SummaryPseudomonas CMR12a is a biocontrol strain that produces phenazine antibiotics and as yet uncharacterized cyclic lipopeptides (CLPs). The CLPs of CMR12a were studied by chemical structure analysis and in silico analysis of the gene clusters encoding the non-ribosomal peptide synthetases responsible for CLP biosynthesis. CMR12a produces two different classes of CLPs: orfamides B, D and E, whereby the latter two represent new derivatives of the orfamide family, and sessilins A-C. The orfamides are made up of a 10 amino acid peptide coupled to a β-hydroxydodecanoyl or β-hydroxytetradecanoyl fatty acid moiety, and are related to orfamides produced by biocontrol strain Pseudomonas protegens Pf-5. The sessilins consist of an 18-amino acid peptide linked to a β-hydroxyoctanoyl fatty acid and differ in one amino acid from tolaasins, toxins produced by the mushroom pathogen Pseudomonas tolaasii. CLP biosynthesis mutants were constructed and tested for biofilm formation and swarming motility. Orfamides appeared indispensable for swarming while sessilin mutants showed reduced biofilm formation, but enhanced swarming motility. The interplay between the two classes of CLPs fine tunes these processes. The presence of sessilins in wild type CMR12a interferes with swarming by hampering the release of orfamides and by co-precipitating orfamides to form a white line in agar.

Phosphorylation of the microtubule-associated Tau protein plays a major role in the regulation of its activity of tubulin polymerization and/or stabilization of microtubule assembly. A dysregulation of the phosphorylation/dephosphorylation balance leading to the hyperphosphorylation of Tau proteins in neurons is thought to favor their aggregation into insoluble filaments. This in turn might underlie neuronal death as encountered in many neurodegenerative disorders, including Alzheimer's disease. Another post-translational modification, the O-linked β-N-acetylglucosaminylation (O-GlcNAcylation), controls the phosphorylation state of Tau, although the precise mechanism is not known. Moreover, analytical difficulties have hampered the precise localization of the O-GlcNAc sites on Tau, except for the S400 site that was very recently identified on the basis of ETD-FT-MS. Here, we identify three O-GlcNAc sites by screening a library of small peptides sampling the proline-rich, the microtubule-associated repeats and the carboxy-terminal domains of Tau as potential substrates for the O-β-N-acetylglucosaminyltransferase (OGT). The in vitro activity of the nucleocytoplasmic OGT was assessed by tandem mass spectrometry and NMR spectroscopy. Using phosphorylated peptides, we establish the relationship between phosphate and O-GlcNAc incorporation at these sites. Phosphorylation of neighboring residues S396 and S404 was found to decrease significantly S400 O-GlcNAcylation. Reciprocally, S400 O-GlcNAcylation reduces S404 phosphorylation by the CDK2/cyclinA3 kinase and interrupts the GSK3β-mediated sequential phosphorylation process.