We use the Gram positive bacterium Bacillus subtilis to study basic cellular processes. B. subtilis is one of the most widely studied and best understood Gram positive bacteria and is remarkably easy to work with. Many cellular processes in B. subtilis are generally representative of and can serve as models for those in several Gram positive pathogens, including Bacillus anthracis, Clostridium species, Enterococcus faecalis, Staphylococcus aureus, and Streptococcus pneumonia. We expect that many of our findings with B. subtilis will be broadly relevant for other bacteria.
Integrative and conjugative elements
Horizontal gene transfer is a driving force in microbial evolution. It is largely mediated by mobile genetic elements, including viruses, conjugative plasmids, and integrative and conjugative elements (ICEs). ICEs contribute to the spread of genes for antibiotic resistances, pathogenesis, symbiosis, metabolism, and more, and are found in all major phylogenetic clades of bacteria. ICEs reside integrated in a host (donor) genome and encode a type IV secretion system that mediates their transfer from donors into new hosts (recipients). When activated, ICE gene expression is induced, the elements excise from the genome and circularize, and can transfer and integrate into a recipient’s chromosome to generate a transconjugant.
Studies of ICEs, especially in Gram positive bacteria, have been limited by low frequencies of element activation and relative difficulty working with many of the common host organisms. However, we discovered the integrative and conjugative element of B. subtilis (ICEBs1), and developed experimental methods to induce ICEBs1 in >90% of cells in a population and achieve relatively high conjugation frequencies. By studying ICEBs1, we can address fundamental issues related to ICE biology, including problems that were previously intractable. We now know a fair amount about its regulation, replication, and genes and sites needed for its normal function. Current areas of interest include: host genes needed for ICE function; interactions between donor and recipient cells; and interactions between ICEs and other mobile genetic elements.
Based on homologies and the conserved lifecycle of ICEs, insights gained from studying ICEBs1 and its host are likely to be generally relevant to many other mobile genetic elements and their hosts. We are now in a position to determine if some of the functions of ICEBs1 apply to other ICEs. For example, Tn916 is the most widespread ICE, has a very broad host range, and is involved in the spread of tetracycline resistance. We are using both ICEBs1 and Tn916 to learn about basic properties of these elements and to engineer them for use as genetic tools in various organisms. Our findings should be relevant to the transfer of genes between bacteria growing in many different environments, including the human microbiome.
Chromosome dynamics and gene expression
Cells have multiple mechanisms for regulating the initiation of replication. Cells also have regulatory responses to perturbations in replication, often called checkpoints, which control gene expression and cell cycle progression. One such pathway is the well-characterized RecA-dependent SOS response to DNA damage and stalled replication forks that de-represses many host genes and lysogenic phages. We defined a RecA-independent response to replication stress that is largely mediated by DnaA, the replication initiator. This connection between replication stress and DnaA led to our more general interest in DnaA as a transcription factor and its role in regulating gene expression in response to replication stress. Coupling gene expression and cell cycle progression to chromosome replication and integrity helps prevent the generation of cells with defective chromosomes. The coordination of genome duplication with cell cycle progression is important for cellular differentiation and preventing uncontrolled cell growth.
DnaA is the replication initiator in virtually all bacteria. It is a member of the AAA+ family of ATPases. It is generally active in the ATP-bound form (DnaA-ATP) and is converted to the inactive DnaA-ADP by nucleotide hydrolysis. Newly synthesized DnaA is predominantly in the ATP-bound form due to the higher concentration of ATP relative to ADP in growing cells. DnaA binds cooperatively to several sites in oriC where it causes melting of DNA (open complex formation) at the DNA unwinding element (DUE). This serves as a platform for loading the replicative helicase and then the rest of the replication machinery. Despite the conservation of DnaA, the mechanisms that regulate its activity are not as broadly conserved. In E. coli, replication initiation appears to be controlled primarily, and perhaps solely, by modulating the nucleotide-bound state of DnaA and accessibility of DnaA to oriC. Many of the regulatory components critical for control in E. coli (e.g., Dam methylase, SeqA, Hda) are limited to proteobacteria. Regulation of DnaA in B. subtilis is quite different from that in E. coli. The emerging view is that most regulation is by proteins that affect the ability of DnaA to bind cooperatively to and form a helix on DNA.
It has long been known that DnaA functions as a transcription factor to repress transcription of its own gene. It is now clear that DnaA binds to several genomic regions outside of oriC and that binding at some of these regions affects gene expression. Furthermore, the activity of DnaA as a transcription factor increases when replication decreases. We are currently studying mechanisms mediating this regulation of DnaA. We use a variety of approaches and methodologies, both in vivo and in vitro, to characterize: the control of replication initiation; regulators of the replication initiator and transcription factor DnaA; and genes controlled in response to perturbations in replication. Understanding these processes in B. subtilis will provide insights regarding similar processes and homologous proteins in a wide variety of organisms, including many Gram positive pathogens. Microbial pathogenesis often depends on normal bacterial replication and growth in the host.