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MalF has been shown to be required for virulence in the important avian pathogen Mycoplasma gallisepticum. To characterise the function of MalF, predicted to be part of putative ABC transporter, we compared metabolite profiles of a mutant with a transposon inserted in malF (MalF-deficient ST mutant 04-1; ΔmalF) with those of wild type bacteria using GC/MS and LC/MS. Of those substrates likely to be transported by an ABC transport system, glycerol was detected at significantly lower abundance in the ΔmalF mutant, when compared to the wild type. Stable isotope labelling using [U-13C] glycerol and RT-qPCR analysis indicated that MalF was responsible for import of glycerol into M. gallisepticum and that, in the absence of MalF, the transcription of gtsA, which encodes a second transporter, GtsA, was upregulated, potentially to increase import of glycerol-3-phosphate into the cell to compensate for the loss of MalF. The loss of MalF appeared to have a global effect on glycerol metabolism, suggesting that it may also play a regulatory role, and cellular morphology was also affected, indicating that the change to glycerol metabolism may have a broader effect on cellular organisation. Overall, this study suggests that the reduced virulence of the ΔmalF mutant is due to perturbed glycerol uptake and metabolism, and that the operon including malF should be reannotated to golABC to reflect its function in glycerol transport. Importance Many mycoplasmas are pathogenic and cause disease in human and animals. M. gallisepticum causes chronic respiratory disease in chickens and infectious sinusitis in turkeys, resulting in economic losses in poultry industries throughout the world. To expand our knowledge about the pathogenesis of mycoplasma infections requires better understanding about the specific gene functions of these bacteria. In this study, we have characterised the metabolic function of a protein involved in pathogenicity of M. gallisepticum, as well as its effect on expression of selected genes, cell phenotype and H2O2 production. This study is a key step forward in understanding why this protein plays a key role in virulence in chickens. This study also emphasises the importance offunctional characterisation of mycoplasma proteins, using tools such as metabolomics, as prediction of function based on homology to other bacterial proteins is not always accurate.

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