Transcription activation by the siderophore sensor Btr is mediated by ligand-dependent stimulation of promoter clearance

Bacterial transcription factors often function as DNA-binding proteins that selectively activate or repress promoters, although the biochemical mechanisms vary. In most well-understood examples, activators function by either increasing the affinity of RNA polymerase (RNAP) for the target promoter, or by increasing the isomerization of the initial closed complex to the open complex. We report that Bacillus subtilis Btr, a member of the AraC family of activators, functions principally as a ligand-dependent activator of promoter clearance. In the presence of its co-activator, the siderophore bacillibactin (BB), the Btr:BB complex enhances productive transcription, while having only modest effects on either RNAP promoter association or the production of abortive transcripts. Btr binds to two direct repeat sequences adjacent to the −35 region; recognition of the downstream motif is most important for establishing a productive interaction between the Btr:BB complex and RNAP. The resulting Btr:BB dependent increase in transcription enables the production of the ferric-BB importer to be activated by the presence of its cognate substrate

Spo0A-stimulated transcription initiation at the bacillus subtilis spoIIG promother

As a last resort to insurmountable environmental stresses, the common soil bacterium Bacillus subtilis undergoes a complicated morphological transformation to produce a metabolically dormant spore. The central regulator of sporulation is a response regulator transcription factor, Spo0A. When activated by phosphorylation, Spo0A~P directly stimulates or represses transcription at a number of promoters encoding genes involved in the early stages of sporulation. This thesis explores the mechanism by which Spo0A~P activates expression at one of these promoters, the spoIIG promoter. At the spoIIG promoter, Spo0A~P binds to 0A boxes and interacts with RNA containing σ[sup A] to facilitate DNA strand separation. Precisely how Spo0A~P stimulated DNA strand separation was not understood. In this thesis, I show that Spo0A~P acts prior to denaturation of the DNA strands and has no effect on transcription from DNA templates in which the -10 element is artificially denatured to expose the non-template strand. Investigation of the thermal dependence of transcription from a series of artificially denatured promoters suggested that strand separation was the primary thermodynamic barrier to transcription initiation but indicated that Spo0A~P did not reduce this energetic barrier. Kinetic assays revealed that Spo0A~P stimulated both the rate of formation of initiated complexes as well as increasing the number of RN AV-spoIIG complexes capable of initiating transcription. These results implied that Spo0A exerted its effect on RNA polymerase prior to the formation of an open complex. I present evidence for a closed intermediate complex formed by RNA polymerase, Spo0A~P and the spoIIG promoter. To isolate the effect of Spo0A~P on events prior to DNA strand separation at the spoIIG promoter I used DNA fragments that contained only promoter sequences 5' to the -10 element in electrophoretic mobility shift assays. RNA polymerase bound to these fragments readily and the RNA polymerase- spoIIG complexes recruited Spo0A~P. This complex was stabilized by Spo0A~P. In addition I investigated the effect of truncating the upstream portions of the spoIIG promoter which contain a pair of Spo0A binding sites at approximately -90 relative to the transcription start site and found that this DNA inhibited the binding of RNA polymerase. Strikingly, Spo0A~P binding depended on the presence of the DNA upstream of the 0A boxes, suggesting a role for the RNA polymerase a subunit carboxy-terminal domains in transcription at this promoter. Finally, Spo0A~P binding to the promoter played two distinct roles. Promoter-distal bound Spo0A~P increased the local concentration of Spo0A~P available to bind to promoter-proximal Spo0A binding sites, while promoter-proximal bound Spo0A~P stabilized and modified pre-formed RNA polymerase-spoIIG complexes. The data is consistent with a model whereby Spo0A~P stabilizes RNA polymerase during the initial stages of open complex formation at the spoIIG promoter, but only until the nontemplate strand of the -10 element is fully single-stranded. These effects account for transcription activation of the spoIIG promoter by Spo0A~P.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

An A257V Mutation in the Bacillus subtilis Response Regulator Spo0A Prevents Regulated Expression of Promoters with Low-Consensus Binding Sites▿

In Bacillus species, the master regulator of sporulation is Spo0A. Spo0A functions by both activating and repressing transcription initiation from target promoters that contain 0A boxes, the binding sites for Spo0A. Several classes of spo0A mutants have been isolated, and the molecular basis for their phenotypes has been determined. However, the molecular basis of the Spo0A(A257V) substitution, representative of an unusual phenotypic class, is not understood. Spo0A(A257V) is unusual in that it abolishes sporulation; in vivo, it fails to activate transcription from key stage II promoters yet retains the ability to repress the abrB promoter. To determine how Spo0A(A257V) retains the ability to repress but not stimulate transcription, we performed a series of in vitro and in vivo assays. We found unexpectedly that the mutant protein both stimulated transcription from the spoIIG promoter and repressed transcription from the abrB promoter, albeit twofold less than the wild type. A DNA binding analysis of Spo0A(A257V) showed that the mutant protein was less able to tolerate alterations in the sequence and arrangement of its DNA binding sites than the wild-type protein. In addition, we found that Spo0A(A257V) could stimulate transcription of a mutant spoIIG promoter in vivo in which low-consensus binding sites were replaced by high-consensus binding sites. We conclude that Spo0A(A257V) is able to bind to and regulate the expression of only genes whose promoters contain high-consensus binding sites and that this effect is sufficient to explain the observed sporulation defect

Reactive oxygen probes - a broad range of colors with easier labeling and compatibility with fixation: novel CellROX® reagents from Molecular Probes® (P3295)

Abstract A natural consequence of aerobic respiration is the generation of highly toxic radicals called reactive oxygen species (ROS). ROS have been implicated in various human pathologies. Although fluorogenic probes such as Aminophenyl fluorescein (APF) and hydroxyphenyl fluorescein (HPF), and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) have been widely used to detect ROS using flow cytometry and imaging platforms, all are excitable by the 488nm laser and emit in the fluorescein channel, and require that labeling occur in a protein-free buffer. In contrast, the novel CellROX® ROS detection reagents from Molecular Probes® offer increased flexibility for multiplex experiments, with fluorescence emission in the fluorescein, PE, or APC channels for CellROX® Green, Orange, and Deep Red. In addition, the CellROX® reagents offer increased ease of use with the ability to label cells in complete growth media, have increased photostability as compared to H2DCFDA, and are compatible with fixation (CellROX® Green and Deep Red). The CellROX® ROS detection reagents are bright and stable ROS sensors that offer significant advantages over existing ROS sensors because they are compatible with labeling in different media and can be used with fixatives. For Research Use Only. Not for use in diagnostic procedures.</jats:p

Zebrafish Mnx proteins specify one motoneuron subtype and suppress acquisition of interneuron characteristics

<p>Abstract</p> <p>Background</p> <p>Precise matching between motoneuron subtypes and the muscles they innervate is a prerequisite for normal behavior. Motoneuron subtype identity is specified by the combination of transcription factors expressed by the cell during its differentiation. Here we investigate the roles of Mnx family transcription factors in specifying the subtypes of individually identified zebrafish primary motoneurons.</p> <p>Results</p> <p>Zebrafish has three Mnx family members. We show that each of them has a distinct and temporally dynamic expression pattern in each primary motoneuron subtype. We also show that two Mnx family members are expressed in identified VeLD interneurons derived from the same progenitor domain that generates primary motoneurons. Surprisingly, we found that Mnx proteins appear unnecessary for differentiation of VeLD interneurons or the CaP motoneuron subtype. Mnx proteins are, however, required for differentiation of the MiP motoneuron subtype. We previously showed that MiPs require two temporally-distinct phases of Islet1 expression for normal development. Here we show that in the absence of Mnx proteins, the later phase of Islet1 expression is initiated but not sustained, and MiPs become hybrids that co-express morphological and molecular features of motoneurons and V2a interneurons. Unexpectedly, these hybrid MiPs often extend CaP-like axons, and some MiPs appear to be entirely transformed to a CaP morphology.</p> <p>Conclusions</p> <p>Our results suggest that Mnx proteins promote MiP subtype identity by suppressing both interneuron development and CaP axon pathfinding. This is, to our knowledge, the first report of transcription factors that act to distinguish CaP and MiP subtype identities. Our results also suggest that MiP motoneurons are more similar to V2 interneurons than are CaP motoneurons.</p

Correction: A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate Expression Constructs with Diverse Research Applications.

[This corrects the article DOI: 10.1371/journal.pone.0159277.]