A method for creating in-frame insertions of fluorescent proteins in non-model gram-negative bacteria

The use of fluorescent proteins to study protein expression and localisation has become common practice in the life sciences. While methods to create gene fusions and replacements with fluorescent proteins in model organisms have rapidly developed, there exist far fewer well-established protocols applicable to non-model bacteria. Here, we present a comprehensive account of an allelic-exchange-based mutagenesis strategy using the I-SceI endonuclease in a clinical strain of S. maltophilia. We demonstrate the use of this strategy for the creation of in-frame insertions of fluorescent proteins and entire gene replacements for the purposes of studying protein localisation and expression. This protocol requires minimal setup, and once optimised, can produce mutants in a matter of weeks. We expect this strategy to be of use for laboratories working with poorly-characterised strains and/or bacteria for which information is scarce.