The Ralstonia solanacearum pathogenicity regulator HrpB induces 3-hydroxy-oxindole synthesis

The transcriptional activator HrpB of the bacterial wilt causing betaproteobacterium Ralstonia solanacearum represents a key regulator for pathogenicity. In particular, it drives expression of hrp genes encoding a type III secretion system (T3SS) as well as effector molecules for delivery into the host cytosol to promote disease. However, the HrpB regulon extends beyond this T3SS. We describe here an HrpB-activated operon of six genes that is responsible for the synthesis of a fluorescent isatin derivative of 149 Amu that we named HDF for HrpB-dependent factor and that we purified from culture supernatants. The structure of the labile molecule was solved by using NMR and CD spectroscopy to be (3S)-3-hydroxy-indolin-2-one and confirmed by its chemical synthesis and MS spectrometry. HDF was found to be present at 20 nM in wild-type cultures grown on minimal medium, and its synthesis increased 15-fold upon overproduction of HrpB, confirming that HrpB activates HDF synthesis. The addition of tryptophan significantly stimulated HDF biosynthesis and was shown to represent the precursor molecule for HDF synthesis. A search for the biological function of the molecule revealed that HDF induces acyl-homoserine lactone receptor-mediated reporter activity of the well studied LuxR transcriptional regulator of Vibrio fischeri. Thus, our results provide evidence that the specificity of acyl-homoserine lactone (acyl-HSL) receptors is clearly broader than previously considered. The failure to detect induction by HDF of the described endogenous quorum-sensing circuits of the pathogen points to a role in interfering with cell–cell signaling of rivalling bacteria

Murine ES-derived pancreatic acinar cells recapitulate features of early pancreatic differentiation

Background & Aims: Acinar cells constitute 90% of the pancreas epithelium, are polarized, and secrete digestive enzymes. These cells play a crucial role in pancreatitis and pancreatic cancer. However, there are limited models to study normal acinar cell differentiation in vitro. The aim of this work was to generate and characterize purified populations of pancreatic acinar cells from embryonic stem (ES) cells. Methods: Reporter ES cells (Ela-pur) were generated that stably expressed both β-galactosidase and puromycin resistance genes under the control of the elastase I promoter. Directed differentiation was achieved by incubation with conditioned media of cultured fetal pancreatic rudiments and adenoviral-mediated co-expression of p48/Ptf1a and Mist1, 2 basic helix-loop-helix transcription factors crucial for normal pancreatic acinar development and differentiation. Results: Selected cells expressed multiple markers of acinar cells, including digestive enzymes and proteins of the secretory pathway, indicating activation of a coordinated differentiation program. The genes coding for digestive enzymes were not regulated as a single module, thus recapitulating what occurs during in vivo pancreatic development. The generated cells displayed transient agonist-induced Ca2+ mobilization and showed a typical response to physiologic concentrations of secretagogues, including enzyme synthesis and secretion. Importantly, these effects did not imply the acquisition of a mixed acinar-ductal phenotype. Conclusions: These studies allow the generation of almost pure acinar-like cells from ES cells, providing a normal cell-based model for the study of the acinar differentiation program in vitro.This study was supported by Spanish Ministry of Education and Science Grants (SAF2001-0432 and GEN2001-4748-C05 to A.S.; GEN2001-4748-C01 and SAF2004-01137 to F.X.R. and SAF2006-4973 to M.A.V.), Instituto de Salud Carlos III grants (02/3053 and PI05/2738 to A.S.; and Red HERACLES to M.A.V.), Catalan Government grants (SGR2005 to M.A.V.), and by a National Institutes of Health grant (DK55489 to S.F.K.). A.S. was supported by the Instituto de Salud Carlos III; M.R., F.D. and M. M. were recipients of a Graduate Fellowship from the Ministry of Education and Science, Instituto de Salud Carlos III, and the Catalan Government, respectively

Biosynthesis of a Complex Yersiniabactin-Like Natural Product via the mic Locus in Phytopathogen Ralstonia solanacearum▿†

A genome mining study in the plant pathogenic bacterium Ralstonia solanacearum GMI1000 unveiled a polyketide synthase/nonribosomal peptide synthetase gene cluster putatively involved in siderophore biosynthesis. Insertional mutagenesis confirmed the respective locus to be operational under iron-deficient conditions and spurred the isolation of the associated natural product. Bioinformatic analyses of the gene cluster facilitated the structural characterization of this compound, which was subsequently identified as the antimycoplasma agent micacocidin. The metal-chelating properties of micacocidin were evaluated in competition experiments, and the cellular uptake of gallium-micacocidin complexes was demonstrated in R. solanacearum GMI1000, indicating a possible siderophore role. Comparative genomics revealed a conservation of the micacocidin gene cluster in defined, but globally dispersed phylotypes of R. solanacearum