Abstract
Cell division is an essential cellular process that requires an array of known and unknown proteins for its spatial and temporal regulation. Here we develop a novel, high-throughput screening method for the identification of bacterial cell division genes and regulators. The method combines the over-expression of a shotgun genomic expression library to perturb the cell division process with high-throughput flow cytometry sorting to screen many thousands of clones. Using this approach, we recovered clones with a filamentous morphology for the model bacterium, Escherichia coli. Genetic analysis revealed that our screen identified both known cell division genes, and genes that have not previously been identified to be involved in cell division. This novel screening strategy is applicable to a wide range of organisms, including pathogenic bacteria, where cell division genes and regulators are attractive drug targets for antibiotic development.
Highlights
Bacterial cell division is essential to bacterial survival, and must be tightly controlled and regulated to ensure the successful generation of two identical daughter cells
To generate populations of varying cell lengths for the development of a flow cytometry analysis and sorting technique, E. coli DH5a cells were treated with the antibiotic cephalexin
We have developed a method for the identification of novel bacterial cell division proteins and regulators using shotgun over– expression of genomic libraries and flow cytometry sorting
Summary
Bacterial cell division is essential to bacterial survival, and must be tightly controlled and regulated to ensure the successful generation of two identical daughter cells. FtsZ, and many other members of the divisome complex (FtsA, FtsN etc.), were identified from temperature sensitive mutations, which result in the formation of filaments (long cells without septa) at the non-permissive temperature [9] Several of these genes are conserved, to varying degrees, and have subsequently been identified in a wide range of bacteria via gene homology [10]. Nucleoid occlusion prevents Z rings forming over the nucleoid or chromosome, while the Min system inhibits the assembly of Z-rings at the cell poles; as the replicated chromosomes segregate, nucleoid occlusion is relieved at midcell allowing formation of a Z ring at this site [14] These proteins alone cannot solely account for the regulation of division site placement, as a recent study has shown that in B. subtilis, Z rings, while much less frequent, are still positioned precisely at midcell in the absence of Noc and Min [15]. The question of how cell division is regulated in bacteria is yet to be completely answered
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