The Spermann organizer induces neural tissue from dorsal ectoderm and dorsalizes lateral and ventral mesoderm in Xenopus. The secreted factor noggin, which is expressed in the organizer, can mimic the dorsalizing signal of the organizer. Data are presented showing that noggin directly induces neural tissue, that it induces neural tissue in the absence of dorsal mesoderm, and that it acts at the appropriate stage to be an endogenous neural inducing signal. Noggin induces cement glands and anterior brain markers, but not hindbrain or spinal cord markers. Thus, noggin has the expression pattern and activity expected of an endogenous neural inducer.
The genetic transformation of plant cells by Agrobacterium tumefaciens is mediated by the genes of the Ti plasmid vir region. To determine the genetic and transcriptional organization of the vir region of pTiA6, vir plasmid clones were saturated with insertion mutations of a Tn3‐lacZ transposon. This element is both an insertion mutagen and a reporter for the expression of the sequences into which it has inserted. One hundred and twenty‐four vir::Tn3‐lac insertions were analyzed for their mutagenic effect on Agrobacterium virulence, and for their expression of beta‐galactosidase activity, the lacZ gene product, in vegetative bacteria and in bacteria cocultivated with plant cells. These data in conjunction with genetic complementation results show that the pTiA6 vir region contains six distinct vir complementation groups: virA, virB, virC, virD, virE and virG. Mutations in these loci eliminate (virA, virB, virD and virG) or significantly restrict (virC and virE) the ability of Agrobacterium to transform plant cells. Each of the vir loci corresponds to a single vir transcription unit: virA is constitutively expressed and non‐inducible; virB, virC, virD and virE are expressed only upon activation by plant cells; and virG is both constitutively expressed and plant‐inducible. The two largest vir operons, virB and virD, are probably polycistronic. The pTiA6 vir region also contains plant‐inducible loci (pin) which are non‐essential for virulence.
SummaryMvfR (PqsR), a Pseudomonas aeruginosa LysR-type transcriptional regulator, plays a critical role in the virulence of this pathogen. MvfR modulates the expression of multiple quorum sensing (QS)-regulated virulence factors; and the expression of the phnAB and pqsA-E genes that encode functions mediating 4-hydroxy-2-alkylquinolines (HAQs) signalling compounds biosynthesis, including 3,4-dihydroxy-2-heptylquinoline (PQS) and its precursor 4-hydroxy-2-heptylquinoline (HHQ). PQS enhances the in vitro DNA-binding affinity of MvfR to the pqsA-E promoter, to suggest it might function as the in vivo MvfR ligand. Here we identify a novel MvfR ligand, as we show that HHQ binds to the MvfR ligand-binding-domain and potentiates MvfR binding to the pqsA-E promoter leading to transcriptional activation of pqsA-E genes. We show that HHQ is highly produced in vivo, where it is not fully converted into PQS, and demonstrate that it is required for MvfR-dependent gene expression and pathogenicity; PQS is fully dispensable, as pqsHmutant cells, which produce HHQ but completely lack PQS, display normal MvfR-dependent gene expression and virulence. Conversely, PQS is required for full production of pyocyanin. These results uncover a novel biological role for HHQ; and provide novel insights on MvfR activation that may aid in the development of therapies that prevent or treat P. aeruginosa infections in humans.
The construction and use of a Tn3‐lac transposon, Tn3‐HoHo1, is described. Tn3‐HoHo1 can serve as a transposon mutagen and provides a new and useful system for the random generation of both transcriptional and translational lacZ gene fusions. In these fusions the production of beta‐galactosidase, the lacZ gene product, is placed under the control of the gene into which Tn3‐HoHo1 has inserted. The expression of the gene can thus be analyzed by monitoring beta‐galactosidase activity. Tn3‐HoHo1 carries a non‐functional transposase gene; consequently, it can transpose only if transposase activity is supplied in trans, and is stable in the absence of this activity. A system for the insertion of Tn3‐HoHo1 into sequences specifically contained within plasmids is described. The applicability of Tn3‐HoHo1 was demonstrated studying three functional regions of the Agrobacterium tumefaciens A6 Ti plasmid. These regions code for octopine catabolism, virulence and plant tumor phenotype. The regulated expression of genes contained within each of these regions was analyzed in Agrobacterium employing Tn3‐HoHo1 generated lac fusions.
Long-term antibiotic use generates pan-resistant super pathogens. Anti-infective compounds that selectively disrupt virulence pathways without affecting cell viability may be used to efficiently combat infections caused by these pathogens. A candidate target pathway is quorum sensing (QS), which many bacterial pathogens use to coordinately regulate virulence determinants. The Pseudomonas aeruginosa MvfR-dependent QS regulatory pathway controls the expression of key virulence genes; and is activated via the extracellular signals 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), whose syntheses depend on anthranilic acid (AA), the primary precursor of 4-hydroxy-2-alkylquinolines (HAQs). Here, we identified halogenated AA analogs that specifically inhibited HAQ biosynthesis and disrupted MvfR-dependent gene expression. These compounds restricted P. aeruginosa systemic dissemination and mortality in mice, without perturbing bacterial viability, and inhibited osmoprotection, a widespread bacterial function. These compounds provide a starting point for the design and development of selective anti-infectives that restrict human P. aeruginosa pathogenesis, and possibly other clinically significant pathogens.
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