A markerless deletion method for genetic manipulation of Burkholderia cenocepacia and other multidrug-resistant gram-negative bacteria

<p>Genetic manipulation of multidrug-resistant bacteria is often difficult and hinders progress in understanding their physiology and pathogenesis. This book chapter highlights advances in genetic manipulation of <em class="a-plus-plus">Burkholderia cenocepacia</em>, which are also applicable to other members of the <em class="a-plus-plus">Burkholderia cepacia</em> complex and multidrug-resistant gram-negative bacteria of other genera. The method detailed here is based on the I-SceI homing endonuclease system, which can be efficiently used for chromosomal integration, deletion, and genetic replacement. This system creates markerless mutations and insertions without leaving a genetic scar and thus can be reused successively to generate multiple modifications in the same strain.</p>