poplar plantations into adjacent natural populations of Populus deltoides and P. balsamifera. The two plantations, both in eastern Canada, contain many different complex hybrid clones with components from exotic species, mostly P. nigra, P. trichocarpa, and P. maximowiczii. We analyzed 12 species-specific single nucleotide polymorphisms from six different genes in 5373 offspring sampled from the natural populations. ? Results: Contributions from all three exotics were found in the offspring, confirming low reproductive barriers among poplar species in these sections. The frequency of hybrid offspring varied among pollen donors, recipient populations, and years. ? Conclusions: The remarkably high rate of hybridization that was found in the smallest natural population sampled suggests that small peripheral populations carry a higher risk of introgression. These results could be used as a starting point for developing regulatory guidelines for the introduction of plants with novel traits.     

A Simple Dynamic Model of Respiratory Pump

To study the interaction of forces that produce chest wall motion, we propose a model based on the lever system of Hillman and Finucane (J Appl Physiol 63(3):951–961, 1987) and introduce some dynamic properties of the respiratory system. The passive elements (rib cage and abdomen) are considered as elastic compartments linked to the open air via a resistive tube, an image of airways. The respiratory muscles (active) force is applied to both compartments. Parameters of the model are identified in using experimental data of airflow signal measured by pneumotachography and rib cage and abdomen signals measured by respiratory inductive plethysmography on eleven healthy volunteers in five conditions: at rest and with four level of added loads. A breath by breath analysis showed, whatever the individual and the condition are, that there are several breaths on which the airflow simulated by our model is well fitted to the airflow measured by pneumotachography as estimated by a determination coefficient R ² ≥ 0.70. This very simple model may well represent the behaviour of the chest wall and thus may be useful to interpret the relative motion of rib cage and abdomen during quiet breathing.