Abstract
Progress in understanding molecular mechanisms contributing to chlamydial pathogenesis has been greatly facilitated by recent advances in genetic manipulation of C. trachomatis. Valuable approaches such as random, chemically induced mutagenesis or targeted, insertion-based gene disruption have led to significant discoveries. We describe herein a technique for generating definitive null strains via complete deletion of chromosomal genes in C. trachomatis. Fluorescence-reported allelic exchange mutagenesis (FRAEM), using the suicide vector pSUmC, enables targeted deletion of desired chromosomal DNA. The protocol provided here describes steps required to produce transformation competent chlamydiae, generate a specific allelic exchange plasmid construct, carry out mutagenesis, and isolate clonal populations of resulting mutant strains.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Nakashima N, Miyazaki K (2014) Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci 15:2773
AbdelRahman Y, Belland R (2005) The chlamydial developmental cycle. FEMS Microbiol Rev 29:949–959
Zhong G (2017) Chlamydial plasmid-dependent pathogenicity. Trends Microbiol 25:141–152
Song L, Carlson JH, Whitmire WM, Kari L, Virtaneva K, Sturdevant DE et al (2013) Chlamydia trachomatis plasmid-encoded Pgp4 is a transcriptional regulator of virulence-associated genes. Infect Immun 81:636–644
Mueller KE, Wolf K, Fields KA (2016) Gene deletion by fluorescence-reported allelic exchange mutagenesis (FRAEM) in Chlamydia trachomatis. MBio 7:e01817–e01815
Bauler LD, Hackstadt T (2014) Expression and targeting of secreted proteins from Chlamydia trachomatis. J Bacteriol 196:1325–1334
Mueller KE, Wolf K, Fields KA (2017) Chlamydia trachomatis transformation and allelic exchange mutagenesis. Curr Protoc Microbiol 45:11A.3.1–11A.3.15
Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison Iii CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343
Wang Y, Kahane S, Cutcliffe LT, Skilton RJ, Lambden PR, Clarke IN (2012) Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector. PLoS Pathog 7:e1002258
Scidmore MA (2005) Cultivation and laboratory maintenance of Chlamydia trachomatis. In: Current protocols in microbiology. Wiley, New York
Silayeva O, Barnes AC (2018) Gibson assembly facilitates bacterial allelic exchange mutagenesis. J Microbiol Methods 144:157–163
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Wolf, K., Rahnama, M., Fields, K.A. (2019). Genetic Manipulation of Chlamydia trachomatis: Chromosomal Deletions. In: Brown, A. (eds) Chlamydia trachomatis. Methods in Molecular Biology, vol 2042. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9694-0_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9694-0_11
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9693-3
Online ISBN: 978-1-4939-9694-0
eBook Packages: Springer Protocols