Colette E. O'Neill, Ian N. Clarke and Derek J. Fisher
from: Chlamydia Biology: From Genome to Disease (Edited by: Ming Tan, Johannes H. Hegemann and Christine Sütterlin). Caister Academic Press, U.K. (2020) Pages: 241-262.
Since the seminal publication in 2011 from Wang and colleagues on plasmid-based gene transfer and the introduction of competence in Chlamydia trachomatis, numerous significant advances have been made in the field of chlamydial genetics using their transformation technique. Collectively, we can now generate targeted insertions and gene deletions in the chromosome, transform four chlamydial species with a variety of different shuttle vectors, construct random chromosomal mutants using chemical and transposon mutagenesis methods, and even generate conditional gene knockdowns using CRISPRi technology. In this chapter, we seek to provide background on how the field of chlamydial genetics grew, where it currently rests, and the needs that remain. While the transformation problem has been overcome, the low transformation frequencies and obligatory intracellular lifestyle are a significant bottleneck. Consequently, mutant construction remains tedious and operon structure and gene essentiality complicate both mutant construction and analysis. Pitfalls that arise when new technologies are brought to bear in a field starved of experimental approaches long practiced for other bacteria are also discussed along with standardization of protocols. It is our hope that this approach will yield significant and reproducible insights into the molecular details underlying the pathogenesis of Chlamydia read more ...