**7.1 Molecular manipulation of** *Chlamydia*

With the recent advances in the molecular genetic manipulation of *Chlamydia,* it is now possible to perform targeted gene inactivation, whole-genome sequencing to identify mutations, and plasmid transformation to generate fluorescent reporter strains to identify proteins involved in the pathogenesis of *Chlamydia* [96]. On the other hand, the ability to express exogenous proteins, epitope tags, and fluorescent and other reporter proteins in *Chlamydia* has expanded the repertoire of possible technologies to study the *Chlamydia*–host interface [97]. High-resolution microscopy has complemented the advances in genetic and biochemical approaches [96].

Molecular manipulation in *Chlamydia* takes advantage of the sequencing of the first *Chlamydiae* genome [98]. *C. trachomatis* can insert exogenous DNA into its genome because it encodes an intact DNA recombination machinery that facilitates the development of a stable transformation system of *Chlamydia* with recombinant DNA [99]. This transformation system has enabled the construction of a series of shuttle vectors for gene inactivation by targeted gene knockouts with versatile multiple-cloning sites (MCS), fluorescent protein reporters, inducible promoters, and new selectable markers. Strategies to mediate targeted genetic modifications, such as gene disruptions and gene replacements, include the Targeting Induced Local Lesions in Genomes (TILLING) technology and TargeTron, based on the transient transformation of *Chlamydia* with a plasmid that encodes an altered group-II intron (Reviewed in Ref. [100]). The recent development of a Fluorescence-reported allelic exchange mutagenesis (FRAEM) using the suicide vector pSUmC has allowed the generation of null mutation strains via the complete deletion of chromosomal genes in *C. trachomatis* [101].

### **7.2 Genome-scale analyses**
