Final Report Summary - COSP (Identification and characterization of factors involved in chromosome organization and septum positioning in bacteria)
Discovery and characterization of important factors that help the model bacterium B. subtilis to organize its DNA
The Marie-Curie IEF project COSP was aimed at the identification of novel factors that help to organize the cellular DNA (the chromosome) and therefore contribute to efficient chromosome segregation (equally dividing of the chromosome copies of the DNA between two daughter cells) during the cell cycle of the soil bacterium Bacillus subtilis. Since B. subtilis is a highly accessible organism in terms of molecular biology and genetics, and there is already a profound understanding of cell cycle-related processes in this bacterium, it was an obvious choice to use it as the object of study for the project. The Errington lab has a long history in investigating the B. subtilis cell cycle, and its ability to form spores that can survive adverse conditions. The molecular processes going on during spore formation make it particularly apt for the analysis of chromosome organization. It was previously shown that a number of protein factors are involved that ensure that the chromosome is efficiently divided between two daughter cells during sporulation.
In this project we set out to develop a genetic screening system based on B. subtilis sporulation. We used a reporter gene that enables visual selection of interesting bacterial colonies on the basis of their colour (blue or white). Spore formation in B. subtilis can be stimulated by growing and then starving a bacterial culture. In the first stages of sporulation the cell divides asymmetrically, with the cell division septum being placed close to one of the cell poles. At this stage, the dividing cell contains two chromosomes, one for the cellular part that eventually becomes the spore (prespore) and one for the mother cell compartment. Initially, only about 1/3 of the chromosome is present in the prespore compartment, comprising a specific region surrounding the origin of chromosome replication (oriC); the remaining part of this chromosome resides in the mother cell compartment. This incorrectly positioned chromosome segments is then moved into the prespore by a transporter protein called SpoIIIE. Mutations abolishing SpoIIIE function trap the cells in a state in which the prespore chromosome is incompletely segregated with only the origin region in the correct compartment. This feature of spoIIIE mutants provides a powerful tool with which to probe the cellular mechanisms that place the oriC region specifically close to the prespore cell pole.
The mother cell and the prespore have distinct genetic programs determining their respective fates. Genetic constructs based on the different genetic programs of prespore and mother cell were designed that allowed us to probe changes in the location of chromosomal regions near to (proximal) or distant from (distal) oriC. Especially fruitful turned out to be a screen that looked for mutations leading to aberrant localization of oriC outside of the prespore, i.e. in the mother cell compartment. A pool of random bacterial mutants was screened to find mutants in which the localization of oriC was perturbed. The genes identified by these mutations lay in the soj gene and two other genes not previously implicated in chromosome segregation. Characterisation of these genes has provided important insights into their functions.
Our results give new insights into the details of chromosome organization in B. subtilis. Understanding this and other aspects of the bacterial cell cycle, which are vital for bacterial growth, may have important implications for antimicrobial drug development.
The Marie-Curie IEF project COSP was aimed at the identification of novel factors that help to organize the cellular DNA (the chromosome) and therefore contribute to efficient chromosome segregation (equally dividing of the chromosome copies of the DNA between two daughter cells) during the cell cycle of the soil bacterium Bacillus subtilis. Since B. subtilis is a highly accessible organism in terms of molecular biology and genetics, and there is already a profound understanding of cell cycle-related processes in this bacterium, it was an obvious choice to use it as the object of study for the project. The Errington lab has a long history in investigating the B. subtilis cell cycle, and its ability to form spores that can survive adverse conditions. The molecular processes going on during spore formation make it particularly apt for the analysis of chromosome organization. It was previously shown that a number of protein factors are involved that ensure that the chromosome is efficiently divided between two daughter cells during sporulation.
In this project we set out to develop a genetic screening system based on B. subtilis sporulation. We used a reporter gene that enables visual selection of interesting bacterial colonies on the basis of their colour (blue or white). Spore formation in B. subtilis can be stimulated by growing and then starving a bacterial culture. In the first stages of sporulation the cell divides asymmetrically, with the cell division septum being placed close to one of the cell poles. At this stage, the dividing cell contains two chromosomes, one for the cellular part that eventually becomes the spore (prespore) and one for the mother cell compartment. Initially, only about 1/3 of the chromosome is present in the prespore compartment, comprising a specific region surrounding the origin of chromosome replication (oriC); the remaining part of this chromosome resides in the mother cell compartment. This incorrectly positioned chromosome segments is then moved into the prespore by a transporter protein called SpoIIIE. Mutations abolishing SpoIIIE function trap the cells in a state in which the prespore chromosome is incompletely segregated with only the origin region in the correct compartment. This feature of spoIIIE mutants provides a powerful tool with which to probe the cellular mechanisms that place the oriC region specifically close to the prespore cell pole.
The mother cell and the prespore have distinct genetic programs determining their respective fates. Genetic constructs based on the different genetic programs of prespore and mother cell were designed that allowed us to probe changes in the location of chromosomal regions near to (proximal) or distant from (distal) oriC. Especially fruitful turned out to be a screen that looked for mutations leading to aberrant localization of oriC outside of the prespore, i.e. in the mother cell compartment. A pool of random bacterial mutants was screened to find mutants in which the localization of oriC was perturbed. The genes identified by these mutations lay in the soj gene and two other genes not previously implicated in chromosome segregation. Characterisation of these genes has provided important insights into their functions.
Our results give new insights into the details of chromosome organization in B. subtilis. Understanding this and other aspects of the bacterial cell cycle, which are vital for bacterial growth, may have important implications for antimicrobial drug development.