The mycobacterial antibiotic resistance determinant WhiB7 acts as a transcriptional activator by binding the primary sigma factor SigA (RpoV).

Tuberculosis therapeutic options are limited by the high intrinsic antibiotic resistance of Mycobacterium tuberculosis. The putative transcriptional regulator WhiB7 is crucial for the activation of systems that provide resistance to diverse antibiotic classes. Here, we used in vitro run-off, two-hybrid assays, as well as mutagenic, complementation and protein pull-down experiments, to characterize WhiB7 as an auto-regulatory, redox-sensitive transcriptional activator in Mycobacterium smegmatis.

Separate mechanisms are involved in rifampicin upmodulated and downmodulated gene expression in Salmonella Typhimurium.

Sub-MIC antibiotics differentially modulate transcription of subsets of genes by unknown mechanisms. Paradoxically, the RNA polymerase inhibitor rifampicin is able to both upmodulate as well as downmodulate transcription when present at sub-MIC levels. In this study, we analyzed DNA sequences required for transcription modulation.

The GtaR protein negatively regulates transcription of the gtaRI operon and modulates gene transfer agent (RcGTA) expression in Rhodobacter capsulatus.

The gtaI gene of Rhodobacter capsulatus encodes an N-acyl-homoserine lactone (acyl-HSL) synthase. Immediately 5' of the gtaI gene is ORF rcc00328 that encodes a potential acyl-HSL receptor protein. A combination of genetic and biochemical approaches showed that rcc00328 (renamed gtaR) modulates the production of a genetic exchange element called the gene transfer agent (RcGTA), and regulates the transcription of gtaI.

Using invention to change how students tackle problems.

Invention activities challenge students to tackle problems that superficially appear unrelated to the course material but illustrate underlying fundamental concepts that are fundamental to material that will be presented. During our invention activities in a first-year biology class, students were presented with problems that are parallel to those that living cells must solve, in weekly sessions over a 13-wk term. We compared students who participated in the invention activities sessions with students who participated in sessions of structured problem solving and with students who did not participate in either activity.

Determinants of RasC specificity during Dictyostelium aggregation.

RasC is required for optimum activation of adenylyl cyclase A and for aggregate stream formation during the early differentiation of Dictyostelium discoideum. RasG is unable to substitute for this requirement despite its sequence similarity to RasC. A critical question is which amino acids in RasC are required for its specific function.

Ras proteins have multiple functions in vegetative cells of Dictyostelium.

During the aggregation of Dictyostelium cells, signaling through RasG is more important in regulating cyclic AMP (cAMP) chemotaxis, whereas signaling through RasC is more important in regulating the cAMP relay. However, RasC is capable of substituting for RasG for chemotaxis, since rasG⁻ cells are only partially deficient in chemotaxis, whereas rasC⁻/rasG⁻ cells are totally incapable of chemotaxis. In this study we have examined the possible functional overlap between RasG and RasC in vegetative cells by comparing the vegetative cell properties of rasG⁻, rasC⁻, and rasC⁻/rasG⁻ cells.

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.

Rap1 activation in response to cAMP occurs downstream of ras activation during Dictyostelium aggregation.

We have used a doubly disrupted rasC(-)/rasG(-) strain of Dictyostelium discoideum, which ectopically expresses the carA gene, to explore the relationship between the activation of RasC and RasG, the two proteins that are necessary for optimum cAMP signaling, and the activation of Rap1, a Ras subfamily protein, that is also activated by cAMP. The ectopic expression of carA restored early developmental gene expression to the rasC(-)/rasG(-) strain, rendering it suitable for an analysis of cAMP signal transduction. Because there was negligible signaling through both the cAMP chemotactic pathway and the adenylyl cyclase activation pathway in the rasC(-)/rasG(-)/[act15]:carA strain, it is clear that RasG and RasC are the only two Ras subfamily proteins that directly control these pathways.

Cyclic AMP signalling in Dictyostelium: G-proteins activate separate Ras pathways using specific RasGEFs.

In general, mammalian Ras guanine nucleotide exchange factors (RasGEFs) show little substrate specificity, although they are often thought to regulate specific pathways. Here, we provide in vitro and in vivo evidence that two RasGEFs can each act on specific Ras proteins. During Dictyostelium development, RasC and RasG are activated in response to cyclic AMP, with each regulating different downstream functions: RasG regulates chemotaxis and RasC is responsible for adenylyl cyclase activation.

Bacillus subtilis RNA polymerase recruits the transcription factor Spo0A approximately P to stabilize a closed complex during transcription initiation.

The Bacillus subtilis response regulator Spo0A approximately P activates transcription from the spoIIG promoter by stimulating a rate-limiting transition between the initial interaction of RNA polymerase with the promoter and initiation of RNA synthesis. Previous work showed that Spo0A exerts its effect on RNA polymerase prior to the formation of an open complex in which the DNA strands at the initiation site have been separated. To isolate the effect of Spo0A approximately P on events prior to DNA strand separation at spoIIG we studied RNA polymerase binding to DNA fragments that were truncated to contain only promoter sequences 5' to the -10 element by electrophoretic mobility shift assays.

Transcription modulation of Salmonella enterica serovar Typhimurium promoters by sub-MIC levels of rifampin.

Promoter-lux fusions that showed rifampin-modulated transcription were identified from a Salmonella enterica serovar Typhimurium 14028 reporter library. The transformation of a subset of fusions into mutants that lacked one of six global regulatory proteins or were rifampin resistant showed that transcription modulation was independent of the global regulators, promoter specific, and dependent on the interaction of rifampin with RNA polymerase.

The world of subinhibitory antibiotic concentrations.

Although antibiotics have long been known to have multiple effects on bacterial cells at low concentrations, it is only with the advent of genome transcription analyses that these activities have been studied in detail at the level of cell metabolism. It has been shown that all antibiotics, regardless of their receptors and mode of action, exhibit the phenomenon of hormesis and provoke considerable transcription activation at low concentrations. These analyses should be of value in providing information on antibiotic side-effects, in bioactive natural product discovery and antibiotic mode-of-action studies.

Delineation of the roles played by RasG and RasC in cAMP-dependent signal transduction during the early development of Dictyostelium discoideum.

On starvation, the cellular slime mold Dictyostelium discoideum initiates a program of development leading to formation of multicellular structures. The initial cell aggregation requires chemotaxis to cyclic AMP (cAMP) and relay of the cAMP signal by the activation of adenylyl cyclase (ACA), and it has been shown previously that the Ras protein RasC is involved in both processes. Insertional inactivation of the rasG gene resulted in delayed aggregation and a partial inhibition of early gene expression, suggesting that RasG also has a role in early development.

An activated Ras protein alters cell adhesion by dephosphorylating Dictyostelium DdCAD-1.

RasG-regulated signal transduction has been linked to a variety of growth-specific processes and appears to also play a role in the early development of Dictyostelium discoideum. In an attempt to uncover some of the molecular components involved in Ras-mediated signalling, several proteins have been described previously, including the cell adhesion molecule DdCAD-1, whose phosphorylation state was affected by the expression of the constitutively activated RasG, RasG(G12T). Here it has been shown that a cadA null strain lacks the phosphoproteins that were tentatively identified as DdCAD-1, confirming its previous designation.

A secondary disruption of the dmpA gene encoding a large membrane protein allows aggregation defective Dictyostelium rasC- cells to form multicellular structures.

The disruption of the gene encoding the Dictyostelium Ras subfamily protein, RasC, results in a strain that does not aggregate and has defects in both cAMP signal relay and cAMP chemotaxis. Disruption of a second gene in the rasC(-) strain by Restriction Enzyme Mediated Integration produced cells that were capable of forming multicellular structures in plaques on bacterial lawns. The disrupted gene (dmpA) encoded a novel membrane protein that was designated Dmp1.

The effect of the disruption of a gene encoding a PI4 kinase on the developmental defect exhibited by Dictyostelium rasC(-) cells.

The disruption of the gene encoding the Dictyostelium Ras subfamily protein, RasC results in a strain that fails to aggregate with defects in both cAMP signal relay and chemotaxis. Restriction enzyme mediated integration disruption of a second gene in the rasC(-) strain resulted in cells that were capable of forming multicellular structures in plaques on bacterial lawns. The disrupted gene, designated pikD(1), encodes a member of the phosphatidyl-inositol-4-kinase beta subfamily.

Loss of the Dictyostelium RasC protein alters vegetative cell size, motility and endocytosis.

In addition to its previously established roles in cAMP relay and cAMP chemotaxis, loss of signal transduction through the RasC protein was found to impact a number of vegetative cell functions. Vegetative rasC- cells exhibited reduced random motility, were less polarized and had altered F-actin distribution. Cells lacking RasC also contained more protein and were larger in size than wild type cells.

Soj antagonizes Spo0A activation of transcription in Bacillus subtilis.

The ParA family protein Soj appears to negatively regulate sporulation in Bacillus subtilis by inhibiting transcription from promoters that are activated by phosphorylated Spo0A. We tested in vitro Soj inhibition of Spo0A-independent variants of a promoter that Soj inhibited (PspoIIG). Transcription from the variants was less sensitive to Soj inhibition, suggesting that inhibition of wild-type PspoIIG was linked to transcription activation by Spo0A.

The identification of Dictyostelium phosphoproteins altered in response to the activation of RasG.

Dictyostelium RasG has been implicated in the regulation of a variety of cellular processes, including the initiation of development, cell movement, and cytokinesis, but the molecular components of the signaling pathways involved are largely unknown. We used a tetracycline-regulated protein expression system to study the effect of activated RasG, RasG(G12T), expression on the phosphorylation state of Dictyostelium proteins. Over 70 vegetative phosphoprotein components were resolved by two-dimensional (2-D) immunoblot analysis and of these 16 phosphothreonine and three phosphotyrosine protein components were found to reproducibly change upon RasG(G12T) expression.

RasC plays a role in transduction of temporal gradient information in the cyclic-AMP wave of Dictyostelium discoideum.

To define the role that RasC plays in motility and chemotaxis, the behavior of a rasC null mutant, rasC-, in buffer and in response to the individual spatial, temporal, and concentration components of a natural cyclic AMP (cAMP) wave was analyzed by using computer-assisted two-dimensional and three-dimensional motion analysis systems. These quantitative studies revealed that rasC- cells translocate at the same velocity and exhibit chemotaxis up spatial gradients of cAMP with the same efficiency as control cells. However, rasC- cells exhibit defects in maintaining anterior-posterior polarity along the substratum and a single anterior pseudopod when translocating in buffer in the absence of an attractant.

Chemoattractant-induced Ras activation during Dictyostelium aggregation.

Ras proteins are highly conserved molecular switches that regulate cellular response to external stimuli. Dictyostelium discoideum contains an extensive family of Ras proteins that function in regulation of mitosis, cytoskeletal function and motility, and the onset of development. Little is known about the events that lead to the activation of Ras proteins in Dictyostelium, primarily owing to a lack of a biochemical assay to measure the levels of activated Ras.

The Bacillus subtilis response regulator Spo0A stimulates sigmaA-dependent transcription prior to the major energetic barrier.

At the spoIIG promoter phosphorylated Spo0A (Spo0A approximately P) binds 0A boxes overlapping the -35 element, interacting with RNA polymerase to facilitate open complex formation. We have compared in vitro transcription from a series of heteroduplex templates containing denatured regions within the promoters. Transcription from heteroduplex templates with 12, 8, or 6 base pairs denatured was independent of Spo0A approximately P, but heteroduplexes with 4 or 2 base pairs denatured required Spo0A approximately P for maximal levels of transcription.

Cytoskeletal regulation by Dictyostelium Ras subfamily proteins.

The Ras subfamily proteins are monomeric GTPases that function as molecular switches in cellular signal transduction. The roles of six of these proteins in regulating actin cytoskeletal functions in Dictyostelium discoideum are discussed in this review.

Roles played by Ras subfamily proteins in the cell and developmental biology of microorganisms.

The Ras subfamily proteins are monomeric GTPases that function as molecular switches in cellular signal transduction pathways. This review describes our current knowledge of the roles that these proteins play in the growth and differentiation of single celled microorganisms.