Person:

Norman, Thomas Maxwell

A model system for investigating how developmental regulatory networks determine cell fate is spore formation in Bacillus subtilis. The master regulator for sporulation is Spo0A, which is activated by phosphorylation via a phosphorelay that is subject to three positive feedback loops. The ultimate decision to sporulate is, however, stochastic in that only a portion of the population sporulates even under optimal conditions. It was previously assumed that activation of Spo0A and hence entry into sporulation is subject to a bistable switch mediated by one or more feedback loops. Here we reinvestigate the basis for bimodality in sporulation. We show that none of the feedback loops is rate limiting for the synthesis and phosphorylation of Spo0A. Instead, the loops ensure a just-in-time supply of relay components for rising levels of phosphorylated Spo0A, with phosphate flux through the relay being limiting for Spo0A activation and sporulation. In addition, genes under Spo0A control did not exhibit a bimodal pattern of expression as expected for a bistable switch. In contrast, we observed a highly heterogeneous pattern of Spo0A activation that increased in a nonlinear manner with time. We present a computational model for the nonlinear increase and propose that the phosphorelay is a noise generator and that only cells that attain a threshold level of phosphorylated Spo0A sporulate.

Many cell types stochastically switch phenotypes under some conditions, so that genetically identical sister cells may behave quite differently in a common environment. This non-genetic variability likely arises from noise in gene expression, which can be co-opted to allow random fate determination. This thesis examines both phenomena from experimental and theoretical perspectives, starting with a phenotypic switch. Cells of Bacillus subtilis grow either as individual, motile cells, or as connected groups of sessile cells called chains. We constructed an array of microfluidic channels in which we could capture and observe single cells in a constant environment over hundreds of generations of growth. These conditions allow unperturbed observation of decision-making driven only by factors internal to the cell. We observe that switching is asymmetric: transitions from motility to chaining occur with constant probability (memorylessly), but the reverse transition is tightly timed (exhibits memory). These properties are explained by dissecting the genetic circuit underlying switching, which can be quantitatively separated into components responsible for initiation and maintenance of the state. We propose that memory enables transgenerational cooperation between a cell founding a biofilm and its progeny, and that a stochastic sequestration mechanism is the source of random switching. Next, we introduce an exact framework for analyzing noise in gene networks that phrases results in terms of compounded parameters with simple interpretations. We uncover a basic identity that relates fluctuations in the production and degradation rates of one component to those of any other component within the cell. Since the result is exact, it applies to whole classes of gene networks. We identify basic constraints on the ability of negative feedback to suppress noise, and show that suppressing noise in one species generally requires introducing it elsewhere. When applied to the most common model of gene expression, the identity reveals a simple connection between the statistics of proteins and their cognate mRNAs. We reanalyze a recent experimental study of stochastic gene expression and show that the data are inconsistent with this prediction. Thus in contrast to early studies of single genes, there is currently discord between models and measurements of stochastic gene expression.

Genetically identical cells sharing an environment can display markedly different phenotypes. It is often unclear how much of this variation derives from chance, external signals, or attempts by individual cells to exert autonomous phenotypic programs. By observing thousands of cells for hundreds of consecutive generations under constant conditions, we dissect the stochastic decision between a solitary, motile state and a chained, sessile state in Bacillus subtilis. The motile state is memoryless, exhibiting no autonomous control over the time spent in the state, whereas chaining is tightly timed. Timing enforces coordination among related cells in the multicellular state. Further, we show that the three-protein regulatory circuit governing the decision is modular, as initiation and maintenance of chaining are genetically separable functions. As stimulation of the same initiating pathway triggers biofilm formation, we argue that autonomous timing allows a trial commitment to multicellularity that external signals could extend.