Bacterial cells are pretty small. In fact, a typical cell is about 10,000 times smaller than an aspirin. Despite this “smallness,” bacteria are genetically wired to make dynamic, sophisticated decisions that regulate cellular processes in both time and space.
How do bacteria encode positional information?
In bacteria, many molecules appear to localize through diffusion-capture mechanisms. At the same time, certain patterns of localization also suggest that the bacterial cell has an underlying architecture that governs the distribution of many macromolecules. We would like to understand how bacterial cells encode spatial information and what the functional implications of this cellular organization are for physiology. Reciprocally, we are investigating the hypothesis that metabolism itself may be the primary determinant “shaping” cellular architecture.
How do bacteria use cellular architecture to coordinate key biological processes?
While we are still learning how bacteria encode positional information, our current studies focus on the synthesis, organization, and interplay between the two largest structures in the cell: the cell envelope and the nucleoid. How are these structures used to organize cellular processes? To address this question, we are currently investigating the following questions:
- How does the nucleoid contribute to the positioning of cell division?
- How is DNA replication regulated by associations with the cell envelope?
How do bacteria make developmental decisions?
Bacteria are capable of many lifestyle choices. As an example, wild-type Bacillus can swim or form chains, generate a biofilm or undergo sporulation. We are interested in investigating the molecular basis of such decisions. Ultimately, our goal is to understand how bacteria potentiate environmental flux and developmental decisions into the requisite changes in cell physiology required for survival.
Cell DivisionClose X
One of the earliest visible steps in Bacillus spore formation is an asymmetric cell division that divides the cell into a large and a small compartment. This polar division is of special interest to us because it breaks two rules of exponential growth division. First, the septation occurs on top of the nucleoid, an event that is normally inhibited to prevent chromosome breakage. Second, the division occurs near the cell quarter position, a place where cell division proteins normally cannot assemble due to inhibition by a regulatory system called Min. We are using the “weird” case of the sporulation division in order to investigate the role of the chromosome in division site selection. More specifically, we are studying how a DNA-binding protein called RefZ coordinates chromosome organization with cell division during sporulation.
DNA ReplicationClose X
During entry into sporulation, B. subtilis ensures that each cell has two copies of the chromosome, one that is pumped and packaged into the developing spore and another that is retained in the nascent “mother” cell. We are studying how Bacillus inhibits new rounds of DNA replication during sporulation by expressing SirA, an inhibitor of the highly conserved DNA replication initiator protein DnaA.
In Bacillus, motility and cell chaining are inversely correlated. Motile cells become unchained, presumably to aid in cell dispersal through swimming. During exponential growth, our lab strain (PY79) is predominantly chained and the decision to switch to the single-cell, motile mode is known to regulated by a sigma factor, SigD. We recently discovered that SigD levels and activity are regulated by intracellular GTP levels through the GTP-sensing protein CodY.
Gene DiscoveryClose X
Post-genomic biology approaches have the potential to transform biology and medicine, but are limited by the fact that we are often unable to link changes in genes or gene products to actual gene function. This is because, even in well-studied organisms like Escherichia coli, 30-50% of ORFs are still experimentally uncharacterized.
To attack the uncharacterized gene problem, we devised a novel approach to systematically identify and characterize new genes involved in cellular organization based on the methodology that enabled us to ascribe functions to both SirA and RefZ. The approach is based on the premise that artificially perturbing normal gene expression (especially by changing expression context) is a highly effective way to obtain distinctive phenotypes that can then be harnessed to generate hypotheses regarding gene function.
Our lab generated the BAGEL (Bacillus Artificial Gene Expression Library), an ordered library consisting of ~1000 uncharacterized genes from B. subtilis, each under the control of an inducible promoter (STAGE I). We are screening the library for growth and morphological phenotypes (STAGE II) and have thus far identified ~20 new genes whose misexpression leads to changes in cell length (shorter or longer cells), cell width, cell shape, nucleoid morphology, or DNA replication status. We are currently performing suppressor selections with a subset of high priority candidates to identify the physical and/or genetic targets for the uncharacterized gene products (STAGE III). This data will be leveraged with the observed phenotypes and known regulatory information to inform testable hypotheses for each gene’s function (STAGE IV) and to assign functions (STAGE V).
We consider the dissemination of the BAGEL as a community research tool a high priority. Potential uses of the BAGEL include:
- Other phenotypic screens (eg: identification of genes that promote biofilm formation)
- Identifying regulators by introducing the pooled library into strains harboring regulatory region reporter constructs
- Generating depletion strains
- Analyzing the cDNA profiles of putative transcriptional regulators
- Screening for complementation of gene deletion phenotypes
With funding, we would like to generate a library that encompasses the entire Bacillus ORFeome.