Jonathan Dworkin

Tunability and Noise Dependence in Differentiation Dynamics

The dynamic process of differentiation depends on the architecture, quantitative parameters, and noise of underlying genetic circuits. However, it remains unclear how these elements combine to control cellular behavior. We analyzed the probabilistic and transient differentiation of Bacillus subtilis cells into the state of competence. A few key parameters independently tuned the frequency of initiation and the duration of competence episodes and allowed the circuit to access different dynamic regimes, including oscillation. Altering circuit architecture showed that the duration of competence events can be made more precise. We used an experimental method to reduce global cellular noise and showed that noise levels are correlated with frequency of differentiation events. Together, the data reveal a noise-dependent circuit that is remarkably resilient and tunable in terms of its dynamic behavior.

A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments.

Bacteria can respond to adverse environmental conditions by drastically reducing or even ceasing metabolic activity. They must then determine that conditions have improved before exiting dormancy, and one indication of such a change is the growth of other bacteria in the local environment. Growing bacteria release muropeptide fragments of the cell wall into the extracellular milieu, and we report here that these muropeptides are potent germinants of dormant Bacillus subtilis spores. The ability of a muropeptide to act as a germinant is determined by the identity of a single amino acid. A well-conserved, eukaryotic-like Ser/Thr membrane kinase containing an extracellular domain capable of binding peptidoglycan is necessary for this response, and a small molecule that stimulates related eukaryotic kinases is sufficient to induce germination. Another small molecule, staurosporine, that inhibits related eukaryotic kinases blocks muropeptide-dependent germination. Thus, in contrast to traditional antimicrobials that inhibit metabolically active cells, staurosporine acts by blocking germination of dormant spores.

Eukaryote-Like Serine/Threonine Kinases and Phosphatases in Bacteria

SUMMARY Genomic studies have revealed the presence of Ser/Thr kinases and phosphatases in many bacterial species, although their physiological roles have largely been unclear. Here we review bacterial Ser/Thr kinases (eSTKs) that show homology in their catalytic domains to eukaryotic Ser/Thr kinases and their partner phosphatases (eSTPs) that are homologous to eukaryotic phosphatases. We first discuss insights into the enzymatic mechanism of eSTK activation derived from structural studies on both the ligand-binding and catalytic domains. We then turn our attention to the identified substrates of eSTKs and eSTPs for a number of species and to the implications of these findings for understanding their physiological roles in these organisms.

Does RNA polymerase help drive chromosome segregation in bacteria

In contrast to eukaryotic cells, bacteria segregate their chromosomes without a conspicuous mitotic apparatus. Replication of bacterial chromosomes initiates bidirectionally from a single site (oriC), and visualization of the region of the chromosome containing oriC in living cells reveals that origins rapidly move apart toward opposite poles of the cell during the cell cycle. The motor that drives this poleward movement is unknown. An attractive candidate is RNA polymerase, which is a powerful and abundant molecular motor. If, as has been suggested for other macromolecular complexes, the movement of RNA polymerase is restricted in the cell, then transcription would translocate the DNA template, thereby providing the motive force to separate replicating chromosomes. A coordinated effect of many transcribing RNA polymerases could result from the widely conserved global bias of gene orientation away from oriC. By using fluorescence microscopy of living Bacillus subtilis cells, we demonstrate that an inhibitor of RNA polymerase acts to inhibit separation of newly duplicated DNAs near the origin of replication. We propose that the force exerted by RNA polymerase contributes to chromosome movement in bacteria, and that this force, coupled with the biased orientation of transcription units, helps to drive chromosome segregation.

Exit from dormancy in microbial organisms

Bacteria can exist in metabolically inactive states that allow them to survive conditions that are not conducive for growth. Such dormant cells may sense when conditions have improved and re-initiate growth, lest they be outcompeted by their neighbours. Growing bacteria turn over and release large quantities of their cell walls into the environment. Drawing from recent work on the germination of Bacillus subtilis spores, we propose that many microorganisms exit dormancy in response to cell wall muropeptides.

Partial penetrance facilitates developmental evolution in bacteria

Development normally occurs similarly in all individuals within an isogenic population, but mutations often affect the fates of individual organisms differently. This phenomenon, known as partial penetrance, has been observed in diverse developmental systems. However, it remains unclear how the underlying genetic network specifies the set of possible alternative fates and how the relative frequencies of these fates evolve. Here we identify a stochastic cell fate determination process that operates in Bacillus subtilis sporulation mutants and show how it allows genetic control of the penetrance of multiple fates. Mutations in an intercompartmental signalling process generate a set of discrete alternative fates not observed in wild-type cells, including rare formation of two viable ‘twin’ spores, rather than one within a single cell. By genetically modulating chromosome replication and septation, we can systematically tune the penetrance of each mutant fate. Furthermore, signalling and replication perturbations synergize to significantly increase the penetrance of twin sporulation. These results suggest a potential pathway for developmental evolution between monosporulation and twin sporulation through states of intermediate twin penetrance. Furthermore, time-lapse microscopy of twin sporulation in wild-type Clostridium oceanicum shows a strong resemblance to twin sporulation in these B. subtilis mutants. Together the results suggest that noise can facilitate developmental evolution by enabling the initial expression of discrete morphological traits at low penetrance, and allowing their stabilization by gradual adjustment of genetic parameters.

The PspA protein of Escherichia coli is a negative regulator of sigma(54)-dependent transcription.

In Eubacteria, expression of genes transcribed by an RNA polymerase holoenzyme containing the alternate sigma factor sigma(54) is positively regulated by proteins belonging to the family of enhancer-binding proteins (EBPs). These proteins bind to upstream activation sequences and are required for the initiation of transcription at the sigma(54)-dependent promoters. They are typically inactive until modified in their N-terminal regulatory domain either by specific phosphorylation or by the binding of a small effector molecule. EBPs lacking this domain, such as the PspF activator of the sigma(54)-dependent pspA promoter, are constitutively active. We describe here the in vivo and in vitro properties of the PspA protein of Escherichia coli, which negatively regulates expression of the pspA promoter without binding DNA directly.

Developmental Commitment in a Bacterium

We investigated developmental commitment during sporulation in Bacillus subtilis. Sporulation is initiated by nutrient limitation and involves division of the developing cell into two progeny, the forespore and the mother cell, with different fates. Differentiation becomes irreversible following division when neither the forespore nor the mother cell can resume growth when provided with nutrients. We show that commitment is governed by the transcription factors sigma(F) and sigma(E), which are activated in the forespore and the mother cell, respectively. We further show that commitment involves spoIIQ, which is under the control of sigma(F), and spoIIP, which is under the control of both sigma(F) and sigma(E). In the presence of nutrients, the forespore can exhibit rodlike, longitudinal growth when SpoIIQ and SpoIIP are absent, whereas the mother cell can do so when SpoIIP alone is absent. Thus, developmental commitment of this single-celled organism, like that of the cells of complex, multicellular organisms, ensures that differentiation is maintained despite changes in the extracellular milieu.

PSICIC: noise and asymmetry in bacterial division revealed by computational image analysis at sub-pixel resolution.

Live-cell imaging by light microscopy has demonstrated that all cells are spatially and temporally organized. Quantitative, computational image analysis is an important part of cellular imaging, providing both enriched information about individual cell properties and the ability to analyze large datasets. However, such studies are often limited by the small size and variable shape of objects of interest. Here, we address two outstanding problems in bacterial cell division by developing a generally applicable, standardized, and modular software suite termed Projected System of Internal Coordinates from Interpolated Contours (PSICIC) that solves common problems in image quantitation. PSICIC implements interpolated-contour analysis for accurate and precise determination of cell borders and automatically generates internal coordinate systems that are superimposable regardless of cell geometry. We have used PSICIC to establish that the cell-fate determinant, SpoIIE, is asymmetrically localized during Bacillus subtilis sporulation, thereby demonstrating the ability of PSICIC to discern protein localization features at sub-pixel scales. We also used PSICIC to examine the accuracy of cell division in Esherichia coli and found a new role for the Min system in regulating division-site placement throughout the cell length, but only prior to the initiation of cell constriction. These results extend our understanding of the regulation of both asymmetry and accuracy in bacterial division while demonstrating the general applicability of PSICIC as a computational approach for quantitative, high-throughput analysis of cellular images.

Differential Gene Expression Governed by Chromosomal Spatial Asymmetry

The activity of the transcription factor sigmaF is confined to one (the forespore) of two cells created by asymmetric division during sporulation in B. subtilis. We show that sigmaF activation is partly governed by the position of the gene for the unstable anti-sigmaF factor SpoIIAB. Because cytokinesis precedes chromosome segregation, most of the chromosome is translocated into the forespore after division. We hypothesize that because spoIIAB enters the forespore late, SpoIIAB lost to proteolysis is temporarily not replenished. Thus, chromosome asymmetry would be translated into the asymmetric distribution of SpoIIAB. Supporting this idea, transposition of spoIIAB to sites present in the forespore at the time of division impaired sporulation when a second pathway that participates in sigmaF activation was disabled.