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. Although gtaI mutants exhibited decreased levels of RcGTA production, mutagenesis of gtaR did not, whereas a gtaR/gtaI double mutant produced wild-type levels of RcGTA. Because mutagenesis of gtaR suppressed the effect of the gtaI mutation, we suggest that the GtaR protein is a negative transcriptional regulator of RcGTA gene expression. We discovered that the gtaR and gtaI genes are co-transcribed, and also negatively regulated by the GtaR protein in the absence of acyl-HSL. A His-tagged GtaR protein was purified, and DNA-binding experiments revealed a binding site in the promoter region of the gtaRI operon. This GtaR protein did not bind to the RcGTA promoter region, and therefore modulation of RcGTA production appears to require at least one additional factor. We found that RcGTA production was stimulated by spent media from other species, and identified exogenous acyl-HSLs that induce RcGTA.

A Ras subfamily GTPase shows cell cycle-dependent nuclear localization.

Previously characterized Ras subfamily proteins have been found to be predominantly associated with the plasma membrane where they function in signal transduction pathways to convey extracellular signals to intracellular targets. Here, we provide evidence that the Dictyostelium Ras subfamily protein RasB has a novel subcellular localization and function. The protein is predominantly localized in the nucleus during most of the cell cycle. Furthermore, during mitosis and cytokinesis RasB assumes a diffuse cellular localization despite the fact that the nuclear membrane stays intact. The linkage between the position of RasB in the cell and division suggests that it may have a role in nuclear division. Consistent with this idea, rasB- cells exhibit severe growth defects and cells overexpressing an activated version of RasB are multinucleate.

RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation.

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC(-) cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC(-) cells stimulated by 2'-deoxy-cAMP, but is produced in response to GTPgammaS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC(-) cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC(-) cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC(-) cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC(-) cells, suggesting that AleA may activate RasC.

Developmental gene expression in Bacillus subtilis crsA47 mutants reveals glucose-activated control of the gene for the minor sigma factor sigma(H).

The presence of excess glucose in growth media prevents normal sporulation of Bacillus subtilis. The crsA47 mutation, located in the gene for the vegetative phase sigma factor (sigma(A)) results in a glucose-resistant sporulation phenotype. As part of a study of the mechanisms whereby the mutation in sigma(A) overcomes glucose repression of sporulation, we examined the expression of genes involved in sporulation initiation in the crsA47 background. The crsA47 mutation had a significant impact on a variety of genes. Changes to stage II gene expression could be linked to alterations in the expression of the sinI and sinR genes. In addition, there was a dramatic increase in the expression of genes dependent on the minor sigma factor sigma(H). This latter change was paralleled by the pattern of spo0H gene transcription in cells with the crsA47 mutation. In vitro analysis of RNA polymerase containing sigma(A47) indicated that it did not have unusually high affinity for the spo0H gene promoter. The in vivo pattern of spo0H expression is not predicted by the known regulatory constraints on spo0H and suggests novel regulation mechanisms that are revealed in the crsA47 background.

RasG regulates discoidin gene expression during Dictyostelium growth.

Activated rasG, rasG(G12T), was expressed in Dictyostelium cells under the control of the folate-repressible discoidin promoter (pVEII-rasG(G12T)) and found to have a unique pattern of expression when cells were transferred to folate-deficient media: an initial increase of RasG(G12T) resulting from the removal of folate, followed by a rapid decline while cells were still in the early exponential phase of growth. Discoidin levels were considerably lower and declined more rapidly in the pVEII-rasG(G12T) transformant than they did in the wild type, suggesting that RasG(G12T) represses discoidin expression. This was independently confirmed by placing the rasG(G12T) gene under the control of the ribonucleotide reductase (rnrB) promoter. Exposure of cells to 10 mM methyl methanesulfonate (MMS) rapidly generated RasG(G12T) and this was accompanied by an equally rapid decrease in discoidin mRNA levels. rasG null cells also contained decreased levels of discoidin under all conditions tested, indicating that RasG is essential for optimum discoidin expression. However, rasG null cells showed normal regulation of discoidin expression in response to PSF, CMF, folate, bacteria, and axenic media, indicating that RasG is not necessary for any of these responses. These results reveal a role for RasG in regulating discoidin gene expression and add a further level of complexity to the regulation of the discoidin promoter.

Lessons and questions from the structure of the Spo0A activation domain.

The carboxy-terminal domain of Spo0A in Bacillus subtilis is one of the few response regulator activation domains for which the structure is known. Here, we discuss some of the mutational data and biological roles of Spo0A in light of its structure.

Expression of activated Ras during Dictyostelium development alters cell localization and changes cell fate.

There is now a body of evidence to indicate that Ras proteins play important roles in development. Dictyostelium expresses several ras genes and each appears to perform a distinct function. Previous data had indicated that the overexpression of an activated form of the major developmentally regulated gene, rasD, caused a major aberration in morphogenesis and cell type determination. We now show that the developmental expression of an activated rasG gene under the control of the rasD promoter causes a similar defect. Our results indicate that the expression of activated rasG in prespore cells results in their transdifferentiation into prestalk cells, whereas activated rasG expression in prestalk causes gross mislocalization of the prestalk cell populations.

Identification of a second region of the Spo0A response regulator of Bacillus subtilis required for transcription activation.

Deletion of the 10 C-terminal amino acids of the Bacillus subtilis response regulator Spo0A or valine substitution at D258 and L260 resulted in a sporulation-negative phenotype and loss of in vivo activation of the spoIIG and spoIIA operon promoters. Repression of the abrB promoter was not affected by the mutations. In combination with the previously characterized mutation (A257V), the results identify amino acids at positions 257, 258, and 260 as being required for transcription activation by Spo0A.

Dictyostelium RasD is required for normal phototaxis, but not differentiation.

RasD, a Dictyostelium homolog of mammalian Ras, is maximally expressed during the multicellular stage of development. Normal Dictyostelium aggregates are phototactic and thermotactic, moving towards sources of light and heat with great sensitivity. We show that disruption of the gene for rasD causes a near-total loss of phototaxis and thermotaxis in mutant aggregates, without obvious effects on undirected movement. Previous experiments had suggested important roles for RasD in development and cell-type determination. Surprisingly, rasD(-) cells show no obvious changes in these processes. These cells represent a novel class of phototaxis mutant, and indicate a role for a Ras pathway in the connections between stimuli and coordinated cell movement.

A role for Asp75 in domain interactions in the Bacillus subtilis response regulator Spo0A.

Spo0A is a two-domain response regulator required for sporulation initiation in Bacillus subtilis. Studies on response regulators have focused on the activity of each domain, but very little is known about the mechanism by which the regulatory domain inhibits the activator domain. In this study, we created a single amino acid substitution in the regulatory domain, D75S, which resulted in a dramatic decrease in sporulation in vivo. In vitro studies with the purified Spo0AD75S protein demonstrated that phosphorylation and DNA binding were comparable with wild type Spo0A. However, the mutant was unable to stimulate transcription by final sigma(A)-RNA polymerase from the Spo0A-dependent spoIIG operon promoter. We suggest that the amino acid Asp(75) and/or the region within which it resides, the alpha3-beta4 loop, are involved in the inhibitory interaction between the regulatory and activator domains of Spo0A.

Functional overlap of the dictyostelium RasG, RasD and RasB proteins.

Disruption of the rasG gene in Dictyostelium discoideum results in several distinct phenotypes: a defect in cytokinesis, reduced motility and reduced growth. Reintroduction of the rasG gene restores all of the properties of the rasG(-) cells to those of the wild type. To determine whether the defects are due to impaired interactions with a single or multiple downstream effectors, we tested the ability of the highly related but non identical Dictyostelium ras genes, rasD and rasB, to rescue the defects. Introduction of the rasD gene under the control of the rasG promoter into rasG null (rasG(-)) cells corrected all phenotypes except the motility defect, suggesting that motility is regulated by a RasG mediated pathway that is different to those regulating growth or cytokinesis. Western blot analysis of RasD protein levels revealed that vegetative rasG(- )cells contained considerably more protein than the parental AX-3 cells, suggesting that RasD protein levels are negatively regulated in vegetative cells by RasG. The level of RasD was enhanced when the rasD gene was introduced under the control of the rasG promoter, and this increase in protein is presumably responsible for the reversal of the growth and cytokinesis defects of the rasG(- )cells. Thus, RasD protein levels are controlled by the level of RasG, but not by the level of RasD. Introduction of the rasB gene under the control of the rasG promoter into rasG(-) cells produced a complex phenotype. The transformants were extremely small and mononucleate and exhibited enhanced motility. However, the growth of these cells was considerably slower than the growth of the rasG(-) cells, suggesting the possibility that high levels of RasB inhibit an essential process. This was confirmed by expressing rasB in wild-type cells; the resulting transformants exhibited severely impaired growth. When RasB protein levels were determined by western blot analysis, it was found that levels were higher in the rasG(- )cells than they were in the wild-type parental, suggesting that RasG also negatively regulates rasB expression in vegetative cells. Overexpression of rasB in the rasG(- )cells also reduced the level of RasD protein. In view of the fact that alternate Ras proteins correct some, but not all, of the defects exhibited by the rasG(-) cells, we propose that RasG interacts with more than one downstream effector. In addition, it is clear that the levels of the various Ras proteins are tightly regulated in vegetative cells and that overexpression can be deleterious.

A mutation that separates the RasG signals that regulate development and cytoskeletal function in Dictyostelium.

The expression of an activated RasG, RasG-G12T, in vegetative cells of Dictyostelium discoideium produced an alteration in cell morphology. Cells underwent a transition between an extensively flattened form that exhibited lateral membrane ruffling to a less flattened form that exhibited prominent dorsal membrane ruffling. These rasG-G12T transformants exhibited a redistribution of F-actin at the cell periphery and did not undergo the rapid contraction upon refeeding that is characteristic of wild-type cells. These results suggest a role for RasG in regulating cytoskeletal rearrangement in D. discoideum. We had shown previously that expression of rasG-G12T inhibited starvation induced aggregation (M. Khosla et al., 1996, Mol. Cell. Biol. 16, 4156-4162). rasG-G12T genes containing secondary mutations were transformed into cells to test whether the effects of rasG-G12T were transmitted through a single downstream effector. Cells expressing rasG-G12T/T35S or rasG-G12T/Y40C (secondary mutations within the effector domain) exhibited normal morphology and underwent normal aggregation, suggesting that signaling through the effector domain was required for both the morphological and the development changes induced by rasG-G12T. In contrast, cells expressing rasG-G12T/T45Q (a secondary mutation in the effector distal flanking domain) exhibited normal aggregation but a morphology indistinguishable from that of rasG-G12T transformants. This result suggests that RasG regulates developmental and cytoskeletal functions by direct interaction with more than one downstream effector.

The small Mr Ras-like GTPase Rap1 and the phospholipase C pathway act to regulate phagocytosis in Dictyostelium discoideum.

The function of the small-Mr Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes in Dictyostelium and the oxidative burst in mammalian neutrophils. To test whether Rap1 regulates phagocytosis, we biochemically analyzed cell lines that conditionally and modestly overexpressed wild-type [Rap1 WT(+)], constitutively active [Rap1 G12T(+)], and dominant negative [Rap1 S17N(+)] forms of D. discoideum Rap1. The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT(+) and Rap1 G12T(+) cells and were reduced in Rap1 S17N(+) cells. The addition of inhibitors of protein kinase A, protein kinase G, protein tyrosine kinase, or phosphatidylinositide 3-kinase did not affect phagocytosis rates in wild-type cells. In contrast, the addition of U73122 (a phospholipase C inhibitor), calphostin C (a protein kinase C inhibitor), and BAPTA-AM (an intracellular Ca2+ chelator) reduced phagocytosis rates by 90, 50, and 65%, respectively, suggesting both arms of the phospholipase C signaling pathways played a role in this process. Other protein kinase C-specific inhibitors, such as chelerythrine and bisindolylmaleimide I, did not reduce phagocytosis rates in control cells, suggesting calphostin C was affecting phagocytosis by interfering with a protein containing a diacylglycerol-binding domain. The addition of calphostin C did not reduce phagocytosis rates in Rap1 G12T(+) cells, suggesting that the putative diacylglycerol-binding protein acted upstream in a signaling pathway with Rap1. Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(+) and Rap1 G12T(+) cells compared with control cells. Together our results suggest that Rap1 and Ca2+ may act together to coordinate important early events regulating phagocytosis.

The Spo0A sof mutations reveal regions of the regulatory domain that interact with a sensor kinase and RNA polymerase.

Spo0A is a two-domain response regulator required for the initiation of sporulation in Bacillus subtilis. Spo0A is activated by phosphorylation of its regulatory domain by a multicomponent phosphorelay. To define the role of the regulatory domain in the activation of Spo0A, we have characterized four of the sof mutations in vitro. The sof mutations were identified previously as suppressors of the sporulation-negative phenotype resulting from a deletion of the gene for one of the phosphorelay components, spo0F. Like wild-type Spo0A, the transcription stimulation properties of all of the Sof proteins were dependent upon phosphorylation. Sof mutants from two classes were improved substrates for direct phosphorylation by the KinA sensor kinase, providing an explanation for their suppression properties. Two other Sof proteins showed a phosphorylation-dependent enhancement of the stability of the Sof approximately P-RNA polymerase-DNA complex. One of these mutants, Sof114, increased the stability of the Sof114 approximately P-RNAP-DNA complex without increasing its own affinity for the spoIIG promoter. A comparison of the location of the sof mutations with mutations in CheY suggests that phosphorylation of Spo0A results in the exposure of a region in the regulatory domain that interacts with RNA polymerase, thereby contributing to the signal transduction mechanism.

Contributions of the domains of the Bacillus subtilis response regulator Spo0A to transcription stimulation of the spoIIG operon.

Spo0A is a response regulator that controls entry into sporulation by specifically stimulating or repressing transcription of critical developmental genes. Response regulators have at least two domains: an output transcription regulation domain and a receiver domain that inhibits the output domain. Phosphorylation of the receiver domain relieves the inhibition. We examined the in vitro transcription activation mechanism for Spo0A, phosphorylated Spo0A (Spo0A approximately P), and a deletion mutant that consists solely of the C-terminal output domain (Spo0ABD). Both Spo0A approximately P and Spo0ABD stimulated transcription from the spoIIG promoter 10-fold more efficiently than Spo0A. Spo0A approximately P and Spo0ABD induced DNA denaturation by RNA polymerase in the -10 recognition region, whereas Spo0A did not. DNase I footprint assays revealed that phosphorylation enhanced binding of intact Spo0A to the 0A boxes, while the binding of Spo0ABD was similar to that of Spo0A. Thus, activation of Spo0A by phosphorylation is not primarily due to enhanced DNA binding. The presence of a phosphorylated N terminus increased the stability of the ternary complex at the spoIIG promoter. We propose that the primary effect of phosphorylation is to expose an RNA polymerase interaction domain to promote transcription from PspoIIG.

The Bacillus subtilis regulator SinR inhibits spoIIG promoter transcription in vitro without displacing RNA polymerase.

Initiation of sporulation in Bacillus subtilis is controlled by several regulators which affect activation by phosphorylation of the key response regulator Spo0A or transcription of Spo0A-P-dependent genes. In vivo overexpression of one of these regulators, sinR , results in suppression of transcription from the Spo0A-P-dependent promoters of spo0A , spoIIA , spoIIE and spoIIG and in vitro SinR binds to the promoters of the spoIIA operon and the spo0A gene. In this study we have demonstrated that in vitro SinR directly repressed Spo0A- P-dependent transcription by B.subtilis RNA polymerase from the spoIIG operon promoter. SinR inhibited transcription prior to formation of heparin-resistant complexes but did not displace RNA polymerase from the spoIIG promoter. DNase I protection studies demonstrated that SinR protected a large region of the spoIIG promoter and induced DNase I hypersensitive sites, particularly around the 0A boxes, at the same positions as those induced by zinc. Since binding of zinc induces bends in the DNA, we concluded that SinR binding also altered the conformation of the spoIIG promoter. We propose that SinR-induced conformational changes in Spo0A-dependent promoters prevent activation of trans-cription by Spo0A-P.

Transcriptional activation of the Bacillus subtilis spoIIG promoter by the response regulator Spo0A is independent of the C-terminal domain of the RNA polymerase alpha subunit.

In vitro transcription from the spoIIG promoter by Bacillus subtilis RNA polymerase reconstituted with wild-type alpha subunits and with C-terminal deletion mutants of the alpha subunit was equally stimulated by the response regulator Spo0A. Some differences in the structure of open complexes formed by RNA polymerase containing alpha subunit mutants were noted, although the wild-type and mutant polymerases appeared to use the same initiation mechanism.

A negative regulator linking chromosome segregation to developmental transcription in Bacillus subtilis.

The SpoOJA and SpoOJB proteins of Bacillus subtilis are similar to the ParA and ParB plasmid-partitioning proteins, respectively, and mutation of spoOJB prevents the expression of stage II genes of sporulation. This phenotype is a consequence of SpoOJA activity in the absence of SpoOJB, and its basis was unknown. In the studies reported here, SpoOJA was found specifically to dissociate transcription initiation complexes formed in vitro by the phosphorylated sporulation transcription factor SpoOA and RNA polymerase with the spollG promoter. This repressor-like activity is likely to be the basis for preventing the onset of differentiation in vivo. SpoOJB is known to neutralize SpoOJA activity in vivo and also to interact with a mitotic-like apparatus responsible for chromosome partitioning. These data suggest that SpoOJA and SpoOJB form a regulatory link between chromosome partition and development gene expression.

DNA strand separation during activation of a developmental promoter by the Bacillus subtilis response regulator Spo0A.

Spo0A is the central regulator of commitment to sporulation in Bacillus subtilis. Spo0A is a member of the response regulator family of proteins and both represses and stimulates transcription from promoters when activated. In vivo Spo0A activation takes place by phosphorylation and in vitro activation can be accomplished by phosphorylation or removal of the N-terminal domain of the protein. We have examined the mechanism of Spo0A stimulation of transcription from the promoter of the spoIIG operon. This operon encodes one of the first compartment specific sigma factors whose appearance regulates sporulation development. When activated Spo0A was incubated with RNA polymerase and a DNA fragment containing the spoIIG promoter, bases between -13 and -3, relative to the start site of transcription, were denatured. Addition of activated Spo0A or RNA polymerase alone did not induce denaturation. Heteroduplex templates that contained the nontemplate sequence of the wild-type promoter on both strands between positions -3 and -13 were efficiently transcribed without activated Spo0A. These data suggest that DNA strand separation is a two-step process and that the activation of Spo0A creates a form that interacts with the polymerase to induce the first of the two steps.

Unique gene organization: alternative splicing in Drosophila produces two structurally unrelated proteins.

The Ub80 gene in eukaryotes produces a ubiquitin fusion protein in which ubiquitin is fused in frame to a tail protein (Redman and Rechsteiner, 1988; Finley et al., 1989; Barrio et al., 1994). The tail protein is incorporated into the ribosome, and ubiquitin is thought to act as a chaperone. The DUb80 gene of Drosophila melanogaster was cloned by Barrio et al. (1994) and contains a 5'-untranslated exon, followed by a large intron and then the first coding exon. We report that the large intron of DUb80 contains an open reading frame, which produces a 259-aa protein (IP259) that is conserved in eukaryotes from yeast to mammals. Transcription of the DUb80 and IP259 mRNAs begins at the same start sites. However, alternate splicing of the primary transcript produces two structurally unrelated proteins. This is the second reported instance of two structurally unrelated proteins being produced via alternate splicing, suggesting that this form of genomic organization may be more common than previously thought.