SUMMARY In cells, specific regulators often compete for limited amounts of a core enzymatic resource. It is typically assumed that competition leads to partitioning of core enzyme molecules among regulators at constant levels. Alternatively, however, different regulatory species could time share, or take turns utilizing, the core resource. Using quantitative time-lapse microscopy, we analyzed sigma factor activity dynamics, and their competition for RNA polymerase, in individual Bacillus subtilis cells under energy stress. Multiple alternative sigma factors were activated in ~1-hr pulses in stochastic and repetitive fashion. Pairwise analysis revealed that two sigma factors rarely pulse simultaneously and that some pairs are anti-correlated, indicating that RNAP utilization alternates among different sigma factors. Mathematical modeling revealed how stochastic time-sharing dynamics can emerge from pulse-generating sigma factor regulatory circuits actively competing for RNAP. Time sharing provides a mechanism for cells to dynamically control the distribution of cell states within a population. Since core molecular components are limiting in many other systems, time sharing may represent a general mode of regulation.

Gene regulatory circuits can receive multiple simultaneous inputs, which can enter the system through different locations. It is thus necessary to establish how these genetic circuits integrate multiple inputs as a function of their relative entry points. Here, we use the dynamic circuit regulating competence for DNA uptake in Bacillus subtilis as a model system to investigate this issue. Specifically, we map the response of single cells in vivo to a combination of (i) a chemical signal controlling the constitutive expression of key competence genes, and (ii) a genetic perturbation in the form of copy number variation of one of these genes, which mimics the level of stress signals sensed by the bacteria. Quantitative timelapse fluorescence microscopy shows that a variety of dynamical behaviors can be reached by the combination of the two inputs. Additionally, the integration depends strongly on the relative locations where the two perturbations enter the circuit. Specifically, when the two inputs act upon different circuit elements, their integration generates novel dynamical behavior, whereas inputs affecting the same element do not. An in silico bidimensional bifurcation analysis of a mathematical model of the circuit offers good quantitative agreement with the experimental observations, and sheds light on the dynamical mechanisms leading to the different integrated responses exhibited by the gene regulatory circuit.signal integration | genetic competence | excitability | bistability | genetic oscillations C ells are usually subject to multiple simultaneous sources of biochemical and physical signals, which they must integrate to respond adequately to their external environment and internal conditions. Previous efforts addressed at understanding signal integration in gene regulation have mainly concentrated on mapping the combinatorial response of single bacterial promoters to multiple transcription factors (1-5), and on quantifying the combined effect of bacterial cell-cell signaling molecules acting upon a shared phosphorelay pathway (6-8). However, in many instances different inputs operate in a distributed manner, acting upon distinct nodes (genes or proteins) of the gene regulation network. In those cases, it is the network itself, and not a particular promoter or phosphorylation reaction, that has to integrate the information at the system's level. It thus becomes necessary to understand how the integrated response of gene regulatory networks depends on the specific entry points of the inputs.Some studies have recently examined the population-level response of complex signaling pathways to ligand combinations (9-12). However, a systematic in vivo study at the single-cell level of the integration of distributed inputs by gene circuits is still lacking. It also remains unclear how these integration maps are related to the underlying regulatory circuitry. Here, we address these issues by means of a combination of time-lapse fluorescence microscopy and mathematical modeling of multiple simultaneous perturbat...

25A diverse set of prophage-mediated mechanisms protecting bacterial hosts from 26 infection has been recently uncovered within Cluster N mycobacteriophages. In that 27 context, we unveil a novel defense mechanism in Cluster N prophage Butters. By using 28 bioinformatics analyses, phage plating efficiency experiments, microscopy, and 29 immunoprecipitation assays, we show that Butters genes located in the central region of 30 the genome play a key role in the defense against heterotypic viral attack. Our study 31 suggests that a two component system articulated by interactions between protein 32 products of genes 30 and 31 confers defense against heterotypic phage infection by 33 PurpleHaze or Alma, but is insufficient to confer defense against attack by the 34 heterotypic phage Island3. Therefore, based on heterotypic phage plating efficiencies 35 on the Butters lysogen, additional prophage genes required for defense are implicated. 36 37 IMPORTANCE 38 Many sequenced bacterial genomes including pathogenic bacteria contain prophages. 39 Some prophages encode defense systems that protect their bacterial host against 40 heterotypic viral attack. Understanding the mechanisms undergirding these defense 41 systems will be critical to development of phage therapy that circumvents these 42 defenses. Additionally, such knowledge will help engineer phage-resistant bacteria of 43 industrial importance.44 45 47they are useful in diagnostics of mycobacterial infections (1), the most notable of which 48 is tuberculosis (TB), and additionally can serve as genetic tools for mycobacteria (2-5). 49Most recently, engineered mycobacteriophages have been used in therapeutic 50 3 applications to combat infections from antibiotic-resistant strains of Mycobacterium 51 abscessus (6). To date over 11,000 mycobacteriophages have been isolated, over 52 1,800 sequenced, and over 1,600 are available in GenBank (7, 8). Mycobacteriophages 53 are a small subset of the estimated 10 31 bacteriophages existing in the biosphere (9). 54 Mycobacteriophages display high levels of genetic diversity and have been divided into 55 29 genomically similar clusters (A-AC) and a group of singletons with no close relatives 56 (10, 7). Although an increase in isolation and genomic characterization of 57 mycobacteriophages has occurred recently, the void in knowledge about gene 58 expression and function of mycobacteriophage gene products remains. 59 Prophages make up a majority of the known bacteriophage population (11). The 60 relationship between prophages and bacterial strains has shown numerous benefits to 61 both the hosts and phages. Prophages confer many advantages to the host upon 62 integration such as enhanced fitness, reduction of mutation rates, selective advantages, 63 and defense against additional viral attack (12). The bacterial host in turn provides the 64 phages with protection from harsh environments (12). In this context, numerous 65 mechanisms of defense have been recently discovered for Pseudomonas, 66 Mycobacterium, and Gordonia prophages...

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Many sequenced bacterial genomes, including those of pathogenic bacteria, contain prophages. Some prophages encode defense systems that protect their bacterial host against heterotypic viral attack. Understanding the mechanisms undergirding these defense systems is crucial to appreciate the scope of bacterial immunity against viral infections and will be critical for better implementation of phage therapy that would require evasion of these defenses. Furthermore, such knowledge of prophage-encoded defense mechanisms may be useful for developing novel genetic tools for engineering phage-resistant bacteria of industrial importance.

The correct functioning of cells requires the orchestration of multiple cellular processes, many of which are inherently dynamical. The conditions under which these dynamical processes entrain each other remain unclear. Here we use synthetic biology to address this question in the case of concurrent cellular oscillations. Specifically, we study at the single-cell level the interaction between the cell division cycle and a robust synthetic gene oscillator in Escherichia coli. Our results suggest that cell division is able to partially entrain the synthetic oscillations under normal growth conditions, by driving the periodic replication of the genes involved in the oscillator. Coupling the synthetic oscillations back into the cell cycle via the expression of a key regulator of chromosome replication increases the synchronization between the two periodic processes. A simple computational model allows us to confirm this effect.

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