The Experience of Time and its Disorders

The chapter first introduces a phenomenological concept of temporality, referring to time as pre-reflectively lived vs. consciously experienced. Lived time is based on the constitutive synthesis of inner time consciousness on the one hand, and on the conative-affective dynamics of life on the other hand. Experienced time, for its part, results from an interruption and desynchronisation of lived time. It unfolds into the dimensions of present, past and future, leading to autobiographical time and finally to narrative identity. On this basis, major psychiatric disorders such as schizophrenia, melancholic depression and obsessive-compulsive disorder will be presented as paradigm cases for a psychopathology of temporality.

Pseudomonas aeruginosaRegulated Intramembrane Proteolysis: Protease MucP Can Overcome Mutations in the AlgO Periplasmic Protease To Restore Alginate Production in Nonmucoid Revertants

ABSTRACTThe progression of cystic fibrosis (CF) from an acute to a chronic disease is often associated with the conversion of the opportunistic pathogenPseudomonas aeruginosafrom a nonmucoid form to a mucoid form in the lung. This conversion involves the constitutive synthesis of the exopolysaccharide alginate, whose production is under the control of the AlgT/U sigma factor. This factor is regulated posttranslationally by an extremely unstable process and has been commonly attributed to mutations in thealgT(algU) gene. By exploiting this unstable phenotype, we isolated 34 spontaneous nonmucoid variants arising from the mucoid strain PDO300, a PAO1 derivative containing themucA22allele commonly found in mucoid CF isolates. Complementation analysis using a minimal tiling path cosmid library revealed that most of these mutants mapped to two protease-encoding genes,algO, also known asprcorPA3257, andmucP. Interestingly, ouralgOmutations were complemented by bothmucPandalgO, leading us to delete, clone, and overexpressmucP,algO,mucE, andmucDin both wild-type PAO1 and PDO300 backgrounds to better understand the regulation of this complex regulatory mechanism. Our findings suggest that the regulatory proteases follow two pathways for regulated intramembrane proteolysis (RIP), where both the AlgO/MucP pathway and MucE/AlgW pathway are required in the wild-type strain but where the AlgO/MucP pathway can bypass the MucE/AlgW pathway in mucoid strains with membrane-associated forms of MucA with shortened C termini, such as the MucA22 variant. This work gives us a better understanding of how alginate production is regulated in the clinically important mucoid variants ofPseudomonas aeruginosa.IMPORTANCEInfection by the opportunistic pathogenPseudomonas aeruginosais the leading cause of morbidity and mortality seen in CF patients. Poor patient prognosis correlates with the genotypic and phenotypic change of the bacteria from a typical nonmucoid to a mucoid form in the CF lung, characterized by the overproduction of alginate. The expression of this exopolysaccharide is under the control an alternate sigma factor, AlgT/U, that is regulated posttranslationally by a series of proteases. A better understanding of this regulatory phenomenon will help in the development of therapies targeting alginate production, ultimately leading to an increase in the length and quality of life for those suffering from CF.

Socially mediated induction and suppression of antibiosis during bacterial coexistence

Despite their importance for humans, there is little consensus on the function of antibiotics in nature for the bacteria that produce them. Classical explanations suggest that bacteria use antibiotics as weapons to kill or inhibit competitors, whereas a recent alternative hypothesis states that antibiotics are signals that coordinate cooperative social interactions between coexisting bacteria. Here we distinguish these hypotheses in the prolific antibiotic-producing genus Streptomyces and provide strong evidence that antibiotics are weapons whose expression is significantly influenced by social and competitive interactions between competing strains. We show that cells induce facultative responses to cues produced by competitors by (i) increasing their own antibiotic production, thereby decreasing costs associated with constitutive synthesis of these expensive products, and (ii) by suppressing antibiotic production in competitors, thereby reducing direct threats to themselves. These results thus show that although antibiotic production is profoundly social, it is emphatically not cooperative. Using computer simulations, we next show that these facultative strategies can facilitate the maintenance of biodiversity in a community context by converting lethal interactions between neighboring colonies to neutral interactions where neither strain excludes the other. Thus, just as bacteriocins can lead to increased diversity via rock–paper–scissors dynamics, so too can antibiotics via elicitation and suppression. Our results reveal that social interactions are crucial for understanding antibiosis and bacterial community dynamics, and highlight the potential of interbacterial interactions for novel drug discovery by eliciting pathways that mediate interference competition.

Self-Resistance and Cell Wall Composition in the Glycopeptide Producer Amycolatopsis balhimycina

ABSTRACTThe prevailing resistance mechanism against glycopeptides in Gram-positive pathogens involves reprogramming the biosynthesis of peptidoglycan precursors, resulting ind-alanyl-d-lactate depsipeptide termini.Amycolatopsis balhimycinaproduces the vancomycin-like glycopeptide balhimycin and therefore has to protect itself from the action of the glycopeptide. We studied the roles of the accessory resistance gene orthologsvanYb,vnlRb, andvnlSb, which are part of the balhimycin biosynthetic gene cluster (represented by the subscript “b”). The VanYbcarboxypeptidase cleaved the terminald-Ala from peptidoglycan precursors, and its heterologous expression enhanced glycopeptide resistance inStreptomyces coelicolor. The VanRS-like two component system VnlRSbwas not involved in glycopeptide resistance or in the expression of thevanHAXglycopeptide resistance genes. MatureA. balhimycinapeptidoglycan contained mainly tri- and tetrapeptides, with only traces of thed-Ala-d-Ala-ending pentapeptides that are binding sites for the antibiotic produced. The structure of the peptidoglycan precursor is consistent with the presence ofvanHAXgenes, which were identified outside the balhimycin synthesis cluster. Both wild-type and non-antibiotic-producing mutant strains synthesized peptidoglycan precursors ending mainly withd-Lac, indicating constitutive synthesis of a resistant cell wall.A. balhimycinacould provide a model for an ancestral glycopeptide producer with constitutively expressed resistance genes.

A newly discovered bacteriocin produced by Enterococcus faecalis 3915

Different Enterococci, isolated from starters for the production of the traditional Bulgarian yellow cheese 'Kashkaval' were screened for bacteriocin production, and one of them, Enterococcus faecalis 3915 demonstrated bacteriocin activity. In this study, we investigated the growth parameters of the producer strain as well as the production kinetics and preliminary characterisation of the produced bacteriocin named enterocin 3915. For the growth modelling, the logistic model was used, while bacteriocin production was monitored. Experiments on inducibility were conducted, and strain was checked for the presence of plasmids. The peptide was crudely purified by ammonium sulphate precipitation followed by preparative PAGE. The approximate molecular mass was determined electrophoretically, and the activity was visualised by electrophoresis and agar overlay technique. It was found that E. faecalis 3915 produces a bacteriocin with constitutive synthesis and chromosomal localisation of its genetic determinants. The peptide revealed to be relatively heat-stable with a molecular mass of about 6.5 kDa. As E. faecalis 3915 originates from cheese starter it can be classified as generally recognised as safe (GRAS). The inhibitory activity of enterocin 3915 comprises commensals or pathogens, so properties generally accepted as probiotic could be attributed to the producer, and potential application in the dairy industry is not to be excluded.

Construction ofBacillus thuringiensiswild-type S76 and Cry–derivatives expressing a green fluorescent protein: two potential marker organisms to study bacteria–plant interactions

Collectively, the species Bacillus thuringiensis , Bacillus cereus , and Bacillus anthracis represent microorganisms of high economic, medical, and biodefense importance. Although the genetic correlation and pathogenic characteristics have been extensively dissected, the ecological properties of these three species in their natural environments remain poorly understood. Thus, a tractable marker for detecting these bacteria under specific environmental and physiological conditions is a valuable tool. With this purpose, a plasmid (pAD43-25) carrying a functional gfp gene sequence (gfpmut3A) was introduced into the wild-type strain Bacillus thuringiensis subsp. kurstaki S76, which bears approximately 11 plasmids, allowing constitutive synthesis of green fluorescent protein (GFP) during vegetative growth (strain S76GFP+). Additionally, this vector was transferred to a plasmid-cured (Cry–) B. thuringiensis host. Bright green cells were detected by fluorescence microscopy in both recombinants by 2 h after inoculation in liquid medium and could be seen throughout the remaining cultivation time until complete sporulation was accomplished. For strain S76GFP+protein profile and plasmid DNA analyses indicate, respectively, that this recombinant maintained Cry proteins expression and resident plasmid outline. Thus, in addition to the potential of strain S76GFP+as a marker organism in bacteria–plant interaction studies, the production and stability of active GFPmut3a make this unique expression system a useful experimental model to study adaptive changes of host–plasmid as well as plasmid–plasmid relationships in a population of cells stressed by the production of a recombinant protein.