|Kevin L. Griffith
Assistant Professor of Microbiology, University of Massachusetts
Ph.D.: University of Maryland Baltimore County
Regulation of Biological Networks by Cell-Cell Signaling
Overview. Cell-cell signaling provides a way for individual bacterial cells to communicate with one another and to coordinate biological processes as a group. The main focus of our research is to understand how the soil bacterium Bacillus subtilis senses and responds to extracellular signaling molecules. We use a combination of systems approaches including genetics, transcriptional profiling, and whole genome DNA binding studies to identify regulatory networks controlled by cell-cell signaling. Biochemical and biophysical approaches are then used to determine the molecular function of regulatory factors and signaling molecules in controlling these physiological processes.
Quorum sensing. Individual bacterial cells secrete signaling molecules that are sensed by neighboring cells and serve as cues to coordinate specific responses with population density, a process referred to as quorum sensing. Quorum sensing is important for a variety of physiological processes including: pathogenesis, antibiotic production, horizontal gene transfer, bioluminescence, competence development, and sporulation.
ComA binding to DNA. Our work predicts that ComA binds to DNA in an unusual manner - two dimers of ComA bind to a tri-partite sequence (Fig. 2). All three recognition elements are required for ComA to bind DNA in vitro and for transcription activation by ComA in vivo. The degeneracy of the binding sequence is an important regulatory feature for maintaining the density-dependent response. Using biochemical and biophysical approaches, we are examining this unusual binding architecture to determine how ComA binds DNA to regulate genes of the quorum response.
Regulation of ComA activity. Two signaling pathways converge to regulate the activity of ComA by modulating its phosphorylation state and by inhibiting its ability to bind DNA via the Rap proteins (Fig. 1). We are dissecting the regulation of ComA by these external signals to determine their effects on DNA binding and transcription activation. Defining the interplay between ComA phosphorylation and the regulation by Raps should provide a better understanding of how the quorum response is coordinated with population density.
Identification of regulatory networks controlled by cell-cell signaling. Our ultimate goal is to map several of the quorum response pathways and to determine the involvement of ComA and co-regulators in controlling gene expression. Many of these pathways are predicted to be controlled directly by ComA, while others are predicted to be controlled indirectly through the action of regulatory factors whose expression is controlled by ComA (e.g., Rap-Phr pairs). Several putative target genes are suspected of requiring co-regulatory factors in addition to ComA. We are using genomic approaches including transcriptional profiling and whole genome binding studies to dissect these regulatory pathways and identify the factors involved in controlling gene expression.
Controllable protein degradation systems. An ongoing interest of our laboratory has been in the development of useful tools for studying regulatory networks in B. subtilis. We engineered a controllable protein degradation system that utilizes small C-terminal peptide tags and heterologous adaptor proteins from E. coli and C. crescentus to deliver tagged proteins to the host B. subtilis protease where they are rapidly degraded (Fig. 3). We are currently using this system to study several proteins with unknown function. Given the conservation of bacterial proteases, this protein degradation system has the potential to be modified for use in other bacteria.
Griffith, K.L., M.M. Fitzpatrick, E.F. Keen, and R.E. Wolf, Jr. 2009. Two functions of the C-terminal domain of Escherichia coli Rob: mediating "sequestration-dispersal" as a novel off-on switch for regulating Rob's activity as a transcriptional activator and preventing degradation of Rob by Lon protease. J. Mol. Biol. 388: 415-30.
Griffith, K.L. and A.D. Grossman. 2008. Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP. Mol. Micro. 70: 1012-1025.
Griffith, K.L. and A.D. Grossman. 2008. A degenerate tripartite DNA-binding site required for activation of ComA-dependent quorum response gene expression in Bacillus subtilis. J. Mol. Biol. 381: 261-75.
Griffith, K.L., S.M. Becker, and R.E. Wolf, Jr. 2005. Characterization of TetD as a transcriptional activator of a subset of genes of the Escherichia coli SoxS/MarA/Rob regulon. Mol. Micro. 56: 1103-1117.
Griffith, K.L., and R.E. Wolf, Jr. 2004. Genetic evidence for "Pre-recruitment" as the mechanism of transcription activation by SoxS of Escherichia coli: the dominance of DNA binding mutations of SoxS. J. Mol. Biol. 344: 1-10.
Griffith, K.L.*, I.M. Shah*, and R.E. Wolf, Jr. 2004. Proteolytic degradation of Escherichia coli transcription activators SoxS and MarA as the mechanism for reversing the induction of the superoxide (SoxRS) and multiple antibiotic resistance (Mar) regulons. Mol. Micro. 51: 1801-1816. * These authors contributed equally.
Griffith, K.L. and R.E. Wolf, Jr. 2002. A comprehensive alanine scanning mutagenesis of the Escherichia coli transcriptional activator SoxS: identifying amino acids important for DNA binding and transcription activation. J. Mol. Biol. 322: 237-257.
Griffith, K.L.*, I.M. Shah*, T.E. Myers, M.C. O'Neill, and R.E. Wolf, Jr. 2002. Evidence for "Pre-recruitment" as a new mechanism of transcription activation in Escherichia coli: the large excess of SoxS binding sites per cell relative to the number of SoxS molecules per cell. Biochem. Biophys. Res. Comm. 291: 979-986. * These authors contributed equally.
Griffith, K.L. and R.E. Wolf, Jr. 2002. Measuring ß-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assay in 96-well arrays. Biochem. Biophys. Res. Comm. 290: 397-402.
Griffith, K.L. and R.E. Wolf, Jr. 2001. Systematic mutagenesis of the DNA binding sites for SoxS in the Escherichia coli zwf and fpr promoters: identifying nucleotides required for DNA binding and transcription activation. Mol. Micro. 40: 1141-1154.
Wood, T.I., K.L. Griffith, W.P. Fawcett, K-W Jair, T.D. Schneider, and R.E. Wolf, Jr. 1999. Interdependence of the position and orientation of SoxS binding sites in the transcriptional activation of the class I subset of Escherichia coli superoxide-inducible promoters. Mol. Micro. 34: 414-430.
Robinson, P.R., K.L. Griffith, J.M. Gross, and M.C. O'Neill. 1999. A back-propagation neural network predicts absorption maxima of chimeric human red/green visual pigments. Vision Research. 39: 1707-1712.