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Relatively few virulence genes have been identified in pathogenic mycoplasmas, so we used signature-tagged mutagenesis to identify mutants of the avian pathogen Mycoplasma gallisepticum with a reduced capacity to persist in vivo and compared the levels of virulence of selected mutants in experimentally infected chickens. Four mutants had insertions in one of the two incomplete oppABCDF operons, and a further three had insertions in distinct hypothetical genes, two containing peptidase motifs and one containing a member of a gene family. The three hypothetical gene mutants and the two with insertions in oppD 1 were used to infect chickens, and all five were shown to have a reduced capacity to induce respiratory tract lesions. One oppD 1 mutant and the MGA_1102 and MGA_1079 mutants had a greatly reduced capacity to persist in the respiratory tract and to induce systemic antibody responses against M. gallisepticum. The other oppD 1 mutant and the MGA_0588 mutant had less capacity than the wild type to persist in the respiratory tract but did elicit systemic antibody responses. Although M. gallisepticum carries two incomplete opp operons, one of which has been acquired by horizontal gene transfer, our results suggest that one of the copies of oppD may be required for full expression of virulence. We have also shown that three hypothetical genes, two of which encode putative peptidases, may be required for full expression of virulence in M. gallisepticum. None of these genes has previously been shown to influence virulence in pathogenic mycoplasmas. KEYWORDS Mycoplasma gallisepticum, oppD, virulence Mycoplasma gallisepticum is the most important mycoplasmal pathogen in poultry (1, 2) and a particularly useful model of mycoplasmal pathogenesis, in part as a typical representative of the Pneumoniae phylogenic group, which includes the important human pathogen M. pneumoniae. There have been relatively few studies attempting to identify virulence genes in mycoplasmas, in part because of the limited availability of tools for such studies. Signature-tagged mutagenesis (STM) is a useful technique for identifying genes likely to be involved in virulence, as it enables the rapid comparison of populations of mutants used to inoculate animals with those recovered from these animals during the course of infection, allowing identification of mutants with a reduced capacity to persist in experimentally infected animals (3, 4). However, the examination of only pools of mutants may result in a failure to distinguish between a mutation in a gene that is essential in vivo and a mutation in a gene that reduces the capacity of the mutant to compete with other mutants in vivo. In addition, it is not possible to fully assess the virulence of individual mutants within populations. Therefore, STM needs to be complemented with direct comparisons of isolated mutants for their capacity to infect, persist, and cause disease.

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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.