Identification of genes involved in Mycoplasma gallisepticum biofilm formation using mini-Tn4001-SGM transposon mutagenesis

Mycoplasma gallisepticum (MG) is an important pathogen that can cause chronic respiratory disease in chickens and infectious sinusitis in turkeys. MG has the ability to form biofilms. The molecular mechanisms underlying MG biofilm formation are complex and poorly understood. To better understand the mechanisms involved in biofilm formation, mini-Tn4001-SGM, a novel transposon vector containing the gentamicin gene was constructed and electroporated into MG strain Rlow. Of the 738 mutants obtained, 12 had significantly reduced capacity to form biofilms in a polystyrene microtiter-plate biofilm assay. Ten different genes were identified as disrupted in these mutants using genomic walking from the transposon insertion sites and Southern bolt hybridization with a transposon-based probe. Four genes were associated with cellular processes, especially synthesis of extracellular polysaccharide and several lipoproteins encoded. Other genes were associated with translation, metabolism and gene regulation, and one had unknown function. Seven genes identified in this study have been previously associated with biofilm formation in MG or other bacterial species. The other three have not been previously reported to play a role in biofilm formation in MG. In conclusion, a new transposon vector was shown to be a powerful tool for future studies of MG pathogenesis. This study adds to our understanding of the molecular mechanisms involved in MG biofilm formation and may shed light on the persistence of MG infections.