George B. Spiegelman

The transition state transcription regulator abrB of Bacillus subtilis is a DNA binding protein.

The product of the abrB gene of Bacillus subtilis is an ambiactive repressor and activator of the transcription of genes expressed during the transition state between vegetative growth and the onset of stationary phase and sporulation. Purified AbrB protein binds specifically in a highly co‐operative fashion to fragments of DNA containing the promoters it affects. DNase I footprints of the binding regions in these promoters revealed large protected areas of 50‐120 nucleotides or more depending on the promoter. Methylation protection experiments gave protected guanine residues on only one face of the DNA helix. A consensus sequence could be deduced around these guanine residues that was not found around non‐protected guanine residues in the footprint region. The results suggested that stationary phase functions and sporulation are repressed during active growth by AbrB and other transition state regulators by binding to the affected promoters in a concentration‐dependent manner.

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. We have adapted an assay, used successfully to measure activated Ras in mammalian cells, to monitor activation of two Dictyostelium Ras proteins, RasC and RasG. We have found that the Ras‐binding domain (RBD) of mammalian Raf1 was capable of binding to the activated form of RasG, but not to the activated form of RasC; however, the RBD of Schizosaccharomyces pombe Byr2 was capable of binding preferentially to the activated forms of both RasC and RasG. Using this assay, we discovered that RasC and RasG showed a rapid and transient activation when aggregation‐competent cells were stimulated with the chemoattractant cAMP, and this activation did not occur in a number of cAMP signalling mutants. These data provide further evidence of a role for both RasC and RasG in the early development of Dictyostelium.

Culture-Independent Molecular Analysis of Microbial Constituents of the Healthy Human Outer Ear

ABSTRACT Molecular-phylogenetic sequence analyses have provided a new perspective on microbial communities by allowing the detection and identification of constituent microorganisms in the absence of cultivation. In this study we used broad-specificity amplification of ribosomal DNA (rDNA) genes to survey organisms present in the human outer ear canal. Samples were obtained from 24 individuals, including members of three extended families, in order to survey the resident microbiota and to examine microbial population structures in individuals related by familial or household associations. To examine the stability of the microbial populations, one individual was sampled four times and another twice over a 14-month period. We found that a distinct set of microbial types was present in the majority of the subjects sampled. The two most prevalent rDNA sequence types that were identified in multiple individuals corresponded closely to those of Alloiococcus otitis and Corynebacterium otitidis, commonly thought to be associated exclusively with infections of the middle ear. Our results suggest, therefore, that the outer ear canal may serve as a reservoir for normally commensal microbes that can contribute to pathogenesis upon introduction into the middle ear. Alternatively, culture analyses of diseases of the middle ear may have been confounded by these contaminating commensal organisms.

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 GTPγS 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.

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

The Spo0JA and Spo0JB proteins of Bacillus subtilis are similar to the ParA and ParB plasmid‐partitioning proteins, respectively, and mutation of spo0JB prevents the expression of stage II genes of sporulation. This phenotype is a consequence of Spo0JA activity in the absence of Spo0JB, and its basis was unknown. In the studies reported here, Spo0JA was found specifically to dissociate transcription initiation complexes formed in vitro by the phosphorylated sporulation transcription factor Spo0A and RNA polymerase with the spoIIG promoter. This repressor‐like activity is likely to be the basis for preventing the onset of differentiation in vivo. Spo0JB is known to neutralize Spo0JA activity in vivo and also to interact with a mitotic‐like apparatus responsible for chromosome partitioning. These data suggest that Spo0JA and Spo0JB form a regulatory link between chromosome partition and development gene expression.

Evidence for a role for the Dictyostelium Rap1 in cell viability and the response to osmotic stress.

The Dictyostelium genome contains a single rapA gene, which encodes a Rap1 monomeric G protein. As attempts at generating rapA-null Dictyostelium cells had been unsuccessful, expression of antisense RNA from the rapA gene under control of the folate repressible discoidin promoter was used to reduce cellular levels of the Rap1 protein. As Rap1 levels gradually decreased following antisense rapA RNA induction, growth rate and cell viability also decreased, a result consistent with the idea that rapA is an essential gene. The Rap1-depleted cells exhibited reduced viability in response to osmotic shock. The accumulation of cGMP in response to 0.4 M sorbitol was reduced after rapA antisense RNA induction and was enhanced in cells expressing the constitutively activated Rap1(G12V) protein, suggesting a role for Rap1 in the generation of cGMP. Dictyostelium Rap1 formed a complex with the Ras-binding domain of RalGDS only when it was in a GTP-bound state. This assay was used to demonstrate that activation of Rap1 in response to 0.4 M sorbitol occurred with initial kinetics similar to those observed for the accumulation of cGMP. Furthermore, the addition of 2 mM EDTA to osmotically shocked cells, a treatment that enhances cGMP accumulation, also enhanced Rap1 activation. These results suggest a direct role for Rap1 in the activation of guanylyl cyclase during the response to hyperosmotic conditions. Rap1 was also activated in response to low temperature but not in response to low osmolarity or high temperature.

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.

Phosphorylation of Spo0A activates its stimulation of in vitro transcription from the Bacillus subtilis spollG operon

The spollG operon of Bacillus subtilis codes for a sporulation‐specjfic sigma factor, σ;E, In vivo expression of the spollG promoter is activated shortly after the onset of sporulation and is dependent on kinA, spo0F, spo0B and spo0A genes. The products of these genes have been shown to participate in a phosphorelay reaction in vitro, culminating in phosphorylation of the transcription factor, Spo0A. The effect of Spo0A phosphorylation on in vitro transcription from the spollG promoter was determined. Aliquots from phos‐phorelay reactions enhanced spollG promoter activity 10‐fold in transcription assays and stimulation of transcription was dependent on Spo0A phosphorylation. Our results provide biochemical evidence that Spo0A and the phosphoreiay form a signal transduction pathway which activates spoil gene expression in development.

Recreating the university from within: Collaborative reflections on the University of British Columbia's engagement with sustainability

Purpose – In 1997, the University of British Columbia (UBC) adopted a sustainable development policy stating that the campus should adhere to sustainable practices in all of its actions and mandates and that all students who attend UBC will be educated about sustainability. The purpose of the paper is to consider how far UBC has moved in the last six years in the direction of sustainability education, what has been accomplished, what lessons have been learned and what challenges lie ahead.Design/methodology/approach – This paper is a collaborative inquiry created by a number of faculty, staff and one doctoral student working on sustainability education issues at UBC.Findings – The shift to sustainability involves: a fundamental thinking‐through of basic issues about the role of the university in society, creating a strong relationship between sustainability principles and the core goals of the university. It also will require a reworking of the design and operation of institutional reward systems, creatin...

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.