Alan Grossman

Bacillus subtilisWe use a combination of genetic, molecular, physiological, biochemical, cell-biological, and genomic approaches to study how cells of Bacillus subtilis sense internal and external conditions and control basic cellular processes in response to these conditions. We are interested in defining signals that are sensed and characterizing signal transduction and the regulatory outputs. We are studying mechanisms controlling cell-cell signaling, gene expression, development, DNA replication, chromosome dynamics, and horizontal gene transfer.

Cell-cell signaling and quorum responses

Many organisms use cell-cell signaling to monitor population density, crowding, and colonization to regulate a range of complex processes, including development, pathogenesis, growth, mating, and transformation. In microbes, this is typically done with secreted signaling molecules and dedicated response pathways. The ability to sense and respond to high population density is a type of cell-cell signaling often referred to as quorum sensing. We have characterized several types of regulatory responses that are controlled by secreted peptides in B. subtilis. Some of these responses are conserved and used generally by gram positive organisms to modulate gene expression, development, pathogenesis, and horizontal gene transfer.

In B. subtilis, one of the major pathways for quorum sensing involves activation of the transcription factor ComA, a response regulator that is active when phosphorylated. The activity of ComA is modulated by at least two different types of peptide signaling molecules that accumulate in culture supernatants as cells grow to high population density. We used DNA microarrays to identify genes controlled by ComA and the peptide signaling pathways, and have characterized the DNA binding site for ComA. We are interested in the regulatory networks affected by combinations of transcription factors, including ComA.

We identified several secreted peptides that influence gene expression and development in B. subtilis. One type of peptide functions outside the cell by interacting with the receptor kinase (ComP) thereby stimulating the activation of the transcription factor ComA. This mechanism has been found to be generally conserved in gram positive bacteria. We found that in B. subtilis, this pathway stimulates the development of genetic competence (the ability to bind and take up exogenous DNA), and stimulates expression of many genes involved in a general response to high population density (quorum sensing).

The second type of peptide signaling pathway that we have characterized involves a family of pentapeptides that are exported, accumulate outside the cell, and then are imported through an oligopeptide permease. Once inside the cell, these peptides interact with a cytoplasmic receptor (called Rap). The receptor normally inhibits the activity of a target transcription factor (often indirectly). When bound to ligand, the Rap protein is antagonized, typically allowing the target transcription factor to be active. We found that one of the pentapeptides stimulates a general quorum response (including the development of genetic competence) and also stimulates sporulation. Another peptide of this type inhibits activation of the integrative and conjugative element ICEBs1.

Cell-cell signaling and horizontal gene transfer

We have found that cell-cell signaling is used to regulate horizontal transfer of the integrative and conjugative element ICEBs1, a conjugative transposon found in the B. subtilis genome. These types of elements are widespread in the microbial world and contribute to horizontal gene transfer, evolution, virulence, and the spread of antibiotic resistance. ICEBs1 is regulated by population density and cell-cell signaling in two ways. 1) At high population density, in the presence of potential mating partners, the element is stimulated to excise from the chromosome and can then transfer to potential recipients. 2) However, if the potential recipients already contain a copy of the element, then excision of the element is inhibited and there is little or no transfer to the potential recipients that already contain the element. We found that the secreted pentapeptide, a product of phrI, that regulates this "recognition of self" is encoded in the element. In the absence of this peptide, as cells grow to high population density, the expression of a gene within the element, rapI, is activated, causing inactivation of the transcriptional repressor of the element, thereby causing increased transcription of ICEBs1 genes and excision and potential mating. The production and accumulation of the PhrI pentapeptide inhibits the function of RapI, thereby preventing ICEBs1 gene expression and preventing excision. The presence of homologous regulatory genes in other mobile elements indicates that the general mechanism is likely to be conserved.

We are interested in the mechanisms used to control horizontal gene transfer. We are characterizing the mechanism of integration and excision, the attachment sites, the origin of transfer, and the conjugation machinery of ICEBs1. We have found that ICEBs1 can transfer to several different types of gram positive bacteria and the integration site is widely conserved. We are using this information to develop ICEBs1 for use in organisms that are otherwise difficult to manipulate genetically.

Integration of physiological signals and stochastic control of development

Gene expression and development are often affected by multiple physiological processes and signals. The mechanisms of signal integration have been active areas of research in many fields. The initiation of sporulation in B. subtilis is activated by nutrient depletion and high population density, inhibited by DNA damage, and requires DNA replication and a functional TCA cycle. Our work demonstrated that all of these conditions and the cognate signals function to regulate the activation (phosphorylation) of Spo0A, the key transcription factor required for the initiation of sporulation. Sporulation starts only when all signals are appropriate for development.

We analyzed gene expression in single cells and showed that activation of the transcription factor is a stochastic process and cells must achieve a threshold level to fully initiate developmental gene expression. If an individual cell falls short of that threshold, then developmental gene expression is essentially off.

Control of transcription, cell division, and development in response to alterations in replication

We have characterized several aspects of transcriptional regulation in response to perturbations in DNA replication. Perturbations in replication inhibit the initiation of sporulation in B. subtilis and also inhibit cell division in many types of bacteria.

We characterized the transcriptional response to perturbations in DNA replication caused by DNA damage and disruptions to the replication complex. Upon inhibition of replication (either initiation or elongation), expression of many genes changes. A significant part of this response is mediated by the conserved replication initiation protein and transcription factor DnaA. DnaA represses expression of some genes and activates others. This is independent of the well characterized RecA-mediated SOS response. We found that the DnaA-mediated response activates an inhibitor of sporulation, and also represses transcription of an essential cell division gene (whose product is unstable), thereby inhibiting sporulation and cell division. Furthermore, many of the genes apparently controlled directly by DnaA are conserved and have putative DnaA binding sites in their regulatory regions in other bacteria, indicating that this response is widespread.

Spatial organization of the chromosome and DNA replication in vivo

Cells invest significant resources in the faithful replication and partitioning of chromosomes. Chromosome duplication involves assembly of a large complex of proteins at an origin (at 0° on the B. subtilis circular chromosome) and processive replication in both directions away from the origin. Our work studying replication in vivo indicated that during replication, the DNA moves to the DNA replication machinery (the replisome), gets duplicated, and then moves away. This is in contrast to models in which the replication machinery moves along the DNA.

To gain insight into the spatial and temporal organization of the replication cycle, we visualized replication origins and the replication machinery (replisomes) inside live cells. Our analysis indicates that the location of the origin at the time of replication initiation establishes the position of the replisome. Also, it appears that sister replication forks are not intimately associated with each other throughout the replication cycle. We also found that the subcelluar location of the 0° region of the chromosome does not require the presence of an active origin of replication, nor does it require sequences within the origin itself. We also found that this positioning is independent of the replication initiation protein DnaA. This work indicates that there as yet uncharacterized factors involved in chromosome positioning and orientation.

Proteins involved in chromosome partitioning and DNA organization

Several proteins are known to be involved chromosome organization and partitioning. Soj (ParA) and Spo0J (ParB) of B. subtilis belong to a family of conserved proteins required for efficient plasmid and chromosome partitioning in many bacterial species. We found that soj null mutations cause a defect in chromosome partitioning when combined with a null mutation in smc. smc encodes the structural maintenance of chromosome protein that is highly conserved and involved in chromosome compaction and partitioning. We also found that soj null mutations caused increased initiation of DNA replication and were partly defective in separation of sister copies of the region around the origin of replication. Null mutations in spo0J are also synthetic with smc and cause defects in separation of sister origin regions. soj and spo0J also contribute to regulation of replication initiation, although the mechanism is not yet known.

Regulation of replication

Just as cells sense internal and external conditions and modulate transcription, they also modulate replication, both initiation and elongation of replication. Sudden decreases in nutrient availability cause a relatively rapid inhibition in replication elongation. We monitored the progression of replication forks using DNA microarrays and found that the inhibition in elongation of replication caused by nutritional downshift is due to accumulation of the signaling molecules pppGpp and ppGpp. We showed that these signaling nucleotides inhibit primase, an essential component of the replication machinery. This regulation is likely to prevent DNA damage or mutagenesis that could occur during replication in the absence of sufficient dNTPs and provides a link between nutrient availability and the preservation of genome integrity, likely forming part of a general bacterial stress response to enhance survival in adverse conditions.

Bose*, B., Auchtung*, J.M., Lee, C.A., and Grossman, A.D. *co-1st authors; A conserved anti-repressor controls horizontal gene transfer by proteolysis.  Mol Microbiol in press (2008).

Griffith, KL, & Grossman, AD (2008) A Degenerate Tripartite DNA-Binding Site Required for Activation of ComA-Dependent Quorum Response Gene Expression in Bacillus subtilis. J Mol Biol. 381:261-275.

Simmons, LA*, Davies, BW, Grossman, AD, & Walker, GC (2008) ß-Clamp directs localization of mismatch repair in Bacillus subtilis. Mol Cell 29, 291-301. *co-advised postdoc with G. Walker

Berkmen, MB, & Grossman, AD (2007) Subcellular positioning of the origin region of the Bacillus subtilis chromosome is independent of sequences within oriC, the site of replication initiation, and the replication initiator DnaA. Mol Microbiol 63, 150-165.

Lee, CA, Auchtung, JM, Monson, RE, & Grossman, AD (2007) Identification and characterization of int (integrase), xis (excisionase) and chromosomal attachment sites of the integrative and conjugative element ICEBs1 of Bacillus subtilis. Mol Microbiol 66, 1356-1369.

Lee, CA, & Grossman, AD (2007) Identification of the origin of transfer (oriT) and DNA relaxase required for conjugation of the integrative and conjugative element ICEBs1 of Bacillus subtilis.  J Bacteriol 189, 7254-7261.

Simmons, LA*, Grossman, AD, & Walker, GC (2007) Replication is required for the RecA localization response to DNA damage in Bacillus subtilis. Proc Natl Acad Sci U S A 104, 1360-1365. *co-advised postdoc with G. Walker

Wang, JD, Berkmen, MB, & Grossman, AD (2007) Genome-wide coorientation of replication and transcription reduces adverse effects on replication in Bacillus subtilis. Proc Natl Acad Sci U S A 104, 5608-5613.

Auchtung, JM, Lee, CA, & Grossman, AD (2006) Modulation of the ComA-dependent quorum response in Bacillus subtilis by multiple Rap proteins and Phr peptides. J Bacteriol 188, 5273-5285.