Person:

Chen, Anna Hang

The spatial and temporal control of chromosome duplication and segregation is crucial for proper cell division. While this process is well studied in eukaryotic and some prokaryotic organisms, relatively little is known about it in prokaryotic polyploids such as Synechococcus elongatus PCC 7942, which is known to possess one to eight copies of its single chromosome. Using a fluorescent repressor-operator system, S. elongatus chromosomes and chromosome replication forks were tagged and visualized. We found that chromosomal duplication is asynchronous and that the total number of chromosomes is correlated with cell length. Thus, replication is independent of cell cycle and coupled to cell growth. Replication events occur in a spatially random fashion. However, once assembled, replisomes move in a constrained manner. On the other hand, we found that segregation displays a striking spatial organization in some cells. Chromosomes transiently align along the major axis of the cell and timing of alignment was correlated to cell division. This mechanism likely contributes to the non-random segregation of chromosome copies to daughter cells.

Background: Cyanobacteria play a significant role in the global carbon cycle. In Synechococcuselongatus, the carbon-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is concentrated into polyhedral, proteinaceous compartments called carboxysomes. Methodology/Principal Findings Using live cell fluorescence microscopy, we show that carboxysomes are first detected as small seeds of RuBisCO that colocalize with existing carboxysomes. These seeds contain little or no shell protein, but increase in RuBisCO content over several hours, during which time they are exposed to the solvent. The maturing seed is then enclosed by shell proteins, a rapid process that seals RuBisCO from the cytosol to establish a distinct, solvent-protected microenvironment that is oxidizing relative to the cytosol. These closure events can be spatially and temporally coincident with the appearance of a nascent daughter RuBisCO seed. Conclusions/Significance: Carboxysomes assemble in a stepwise fashion, inside-to-outside, revealing that cargo is the principle organizer of this compartment’s biogenesis. Our observations of the spatial relationship of seeds to previously formed carboxysomes lead us to propose a model for carboxysome replication via sequential fission, polymerization, and encapsulation of their internal cargo.

The goal of synthetic biology is to engineer biological systems in order to solve industrial and medical challenges, as well as to learn about these systems by building. Cyanobacteria, a chassis for such engineering, are major players in the global carbon cycle and their ability to fix carbon has been harnessed to produce various chemicals, including biofuels. In addition, cyanobacteria possess remarkable spatial and temporal organization in the cell. In this dissertation, I monitor, break down, and rebuild the molecular components necessary for this spatial and temporal coordination of cyanobacterial growth. These studies give us a better understanding of basic cyanobacterial biology and enable the further development of cyanobacteria for synthetic biology applications. In chapter 1, I describe the arrangement of the cyanobacterial chromosomes over time, showing the mechanisms regulating chromosome duplication and segregation. The polyploid nature of cyanobacteria make this study relevant to their efficient genome engineering. In chapter 2, I elucidate the assembly of the primary carbon fixation machinery, carboxysomes. I show that the internal cargo of carboxysomes, RuBisCO, seeds assembly, followed by the recruitment of shell proteins, which form a solvent-protected microenvironment. Finally, in chapter 3, I engineer a synthetic circadian clock from cyanobacterial components in a heterologous organism, E. coli. I demonstrate the clock's modularity and pave the way for its use in medical and industrial applications. Taken together, this work furthers our understanding of cyanobacterial physiology and forms a foundation for their efficient engineering to increase their carbon fixation capabilities. Furthermore, the fundamental spatial and temporal organization strategies elucidated here can serve as inspiration for the engineering of heterologous systems that can serve similar purposes in different contexts.