Improved transformation efficiency in enables disruption of the MIB-MIP system targeting human immunoglobulins.

The pathogenicity of is poorly understood, mainly due to the absence of efficient genetic tools. A polyethylene glycol-mediated transformation protocol was recently developed for the reference strain M132 using the pMT85-Tet plasmid. The transformation efficiency remained low, hampering generation of a large mutant library. In this study, we improved transformation efficiency by designing -specific pMT85 derivatives. Using the Gibson Assembly, the -derived (M) gene of the pMT85-Tet plasmid was replaced by that of a clinical isolate. Next, the -derived spiralin gene promoter driving (M) expression was substituted by one of three putative regulatory regions (RRs): the arginine deiminase RR, the elongation factor Tu RR, or the 68 bp SynMyco synthetic RR. SynMyco-based construction led to a 100-fold increase in transformation efficiency in M132. This construct was also transformed into the PG21 reference strain and three other clinical isolates. The transposon insertion locus was determined for 128 M132-transformants. The majority of the impacted coding sequences encoded lipoproteins and proteins involved in DNA repair or in gene transfer. One transposon integration site was in the mycoplasma immunoglobulin protease gene. Phenotypic characterization of the mutant showed complete disruption of the human antibody cleavage ability of the transformant. These results demonstrate that our -optimized plasmid can be used to generate large random transposon insertion libraries, enabling future studies of the pathogenicity of . IMPORTANCE is an opportunistic human pathogen, whose physiopathology is poorly understood and for which genetic tools for transposition mutagenesis have been unavailable for years. A PEG-mediated transformation protocol was developed using the pMT85-Tet plasmid, but the transformation efficiency remained low. We designed a modified pMT85-Tet plasmid suitable for . The use of a synthetic regulatory region upstream of the antibiotic resistance marker led to a 100-fold increase in the transformation efficiency. The generation and characterization of large transposon mutagenesis mutant libraries will provide insight into pathogenesis. We selected a transformant in which the transposon was integrated in the locus encoding the immunoglobulin cleavage system MIB-MIP. Phenotypic characterization showed that the wild-type strain has a functional MIB-MIP system, whereas the mutant strain had lost the ability to cleave human immunoglobulins.