<p>Cell division in the Gram-positive bacterium <i>Streptomyces coelicolor</i> starts with the assembly of the tubulin homologue FtsZ into a cytokinetic ring (the Z ring) at the site of septation. In stark contrast to the binary fission of most bacteria, the syncytial hyphal cells of <i>S. coelicolor</i> exploit two types of cell division with strikingly different outcomes depending on the developmental stage. </p><p>The main goal of this study has been to identify developmental mechanisms that modulate this differential performance of the basic cell division machinery.</p><p>By isolation and characterization of a non-sporulating <i>ftsZ</i> mutant, we demonstrated that the requirements for Z-ring formation differ between the two types of septation. The <i>ftsZ17</i>(Spo) mutation abolished septation without overtly affecting vegetative growth. This mutant was defective in the assembly of FtsZ into regularly spaced Z rings in sporogenic hyphae, suggesting that the assembly of Z rings is developmentally controlled during sporulation.</p><p>An FtsZ-EGFP translational fusion was constructed and used to visualize the progression of FtsZ ring assembly in vivo. This revealed that polymerization of FtsZ occurred throughout the sporogenic cell, with no evidence for pre-determined nucleation sites, and that the placement of multiple Z rings is a dynamic process and involves remodeling of spiral-shaped FtsZ intermediates into regularly spaced rings. </p><p>The dynamics of the multiple Z-rings assembly during sporulation was perturbed by the action of the protein CrgA, which is important for coordinating growth and cell division in sporogenic hyphae. CrgA was also found to affect the timing of <i>ftsZ</i> expression and the turnover of the FtsZ protein. </p><p><i>S. coelicolor</i> is the main genetic model of the streptomycetes, which are major industrial antibiotic producers. The control of cell division in these organisms differs from that of other bacteria like <i>Escherichia coli</i>. Thus, it is of fundamental importance to clarify how the streptomycetes reproduce themselves. </p>

Cell division in the Gram-positive bacterium Streptomyces coelicolor starts with the assembly of the tubulin homologue FtsZ into a cytokinetic ring (the Z ring) at the site of septation. In stark contrast to the binary fission of most bacteria, the syncytial hyphal cells of S. coelicolor exploit two types of cell division with strikingly different outcomes depending on the developmental stage. The main goal of this study has been to identify developmental mechanisms that modulate this differential performance of the basic cell division machinery. By isolation and characterization of a non-sporulating ftsZ mutant, we demonstrated that the requirements for Z-ring formation differ between the two types of septation. The ftsZ17(Spo) mutation abolished septation without overtly affecting vegetative growth. This mutant was defective in the assembly of FtsZ into regularly spaced Z rings in sporogenic hyphae, suggesting that the assembly of Z rings is developmentally controlled during sporulation. An FtsZ-EGFP translational fusion was constructed and used to visualize the progression of FtsZ ring assembly in vivo. This revealed that polymerization of FtsZ occurred throughout the sporogenic cell, with no evidence for pre-determined nucleation sites, and that the placement of multiple Z rings is a dynamic process and involves remodeling of spiral-shaped FtsZ intermediates into regularly spaced rings. The dynamics of the multiple Z-rings assembly during sporulation was perturbed by the action of the protein CrgA, which is important for coordinating growth and cell division in sporogenic hyphae. CrgA was also found to affect the timing of ftsZ expression and the turnover of the FtsZ protein. S. coelicolor is the main genetic model of the streptomycetes, which are major industrial antibiotic producers. The control of cell division in these organisms differs from that of other bacteria like Escherichia coli. Thus, it is of fundamental importance to clarify how the streptomycetes reproduce themselves.

The stochastic nature of intracellular chemistry guarantees that even genetically identical cells sharing an environment will differ in composition. The question of whether this chemical diversity translates into significant phenotypic individuality is tied to the relative timescales of the processes involved. In order for cells in a population to have distinct functional identities, they must maintain their states for an appreciable period of time. Quantification of these timescales requires accurate time-lapse measurements covering tens or even hundreds of generations, a technical hurdle that has left these questions largely underexplored. In this thesis I present three pieces of work that aim to provide a foundation for the study of fluctuation timescales in bacteria. In the first part, I describe modifications to a recently developed microfluidic platform for continuous culture of cells under constant conditions. These revised devices enable the high-throughput, long-term measurement of gene expression dynamics while eliminating several confounding experimental factors that interfere with timescale measurements. In the second part, I employ one of these devices to survey fluctuation timescales in ~50 reporters for Eshcerichia coli gene expression. Under rich conditions, all reporters exhibited nearly identical, rapid fluctuation dynamics that were captured by a simple model of gene expression. In contrast, under poor nutritional conditions gene expression states became correlated over several cell divisions. However, accounting for instantaneous growth rate fluctuations eliminated these slow timescales, revealing an exceedingly simple behavior. In the third part, I describe our work to dissect the stochastic transition between the solitary motile state and sessile multicellular state in exponentially growing Bacillus subtilis</italic.. By enforcing static environmental conditions, we uncover the cell's internal strategies for state switching. The transition to the multicellular state occurs without regard to the cell's state history, whereas commitment to the multicellular state is tightly timed. By manipulating the genetic circuit responsible for the switch, we also expose surprising functional modularity in the commitment. I believe that the striking range of gene expression timescales we observe--from the fast fluctuations in E. coli gene expression to the feedback-amplified noise in B. subtilis--will serve as a useful starting point for future studies.

Chlamydia are obligate intracellular bacterial pathogens that are responsible for infectious blindness, sexually transmitted infections, and acute respiratory disease in humans. These pathogens undergo an essential biphasic developmental cycle differentiating between two functionally distinct forms known as the infectious elementary body (EB) and the replicative reticulate body (RB). Identifying the signals and regulatory mechanisms that enable Chlamydia to establish infection, differentiate between the two developmental forms, and survive within the host cell is critical to understanding chlamydial pathogenesis and developing future therapeutic strategies. In pathogenic bacteria, serine, threonine, and tyrosine (Ser/Thr/Tyr) protein kinases and phosphatases are critical for development, metabolism, and virulence. Chlamydia encode two validated protein kinases (pkn1 and pknd), a putative protein phosphatase (ctl0511; CppA), and appear capable of global phosphorylation that differs between the developmental forms. While these findings support a role for protein phosphorylation in chlamydial pathogenesis, a validated cognate protein phosphatase for Pkn1 and PknD mediating reversible phosphorylation was lacking. We hypothesized that CppA is the partner phosphatase for the chlamydial protein kinases, and in this study we validated and characterized CppA as a broad specificity protein phosphatase type 2C. Using in vivo and in vitro approaches we demonstrated that CppA acts on P-Ser/Thr/Tyr residues and can dephosphorylate multiple chlamydial protein substrates including PknD and the FHA 2 domain of CdsD, a component of the type 3 secretion apparatus. The importance of CppA for chlamydial growth and development was determined using a chemical “knock-out” approach and study of CppA missense mutations identified in slow growing C. trachomatis L2 chemical mutants. Treatment of C. trachomatis L2, C. trachomatis D, and C. muridarum with CppA inhibitors significantly reduced progeny levels and inclusion size in a time dependent manner with more significant growth inhibition in the first 12 hours post infection. Collectively, our findings support that CppA works in conjunction with PknD, and likely Pkn1, to mediate reversible phosphorylation of multiple protein substrates leading to changes in chlamydial physiology that appear to be key for early steps in development.