Rational engineering of Mycoplasma pneumoniae as attenuated chassis. Genome sequencing of Mycoplasma pneumoniae CV2

M. pneumoniae is generally considered as a mild infectious agent, and its few pathogenic determinants are well characterized. Its reduced genome and simplified gene and metabolic networks makes it an ideal organism for synthetic biology applications. In order to generate an attenuated chassis strain based on M. pneumoniae, we removed the different pathogenic genes using recently developed genome editing tools for this bacterium: the Community-Acquired Respiratory Distress Syndrome (CARDS) toxin (mpn372) and the Ca2+-dependent cytotoxic membrane-associated nuclease (mpn133). The genome of the resulting ∆mpn133∆mpn372 strain, called CV2, was sequenced on Illumina MiSeq platform and assembled to confirm the genetic modifications.